Albert: Albert & Jakobiec's Principles &

Practice of Ophthalmology

THIRD EDITION

Daniel M. Albert, MD MS
Chair Emeritus, F. A. Davis Professor and Lorenz F. Zimmerman Professor, Department of
Ophthalmology and Visual Sciences, Retina Research Foundation Emmett A. Humble Distinguished
Director, of the Alice R. McPherson, MD, Eye Research Institute, University of Wisconsin
Medical School, Madison, Wisconsin, USA
Joan W. Miller, MD
Henry Willard Williams Professor of Ophthalmology, Chief and Chair, Department of
Ophthalmology, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston,
Massachusetts, USA
Associate Editors:
Dimitri T. Azar, MD
B.A. Field Chair of Ophthalmologic Research, Professor and Head, Department of Ophthalmology
and Visual Sciences, University of Illinois Eye and Ear Infirmary, Chicago, Illinois, USA
Barbara A. Blodi, MD
Associate Professor, Department of Ophthalmology and Visual Sciences, University of Wisconsin
Medical School, Madison, Wisconsin, USA
Managing Editors:
Janet E. Cohan
Administrative Manager, Department of Ophthalmology, Massachusetts Eye and Ear Infirmary,
Harvard Medical School, Boston, Massachusetts, USA
Tracy Perkins, MPH
Administrative Director, Alice R. McPherson, MD Eye Research Institute, University of
Wisconsin Medical School, Madison, Wisconsin, US
DEDICATION

To CLAES H. DOHLMAN
Superb surgeon, mentor, teacher, innovator and friend.
D.M.A & J.W.M
SAUNDERS ELSEVIER
SAUNDERS is an imprint of Elsevier Inc.
? 2000, 1994 by W.B Saunders Company
? 2008, Elsevier Inc. All rights reserved.
First published 2008
First edition 1994
Second edition 2000
Third edition 2008
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ISBN: 978-1-4160-0016-7
Notice
Medical knowledge is constantly changing. Standard safety
precautions must be followed, but as new research and clinical
experience broaden our knowledge, changes in treatment and drug
therapy may become necessary or appropriate. Readers are
advised to check the most current product information provided
by the manufacturer of each drug to be administered to verify
the recommended dose, the method and duration of
administration, and contraindications. It is the responsibility
of the practitioner, relying on experience and knowledge of the
patient, to determine dosages and the best treatment for each
individual patient. Neither the Publisher nor the author assume
any liability for any injury and/or damage to persons or
property arising from this publication.
The Publisher
Preface to the 3rd Edition
Do clinicians and trainees really need textbooks anymore? In an
era of ever-expanding connectivity and immediate access to
published articles, why would anyone consult a textbook, which
by its very nature is incomplete before it is even published? No
doubt these are strange questions coming from the editors of
the third edition of the most popular multi-volume ophthalmic
textbook, but they must be asked and answered. Our answer
is an unequivocal “yes”! Books like this serve an extremely
important function – that of a repository for expert reviews of
our current understanding of ophthalmic health and disease.
The chapters and sections in Albert and Jakobiec are an
important resource for the clinician and student, providing a
comprehensive information base on an extensive list of topics.
Of course journal articles continue to be the most useful source
of information about new developments in the field but they do
not replace books. Constraints on the length of journal articles,
inattention to the provenance of the ideas they contain, and an
understandable tendency to self-promote the authors’ thesis,
limit the value of many “original contributions.” Readers
of journal articles forearmed with information found in an
encyclopedic text can place these articles into perspective.
Thus, the two sources are complimentary. In a very real sense
this textbook serves as a springboard to the constantly
expanding universe of published scientific literature.
What is new in the third edition? The second edition (2002)
was a reworking of the very successful first edition (1996) of
Albert and Jakobiec’s Principles and Practice of Ophthalmology.
For the third edition we undertook a critical evaluation of each
section and chapter to ensure that topics were well-covered with
minimal redundancy, that new areas of practice and research
were adequately described, and that topics that were over-
represented could be substantially shortened or deleted. This
evaluation involved all of the editors (Dan Albert, Joan Miller,
Barbara Blodi and Dimitri Azar) as well as new and returning
section editors. As an example, under the direction of Dimitri
Azar, we incorporated a new section on refractive surgery that
provides the principles of refractive surgery as well as useful
descriptions of evaluation techniques and procedures. The
Oncology section was substantially expanded and revised under
the section editorship of Evangelos Gragoudas and Joan
O’Brien. Pediatrics was also extensively revised by David
Hunter and Monte Mills, and the Pharmacology and Toxicology
sections were combined and revised under the direction of Mark
Abelson. Barbara Blodi and Joan Miller reworked the extensive
retina section, to include current techniques, new diagnostic
modalities (including OCT), and new drug therapies. The
human genome project and modern genetics are revolutionizing
medicine, and genetics information has been incorporated into
all sections. Finally, the last section of the textbook headed by
Kathy Colby and Nancy Holekamp is a section on Ethics and
Professionalism topics that are increasingly important to
practicing clinicians, and an ACGME requirement for resident
training. A concerted effort was made throughout the third
edition to complement the text with diagrams, line drawings
and color figures. In addition, each chapter contains a key
points section. Overall, the third edition has exceeded the
expectations of all of the editors. We were pleased by the
enthusiasm of new and returning authors, more than 600 in
total, as well as new and returning section editors, and were
excited by the teamwork and cooperation shown in upgrading
and improving this important project. The result is a definitive
textbook in ophthalmology, available in hardcover and by web
access.
The editorial team has been a wonderful collaboration and
the senior editors are very grateful for the prodigious efforts of
Drs. Dimitri Azar and Barbara Blodi. We were saddened that Dr.
Frederick Jakobiec, a co-founder of this project and co-editor on
editions 1 and 2, was unable to participate as an editor in the
third edition, although still contributing as a co-author. We look
forward to his return to the ophthalmology community, and we
can report that Dr. Jakobiec is pleased and supportive of the
upcoming 3rd edition of the textbook named for him and Dr.
Albert. All of the editorial team is most appreciative of the
unstinting and generous support of Elsevier Publishing; in
particular the leadership of the senior editor, Russell Gabbedy,
and the hard work and diligence of Zak Knowles, contributing
editor, whose efforts in collecting and coordinating chapters, as
well as initial editing of chapters were unsurpassed. The
managing editors, Tracy Perkins and Janet Cohan, provided
important coordination between the authors, section editors,
editors and publisher, and handled all of their responsibilities
with aplomb. Above all, the contributing authors who wrote the
chapters and the section editors who delineated the section
content and edited the component chapters deserve the greatest
credit for the superb quality of the textbook.
We sincerely hope that the third edition of Albert and
Jakobiec’s Principles and Practice in Ophthalmology provides
ophthalmologists and trainees with a gateway into the
wonderful science and art of ophthalmology in order to provide
the best care for our patients, and to continually advance
our field.
Daniel M. Albert and Joan W. Miller
xvii
Preface to the 1st Edition
“INCIPIT.” The medieval scribe would write this Latin word,
meaning so it begins, to signal the start of the book he was
transcribing. It was a dramatic word that conveyed promise of
instruction and delight. In more modern times INCIPIT has
been replaced by the PREFACE. It may be the first thing the
reader sees, but it is, in fact, the last thing the author writes
before the book goes to press. I appreciate the opportunity to
make some personal comments regarding Principles and
Practice of Ophthalmology.
One of the most exciting things about writing and editing a
book in a learned field is that it puts the authors and editors in
touch with those who have gone before. Each author shares
with those who have labored in past years and in past centuries
the tasks of assessing the knowledge that exists in his or her
field, of determining what is important, and of trying to convey
it to his or her peers. In the course of the work the author
experiences the same anticipation, angst, and ennui of those
who have gone before. He or she can well envision the various
moments of triumph and despair that all authors and editors
must feel as they organize, review, and revise the accumulating
manuscripts and reassure, cajole, and make demands of their
fellow editors, authors, and publisher.
This feeling of solidarity with early writers becomes even
more profound when one is a collector and reviewer of books,
and conversant with the history of one’s field. In Ecclesiastes
it is stated, “of the making of books, there is no end” (12:12).
Indeed, there are more books than any other human artifact on
earth. There is, however, a beginning to the “making of books”
in any given field. The first ophthalmology book to be published
was Benvenuto Grassi’s De Oculis in Florence in 1474. Firmin
Didot in his famous Bibliographical Encyclopedia wrote that
Grassus, an Italian physician of the School of Solerno, lived in
the 12th century and was the author of two books, the Ferrara
Quarto (1474) and the Venetian Folio (1497). Eye care in the
15th century was in the hands of itinerant barber surgeons and
quacks, and a treatise by a learned physician was a remarkable
occurrence. The next book on the eye to appear was an anony-
mous pamphlet written for the layperson in 1538 and entitled
Ein Newes Hochnutzliches Büchlin von Erkantnus der
Kranckheyten der Augen. Like Principles and Practice of
Ophthalmology, the Büchlin stated its intention to provide
highly useful knowledge of eye diseases, the anatomy of the eye,
and various remedies. It was illustrated with a fullpage woodcut
of the anatomy of the eye (Fig. 1). At the conclusion of the book,
the publisher, Vogtherr, promised to bring more and better
information to light shortly, and indeed, the next year he
published a small book by Leonhart Fuchs (1501–1566) entitled
Alle Kranckheyt der Augen.
Fuchs, a fervent Hippocratist, was Professor first of Philo-
sophy and then of Medicine at Ingolstadt, Physician of the
Margrave Georg of Brandenburg, and finally Professor at
Tübingen for 31 years. Like the earlier Büchlin, his work begins
with an anatomic woodcut (Fig. 2) and then lists in tabular
form various eye conditions, including strabismus, paralysis,
amblyopia, and nictalops. The work uses a distinctly Greco
Roman terminology, presenting information on the parts of the
eye and their affections, including conjunctivitis, ophthalmia,
carcinoma, and “glaucoma.” The book concludes with a remedy
collection similar to that found in the Büchlin. Most significant
in the association of Leonhart Fuchs with this book is the fact
that a properly trained and well recognized physican addressed
the subject of ophthalmology.
Julius Hirschberg, the ophthalmic historian, noted that
Fuch’s Alle Kranckheyt, along with the anonymous Büchlin,
apparently influenced Georg Bartisch in his writing of Das Ist
Augendienst. This latter work, published in 1583, marked the
founding of modern ophthalmology. Bartisch (1535–1606) was
an itinerant barber surgeon but nonetheless a thoughtful and
skillful surgeon, whose many innovations included the first
procedure for extirpation of the globe for ocular cancer. Bartisch
proposed standards for the individual who practices eye surgery,
noting that rigorous training and concentration of effort were
needed to practice this specialty successfully.
By the late 16th century, eye surgery and the treatment of eye
disease began to move into the realm of the more formally
trained and respected surgeon. This is evidenced by Jacques
Guillemeau’s Traité des Maladies de L’Oeil, published in 1585.
Guillemeau (1550–1612) was a pupil of the surgical giant
Ambroise Paré, and his book was an epitome of the existing
knowledge on the subject.
The transition from couching of cataracts to the modern
method of treating cataracts by extraction of the lens, as
introduced by Jacques Daviel in 1753, further defined the skill
and training necessary for the care of the eyes. The initiation
of ophthalmology as a separate specialty within the realm of
medicine and surgery was signaled by the publication of George
Joseph Beer’s two volume Lehre von den Augenkrankheiten in
1813–1817. Beer (1763–1821) founded the first eye hospital in
1786 in Vienna, and his students became famous ophthalmic
surgeons and professors throughout Europe.
In England, it was not only the demands of cataract surgery
but also the great pandemic of trachoma following the Napo-
leonic wars that led to the establishment of ophthalmology as a
recognized specialty. Benjamin Travers (1783–1858) published
the earliest treatise in English on diseases of the eye, A Synopsis
of the Diseases of the Eye, in 1820. In the United States,
acceptance of ophthalmology as a specialty had to await the
description of the ophthalmoscope by Helmholtz in 1851,
and the additional special skills that using the early primitive
“Augenspiegel” required.
As the complexity of ophthalmology increased and as sub-
specialization began to develop in the 19th century, multi-
authored books began to appear. This culminated in the
appearance in 1874 of the first volume of the GraefeSaemisch xix
 Preface to the 1st Edition
FIGURE 1.
Handbuch. The final volume of this great collective work, of
which Alfred Carl Graefe (1830–1899) and Edwin Theodor
Saemisch (1833–1909) were editors, appeared in 1880. The
definitive second edition, which for more than a quarter of a
century remained the most comprehensive and authoritative
work in the field, appeared in 15 volumes between 1899 and
1918. The great French counterpart to the Graefe Saemisch
Handbuch was the Encyclopédie Française d’Ophtalmologie,
which appeared in nine volumes (1903–1910), edited by Octave
Doin, and filled a similar role for the French speaking
ophthalmologist.
In 1896, the first of four volumes of Norris and Oliver’s
System of Diseases of the Eye was published in the United
States. The senior editor, Dr. William Fisher Norris
(1839–1901), was the first Clinical Professor of Diseases of
the Eye at the University of Pennsylvania. Charles A. Oliver
(1853–1911) was his student. Norris considered the System
to be his monumental work. For each section he chose an
outstanding authority in the field, having in the end more than
60 American, British, Dutch, French, and German ophthal-
mologists as contributors. Almost 6 years of combined labor on
the part of the editors was needed for completion of the work.
In 1913, Casey A. Wood (1856–1942) introduced the first of
his 18 volumes of the American Encyclopedia and Dictionary
of Ophthalmology. The final volume appeared in 1921. Drawn
largely from the Graef Saemisch Handbuch and the Encyclo-
pédie Française d’Ophtalmologie, Wood’s Encyclopedia
provided information on the whole of ophthalmology through a
strictly alphabetic sequence of subject headings.
The book from which the present work draws inspiration
is Duke Elder’s Textbook of Ophthalmology (7 volumes; 1932)
and particularly the second edition of this work entitled System
of Ophthalmology (15 volumes, published between 1958 and
1976). The System of Ophthalmology was written by Sir
Stewart Duke Elder (1898–1978) in conjunction with his
colleagues at the Institute of Ophthalmology in London. In
1976, when the last of his 15 volumes appeared, Duke Elder
xx wrote in the Preface:
FIGURE 2.
The writing of these two series, the Textbook and the System,
has occupied all my available time for half a century. I cannot
deny that its completion brings me relief on the recovery of my
freedom, but at the same time it has left some sadness for I have
enjoyed writing it. As Edward Gibbon said on having written
the last line of The Decline and Fall of the Roman Empire:
“A sober melancholy has spread over my mind by the idea
that I have taken everlasting leave of an old and agreeable
companion.”
Duke Elder adds a final line that I hope will be more àpropos
to the present editors and contributors. “At the same time
the prayer of Sir Francis Drake on the eve of the attack of the
Spanish Armada is apposite: ‘Give us to know that it is not the
beginning but the continuing of the same until it is entirely
finished which yieldeth the true glory.”’ The void that developed
as the Duke Elder series became outdated has been partially
filled by many fine books, notably Thomas Duane’s excellent 5
volume Clinical Ophthalmology.
Inspiration to undertake a major work such as this is derived
not only from the past books but also from teachers and role
models. For me, this includes Francis Heed Adler, Harold
G. Scheie, William C. Frayer, David G. Cogan, Ludwig von
Sallmann, Alan S. Rabson, Lorenz E. Zimmerman, Frederick C.
Blodi, Claes H. Dohlman, and Matthew D. Davis.
Whereas the inspiration for the present text was derived from
Duke Elder’s Textbook and System and from teachers and role
models, learning how to write and organize a book came for
me from Adler’s Textbook of Ophthalmology, published by
W.B. Saunders. This popular textbook for medical students and
general practitioners was first produced by Dr. Sanford Gifford
(1892–1945) in 1938. Francis Heed Adler (1895–1987), after
writing the 6th edition, published in 1962, invited Harold G.
Scheie (1909–1989), his successor as Chairman of Ophthal-
mology at the University of Pennsylvania, and myself to take
over authorship. We completely rewrote this book and noted
in the Preface to the 8th edition, published in 1969: “This
book aims to provide the medical student and the practicing
physician with a concise and profusely illustrated current text,

organized in a convenient and useable manner, on the eye and
its disorders. It is hoped that the beginning, or even practicing,
ophthalmologist may find it of value.”
In 1969 it was apparent that even for the intended audience,
contributions by individuals expert in the subspecialties of
ophthalmology were required. The book was published in
Spanish and Chinese editions and was popular enough to
warrant an updated 9th edition, which appeared in 1977. One
of the high points of this work was interacting with John
Dusseau, the Editor in Chief for the W.B. Saunders Company.
As a 10th edition was contemplated, I became increasingly
convinced that what was needed in current ophthalmology was
a new, comprehensive, well illustrated set of texts intended
for the practicing ophthalmologist and written by outstanding
authorities in the field. I envisioned a work that in one series of
volumes would provide all of the basic clinical and scientific
information required by practicing ophthalmologists in their
everyday work. For more detailed or specialized information,
this work should direct the practitioner to the pertinent journal
articles or more specialized publications. As time progressed, a
plan for this work took shape and received support from the
W.B. Saunders Company.
Memories of the formative stages of the Principles and
Practice of Ophthalmology remain vivid: Proposing the project
to Frederick Jakobiec in the cafeteria of the Massachusetts
Eye and Ear Infirmary in early 1989. Having dinner with Lewis
Reines, President and Chief Executive Officer, and Richard
Zorab, Senior Medical Editor, at the Four Seasons Hotel in
May 1989, where we agreed upon the scope of the work. My
excitement as I walked across the Public Garden and down
Charles Street back to the Infirmary, contemplating the work
we were to undertake. Finalizing the outline for the book in
Henry Allen’s well stocked “faculty lounge” in a dormitory at
Colby College during the Lancaster Course. Meeting with
members of the Harvard Faculty in the somber setting of the
rare book room to recruit the Section Editors. Persuading Nancy
Robinson, my able assistant since 1969, to take on the job of
Managing Editor. The receipt of our first manuscript from Dr.
David Cogan.
We considered making this work a departmental under-
taking, utilizing the faculty and alumni of various Harvard
programs. However, the broad scope of the series required
recruitment of outstanding authors from many institutions.
Once the Section Editors were in place, there was never any
doubt in my mind that this work would succeed. The Section
Editors proved a hardworking and dedicated group, and their
choice of authors reflects their good judgment and persuasive
abilities. I believe that you will appreciate the scope of
knowledge and the erudition.
The editorship of this book provided me not only with an
insight into the knowledge and thinking of some of the finest
minds in ophthalmology but also with an insight into their
lives. What an overwhelmingly busy group of people! Work was
completed not through intimidation with deadlines but by
virtue of their love of ophthalmology and their desire to share
their knowledge and experience. The talent, commitment,
persistence, and good humor of the authors are truly what made
this book a reality.
It was our intent to present a work that was at once scholarly
and pragmatic, that dealt effectively with the complexities
and subtleties of modern ophthalmology, but that did not
overwhelm the reader. We have worked toward a series of
volumes that contained the relevant basic science information
to sustain and complement the clinical facts. We wanted a
well illustrated set that went beyond the illustrations in any
Preface to the 1st Edition
textbook or system previously published, in terms of quantity
and quality and usefulnesss of the pictures.
In specific terms, in editing the book we tried to identify
and eliminate errors in accuracy. We worked to provide as
uniform a literary style as is possible in light of the numerous
contributors. We attempted to make as consistent as possible
the level of detail presented in the many sections and chapters.
Related to this, we sought to maintain the length according to
our agreed upon plan. We tried, as far as possible, to eliminate
repetition and at the same time to prevent gaps in information.
We worked to direct the location of information into a logical
and convenient arrangement. We attempted to separate the
basic science chapters to the major extent into the separate
Basic Sciences volume, but at the same time to integrate basic
science information with clinical detail in other sections as
needed. These tasks were made challenging by the size of the
work, the number of authors, and the limited options for
change as material was received close to publishing deadlines.
We believe that these efforts have succeeded in providing
ophthalmologists and visual scientists with a useful resource in
their practices. We shall know in succeeding years the level of
this success and hope to have the opportunity to improve all
these aspects as the book is updated and published in future
editions. Bacon wrote: “Reading maketh a full man, conference
a ready man, and writing an exact man.” He should have added:
Editing maketh a humble man.
I am personally grateful to a number of individuals for
making this book a reality. Nancy Robinson leads the list. Her
intelligent, gracious, and unceasing effort as Managing Editor
was essential to its successful completion. Mr. Lewis Reines,
President of the W.B. Saunders Company, has a profound
knowledge of publishing and books that makes him a worthy
successor to John Dusseau. Richard Zorab, Senior Medical
Editor, and Hazel N. Hacker, Developmental Editor, are
thoroughly professional and supportive individuals with whom
it was a pleasure to work. Many of the black and white
illustrations were drawn by Laurel Cook Lhowe and Marcia
Williams; Kit Johnson provided many of the anterior segment
photographs. Archival materials were retrieved with the aid
of Richard Wolfe, Curator of Rare Books at the Francis A.
Countway Library of Medicine, and Chris Nims and Kathleen
Kennedy of the Howe Library at the Massachusetts Eye and Ear
Infirmary.
The most exciting aspect of writing and editing a work of
this type is that it puts one in touch with the present day
ophthalmologists and visual scientists as well as physicians
training to be ophthalmologists in the future. We hope that this
book will establish its own tradition of excellence and useful-
ness and that it will win it a place in the lives of ophthal-
mologists today and in the future.
“EXPLICIT,” scribes wrote at the end of every book.
EXPLICIT means it has been unfolded. Olmert notes in The
Smithsonian Book of Books, “the unrolling or unfolding of
knowledge is a powerful act because it shifts responsibility from
writer to reader.... Great books endure because they help us
interpret our lives. It’s a personal quest, this grappling with the
world and ourselves, and we need all the help we can get.” We
hope that this work will provide such help to the professional
lives of ophthalmologists and visual scientists.
DANIEL M. ALBERT, M.D., M.S.
MADISON, WISCONSIN
xxi
List of Contributors

Juan-Carlos Abad MD Lloyd P Aiello MD PhD Ibrahim A Al Jadaan MD

Clinica Oftalmologica de Medellin Director of Beetham Eye Institute Chief
Medellin Section Head of Eye Research Glaucoma Division
Colombia Joslin Diabetes Center King Khaled Eye Specialist Hospital
Beetham Eye Institute Riyadh
Mark B Abelson MD CM FRCS Boston MA Kingdom of Saudi Arabia
Associate Clinical Professor of USA
Ophthalmology Sabah Al-Jastaneiah MD
Massachusetts Eye and Ear Infirmary Levent Akduman MD Consultant Ophthalmologist
Harvard Medical School Assistant Professor of Ophthalmology Anterior Segment and Refractive Surgery
Clinical Senior Scientist Department of Ophthalmology Division
Schepens Eye Research Institute St Louis University School of Medicine King Khaled Eye Specialist Hospital
Boston MA St Louis MO Riyadh
USA USA Kingdom of Saudi Arabia
David H Abramson MD Marissa L Albano MD Calliope E Allen MD
Chief c/o Robert P Murphy Fellow
Ophthalmic Oncology Service The Retina Group of Washington Eye Plastics, Orbital and Cosmetic Surgery
Department of Surgery Fairfax VA Massachusetts Eye & Ear Infirmary
Memorial Sloane Kettering Cancer Center USA Boston MA
New York NY USA
USA Daniel M. Albert MD MS
Chair Emeritus, F. A. Davis Professor and David Allen BSc FRCS FRCOphth
Martin A Acquadro MD Lorenz F. Zimmerman Professor Consultant Ophthamologist
Perioperative Medical Doctor Department of Ophthalmology and Visual Sunderland Eye Infirmary
Director Sciences Sunderland
Department of Anesthesiology and Pain Retina Research Foundation Emmett A. United Kingdom
Caritas Carney Hospital Humble Distinguished Director
Dorchester MA Alice R. McPherson, MD, Eye Research Robert C Allen MD (deceased)
USA Institute Formerly Professor of Ophthalmology and
University of Wisconsin Pharmacology
Anthony P Adamis MD Madison WI Formerly Chairman, Department of
Chief Scientific Officer USA Ophthalmology
Executive Vice President, Research & Virginia Commonwealth University
Development Terry J Alexandrou MD Richmond VA
(OSI) Eyetech Pharmaceuticals Chief Resident USA
New York NY Department of Ophthalmology and Visual
USA Science Albert Alm MD PhD
University of Chicago Professor
Wesley H Adams MD Chicago IL Department of Neuroscience, Ophthalmology
Ophthalmology Resident USA University Hospital
Department of Ophthalmology Uppsala
Wake Forest University Eye Center Eduardo C Alfonso MD Sweden
Winston-Salem NC Professor, Edward W D Norton Chair in
USA Ophthalmology Samar Al-Swailem MD
Medical Director Consultant Ophthalmologist
Natalie A Afshari MD Ocular Microbiology Laboratory Anterior Segment Division
Associate Professor of Ophthalmology Bascom Palmer Eye Institute King Khaled Eye Specialist Hospital
Department of Ophthalmology University of Miami Riyadh
Duke University Eye Center Miami FL Kingdom of Saudi Arabia
Durham NC USA
USA Abigail K Alt BA
Jorge L Alió MD PhD c/o Thaddeus P Dryja MD
Everett Ai MD Professor and Chairman of Ophthalmology, Massachusetts Eye and Ear Infirmary
Director Miguel Hernandez University Harvard Medical School
Retina Unit Medical Director, VISSUM Boston MA
California Pacific Medical Center Instituto Oftalmológico de Alicante USA
San Francisco CA Alicante
USA Michael M Altaweel MD FRCS(C)
Spain Assistant Professor & Co-Director, Fundus
Lloyd M Aiello MD Hassan Alizadeh PhD Photograph Reading Center
Clinical Professor of Medicine Assistant Professor of Ophthalmology Department of Ophthalmology and Visual
Joslin Diabetes Center – Beetham Eye Department of Ophthalmology Science
Institute University of Texas Southwestern Medical University of Wisconsin
Harvard Medical School Center Madison WI
Boston MA Dallas TX USA xxiii
USA USA
 List of Contributors

Russell Anderson BA Isabelle Audo MD PhD George B Bartley MD

Medical Writer Ophthalmologist Professor of Ophthalmology
Dry Eye Department Laboratory of Cellular Physiopathology and Mayo Medical School
Ophthalmic Research Associates Retinal Molecules Chief Executive Officer
North Andover MA Faculty of Medicine Mayo Clinic
USA INSERM Jacksonville FL
Université Pierre et Marie Curie USA
Christopher M Andreoli MD Hôpital St Antoine
Ophthalmologist Paris Jason J S Barton MD PhD FRCPC
Department of Ophthalmology France Director of Neuro-Ophthalmology
Massachusetts Eye and Ear Infirmary Professor and Canada Research Chair
Harvard Medical School Gerd U Auffarth Priv-Doz Dr med Neuro-Ophthalmology
Boston MA Research Group Leader VGH Eye Care Center
USA Heidelberg IOL & Refractive Surgery Vancouver BC
Research Group Canada
Sofia Androudi MD Department of Ophthalmology
First Department of Ophthalmology University of Heidelberg Irmgard Behlau MD
Aristotle University of Thessaloniki Heidelberg Department of Ophthalmology
Thessaloniki Germany Massachusetts Eye and Ear Infirmary
Greece Instructor In Medicine, Harvard Medical
Robin K Avery MD School
Leonard P K Ang MD MMed(Ophth) FRCS(Ed) Section Head, Transplant Infectious Disease Boston MA
MRCOphth Department of Infectious Diseases USA
Consultant Cleveland Clinic Foundation
Department of Cataract and Comprehensive Cleveland OH Jose I Belda MD PhD EBO
Ophthalmology USA Chairman
Singapore National Eye Centre Department of Ophthalmology
Singapore Dimitri T Azar MD Hospital de Torrevieja
B A Field Chair of Ophthalmologic Research Alicante
Fahd Anzaar MD Professor and Head, Department of Spain
Research Coordinator Ophthalmology and Visual Sciences
Massachusetts Eye Research and Surgery University of Illinois Eye and Ear Infirmary Jeffrey L Bennett MD PhD
Institute Chicago IL Associate Professor of Neurology &
Cambridge MA USA Ophthalmology
USA Department of Neurology
Ann S Baker MD (deceased) University of Colorado Health Sciences
David J Apple MD Formerly Director of the Infectious Disease Center
Professor of Ophthalmology and Pathology Service Denver CO
Director of Research Pawek-Vallotton Massachusetts Eye and Ear Infirmary USA
University of South Carolina Formerly Associate Professor of
Charleston SC Ophthalmology Timothy J Bennett CRA FOPS
USA Harvard Medical School Ophthalmic Photographer
Boston MA Department of Ophthalmology
Claudia A Arrigg MD MEd Penn State Milton S Hershey Medical Center
Senior Surgeon USA
Hershey PA
Lawrence General Hospital Mark Balles MD USA
Lawrence MA Retina Center of Maine
USA South Portland ME Gregg J Berdy MD FACS
USA Assistant Professor of Clinical Ophthalmology
Pablo Artal PhD & Visual Science
Professor of Optics Scott D Barnes MD Department of Ophthalmology and Visual
Centro de Investigacion en Optica y Fellow, Cornea Service, Massachusetts Eye Science
Nanofisica (CiOyN) and Ear Infirmary and Harvard Medical Washington University School of Medicine
Universidad de Murcia School St Louis MO
Murcia Chief, Ophthalmology and Refractive Surgery USA
Spain Department of Ophthalmology
Womack Army Medical Center Carlo Roberto Bernardino MD FACS
Penny Asbell MD Associate Professor of Ophthalmology
Professor of Ophthalmology Fort Bragg NC
USA Yale University School of Medicine
Department of Ophthalmology New Haven CT
Mount Sinai Medical Center Donald M Barnett MD USA
New York NY Assistant Clinical Professor of Medicine
USA Joslin Diabetes Center Vitaliano Bernardino MD
Beetham Eye Institute Ophthalmologist
George K Asdourian MD Private Practice
Chief, Division of Ophthalmology Harvard Medical School
Boston MA Langhorne PA
University of Massachusetts Memorial USA
Medical Center USA
Worcester MA Neal P Barney MD Eliot L Berson MD
USA Associate Professor of Ophthalmology Director, Electroretinography Service
Department of Ophthalmology and Visual Massachusetts Ear and Eye Infirmary
Neal Atebara MD William F Chatlos Professor of
Ophthalmologist Sciences
University of Wisconsin School of Medicine Ophthalmology
Retina Center of Hawaii Harvard Medical School
Honolulu HI Madison WI
USA Boston MA
USA USA
Pelin Atmaca-Sonmez Fina C Barouch MD
Research Fellow Assistant Professor of Ophthalmology
Department of Ophthalmology Eye Institute
University of Michigan Lahey Clinic Medical Center
Peabody MA
xxiv Ann Arbor MI
USA
USA
 List of Contributors

Amitabh Bharadwaj MD Luigi Borrillo MD Alfred Brini MD

Ophthalmologist Retina-Vitreous Associates Inc Emeritus Professor of Ophthalmology
Department of Ophthalmology El Camino Hospital Louis Pasteur University
Wills Eye Hospital Mountain View, CA Strasbourg
Philadelphia PA USA France
USA
Gary E Borodic MD Donald L Budenz MD MPH
Robert Bhisitkul MD PhD Ophthalmologist Associate Professor
Assistant Professor of Clinical Ophthalmology Department of Ophthalmology Epidemiology and Public Health
Department of Ophthalmology Massachusetts Eye and Ear Infirmary Bascom Palmer Eye Institute
UCSF Beckman Vision Center Harvard Medical School Miami FL
San Francisco CA Boston MA USA
USA USA
Angela N Buffenn MD MPH
Ravinder D Bhui BApSc in Elec Eng S Arthur Boruchoff MD Assistant Professor of Clinical Ophthalmology
Senior Medical Student Professor (Retired) Childrens Hospital Los Angeles
Schulich School of Medicine and Dentistry Department of Ophthalmology Department of Ophthalmology
The University of Western Ontario Boston University School of Medicine Los Angeles CA
London ON Boston MA USA
Canada USA
Scott E Burk MD PhD
Jurij Bilyk MD Swaraj Bose MD Ophthalmologist
Attending Surgeon Associate Professor Department of Ophthalmology
Oculoplastic and Orbital Surgery Service Department of Ophthalmology Cincinnati Eye Institute
Wills Eye Institute University of California, Irvine Cincinnati OH
Philadelphia PA Irvine CA USA
USA USA
Salim Butrus MD
Valérie Biousse MD Michael E Boulton PhD Associate Clinical Professor
Associate Professor of Ophthalmology and Director of AMD Center Department of Ophthalmology
Neurology Department of Ophthalmology and Visual George Washington University
Emory Eye Center Sciences Washington DC
Emory University School of Medicine University of Texas Medical Branch USA
Atlanta GA Galveston TX
USA USA David Callanan MD
Vitreoretinal Specialist
Alan C Bird MD FRCS FRCOphth R W Bowman MD Texas Retina Associates
Professor Professor Arlington TX
Department of Clinical Ophthalmology Department of Ophthalmology USA
Moorfields Eye Hospital University of Texas Southwestern Medical
London Center J Douglas Cameron MD
United Kingdom Dallas TX Professor of Ophthalmology
USA Clinical Ophthalmology
Norman Paul Blair MD Scheie Eye Institute
Professor of Ophthalmology, Director of Elizabeth A Bradley MD University of Pennsylvania
Vitreoretinal Service Assistant Professor of Ophthalmology Philadelphia PA
Department of Ophthalmology and Visual Department of Ophthalmology USA
Sciences Mayo Clinic
University of Illinois Rochester MN Louis B Cantor MD
Chicago IL USA Professor of Ophthalmology
USA Department of Ophthalmology
Periklis D Brazitikos MD Indiana University School of Medicine
Barbara A Blodi MD Associate Professor of Ophthalmology Indianapolis IN
Associate Professor, Specialist in Retinal Department of Ophthamology USA
Disease Aristotle University of Thessaloniki
Department of Ophthalmology & Visual Thessaloniki William A Cantore MD
Sciences Greece Associate Professor of Ophthalmology and
University of Wisconsin-Madison Neurology
Madison WI Robert Breeze MD Department of Ophthalmology
USA Professor and Vice Chair Penn State University College of Medicine
Deparment of Neurosurgery Hershey PA
Mark S Blumenkranz MD University of Colorado Health Sciences USA
Professor and Chairman Center
Department of Ophthalmology Aurora CO Jorge Cantu-Dibildox MD
Stanford University School of Medicine USA Centro de Oftalmologia San Jose, S C
Stanford CA Fundación de Ojos Vidaurri, A C
USA Neil M Bressler MD Monterrey NL
Professor of Ophthalmology Mexico
H Culver Boldt MD The Wilmer Eye Institute
Professor of Ophthalmology The Johns Hopkins University School of Victoria Casas MD
Department of Ophthalmology Medicine Research Fellow
University of Iowa Baltimore MD Ocular Surface Research & Education
Iowa City IA USA Foundation
USA Miami FL
Susan B Bressler MD USA
Mark S Borchert MD Professor of Ophthalmology
Associate Professor of Ophthalmology Department of Ophthalmology Miriam Casper MD
Department of Ophthalmology John Hopkins Hospital c/o David J Apple MD
Children’s Hospital Los Angeles Baltimore MD University of South Carolina
Los Angeles CA USA Charleston SC
USA USA
xxv
 List of Contributors

Robin J Casten PhD Joe Chen MD Antonio P Ciardella MD

Assistant Professor c/o Keith L Lane MD Chief, Department of Ophthalmology
Department of Psychiatry and Human ORA Clinical Research and Development Denver Health Medical Center
Behaviour North Andover MA Denver CO
Thomas Jefferson University USA USA
Philadelphia PA
USA Julie A Chen MD Mortimer Civan MD
c/o Joan M O’Brien MD Professor of Physiology
Yara P Catoira MD Division of Ophthalmology Department of Physiology
Assistant Professor of Clinical Ophthalmology University of California San Francisco University of Pennsylvania Health System
Department of Ophthalmology Medical Center Philadelphia PA
Indiana University School of Medicine San Francisco CA USA
Indianapolis IN USA
USA Liane Clamen MD
Teresa C Chen MD Harvard Medical School
Jerry Cavallerano OD PhD Assistant Professor of Medicine Boston MA
Assistant to the Director Glaucoma Service USA
Joslin Diabetes Center Massachusetts Eye and Ear Infirmary
Beetham Eye Institute Assistant Professor of Medicine, Harvard John I Clark PhD
Boston MA Medical School Professor, Biological Structure
USA Boston MA School of Medicine
USA University of Washington
Samantha J Chai MD Seattle WA
Medical Resident Zhou Chen PhD USA
Department of Ophthalmology Senior Pharmacologist and Toxicologist
Cullen Eye Institute Center for Drug Evaluation and Research Glenn Cockerham MD
Baylor College of Medicine Food & Drug Administration Clinical Associate Professor
Houston TX Silver Spring MD Department of Ophthalmology
USA USA Stanford University
Stanford CA
Maria R Chalita MD PhD Patricia Chévez-Barrios MD USA
Director of Cornea and Refractive Surgery Clinical Assistant Professor
Department of Ophthalmology Departments of Ophthalmology & Pathology Andre Cohen MD
Federal University of Brazil Baylor College of Medicine and the Texas Ophthalmologist
Sao Paulo Children’s Cancer Center Marietta Eye Consultants
Brazil Houston TX Marietta GA
USA USA
Sherman M Chamberlain MD FACP FACG
Assistant Professor of Medicine Emily Y Chew MD Elisabeth J Cohen MD
Gastroenterology and Hepatology Medical Officer, Division of Biometry and Director Cornea Service, Attending Surgeon,
Medical College of Georgia Epidemiology Wills Eye Hospital
Augusta GA National Eye Institute Professor, Department of Ophthalmology
USA National Institutes of Health Jefferson Medical College of Thomas
Bethseda MD Jefferson University
Audrey S Chan MD USA Philadelphia PA
Cornea and Refractive Surgery Fellow USA
Massachusetts Eye and Ear Infirmary Mark Chiang MBBS
Harvard Medical School Birmingham and Midland Eye Centre Kathryn A Colby MD PhD
Boston MA Birmingham Director, Joint Clinical Research Center
USA United Kingdom Attending Surgeon, Cornea Service
Massachusetts Eye and Ear Infirmary
Chi-Chao Chan MD James Chodosh MD Assistant Professor of Ophthalmology,
Head, Immunopathology Section Professor of Ophthalmology Harvard Medical School
National Eye Institute Department of Ophthalmology Boston MA
National Institutes of Health University of Oklahoma Health Sciences USA
Bethesda MD Center
USA Oklahoma City OK Anne L Coleman MD PhD
USA Professor of Ophthalmology and
Paul Chan MD Epidemiology
Assistant Professor of Ophthalmology Eva-Marie Chong MBBS Departments of Ophthalmology and
New York Presbyterian Physician Epidemiology
Wiell Medical College of Cornell University Department of Ophthalmology Jules Stein Eye Institute
New York NY Arizona Medical Center Los Angeles CA
USA Peoria AZ USA
USA
Matthew J Chapin MD Hanna R Coleman MD
Ophthalmic Research Associates, Inc Denise Chun BS Staff Clinician
North Andover MA Doctoral Candidate in Genetics, Harvard Department of Ophthalmology
USA Medical School New York Presbyterian Hospital
Department of Molecular Biology Columbia University Medical Center
Karen L Chapman MD Massachusetts General Hospital New York NY
University of South Florida Boston MA USA
Sarasota Memorial Hospital USA
Sarasota FL Joseph Colin MD
USA Leo T Chylack Jr MD Professor of Ophthalmology
Director of Research Department of Ophthalmology
Eric Chen MD Center for Ophthalmic Research C H U Morvan
Retina Research Center Brigham & Women’s Hospital Brest
Austin TX Boston MA France
USA USA

xxvi
 List of Contributors

J Michael Collier PhD Donald J D’Amico MD Adam G de la Garza MD

Instructor, Harvard Medical School Professor and Chairman Chief Resident, Wake Forest University Eye
Senior Medical Physicist Ophthalmologist-in-Chief Center
Department of Radiation Oncology Department of Ophthalmology Wake Forest University School of Medicine
Massachusetts General Hospital Weill Cornell Medical College Winston-Salem NC
Boston MA New York NY USA
USA USA
Margaret M DeAngelis PhD
Grant M Comer MD Reza Dana MD MSc MPH Instructor of Ophthalmology
Assistant Professor Director, Cornea and Refractive Surgery Massachusetts Eye & Ear Infirmary
Kellogg Eye Center Services Boston MA
University of Michigan Massachusetts Eye and Ear Infirmary USA
Ann Arbor MI Professor, Harvard Medical School
USA Senior Scientist & W Clement Stone Scholar Sheri L DeMartelaere MD
Schepens Eye Research Institute Director of Orbital and Ocular Trauma
M Ronan Conlon MD FRCSC Boston MA Ophthalmology Service
Eye Physician and Surgeon USA Brooke Army Medical Center
Midwest Eye Care Institute Fort Sam Houston TX
Saskatoon SK Aude Danan-Husson MD USA
Canada Service d’ophtalmologie
Centre Hospitalier National d’Ophtalmologie Joseph L Demer MD PhD
Kim E Cooper MD des Quinze-vingts Leonard Apt Professor of Ophthalmology
Associate Professor Paris Departments of Ophthalmology and
Southwest College of Naturopathic Medicine France Neurology
Tempe AR Jules Stein Eye Institute
USA Helen B Danesh-Meyer MBChB MD Los Angeles CA
FRANZCO USA
James J Corbett MD Associate Professor of Ophthalmology
McCarty Professor and Chairman for Department of Ophthalmology Avninder Dhaliwal MD
Neurology University of Auckland Medical School University of Minnesota Medical School
Department of Neurology Auckland Minneapolis MN
University of Mississippi Medical Center New Zealand USA
Jackson MS
USA Ronald P Danis MD J Paul Dieckert MD
Professor of Ophthalmology and Visual Center Director, Division of Ophthalmology
Miguel C Coma MD FEBOphth Science Scott and White Memorial Hospital
Massachusetts Eye Research and Surgery Director, Fundus Photograph Reading Center Temple TX
Institute Department of Ophthalmology and Visual USA
Cambridge MA USA Science
Department of Ophthalmology Diana V Do MD
University of Wisconsin Fellow in Advanced Speciality Training in
Hospital de León, León, Spain Madison WI Medical and Surgical Diseases of the Retina
Marshall N Cyrlin MD USA Assistant Professor of Ophthalmology
Clinical Professor of Biomedical Sciences Jason K Darlington MD The Johns Hopkins University School of
Eye Research Institute Department of Ophthalmology Medicine
Oakland University University of California at Davis The Wilmer Eye Institute
Rochester MN Sacramento CA Baltimore MD
USA USA USA
Linda R Dagi MD Stefanie L Davidson MD Marshall G Doane PhD
Director of Adult Strabismus, Instructor in Assistant Clinical Professor, University of Emeritus Senior Scientist
Ophthalmology Pennsylvania Department of Ophthalmology
Department of Ophthalmology Division of Ophthalmology Schepens Eye Research Institute
Childrens Hospital Childrens Hospital of Philadelphia Harvard Medical School
Boston MA Philadelphia PA Boston MA
USA USA USA
Matthew A Dahlgren MD Janet L Davis MD MA Christopher Dodds MBBS MRCGP FRCA
Fellow, Cornea and Anterior Segement, Associate Professor of Ophthalmology Professor of Anaesthesia
Department of Ophthalmology Division of Ophthalmology Academic Anaesthetic Department
University of Minnesota Medical School University of Miami James Cook University Hospital
Minneapolis MN Miami FL Middlesbrough
USA USA United Kingdom
Timothy J Daley BS Elizabeth A Davis MD FACS Claes H Dohlman MD PhD
University of Wisconsin Hospital and Clinics Adjunct Clinical Assistant Professor, Professor of Ophthalmology, Harvard Medical
Madison WI University of Minnesota School, Chief Emeritus
USA Director, Minnesota Eye, Laser and Surgery Cornea Service
Center Massachusetts Eye and Ear Infirmary
Andrea P Da Mata MD Harvard Medical School
Ocular Immunology and Uveitis Foundation Bloomington MN
USA Boston MA
Massachusetts Eye Research and Surgery USA
Institute Jose J de la Cruz MD
Cambridge MA Cornea Fellow, Department of Guy Donati MD
USA Ophthalmology and Visual Science Chare D’Ensign
University of Illinois at Chicago Department of Pathology
Bertil Damato MD PhD FRCOphth University of Geneva
Professor of Ophthalmology Chicago IL
USA Geneva
Ocular Oncology Service Switzerland
Royal Liverpool University Hospital
Liverpool
United Kingdom xxvii
 List of Contributors

Eric D Donnenfeld MD FACS Chiara M Eandi MD Frederick L Ferris III MD

Co-director, Cornea Division The LuEsther T Mertz Retinal Research Director, Division of Epidemiology and
Ophthalmic Consultants of Long Island Fellow Clinical Research
New York NY Manhattan Eye, Ear and Throat Hospital National Eye Institute
USA New York NY National Institutes of Health
USA Bethesda MD
Arlene Drack MD USA
Chief of Ophthalmology, Children’s Hospital Deepak P Edward MD
Associate Professor Professor and Chairman Howard F Fine MD MHSc
Department of Ophthalmology Department of Ophthalmology - Suma Health Vitreoretinal surgical fellow
University of Colorado Health Sciences Systems Vitreous Retina Macula New York
Center Northeastern Ohio University School of New York NY
Aurora CO Medicine USA
USA Akron OH
USA Donald C Fletcher MD
Thaddeus P Dryja MD Medical Director
Director, David C Cogan Eye Pathology Robert A Egan MD Frank Stein & Paul May Center for Low Vision
Laboratory Assistant Professor of Ophthalmology and Rehabilitation
Massachusetts Eye and Ear Infirmary Neurology California Pacific Medical Center
Harvard Medical School Departments of Ophthalmology and Scientist, Smith-Kettlewell Eye Research
Boston MA Neurology Institute
USA Casey Eye Institute San Francisco CA
Portland OR USA
David Dueker MD USA
Professor of Ophthalmology Paul Flikier MD
The Eye Institute David A Eichenbaum MD Farmacia Alvarez, Heredia
Medical College of Wisconsin Associate Director, Centro Medico de la Vision
Milwaukee WI Retina-Vitrous Associates of Florida San Jose
USA St Petersburg FL Costa Rica
USA
Jay S Duker MD Richard P Floyd MD
Director New England Eye Center Susan E Eklund BA Clinical Instructor
Chairman and Professor of Ophthalmology Assistant, Department of Ophthalmology Department of Ophthalmology
Tufts University School of Medicine Children’s Hospital Harvard Medical School
Tufts New England Medical Center Boston MA Boston MA
Boston MA USA USA
USA
Elizabeth C Engle MD Harry W Flynn Jr, MD
Jennifer A Dunbar MD Associate Professor of Neurology Professor, The J Donald M Gass
Director of Pediatric Ophthalmology Harvard Medical School Distinguished Chair of Ophthalmology
Department of Ophthalmology Department of Neurology, Program in Bascom Palmer Eye Institute
Loma Linda University Genomics, Children’s Hospital The University of Miami Miller School of
Loma Linda CA Boston MA Medicine
USA USA Miami FL
USA
James P Dunn MD Kristine Erickson OD PhD
Associate Professor of Ophthalmology Senior Director Clinical Affairs Donald S Fong MD MPH
The Wilmer Eye Institute Unigene Corporation Director, Cinical Trials Research
John Hopkins School of Medicine Boonton NJ Kaiser Permanente Southern California
Baltimore MD USA Pasadena CA
USA USA
Bita Esmaeli MD FACS
William J Dupps Jr, MD PhD Associate Professor of Ophthalmology; Ramon L Font MD
Associate Staff, Ophthalmology and Director of Ophthalmic Plastic and Professor of Pathology and Ophthalmology
Biomedical Engineering Reconstructive and Orbital Surgery The Sarah Campbell Blaffer Chair of
Cole Eye Institute Fellowship Ophthalmology
Cleveland Clinic and Lerner Research Department of Medicine The Neurosensory Center
Institute The University of Texas Houston TX
Cleveland OH Houston TX USA
USA USA
Brian J R Forbes MD PhD
Marlene L Durand MD Aaron Fay MD Assistant Professor of Ophthalmology
Director of Infectious Diseases, Interim Director, Ophthalmic Plastic Surgery Department of Ophthalmology
Massachusetts Eye and Ear Infirmary Massachusetts Eye and Ear Infirmary The Childrens Hospital of Philadelphia
Assistant Professor of Medicine, Harvard Assistant Clinical Professor of Wallingford PA
Medical School; Ophthalmology, Department of USA
Infectious Diseases Unit Ophthalmology, Harvard Medical School
Massachusetts General Hospital Boston MA Rod Foroozan MD
Boston MA USA Assistant Professor of Ophthalmology
USA Department of Ophthalmology
Leonard Feiner MD PhD Baylor College of Medicine
Jonathan J Dutton MD PhD Ophthalmology Department Houston TX
Professor and Vice Chair Montefiore Medical Center USA
Department of Ophthalmology Lawrence NY
University of North Carolina USA Bradley S Foster MD
Chapel Hill NC Assistant Clinical Professor of Ophthalmology
USA Sharon Fekrat MD New England Retina Consultants
Assistant Professor West Springfield MA
Department of Ophthalmology USA
Vitreoretinal Surgery
Duke Eye Center
xxviii Durham NC
USA
 List of Contributors

C Stephen Foster MD FACS David Friedman MD James A Garrity MD

Founder and President Assistant Professor Professor of Ophthalmology
The Massachusetts Eye Research Institute Ophthalmology Department Department of Ophthalmology
Clinical Professor of Ophthalmology Wilmer Eye Institute Mayo Clinic
Harvard Medical School John Hopkins University School of Medicine Rochester MN
Cambridge MA Baltimore MD USA
USA USA
Damien Gatinel MD
Jill A Foster MD Deborah I Friedman MD FAAN Assistant Professor
Assistant Clinical Professor Associate Professor of Ophthalmology and Ophthalmology Department
The William H Havener Eye Institute Neurology Fondation Ophtalmologique A de Rothschild
The Ohio State University Departments of Ophthalmology and Paris
Columbus OH Neurology France
USA University of Rochester School of Medicine
and Dentistry Steven J Gedde MD
Gary N Foulks MD FACS Rochester NY Professor of Ophthalmology and Residency
Arthur & Virginia Keeney Professor of USA Program Director
Ophthalmology Department of Ophthalmology
Department of Ophthalmology Ephraim Friedman MD Bascom Palmer Eye Institute
University of Louisville School of Medicine Former Chief, Department of Ophthalmology Miami FL
Louisville KY Massachusetts Eye and Ear Infirmary USA
USA Harvard Medical School Retina Service
Boston MA Craig E Geist MD FACS
Tamara R Fountain MD USA Chairman, Department of Ophthalmology
Associate Professor Associate Professor, Ophthalmology,
Department of Ophthalmology Arthur D Fu MD Neurology, Neurological Surgery
Rush University in Chicago Ophthalmologist Director, Oculoplastics, Orbit, Lacrimal
Northbrook IL Pacific Vision Foundation Director, Neuro-Ophthalmology
USA California Pacific Medical Center The George Washington University
San Francisco CA Washington DC
Gregory M Fox MD USA USA
Clinical Instructor of Ophthalmology
Department of Ophthalmology Anne B Fulton MD Steve Gerber MD
Allegheny University Associate Professor of Ophthalmology and Chairman
Wilmington DE Senior Associate in Ophthalmology Department of Ophthalmology
USA Department of Ophthalmology Memorial Hospital
Children’s Hospital South Bend IN
Thomas F Freddo OD PhD FAAO Boston MA USA
Professor and Director USA
School of Optometry Ramon C Ghanem MD
University of Waterloo Ahmed Galal MD PhD Sadalla Amin Ghanem
Waterloo ON Department of Refractive Surgery Hospital de Olhos
Canada Vissum/Instituto Oftalmologico de Alicante Batista
Alicante Joinville - SC
Sharon F Freedman MD Spain Brazil
Associate Professor of Ophthalmology
Associate Professor of Pediatrics Steven Galetta MD Jon P Gieser MD
Department of Pediatric Ophthalmology & Director, Neuro-Ophthalmology Services Wheaton Eye Clinic
Strabismus Hospital of the University of Pennsylvania Wheaton IL
Duke University Eye Center Philadelphia PA USA
Durham NC USA
USA Michael S Gilmore PhD
Mark Gallardo MD Charles L Schepens Professor of
K Bailey Freund MD Resident Physician Ophthalmology
Retina Specialist Office of Border Health President and Ankeny Director of Research
Vitreous-Retina-Macula Consultants of Texas Tech University Health Sciences Center The Schepens Eye Research Institute
New York El Paso TX Harvard Medical School
New York NY USA Boston MA
USA USA
Brenda Gallie MD FRCS(C)
Thomas R Friberg MD Professor of Ophthalmology Howard V Gimbel MD MPH FRCSC FACS
Professor of Ophthalmology, Professor of Departments of Medical Biophysics and Chair and Professor of The Department of
Bioengineering Molecular and Medical Genetics Ophthalmology
Director of the Retina Service University of Toronto Refractive Surgery, Department of
Departments of Ophthalmology and Head of Cancer Informatics Ophthalmology
Bioengineering University Health Network Loma Linda University
UPMC Eye Center Ontario Cancer Institute Loma Linda CA
Pittsburgh PA Princess Margaret Hospital USA
USA Toronto ON
Canada Ilene K Gipson PhD
Alan H Friedman MD Senior Scientist and Professor of
Department of Ophthalmology Alec Garner MD Ophthalmology
Mount Sinai School of Medicine Head of Department Department of Ophthalmology
New York NY Department of Pathology Schepens Eye Research Institute
USA Institute of Ophthalmology Boston MA
London USA
United Kingdom

xxix
 List of Contributors

Tyrone Glover MD David B Granet MD FACS FAAP FAAO Vamsi K Gullapalli MD PhD
Clinical Professor, Ophthalmology Anne F Ratner Professor of Ophthalmology & Resident
Oculoplastic Surgery Pediatrics Department of Ophthalmology and Visual
Kaiser Permanente Director, Pediatric Ophthalmology & Adult Science
Sacramento CA Re-Alignment Services Institute of Ophthalmology and Visual
USA Anne F & Abraham Ratner Children’s Eye Center Science
Shiley Eye Center University of Medicine and Dentistry of
Robert A Goldberg MD FACS University of California, San Diego New Jersey
Associate Professor of Ophthalmology La Jolla CA Newark NJ
Chief, Division of Orbital and Ophthalmic USA USA
Plastic Surgery
Jules Stein Eye Institute Michael J Greaney Padma Gulur MD
Los Angeles CA Senior Clinical Lecturer, Department of Instructor in Anaesthesia, Harvard Medical
USA Ophthalmology, University of Bristol School
Senior Consultant Pain Center Department of Anesthesia and
Mordechai Goldenfeld MD
Bristol Eye Hospital Critical Care
Senior Attending Ophthalmologist
Bristol Massachusetts General Hospital
The Sam Rothberg Glaucoma Centre
United Kingdom Boston MA
Goldschleger Eye Institute
USA
Sheba Medical Center Daniel G Green PhD
Tel-Hashomer Professor Emeritus, Ophthalmology and Jonathan Gunther MD
Israel Visual Sciences Department of Ophthalmology and Visual
Scott M Goldstein MD Professor, Biomedical Engineering Sciences
Clinical Associate The University of Michigan Kellogg Eye University of Wisconsin Medical School
Childrens Hospital of Philadelphia Center Madison WI
Tricounty Eye Physicians & Surgeons Ann Arbor MI USA
Southampton PA USA
Manish Gupta DNB FRCS(Glasg), MRCS(Ed)
USA Franz Grehn Dr h.c. NHS Greater Glasgow and Clyde
Cintia F Gomi MD Professor of Ophthalmology Stobhill and Gartnevel Hospital
Hamilton Glaucoma Center Chairman, Department of Ophthalmology Glasgow
University of California, San Diego, University of Würzburg United Kingdom
La Jolla CA Würzburg
Germany Mayank Gupta
USA
c/o Deepak P Edward MD
Haiyan Gong MD MS PhD Jack V Greiner DO PhD Northeastern Ohio University School of
Research Assistant Professor Instructor of Ophthalmology Medicine
Department of Ophthalmology Schepens Eye Research Institute Akron OH
Boston University School of Medicine Harvard Medical School USA
Boston MA Boston MA
USA USA David R Guyer MD
Clinical Professor
John A Gonzales MD Craig M Greven MD FACS Department of Ophthalmology
Physician Director, Professor and Chairman NYU Medical Center
Immunopathology Section Department of Ophthalmology New York NY
Laboratory of Immunology Wake Forest University Eye Center USA
National Eye Institute Wake Forest University School of Medicine
National Institutes of Health Winston-Salem NC Darin R Haivala MD
Besthesda MD USA Clinical Assistant Professor
USA Department of Ophthalmology
Gregory J Griepentrog MD University of Oklahoma
John Goosey MD Chief Resident Associate Dean A McGee Eye Institute
Director Mayo Clinic Oklahoma City OK
Houston Eye Associates Rochester MN USA
Houston TX USA
USA Julia A Haller MD
Carl Groenewald MD Robert Bond Welch Professor of
Justin L Gottlieb MD
Consultant Vitreoretinal Surgeon Ophthamology
Associate Professor
St Paul’s Eye Unit Wilmer Ophthalmological Institute
Department of Ophthalmology and Visual
Royal Liverpool University Hospital Johns Hopkins Medical Institutions
Sciences
Liverpool Baltimore MD
University of Wisconsin
United Kingdom USA
Madison WI
USA Cynthia L Grosskreutz MD PhD G M Halmagyi MD BSc FACS DCH
Joshua Gould DO Co-Director, Glaucoma Service Professor of Neurology
Physician Massachusetts Eye and Ear Infirmary Department of Neurology
Eye Care Center of New Jersey Associate Professor of Ophthalmology Royal Prince Albert Hospital
Bloomingfield NJ Harvard Medical School Sydney NSW
USA Boston MA Australia
USA
Evangelos S Gragoudas MD Lawrence S Halperin MD FACS
Director, Retina Service Lori Latowski Grover OD Physician
Massachusetts Eye and Ear Infirmary Assistant Professor of Ophthalmology Retina Vitreous Consultants of South Florida
Professor of Ophthalmology, Harvard Medical Department of Ophthalmology Fort Lauderdale FL
School Lions Vision Research and Rehabilitation USA
Boston MA Center
Baltimore MD Islam M Hamdi FRCS MD
USA Magrabi Center
USA
Jeddah
Kingdom of Saudi Arabia

xxx
 List of Contributors

Steven R Hamilton MD Yasutaka Hayashida MD PhD Ahmed A Hidayat MD

Clinical Associate Professor of Research Fellow Chief, Ophthalmic Pathology
Ophthalmology and Neurology Ocular Surface Research and Education Armed Forces Institute of Pathology
Department of Ophthalmology and Neurology Foundation Rockville MD
University of Washington Miami FL USA
Neuro-Ophthalmic Associates Northwest USA
Seattle WA Eva Juliet Higginbotham MD
John R Heckenlively MD FRCOpath Professor of Ophthalmology and Chair
USA
Paul R Lichter Professor of Ophthalmic Department of Ophthalmology
Kristin M Hammersmith MD Genetics University of Maryland Medicine
Assistant Surgeon, Cornea Service, Wills Eye Professor of Ophthalmology and Visual Baltimore MD
Hospital Science USA
Instructor, Thomas Jefferson Medical College Kellogg Eye Centre
Wills Eye Institute University of Michigan Tatsuo Hirose MD
Thomas Jefferson University Ann Arbor MI Clinical Professor of Ophthalmology
Philadelphia PA USA Schepens Retina Associates
USA Boston MA
Thomas R Hedges III, MD USA
Dennis P Han MD Director, Neuro-Ophthalmology Service
Jack A and Elaine D Klieger Professor of Co-Director, Electrophysiology Service Allen C Ho MD
Ophthalmology, Vitreoretinal Section Head Director, Neuro-Ophthalmology Fellowship Professor of Ophthalmology
Department of Ophthalmology Program Retina Service
Medical College of Wisconsin New England Eye Center Thomas Jefferson University
Milwaukee WI Boston MA Philadelphia PA
USA USA USA

Ronald M Hansen PhD Alfred D Heggie MD ThucAnh T Ho MD

Instructor Professor Emeritus of Pediatrics Vitreoretinal Fellow
Department of Ophthalmology Departments of Pediatrics, Preventive Illinois Retina Associates
Children’s Hospital and Harvard Medical Medicine, and Obstetrics and Gynecology Rush University Medical Center
School Case Western Reserve University School of Chicago IL
Boston MA Medicine USA
USA Cleveland OH R Nick Hogan MD PhD
J William Harbour MD USA Associate Professor of Ophthalmology
Distinguished Professor of Ophthalmology, Department of Ophthalmology
Katrinka L Heher MD
Cell Biology, and Medicine (Molecular University of Texas South Western Medical
Director, Aesthetic Eyelid & Facial Surgery
Oncology) Center
Director, Oculoplastic & Orbital Surgery
Director, Ocular Oncology Service Dallas TX
Service
Department of Ophthalmology USA
Director, Ophthalmic Plastics and
Washington University School of Medicine Reconstructive Surgery Fellowship Program David E Holck MD
St Louis MO New England Eye Center Director, Oculoplastic, Reconstructive, Orbit,
USA Tufts University School of Medicine and Ocular Oncology Service
Seenu M Hariprasad MD Boston MA Department of Ophthalmology
Assistant Professor and Director of Clinical USA Wilford Hall Medical Center
Research Assistant Professor of Surgery, USUHS
Jeffrey S Heier MD Assistant Professor of Ophthalmology
Chief, Vitreoretinal Service Vitreoretinal Specialist
Department of Ophthalmology and Visual University of Texas
Ophthalmic Consultants of Boston San Antonio TX
Science Boston MA
University of Chicago USA
USA
Chicago IL Nancy M Holekamp MD
USA J Fielding Hejtmancik MD PhD Associate Professor of Clinical
Medical Officer Ophthalmology
Mona Harissi-Dagher MD Ophthalmic Genetics and Visual Function
Assistant in Ophthalmology Department of Ophthalmology and Visual
Branch Science
Massachusetts Eye and Ear Infirmary National Eye Institute
Harvard Medical School Washington University School of Medicine
National Institutes of Health Barnes Retina Institute
Boston MA Bethesda MD
USA St Louis MO
USA USA
Shirin E. Hassan PhD
Bonnie A Henderson MD FACS Peter G Hovland MD PhD
c/o David Friedman
Assistant Clinical Professor Physician
Assistant Professor
Department of Ophthalmology Colorado Retina Associates
Wilmer Eye Institute
Harvard Medical School Denver CO
John Hopkins University School of Medicine
Boston MA USA
Baltimore MD
USA
USA Thomas C Hsu MD
Mark P Hatton MD Peter S Hersh MD FACS Tufts University School of Medicine
Clinical Instructor, Harvard Medical School Professor of Ophthalmology New England Eye Center
Adjunct Clinical Scientist Director, Cornea and Laser Eye Institute - Boston MA
Schepens Eye Research Institute Hersh Vision Group USA
Ophthalmic Consultants of Boston Clinical Professor of Ophthalmology
Chief, Cornea and Refractive Surgery William C Hsu MD
Boston MA Assistant Professor of Medicine
USA University of Medicine and Dentistry New
Jersey Joslin Diabetes Center
Pamela Hawley MS Teaneck NJ Beetham Eye Institute
Genetic Counseling Center USA Harvard Medical School
Children’s Hospital Boston MA
Harvard USA
Boston MA xxxi
USA
 List of Contributors

Andrew J W Huang MD MPH Fei Ji PhD Alon Kahana MD PhD

Director of Cornea and Refractive Surgery Research Associate Assistant Professor; Eye Plastics, Orbit and
Department of Ophthalmology Laboratory of Statistical Genetics Facial Cosmetic Surgery
University of Minnesota Rockefeller University Department of Ophthalmology and Visual
Minneapolis MN New York NY Sciences
USA USA Kellogg Eye Center
Ann Arbor MI
Mark S Hughes MD David L Johnson MD USA
Adjunct Assistant Clinical Scientist Clinical Instructor/Vitreoretinal Fellow
The Schepens Eye Research Institute Department of Ophthalmology and Visual Malik Y Kahook MD
Boston MA Sciences Assistant Professor and Director of Clinical
USA University Of Wisconsin Medical School Research
Jennifer Hui MD Madison WI Rocky Mountain Lions Eye Institute
Ophthalmology Resident USA University of Colorado at Denver Health
Department of Ophthalmology Sciences Center
Douglas H Johnson MD (deceased) Aurora CO
Bascom Palmer Eye Institute Formerly Professor of Ophthalmology
Miami FL USA
Department of Ophthalmology
USA Mayo Clinic Elliott Kanner MD PhD
David G Hunter MD PhD Rochester MN Assistant Professor of Ophthalmology
Associate Professor of Ophthalmology, USA Hamilton Eye Institute
Harvard Medical School University of Tennessee Health Science
Mark W Johnson MD Center
Ophthalmologist-in-Chief Professor
Richard Robb Chair in Ophthalmology Memphis TN
Kellogg Eye Center USA
Department of Ophthalmology University of Michigan
Children’s Hospital Boston Ann Arbor MI Kevin Kalwerisky MD
Boston MA USA Department of Otolaryngology, Head & Neck
USA Surgery
R Paul Johnson MD The New York Presbyterian Hospital
Laryssa A Huryn MD Associate Professor of Medicine
Bascom Palmer Eye Institute Weill Medical College of Cornell University
Infectious Diseases Unit New York NY
Miami FL Massachusetts General Hospital
USA USA
Charlestown MA
Deeba Husain MD USA Henry J Kaplan MD
Assistant Professor of Ophthalmology Professor and Chairman
Robert N Johnson MD Department of Ophthalmology and Visual
Retina Service - Dept of Ophthalmology Assistant Clinical Professor of Ophthalmology
Boston University School of Medicine Sciences
Department of Ophthalmology University of Louisville
Boston MA University of California
USA Louisville KY
West Coast Retina Medical Group USA
Robert A Hyndiuk MD San Francisco CA
The Eye Institute USA Ekaterini C Karatza MD
Medical College of Wisconsin Staff Ophthalmologist
Karen M Joos MD PhD Cincinnati Eye Institute
Milwaukee WI Associate Professor
USA Cincinnati OH
Department of Ophthalmology and Visual USA
Michael Ip MD Sciences
Associate Professor of Ophthalmology Vanderbilt University Randy Kardon MD PhD
Department of Ophthalmology and Visual Nashville TN Associate Professor of Ophthalmology
Sciences USA Director of Neuro-ophthalmology
Fundus Photograph Reading Center The University of Iowa Hospitals and Clinics
Nancy C Joyce PhD Iowa City IA
Madison WI Schepens Eye Research Institute
USA USA
Senior Scientist Associate Professor, Harvard
Brian J Jacobs MD Medical School James A Katowitz MD
Assistant Professor of Ophthalmology Boston MA Attending Surgeon
Rush University Medical Center USA Ophthalmology
Chicago IL Childrens Hospital of Philadelphia
J Michael Jumper MD Philadelphia PA
USA Assistant Clinical Professor of Ophthalmology USA
Frederick A Jakobiec MD DSc(Med) University of California
Former Henry Willard Williams Professor and Director, Retina Service William R Katowitz MD
Former Chief of Ophthalmology West Coast Retina Medical Group Department of Ophthalmology
Departments of Pathology and San Francisco CA University of Rochester School of Medicine
Ophthalmology USA and Dentistry
Harvard Medical School Rochester NY
Ula V. Jurkunas MD USA
Boston MA Instructor in Ophthalmology
USA Massachusetts Eye and Ear Infirmary Melanie Kazlas MD
Lee M Jampol MD Clinical Scientist Acting Director; Instructor
Louis Feinberg Professor and Chairman Schepens Eye Research Institute Pediatric Ophthalmology & Strabismus
Department of Ophthalmology Harvard Medical School Massachusetts Eye & Ear Infirmary
Northwestern University Medical School Boston MA Boston MA
Chicago IL USA USA
USA Kelly S Keefe CAPT MC USN
Harold G Jensen PhD Staff Ophthalmic Pathologist
Clinical Project Manager Comprehensive Ophthalmologist
Allergan, Inc Naval Medical Center
Irvine CA San Diego CA
xxxii USA USA
 List of Contributors

Lara Kelley MD Jonathan W Kim MD Thomas Kohnen MD

Assistant Professor, Dermatology Physician Professor of Ophthalmology
Harvard Medical School Memorial Sloan-Kettering Cancer Center Deputy Chairman
Beth Israel Deaconess Medical Center New York NY Klinik fur Augenheilkunde
Boston MA USA Johann Wolfgang Goethe University
USA Frankfurt
Rosa Y Kim MD Germany
Charles J Kent MD Physician
Fellowship Training in Ocuplastics and Ocular Vitreoretinal Consultants Takeshi Kojima MD PhD
Pathology Houston TX Research Group for Environmental
Everett & Hurite Ophthalmology Associates USA Conservation Processing
Pittsburgh PA Department of Material Development
USA Stella K Kim MD Takasaki Radiation Chemistry Research
Assistant Professor of Ophthalmology Establishment
Kenneth R Kenyon MD FACS Section of Ophthalmology Japan Atomic Energy Research Institute
Associate Clinical Professor MD Anderson Cancer Center Takasaki-shi
Harvard Medical School; Houston TX Japan
Eye Health Vision Centers USA
North Dartmouth MA Tobias Koller MD
USA Tae-Im Kim MD PhD Refractive Surgeon
Department of Ophthalmology Institute of Ophthalmic and Refractive
Bilal F Khan MD Yonsei University Health System Surgery
Assistant in Ophthalmology Seoul Zurich
Massachusetts Eye and Ear Infirmary South Korea Switzerland
Harvard Medical School
Boston MA Christina M Klais MD David A Kostick MD
USA Retina Fellow Assistant Professor of Ophthalmology
LuEsther T Mertz Retinal Research Center Department of Ophthalmology
Jemshed A Khan MD Manhattan Eye, Ear and Throat Hospital Mayo Clinic College of Medicine
Clinical Professor of Ophthalmology New York NY Jacksonville FL
Kansas University USA USA
Kansas City MO
USA Stephen R Klapper MD FACS Joel A Kraut MD
Ophthalmologist Medical Director
Naheed W Khan PhD Klapper Eyelid & Facial Plastic Surgery Vision Rehabilitation Service
Electrophysiologist Carmel IN Massachusetts Eye and Ear Infirmary
Department of Ophthalmology and Visual USA Harvard Medical School
Sciences Boston MA
W K Kellogg Eye Center Barbara E K Klein MD MPH
USA
University of Michigan Professor of Medicine
Ann Arbor MI Department of Ophthalmology and Visual Chandrasekharan Krishnan MD
USA Sciences Assistant Professor of Ophthalmology
University of Wisconsin Medical School Tufts University School of Medicine
Peng Tee Khaw PhD FRCP FRCS FRCOphth Madison WI Glaucoma and Cataract Service
FIBiol FRCPath FMedSci USA New England Eye Center
Professor of Glaucoma and Ocular Healing Boston MA
and Consultant Ophthalmic Surgeon Guy Kleinmann MD
USA
Biomedical Research Centre (Ophthalmology) Adjunct Assistant Professor of
UCL Institute of Ophthalmology and Ophthalmology Ronald R Krueger MD MSE
Moorfields Eye Hospital Department of Ophthalmology Director of Refractive Surgery, Cleveland
London Herman Eye Center Clinic Foundation, Cleveland, OH, USA
United Kingdom Houston TX Saint Louis University Eye Institute
USA Saint Louis University School of Medicine
Femida Kherani MD FRCSC St Louis MO
Ophthalmic Cosmetic Surgeon Thomas Klink DrMed USA
Heights Laser Centre Scientific Assistant
Burnaby BC Department of Ophthalmology Joseph H Krug Jr, MD
Canada University of Würzburg Assistant Director of Glaucoma Consultation
Würzburg Service
Eva C Kim MD Germany Massachusetts Eye and Ear Infirmary
Fellow in Ocular Inflammation/Uveitis Harvard Medical School
The Proctor Foundation Dino D Klisovic MD Boston MA
University of California San Francisco Department of Ophthalmology USA
San Francisco CA Nationwide Children’s Hospital
USA Midwest Retina Inc Sara Krupsky MD
Columbus OH Goldschleger Eye Institute
Hee Joon Kim MD USA Sheba Medical Center
Resident Tel Hashomer
Department of Ophthalmology and Visual Stephen D Klyce Israel
Science Executive Editor
Department of Ophthalmology Rachel W Kuchtey MD PhD
University of Texas Health Science Center at
Louisiana State University Eye Institute Clinical Ophthalmologist, Glaucoma
Houston
New Orleans LA Vanderbilt University of Ophthalmology &
Houston TX
USA Visual Sciences
USA
Nashville TN
Ivana K Kim MD Tolga Kocaturk MD USA
Instructor of Ophthalmology Department of Ophthalmology
Retina Service Adnan Menderes University Medical School
Massachusetts Eye and Ear Infirmary Aydin
Harvard Medical School Turkey
Boston MA
USA xxxiii
 List of Contributors

Ramsay S Kurban MD Jonathan H Lass MD Andrea Leonardi MD

Clinical Assistant Professor Charles I Thomas Professor and Chairman Assistant Professor in Ophthalmology
Department of Dermatology CWRU Department of Ophthalmology and Department of Neuroscience, Ophthalmology
Penn State University Visual Sciences Chairman Unit
Milton S Hershey Medical Center Department of Ophthalmology and Visual University of Padua
Hershey PA Sciences Padua
USA University Hospitals Case Medical Center Italy
Cleveland OH
Paul A Kurz MD USA Simmons Lessell MD
Instructor of Ophthalmology Director, Neuro-Ophthalmology Service
Casey Eye Institute Mary G Lawrence MD MPH Massachusetts Eye and Ear Infirmary
Oregon Health & Science University Associate Professor, Glaucoma, Cataract and Professor, Harvard Medical School
Portland OR Visual Rehabilitation Boston MA
USA Glaucoma Service USA
University of Minnesota Medical School
J R Kuszak PhD Minneapolis MN Leonard A Levin MD PhD
Departments of Ophthalmology and USA Professor of Ophthalmology and Visual
Pathology Sciences, Neurology, and Neurological
Rush University Medical Center Andrew G Lee MD Surgery
Chicago IL Professor of Ophthalmology, Neurology and University of Wisconsin School of Medicine
USA Neurosurgery and Public Health
Departments of Ophthalmology, Neurology Madison WI
Young H Kwon MD PhD and Neurosurgery USA
Associate Professor of Ophthalmology University of Iowa Hospitals Canada Research Chair of Ophthalmology
Department of Ophthalmology Iowa City IA and Visual Sciences
University of Iowa USA University of Montreal
Iowa City IA Montreal QC
USA Carol M Lee MD Canada
Clinical Professor, Department of
Thad A Labbe MD Ophthalmology Grace A Levy-Clarke MD
Glaucoma Specialist NYU Medical Center Fellowship Program Director
Ophthalmologist New York NY Uveitis and Ocular Immunology
Eye Associates of Central Texas USA Laboratory of Immunology
Austin TX National Eye Institute
USA Michael S Lee MD National Institutes of Health
Associate Professor Bethesda MD
Deborah L Lam MD Departments of Ophthalmology, Neurology,
Pacific Northwest Eye Associates USA
and Neurosurgery
Tacoma WA University of Minnesota Julie C Lew MD
USA Minneapolis MN Assistant Clinical Professor
Jeffrey C Lamkin MD USA Suny Downstate Medical Centre
Department of Ophthalmology Department of Ophthalmology
Paul P Lee MD JD Brooklyn NY
Akron City Hospital Professor of Ophthalmology
The Retina Group of NE Ohio Inc USA
Department of Ophthalmology
Akron OH Duke University Eye Center Craig Lewis MD
USA Durham NC Cole Eye Institute
Kathleen A Lamping MD USA Cleveland Clinic
Associate Clinical Professor Cleveland OH
William B Lee MD USA
Department of Ophthalmology Eye Consultant
Case Western Reserve University Eye Consultants of Atlanta Wei Li MD PhD
South Euclid OH Piedmont Hospital Research Fellow
USA Atlanta GA Ocular Surface Center
Anne Marie Lane MPH USA Miami FL
Clinical Research Manager, Retina Service USA
Igal Leibovitch MD
Massachusetts Eye and Ear Infirmary Oculoplastic and Orbital Division Laurence S Lim MBBS
Instructor in Ophthalmology, Harvard Medical Ophthalmology Department Principal Investigator
School Tel-Aviv Medical Center Singapore National Eye Centre
Boston MA Tel-Aviv Singapore
USA Israel
Lyndell L Lim MBBS FRANZCO
Katherine A Lane MD Bradley N Lemke MD FACS Mankiewicz-Zelkin Crock Fellow
Resident, Oculoplastic and Orbital Surgery Clinical Professor of Oculofacial Surgery Centre for Eye Research Australia
Service Department of Ophthalmology and Visual University of Melbourne
Wills Eye Hospital Sciences East Melbourne VIC
Philadelphia PA University of Wisconsin - Madison Australia
USA Madison WI
USA Wee-Kiak Lim FRCOphth FRCS(Ed) MMED
Keith J Lane MD Associate Consultant
Senior Manager, Research and Development Craig A Lemley MD Ocular Inflammation and Immunology
/Preclinical The Eye Institute Singapore National Eye Centre
ORA Clinical Research and Development Medical College of Wisconsin Singapore
North Andover MA Milwaukee WI
USA USA Grant T Liu MD
Neuro-ophthalmologist
Children’s Hospital of Philadelphia
Philadelphia PA
USA

xxxiv
 List of Contributors

John I Loewenstein MD Robert E Marc PhD Michael McCrakken

Associate Professor of Ophthalmology Director of Research Clinical Instructor
Retina Service John A Moran Eye Center Department of Ophthalmology
Massachusetts Eye and Ear Infirmary Salt Lake City UT University of Colorado Health Sciences
Harvard Medical School USA Center
Boston MA Denver CO
USA Mellone Marchong USA
Department of Applied Molecular Oncology
McGregor N Lott MD Ontario Cancer Institute - University Health James P McCulley MD
Department of Ophthalmology Network Professor & Chairman of Ophthalmology
Medical College of Georgia Princess Margaret Hospital Department of Ophthalmology
Augusta GA Toronto ON University of Texas Southwestern Medical
USA Canada Center
Dallas TX
Jonathan C Lowry MD Dennis M Marcus MD USA
Ophthalmologist Professor of Clinical Ophthalmology
Morganton Eye Physicians Department of Clinical Ophthalmology John A McDermott
Morganton NC Southeast Retina Center Assistant Clinical Professor of Ophthalmology
USA Augusta GA Department of Ophthalmology
USA New York Eye and Ear Infirmary
David B Lyon MD FACS New York NY
Associate Professor Julie A Mares PhD USA
Department of Ophthalmology Professor
University of Missouri-Kansas City School of Department of Ophthalmology & Visual H Richard McDonald MD
Medicine Sciences Director, San Francisco Retina Foundation
Prairie Village KS WARF Co-Director, Vitreoretinal Fellowship
USA Madison WI California Pacific Retina Center
USA West Coast Retina Medical Group
Robert E Lytle MD San Francisco CA
Ophthalmologist Brian P Marr MD USA
Massachusetts Eye and Ear Infirmary Oncology Service
Harvard Medical School Wills Eye Institute Marguerite B McDonald MD FACS
Boston MA Thomas Jefferson University Ophthalmic Consultants of Long Island
USA Philadelphia PA Lynbrook NY
USA USA
Mathew MacCumber MD PhD
Associate Professor Carlos E Martinez MS MD Peter J McDonnell MD
Associate Chairman of Research Eye Physicians of Long Beach William Holland Wilmer Professor of
Rush University Medical Center Long Beach CA Ophthalmology
Chicago IL USA Director, Wilmer Ophthalmological Institute
USA Johns Hopkins University School of Medicine
Robert W Massof PhD Baltimore MD
Bonnie T Mackool MD MSPH Professor of Ophthalmology, Professor of USA
Director of Dermatology Neuroscience
Consultation Service Director, Lions Vision Research and Robert McGillivray BSEE CLVT
Massachusetts General Hospital Rehabilitation Center Director
Boston MA Wilmer Ophthalmological Institute Low Vision Services
USA Johns Hopkins University School of Medicine The Carroll Center for the Blind
Baltimore MD Low Vision Engineering Consultant
Nalini A Madiwale MD USA Massachusetts Commission for the Blind
Physician Newton MA
Albany-Troy Cataract & Laser Associates Yukihiro Matsumoto USA
Troy NY Research Fellow
USA Ocular Surface Research and Education Craig A McKeown MD
Foundation Associate Professor of Clinical
Francis Mah MD Miami FL Ophthalmology
Assistant Professor of Ophthalmology USA Bascom Palmer Eye Institute
Department of Ophthalmology Miller School of Medicine
University of Pittsburgh Medical Center Cynthia Mattox MD University of Miami
Pittsburgh PA Assistant Professor of Ophthalmology Miami FL
USA Ophthalmology - New England Eye Center USA
Tufts-New England Medical Center
Martin A Mainster PhD MD FRCOphth Boston MA James McLaughlin MD
Fry Endowed Professor and Vice Chairman of USA Medical Writer
Ophthalmology Ophthalmic Research Associates, Inc
Department of Ophthalmology Marlon Maus MD North Andover MA
University of Kansas School of Medicine DrPH Candidate USA
Kansas City MO University of California at Berkeley
USA Berkeley CA W Wynn McMullen MD
USA Vitereoretinal Consultant
Michael H Manning Jr Coastal Eye Associates
c/o Sherman M Chamberlain MD FACP Cathleen M McCabe MD Houston TX
FACG Indiana LASIK Center USA
Medical College of Georgia Fort Wayne IN
Augusta GA USA Shlomo Melamed MD
USA The Sam Rothberg Glaucoma Centre
Steven A McCormick MD Goldschleger Eye Institute
Steven L Mansberger MD MPH Director of Pathology and Laboratory Sheba Medical Center
Associate Scientist Medicine Tel-Hashomer
Devers Eye Institute The New York Eye and Ear Infirmary Israel
Portland OR New York NY
USA USA xxxv
 List of Contributors

George Meligonis FRCPath Neil R Miller MD A Linn Murphree MD

Corneoplastic Unit Professor of Ophthalmology, Neurology and Director
Queen Victoria Hospital Neuro-Ophthalmology The Retinoblastoma Centre
East Grinstead Departments of Ophthalmology, Neurology Childrens Hospital of Los Angeles
East Sussex and Neuro-Ophthalmology Los Angeles CA
United Kingdom Wilmer Eye Institute USA
Johns Hopkins Hospital
Efstratios Mendrinos MD Baltimore MD Robert P Murphy MD
Ophthalmic Fellow USA The Retina Group of Washington
Ophthalmic Service Fairfax VA
Geneva University David M Mills MD USA
Geneva Oculofacial Plastic, Reconstructive, and
Switzerland Cosmetic Surgeon Timothy G Murray MD MBA FACS
Nicolitz Eye Consultants Professor of Ophthalmology
Dale R Meyer MD Jacksonville FL Department of Ophthalmology
Director, Ophthalmic Plastic Surgery USA Bascom Palmer Eye Institute
Professor of Ophthalmology Miami FL
Lions Eye Institute Monte D Mills MD USA
Albany Medical Center Chief, Division of Ophthalmology
Albany NY Children’s Hospital of Philadelphia Philip I Murray PhD FRCP FRCS FRCOphth
USA Philadelphia PA Professor of Ophthalmology
USA Academic Unit of Ophthalmology
Catherine B Meyerle MD Birmingham and Midland Eye Centre
Retinal Physician Tatyana Milman MD City Hospital NHS Trust
National Eye Institute Assistant Professor of Ophthalmology Birmingham
National Institutes of Health Co-director, Ophthalmic Pathology Division United Kingdom
Bethesda MD Institute of Ophthalmology and Visual
USA Science Karina Nagao MD
UMDNJ-New Jersey Medical School Harvard Medical School
William F Mieler MD Newark NJ Boston MA
Professor and Chairman USA USA
Department of Ophthalmology and Visual
Science Lylas Mogk MD Jay Neitz PhD
University of Chicago Director R D and Linda Peters Professor
Chicago IL Visual Rehabilitation and Research Center Department of Ophthalmology
USA Henry Ford Health System Medical College of Wisconsin
Livonia MI Milwaukee WI
Michael Migliori MD USA USA
Clinical Associate Professor
The Warren Alpert Medical School Marja Mogk PhD Maureen Neitz PhD
Brown University Assistant Professor of English Richard O Schultz-Ruth A
Providence RI California Lutheran University Works-Ophthalmology Research Professor
USA Los Angeles CA The Eye Institute
USA Medical College of Wisconsin
Martin C Mihm Jr, MD Milwaukee WI
Clinical Professor of Pathology Jordi Monés MD USA
Senior Dermatopathologist Associate Professor of Ophthalmology
The Pigmented Lesion Clinic Institut de la Macula i de la Retina Peter A Netland MD PhD
Massachusetts General Hospital Barcelona Siegal Professor of Ophthalmology, Director
Boston MA Spain of Glaucoma, Academic Vice-Chair
USA Department of Ophthalmology
Robert Montes-Micó OD MPhil Hamilton Eye Institute
Darlene Miller DHSc MPH SM CIC Optica University of Tennessee Health Science
Research Assistant Professor Facultat de Fisica Center
Scientific Director Universidad de Valencia Memphis TN
Abrams Ocular Microbiology Laboratory Valencia USA
Bascom Palmer Eye Institute Spain
Anne Bates Leach Eye Hospital Arthur H Neufeld PhD
Miller School of Medicine Christie L Morse MD Professor of Ophthalmology
University of Miami Concord Eye Care Forsythe Laboratory for the Investigation of
Miami FL Concord NH Aging Retina
USA USA Northwestern University Fienberg School of
Medicine
David Miller MD Asa D Morton MD Chicago IL
Associate Clinical Professor of Ophthamology Eye Care of San Diego/CA Laser Vision, Inc USA
Department of Ophthalmology Escondido CA
Harvard Medical School USA Nancy J Newman MD
Jamaica Plain MA Professor of Ophtalmology and Neurology
Anne Moskowitz OD PhD Neuro-Ophthalmology Unit
USA Research Associate in Ophthalmology Emory Eye Center
Joan W Miller MD Children’s Hospital, Boston Atlanta GA
Henry Willard Williams Professor of Instructor of Ophthalmology USA
Ophthalmology Harvard Medical School
Chief and Chair, Department of Boston MA Eugene W M Ng MD
Ophthalmology USA Eyetech Pharmaceuticals, Inc
Massachusetts Eye and Ear Infirmary New York NY
Shizuo Mukai MD USA
Harvard Medical School Assistant Professor of Ophthalmology
Boston MA Massachusetts Eye and Ear Infirmary
USA Harvard Medical School
Boston MA
xxxvi USA
 List of Contributors

Quan Dong Nguyen MD MSc Yen Hoong Ooi MD Louis R Pasquale MD

Assistant Professor of Ophthalmology c/o Douglas Rhee MD Co-Director, Glaucoma Service
Diseases of the Retina and Vitreous, and Massachusetts Eye and Ear Infirmary Assistant Professor of Ophthalmology
Uveitis Harvard Medical School Department of Ophthalmology
Wilmer Eye Institute Boston MA Massachusetts Eye and Ear Infirmary
Johns Hopkins Hospital USA Harvard Medical School
Baltimore MD Boston MA
USA E Mitchel Opremcak MD USA
Clinical Associate Professor
Jerry Y Niederkorn PhD Department of Ophthalmology Neha N Patel MD
Professor of Ophthalmology Ohio State University College of Medicine Resident
Department of Ophthalmology Columbus OH Department of Ophthalmic and Visual
University of Texas Southwestern Medical USA Science
Center University of Chicago
Dallas TX George Ousler BS Chicago IL
USA Director USA
Dry Eye Department
Robert J Noecker MD Ophthalmic Research Associates Sayjal J Patel MD
Vice Chair, Clinical Affairs North Andover MA Wilmer Eye Institute
Eye and Ear Institute USA Baltimore MD
Associate Professor USA
University of Pittsburgh School of Medicine Randall R Ozment MD
Pittsburgh PA Physician Thomas D Patrianakos DO
USA Dublin Eye Associates Attending Physician
Dublin GA Division of Ophthalmology
Robert B Nussenblatt MD MPH USA John H Stroger Hospital of Cook County
Scientific Director and Chief, Laboratory of Chicago IL
Immunology, Intramural Program Samuel Packer MD USA
Section Head, Clinical Immunology Section Professor of Clinical Ophthalmology,
National Eye Institute New York University School of Medicine James R Patrinely MD FACS
National Institutes of Health Chair, Department of Ophthalmology Plastic Eye Surgery Associates PLLC
Bethesda MD North Shore Long Island Jewish Health Houston TX
USA System USA
New York NY
Joan M O’Brien MD USA Deborah Pavan-Langston MD FACS
Professor of Ophthalmology and Pediatrics Associate Professor of Ophthalmology
Director of Ocular Oncology Millicent L Palmer MD Surgeon and Director of Clinical Virology
Division of Ophthalmology Associate Professor, Department of Surgery Massachusetts Eye and Ear Infirmary
University of California San Francisco Creighton University Medical School Harvard School of Medicine
Medical Center Division of Ophthalmology Boston MA
San Francisco CA Creighton University Medical Center USA
USA Omaha NE
USA Eli Peli MSc OD
Paul D O’Brien FRCSI MRCOphth MMedSci Professor of Ophthalmology
Specialist Registrar in Ophthalmology George N Papaliodis MD Harvard Medical School
Royal Victoria Eye and Ear Hospital Instructor in Ophthalmology and Internal Moakley Scholar in Aging Eye Research
Dublin Medicine Schepens Eye Research Institute
Ireland Massachusetts Eye and Ear Infirmary Boston MA
Harvard Medical School USA
Terrence P O’Brien MD Boston MA
Professor of Ophthalmology USA Susan M Pepin MD
Charlotte Breyer Rodgers Distinguished Chair Assistant Professor of Surgery
in Ophthalmology D J John Park MD Section of Ophthalmology
Director of the Refractive Surgery Service Resident Dartmouth Hitchcock Medical Center
Bascom Palmer Eye Institute Department of Plastics and Reconstructive Lebanon NH
Palm Beach FL Surgery USA
USA University of California
Victor L Perez MD
Irvine CA
Assistant Professor
Denis O’Day MD FACS USA
Bascom Palmer Eye Institute
Professor of Ophthalmology
David W Parke II MD University of Miami School of Medicine
Department of Ophthalmology
Edward L Gaylord Professor and Chairman Miami FL
Vanderbilt Eye Institute
Department of Ophthalmology USA
Nashville TN
USA President and CEO Juan J Pérez-Santonja MD PhD
The Dean A McGee Eye Institute Instituto Oftalmológico de Alicante
R Joseph Olk MD Oklahoma City OK Alicante
Bond Eye Associates USA Spain
Peoria IL
USA Cameron F Parsa MD John R Perfect MD
Assistant Professor of Ophthalmology Director, Duke University Mycology Research
Karl R Olsen MD Krieger Children’s Eye Center Unit (DUMRU)
Clinical Assistant Professor of Ophthalmology The Wilmer Eye Institute Division of Infectious Diseases
University of Pittsburgh School of Medicine Baltimore MD Department of Medicine
Retina Vitreous Consultants USA Duke University
Pittsbrugh PA Winston-Salem NC
USA M Andrew Parsons FRCPath
Honorary Consultant in Ophthalmic USA
Sumru Onal MD Pathology
Department of Ophthalmology Academic Unit of Pathology
Marmara University School of Medicine Royal Hallamshire Hospital
Istanbul Sheffield
Turkey United Kingdom xxxvii
 List of Contributors

Henry D Perry MD FACS Valerie Purvin MD Elias Reichel MD

Founding Partner Clinical Professor of Ophthalmology & Associate Professor of Ophthalmology
Director: Cornea Division Neurology Vitreoretinal Diseases
Ophthalmic Consultants of Long Island Departments of Ophthalmology and New England Eye Center
Rockville Center NY Neurology Tufts University School of Medicine
USA Indiana Medical Center Boston MA
Indianapolis IN USA
Joram Piatigorsky PhD USA
Chief Martin H Reinke MD
Laboratory of Molecular and Developmental David A Quillen MD Private Practice
Biology George and Barbara Blankenship Professor Southlake TX
National Eye Institute - National Institute of and Chair USA
Health Department of Ophthalmology
Bethesda MD Penn State College of Medicine Douglas Rhee MD
USA Hershey PA Assistant Professor of Ophthalmology
USA Department of Ophthalmology
Dante Pieramici MD Massachusetts Eye and Ear Infirmary
Co-Director Graham E Quinn MD Harvard Medical School
California Retina Consultants Attending Surgeon, Research Fellow Boston MA
Santa Barbara CA Department of Ophthalmology USA
USA The Childrens Hospital of Philadelphia
Philadelphia PA Claudia U Richter MD
Eric A Pierce MD PhD Ophthalmic Consultants of Boston
Assistant Professor of Ophthalmology USA
Boston MA
F.M. Kirby Center for Molecular Melvin D Rabena BSc USA
Ophthalmology Director of Research
Scheie Eye Institute California Retina Consultants Joseph F Rizzo lll MD
University of Pennsylvania School of Medicine Santa Barbara CA Associate Professor of Ophthalmology
Philadelphia PA USA Massachusetts Eye and Ear Infirmary
USA Harvard Medical School
James L Rae PhD Boston MA
Roberto Pineda II MD Professor of Ophthalmology and Physiology USA
Assistant Professor Physiology and Biomedical Engineering
Massachusetts Eye and Ear Infirmary Mayo Clinic Richard M Robb MD
Chief of Ophthalmology, Brigham & Women’s Rochester MN Associate Professor of Ophthalmology
Hospital, Boston USA Harvard Medical School
Assistant Professor, Department of Department of Ophthalmology
Ophthalmology, Harvard Medical School Michael B Raizman MD Children’s Hospital Boston
Boston MA Ophthalmic Consultant Boston MA
USA Ophthalmic Consultants Of Boston USA
Associate Professor of Ophthalmology
Misha L Pless MD Tafts University School of Medicine Anja C Roden MD
Director, Division of General Neurology Boston MA c/o Diva R Salomao MD
Massachusetts General Hospital USA Department of Pathology
Boston MA Mayo Clinic
USA Alessandro Randazzo MD Rochester MN
Department of Ophthalmology USA
Howard D Pomeranz MD PhD Istituto Clinico Humanitas Rozzano
Clinical Associate Professor Milano University I Rand Rodgers MD
Department of Ophthalmology Milan Assistant Clinical Professor, Mount Sinai
North Shore Long Island Jewish Health Italy Medical Center
System Director of Ophthalmic Facial and Plastic
Great Neck NY Narsing A Rao MD Surgery
USA Professor of Ophthalmology and Pathology North Shore University Hospital NYU
Doheny Eye Institute Private Practice
Constantin J Pournaras MD University of California New York NY
Department of Ophthalmology Los Angeles CA USA
Geneva University Hospitals USA
Geneva Merlyn M Rodrigues MD PhD
Switzerland Christopher J Rapuano MD c/o Kelly S Keefe MD
Co-Director Cornea Service Naval Medical Center
William Power MBBCH FRCS FRCOphth Co-Director Professor of Ophthalmology, San Diego CA
Consultant Ophthalmic Surgeon Jefferson Medical College USA
Blackrock Clinic Thomas Jefferson University
Blackrock Co-Director, Cornea Service Yonina Ron MD
Co Dublin Refractive Surgery Department Department of Ophthalmology
Ireland Wills Eye Hospital Rabin Medical Center
Philadelphia PA Beilinson Campus
Manvi Prakash MD Petah Tiqva
Postdoctoral Fellow USA
Israel
Joslin Diabetes Center Sherman W Reeves MD MPH
Beetham Eye Institute Cornea, External Disease and Retractive Geoffrey E Rose DSC MS MRCP FRCS
Harvard Medical School Surgery FRCOphth
Boston MA Minnesota Eye Consultants Consultant Ophthalmic Surgeon
USA Minneapolis MN Adnexal Department
USA Moorfields Eye Hospital
Anita G Prasad MD London
Department of Ophthalmology and Visual Carl D Regillo MD FACS United Kingdom
Sciences Professor of Ophthalmology
Washington University Medical School Wills Eye Hospital
St Louis MO Philadelphia PA
xxxviii USA USA
 List of Contributors

Emanuel S Rosen MD FRCS FRCOphth Mark S Ruttum MD Michael A Sandberg PhD

Consultant Ophthalmic Surgeon Professor of Ophthalmology Associate Professor of Ophthalmology
Manchester Central Health Care Authority Head, Pediatric Ophthalmology and Adult Berman-Gund Laboratory
Manchester Strabismus Section Massachusetts Eye and Ear Infirmary
United Kingdom Medical College of Wisconsin Harvard Medical School
Milwaukee WI Boston MA
James T Rosenbaum MD USA USA
Professor of Medicine, Ophthalmology and
Cell Biology Allan R Rutzen MD FACS Virender S Sangwan MD
Chief, Division of Arthritis and Rheumatic Associate Professor of Ophthalmology Head, Cornea and Anterior Segment Services
Diseases Department of Ophthalmology L V Prasad Eye Institute
Director, Uveitis Clinic Casey Eye Institute University of Maryland Hyderabad
Oregon Health and Science University Baltimore MD India
Portland OR USA
Maria A Saornil MD
USA
Edward T Ryan MD Ocular Pathology Unit
Perry Rosenthal MD Director, Tropical & Geographic Medicine Hospital Clinico Universitario
Assistant Clinical Professor of Ophthalmology Center Valladolid
Department of Ophthalmology Massachusetts General Hospital Spain
Boston Foundation for Sight Associate Professor of Medicine Joseph W Sassani MD
Boston MA Harvard Medical School Professor of Ophthalmology and Pathology
USA Assistant Professor Pennsylvania State University
Dept of Immunology and Infectious Diseases Hershey Medical Center
Strutha C Rouse II MD Harvard School of Public Health
Horizon Eye Care Hershey PA
Boston MA USA
Charlotte NC USA
USA Rony R Sayegh MD
Alfredo A Sadun MD PhD Research Fellow
Barry W Rovner MD Thornton Professor of Ophthalmology and
Professor & Medical Director Cornea and Refractive Surgery Service
Neurosurgery Massachusetts Eye and Ear Infirmary
Department of Psychiatry and Human Doheny Eye Institute
Behavior Department of Ophthalmology
Kech School of Medicine Boston MA
Thomas Jefferson University University of California
Philadelphia PA USA
Los Angeles CA
USA USA Andrew P Schachat MD
Malgorzata Rozanowska PhD Vice Chairman for Clinical Affairs
José-Alain Sahel MD Cole Eye Institute
Lecturer Professor of Ophthalmology
School of Optometry and Vision Sciences Cleveland Clinic Foundation
Head, Laboratory of Retinal Pathobiology Cleveland OH
Cardiff University University Louis Pasteur
Cardiff USA
Strasbourg
United Kingdom France Wiley A Schell MD
Michael P Rubin MD Director, Medical Mycology Research Center
Leorey Saligan MD Assistant Professor of Medicine Department
Fellow in Vitreoretinal Diseases and Surgery Nurse Practitioner
Massachusetts Eye and Ear Infirmary, of Medicine
National Eye Institute Division of Infectious Diseases and
Harvard Medical School National Institutes of Health
Boston MA International Health
Bethesda MD Duke University Medical Center
USA USA Durham NC
Peter A D Rubin MD FACS Sarwat Salim MD FACS USA
Eye Plastics Consultant Assistant Clinical Professor of Ophthalmology
Brookline MA Amy C Schefler MD
Yale Eye Center Resident in Ophthalmology
Associate Clinical Professor Yale University School of Medicine
Harvard Medical School Bascom Palmer Eye Institute
New Haven CT Miami FL
USA USA USA
Shimon Rumelt MD John F Salmon MD FRCS FRCOphth
Attending Physician Tina Scheufele MD
Consultant Ophthalmic Surgeon Vitreoretinal Surgeon
Ophthalmology Department The Radcliffe Infirmary
Western Galilee - Nahariya Medical Center Ophthalmic Consultants of Boston
Oxford Eye Hospital Boston MA
Nahariya Oxford
Israel USA
United Kingdom
Anil K Rustgi MD Vivian Schiedler MD
Diva R Salomão MD Oculoplastic and Orbital Surgeon,
Professor of Medicine and Genetics Associate Professor of Pathology
Chief of Gastroenterology Charlottesville, VA
Department of Pathology Ophthalmic Plastic & Reconstructive Surgery
University of Pennsylvania Medical Center Mayo Clinic
Philadelphia PA Fellow
Rochester MN Department of Ophthalmology
USA USA University of Washington
Tina Rutar MD David Sami MD Seattle WA
Resident Division Chief for PSF Ophthalmology USA
Department of Ophthalmology CHOC Children’s Hospital
University of California San Francisco Gretchen Schneider MD
Orange CA Adjunct Assistant Professor in the Genetic
San Francisco CA USA
USA Counseling program
Genetic Counseling Faculty
Brandeis University
Waltham MA
USA
xxxix
 List of Contributors

Alison Schroeder BA Irina Serbanescu BA Bradford J Shingleton MD

Laboratory Manager Research Assistant Clinical Professor of
Department of Ophthalmology Division of neurology Ophthalmology, Harvard Medical School
Boston University School of Medicine The Hospital for Sick Children Ophthalmic Consultants of Boston
Boston MA Toronto ON Boston MA
USA Canada USA
Ronald A Schuchard PhD Briar Sexton MD FRCSC John W Shore MD FACS
Director of Rehabilitation Research and Fellow in Neuro-Ophthalmology Texas Oculoplastics Consultants
Development Center VGH Eye Care Center Austin TX
Associate Professor Vancouver BC USA
Department of Neurology Canada
Emory University School of Medicine Lesya M Shuba MD PhD
Atlanta GA Tarek M Shaarawy MD Assistant Professor
USA Chef Department of Ophthalmology & Visual
Clinique d’ophtalmologie Sciences
Joel S Schuman MD Secteur du Glaucome Dalhousie University
Eye and Ear Foundation Professor and Hôpitaux Universitaires de Génève Halifax NS
Chairman Génève Canada
Department of Ophthalmology Switzerland
University of Pittsburgh School of Medicine Guy J Ben Simon MD
Pittsburgh PA Peter Shah BSc (Hons) MBChB FRCOphth Goldschleger Eye Institute
USA Consultant Sheba Medical Center
Birmingham and Midland Eye Centre Tel Hashomer
Ivan R Schwab MD FACS City Hospital Israel
Professor of Ophthalmology Birmingham
Department of Ophthalmology United Kingdom Richard J Simmons MD
University of California at Davis Emeritus Ophthalmic Surgeon
Sacramento CA Aron Shapiro BS Harvard Medical School
USA Director Boston MA
Anti-inflammatory/Anti-infectives Department USA
Adrienne Scott MD Ophthalmic Research Associates
Clinical Associate North Andover MA Michael Simpson
Vitreoretinal Surgery USA c/o David Miller MD
Duke University Eye Center Department of Ophthalmology
Durham NC Savitri Sharma MD MAMS Harvard Medical School
USA Associate Director, Laboratory Services Jamaica Plain MA
L V Prasad Eye Institute USA
Ingrid U Scott MD MPH Bhubaneswar, Orissa
Professor of Ophthalmology and Health India Arun D Singh MD
Evaluation Sciences Director
Department of Ophthalmology Jean Shein MD Department of Ophthalmic Oncology
Penn State College of Medicine Attending Physician Cole Eye Institute and Taussing Cancer Center
Hershey PA Crane Eye Care Hana Kukui Center Cleveland OH
USA Lihue HI USA
USA
Marvin L Sears MD Omah S Singh MD
Professor and Chairman Emeritus Debra J Shetlar MD Director
Department of Ophthalmology and Visual Associate Professor of Ophthalmology New England Eye Center
Science Baylor College of Medicine Beverley MA
Yale University School of Medicine Staff Physician USA
New Haven CT Michael E DeBakey V A Medical Center
Houston TX Karen Sisley BSc PhD
USA Non-Clinical Lecturer Ocular Oncology
USA
Johanna M Seddon MD ScD Academic Unit of Ophthalmology and
Professor of Ophthalmology M Bruce Shields MD Orthoptics
Tufts University School of Medicine Professor of Ophthalmology and Visual University of Sheffield
Director, Ophthalmic Epidemiology and Science Sheffield
Genetics Service Yale Eye Center United Kingdom
New England Eye Center New Haven CT
USA Arthur J Sit MD
Boston MA Assistant Professor of Ophthalmology
USA Carol L Shields MD Mayo Clinic
Theo Seiler MD PhD Professor of Ophthalmology, Thomas Rochester MN
Professor Jefferson Medical College USA
Institut für Refractive und Ophthalmochirurgie Attending Surgeon and Associate Director
David Smerdon FRCSEd FRCOphth
(IROC) Wills Eye Hospital
Consultant Ophthalmologist
Zürich Philadelphia PA
James Cook University Hospital
Switzerland USA
Middlesbrough
Jerry A Shields MD United Kingdom
Robert P Selkin MD
Private Practice Professor of Ophthalmology, Thomas William E Smiddy MD
Plano TX Jefferson University Professor of Ophthalmology
USA Director Department of Ophthalmology
Oncology Services Bascom Palmer Eye Institute
Richard D Semba MD MA MPH Wills Eye Hospital Miami FL
W Richard Green Professor of Philadelphia PA USA
Ophthalmology USA
Wilmer Eye Institute
Baltimore MD
USA
xl
 List of Contributors

Ronald E Smith MD Tomy Starck MD Timothy J Sullivan FRANZCO FRACS

Professor and Chair Director Eyelid, Lacrimal and Orbital Clinic
Department of Ophthalmology UltraVision Center Department of Ophthalmology
Keck School of Medicine of USC San Antonio TX Royal Brisbane Hospital
Los Angeles CA USA Herston QLD
USA Australia
Walter J Stark MD
Terry J Smith MD Professor of Ophthalmology Jennifer K Sun MD
Professor and Head Director of the Stark-Mosher Center Lecturer
Division of Molecular Medicine The John Hopkins Hospital, Wilmer Eye Joslin Diabetes Center
David Geffen School of Medicine Institute Beetham Eye Institute
Harbor-UCLA Medical Center Baltimore MD Harvard Medical School
Torrance CA USA Boston MA
USA USA
Joshua D Stein MD MS
Neal G Snebold MD Assistant Professor Janet S Sunness MD
Ophthalmologist Department of Ophthalmology and Visual Medical Director
Massachusetts Eye and Ear Infirmary Sciences Richard E Hoover Rehabilitation Services for
Harvard Medical School Kellogg Eye Center Low Vision and Blindness
Boston MA Ann Arbor MI Greater Baltimore Medical Center
USA USA Baltimore MD
Lucia Sobrin MD USA
Roger F Steinert MD
Instructor of Ophthalmology Professor of Ophthalmology and Biomedical Francis C Sutula MD
Retina and Uvetis Services Engineering Milford Eye Care
Massachusetts Eye and Ear Infirmary Director of Cornea, Refractive and Cataract Milford MA
Harvard Medical School Surgery USA
Boston MA Vice Chair of Clinical Ophthalmology
USA Department of Ophthalmology Nasreen A Syed MD
University of California Irvine Assistant Professor, Ophthalmology and
John A Sorenson MD Pathology
Attenting Surgeon Irvine CA
USA Department of Ophthalmology and Visual
Vitreoretinal Service Sciences
Manhattan Eye, Ear, and Throat Hospital Leon Strauss MD University of Iowa
New York NY Instructor Iowa City IA
USA Wilmer Eye Institute USA
Sarkis H Soukiasian MD John Hopkins University School of Medicine
Baltimore MD Christopher N Ta MD
Director: Cornea and External Disease
USA Associate Professor of Ophthalmology
Director: Ocular Inflammation and Uveitis
Department of Ophthalmology
Lahey Clinic
Barbara W Streeten MD Stanford University
Burlington MA
Professor of Ophthalmology and Pathology Palo Alto CA
USA
State University of New York USA
George L Spaeth MD FRCO FACS Upstate Medical University
Louis Esposito Research Professor of Syracuse NY Hidehiro Takei MD
Ophthalmology USA Staff Pathologist
Jefferson Medical College Department of Pathology
Director of the William & Anna Goldberg J Wayne Streilein MD (deceased) The Methodist Hospital
Glaucoma Service Formerly Senior Scientist, President, Charles Houston TX
Wills Eye Institute L Schepens Professor of Ophthalmology, USA
Philadelphia PA Professor of Dermatology
Formerly Vice Chair for Research, Jonathan H Talamo MD
USA Associate Clinical Professor of
Department of Ophthalmology
Richard F Spaide MD Harvard Medical School Ophthalmology
Associate Clinical Professor of Ophthalmology Boston MA Department of Ophthalmology
Manhattan Eye, Ear, and Throat Hospital USA Harvard Medical School
New York NY Waltham MA
USA James D Strong CRA USA
Senior Ophthalmic Imager
Monika Srivastava MD Department of Ophthalmology Richard R Tamesis MD
Clinical Assistant Professor Penn State Milton S Hershey Medical Center Department of Ophthalmology
Department of Dermatology Hershey PA Loma Linda University Medical Center
New York University USA Loma Linda CA
New York NY USA
USA Ilene K Sugino MS
Director, Ocular Cell Transplantation Madhura Tamhankar MD
Sunil K Srivastava MD Laboratory Associate Professor
Assistant Professor of Ophthalmology Institute of Ophthalmology and Visual Science Department of Ophthalmology
Section of Vitreoretinal Surgery & Disease New Jersey Medical School University of Pennsylvania Medical School
Emory Eye Center Newark NJ Philadelphia PA
Atlanta GA USA USA
USA Kristen J Tarbet MD SACS
Eric B Suhler MD MPH
Alexandros N Stangos MD Chief of Ophthalmology Private Practice
Division of Ophthalmology Portland VA Medical Center Bellevue WA
Department of Clinical Neurosciences Assistant Professor of Ophthalmology and USA
University Hospitals of Geneva Co-director
Geneva Department of Ophthalmology
Switzerland Casey Eye Institute
Portland OR
USA
xli
 List of Contributors

Michelle Tarver-Carr MD PhD Gail Torkildsen MD Russell N Van Gelder MD PhD

Assistant, Ocular Immunology Physician Associate Professor of Ophthalmology and
Wilmer Eye Institute Andover Eye Associates Visual Sciences
Departments of Medicine and Epidemiology Andover MA Department of Ophthalmology and Visual
Johns Hopkins University School of Medicine USA Sciences
Baltimore MD Washington University School of Medicine
USA Cynthia A Toth MD St Louis MO
Associate Professor of Ophthalmology and USA
Mark A Terry MD Biomedical Engineering
Director, Corneal Services Duke Eye Center Gregory P Van Stavern MD
Clinical Professor, Department of Durham NC Assitant Professor of Ophthalmology,
Ophthalmology USA Neurology and Nerosurgery
Devers Eye Institute Kresge Eye Institute
Oregon Health Sciences University Elias I Traboulsi MD Wayne State University
Portland OR Professor of Ophthalmology Detroit MI
USA The Cole Eye Institute USA
Cleveland OH
Joseph M Thomas MD USA Deborah K Vander Veen MD
Associate Clinical Professor Assistant Professor
Department of Neurology Michele Trucksis PhD MD Department of Ophthalmology
Case Western Reserve University School of Associate Clinical Professor Children’s Hospital and Harvard Medical
Medicine Harvard Medical School School
Cleveland OH Associate Director Clinical Pharmacology Boston MA
USA Merck & Co. Inc USA
Boston MA
Vance Thompson MD USA Demetrios Vavvas MD PhD
Assistant Professor of Medicine Instructor in Ophthalmology
University of South Dakota School of James C Tsai MD Retina Service Department of Ophthalmology
Medicine Robert R Young Professor and Chairman Massachusetts Eye and Ear Infirmary
Director of Refractive Surgery Department of Ophthalmology and Visual Harvard Medical School
Sioux Valley Clinic Science Boston MA
Vance Thompson Vision Yale University School of Medicine USA
Sioux Falls SD New Haven CT
USA USA David H Verity MA FRC Ophth
Consultant Ophthalmic Surgeon
Jennifer E Thorne MD PhD Julie H Tsai MD Adnexal Departments
Assistant Professor of Ophthalmology Assistant Professor Moorfields Eye Hospital
Division of Ocular Immunology Department of Ophthalmology London
Wilmer Eye Institute University of South Carolina School of United Kingdom
Baltimore MD Medicine
USA Columbia SC Paolo Vinciguerra MD
USA Medical Director
Matthew J Thurtell BSc(Med) MBBS MScMed Studio Oculistico Vincieye SRL
Neuro-Ophthalmology Fellow David T Tse MD FACS Milan
Department of Neurology Professor of Ophthalmology Italy
Royal Prince Albert Hospital Department of Ophthalmology
Sydney NSW Bascom Palmer Eye Institute Paul F Vinger MD
Australia Miami FL Clinical Professor
USA Ophthalmology
David P Tingey MD FRCSC Tufts University School of Medicine
Associate Professor Scheffer C G Tseng MD PhD New England Medical Center
Ivey Eye Institute Research Director Boston MA
London Health Sciences Center Ocular Surface Center USA
London ON Miami FL
Canada USA Nicholas J Volpe MD
Professor of Ophthalmology and Neurology
King W To MD Elmer Y Tu MD Vice Chair and Residency Program Director
Clinical Professor of Ophthalmology Associate Professor of Clinical Department of Ophthalmology
Brown University School of Medicine Ophthalmology PENN Eye Care
Barrington RI Director of the Cornea and External Disease Philadelphia PA
USA Service USA
Department of Ophthalmology
Faisal M Tobaigy MD University of Illinois at Chicago Werner Wackernagel MD
Department of Ophthalmology Chicago IL Physician
Massachusetts Eye and Ear Infirmary and the USA Department of Ophthalmology
Schepens Eye Research Institute Medical University Graz
Harvard Medical School Ira J Udell MD Graz
Boston MA Professor of Ophthalmology Austria
USA Albert Einstein College of Medicine
New York NY Sonal Desai Wadhwa MD
Michael J Tolentino MD USA Assistant Professor of Ophthalmology
Director of Research, Center for Retina and Division of Ophthalmology
Macular Disease Alejandra A Valenzuela MD University of Maryland
Center for Retina and Macular Disease Assistant Professor Baltimore MD
Winter Haven FL Department of Ophthalmology and Visual USA
USA Sciences
Dalhousie University
Melissa G Tong BSc Halifax NS
Department of Medicine Canada
Jefferson Medical College
Philadelphia PA
xlii USA
 List of Contributors

Michael D Wagoner MD Scott M Whitcup MD Jules Winokur MD

Professor of Ophthalmology Executive Vice President North Shore Long Island Jewish Health
Department of Ophthalmology and Visual Head of Research and Development System
Sciences Allegran Inc New York NY
University of Iowa Hospitals and Clinics Irvine CA USA
Iowa City IA USA
USA William J Wirostko MD
Valerie A White MD FRCPC Associate Professor of Ophthalmology
Nadia K Waheed MD Professor The Eye Institute
Fellow Department of Pathology & Laboratory Medical College of Milwaukee
Immunology and Uveitis Service Medicine, Milwaukee WI
Department of Ophthalmology University of British Columbia USA
Massachusetts Eye and Ear Infirmary Vancouver General Hospital
Harvard Medical School Vancouver BC Gadi Wollstein MD
Boston MA Canada Assistant Professor and Director
USA Ophthalmic Imaging Research Laboratories
William L White MD The Eye & Ear Institute
David S Walton MD Department of Ophthalmology Dept of Ophthalmology
Clinical Professor of Ophthalmology The Eye Foundation UPMC Eye Center
Harvard Medical School University of Missouri-Kansas City Pittsburgh PA
Boston MA Kansas City MO USA
USA USA
Albert Chak Ming Wong FCOph(HK)
Martin Wand MD Jason Wickens MD FHKAM(Ophth)
Clinical Professor of Ophthalmology Barnes Retina Institute Associate Consultant
University of Connecticut School of Medicine Department of Ophthalmology Caritas Medical Center
Farmington CT Washington University School of Medicine Shamshuipo, Kowloon
USA St Louis MO Hong King
USA China
Jie Jin Wang MMed PhD
Associate Professor of Epidemiology Janey L Wiggs MD PhD Tien Y Wong MBBS MMED (Ophth) FRCSE
Westmead Millennium Institute Associate Professor of Ophthalmology FRANZCO FAFPHM MPH PhD
University of Sydney Massachusetts Eye and Ear Infirmary Professor of Ophthalmology
Sydney NSW Harvard Medical School Department of Ophthalmology & Centre for
Australia Boston MA Eye Research Australia
USA University of Melbourne
Scott M Warden MD East Melbourne VIC
Retina Service Jacob T Wilensky MD Australia
Massachusetts Eye and Ear Infirmary Professor of Ophthalmology
Department of Ophthalmology Director, Glaucoma Service John J Woog MD FACS
Harvard Medical School University of Illinois College of Medicine Associate Professor of Ophthalmology,
Boston MA Chicago IL Ophthalmic Plastic and Reconstructive
USA USA Surgery
Department of Ophthalmology
Lennox Webb FRCOphth FRCS(Ed) Charles P Wilkinson MD Mayo Clinic
Consultant Ophthalmic Surgeon Chairman, Department of Ophthalmology Rochester MN
Royal Alexandra Hospital Greater Baltimore Medical Center USA
Paisley Professor, Department of Ophthalmology
United Kingdom John Hopkins University Michael Wride PhD
Baltimore MD Lecturer
David Weber MD USA School of Optemetry and Vision Sciences
Assistant Professor Cardiff University
Department of Physical Medicine & Patrick D Williams MD Cardiff
Rehabilitation Vitreo Retinal Specialist United Kingdom
Mayo Clinic College of Medicine Texas Retina Associates
Rochester MN Arlington TX Carolyn S Wu MD
USA USA Instructor of Ophthalmology
Harvard Medical School
Daniel Wee MD David J Wilson MD Boston MA
Department of Ophthalmology Associate Professor USA
The Palmetto Health/ University of South Department of Ophthalmology;
Carolina School of Medicine Director, Christensen Eye Pathology Darrell WuDunn MD PhD
Columbia SC Laboratory Associate Professor of Ophthalmology
USA Casey Eye Institute Indiana University School of Medicine
Oregon Health Sciences University Indianapolis IN
Corey B Westerfeld MD Portland OR USA
Research Fellow USA
Department of Ophthalmology Jean Yang MD
Massachusetts Eye and Ear Infirmary M Roy Wilson MD MS Department of Ophthalmology
Harvard Medical School Chancellor North Shore-Long Island Jewish Medical
Boston MA University of Colorado and Health Sciences Center
USA Center Great Neck NY
Denver CO USA
Christopher T Westfall MD USA
Professor of Ophthalmology Lawrence A Yannuzzi MD
Jones Eye Institute & Arkansas Children’s Steven E Wilson MD Vice-Chairman, Department of
Hospital Director of Corneal Research and Professor Ophthalmology
University of Arkansas for Medical Sciences of Ophthalmology Director of Retinal Services
Little Rock AR The Cleveland Clinic Foundation Manhattan Eye, Ear and Throat Hospital
USA Cole Eye Institute New York NY
Cleveland OH USA
USA xliii
 List of Contributors

Michael J Yaremchuk MD Jenny Y Yu MD Marco Zarbin MD PhD FACS

Clinical Professor of Surgery Consulting Physician Professor of Ophthalmology and
Harvard Medical School Department of Ophthalmology Neuroscience
Boston MA UPMC Children’s Hospital of Pittsburgh Department of Ophthalmology
USA Pittsburgh PA Institute of Ophthalmology and Visual
USA Science
R Patrick Yeatts MD FACS University of Medicine and Dentistry,
Professor and Vice Chairman Beatrice Y J T Yue PhD New Jersey
Department of Ophthalmology Thanis A Field Professor of Ophthamology Newark NJ
Wake Forest University Eye Center Department of Ophthalmology & Visual USA
Winston-Salem NC Sciences
USA University of Illinois at Chicago Leonidas Zografos MD
Chicago IL Professor and Chairman
Richard W Yee MD USA Jules Gonin Eye Hospital
Medical Director LADARVISION Center Lausanne
Hermann Eye Center Charles M Zacks MD Switzerland
Memorial Hermann Hospital Corneal Specialist
Houston TX Maine Eye Center Christopher I Zoumalan MD
USA Portland ME Resident in Ophthalmology
USA Department of Ophthalmology
Steven Yeh MD Stanford University Medical Center
Clinical Fellow Bruce M Zagelbaum MD FACS Stanford CA
Uveitis and Ocular Immunology Associate Clinical Professor of USA
Laboratory of Immunology Ophthalmology
National Eye Institute New York University School of Medicine
National Institute of Health New York NY
Bethesda MD USA
USA
Maryam Zamani MD
Lucy H Y Young MD PhD FACS Oculoplastic Fellow
Associate Professor London
Massachusetts Eye and Ear Infirmary United Kingdom
Harvard Medical School
Boston MA
USA

xliv
 SECTION 1 GENETICS
Edited by Janey L. Wiggs and Thaddeus P. Dryja
CHAPTER
1 Fundamentals of Genetics
Thaddeus P. Dryja
A GENE IS DEFINED BY A PHENOTYPE
Genes are the fundamental units used in the study of inherited
traits or diseases. A gene is classically defined by the phenotype
that is associated with it. For example, the gene causing
choroideremia is the choroideremia gene, and the gene causing
retinoblastoma is the retinoblastoma gene. However, in more
recent years, many genes have been defined on the basis of the
encoded protein product, irrespective of any phenotypes known
to be associated with variations or mutations. For instance, a
gene on chromosome 3 is named the ‘rhodopsin gene’ because
it encodes rhodopsin. Years after the isolation and characteriza-
tion of the rhodopsin gene, it was discovered that mutations at
this gene can cause retinitis pigmentosa or stationary night
blindness. Rather than renaming the locus as the retinitis
pigmentosa gene or otherwise, this gene retains its name as the
rhodopsin gene.
The term ‘gene’ is actually somewhat ambiguous, because it
can refer to the position on a chromosome (a locus) that governs
a heritable trait or to a form of the DNA sequence at the locus
(an allele) that is associated with a particular phenotype. There-
fore, in common usage, one might state that a variation in iris
color is due to a ‘gene’, and it is also correct to state that a
brown-eyed person has the ‘gene’ for a brown iris. In the first
case, one is stating that a genetic locus has alleles that specify
iris color, and in the second case, one is referring to a particular
allele at the iris color locus. To be more specific and unam-
biguous, one should state that a genetic locus controls iris color
and that an individual with brown eyes carries a brown allele
at that locus. The distinction is important, especially when one
counsels a family with a hereditary disease such as retino-
blastoma. The family may speak of the affected child as having
the ‘retinoblastoma gene’. They will be surprised to learn from
the ophthalmologist that all family members have the ‘retino-
blastoma gene’, but that some relatives have normal versions of
the gene that do not predispose to the cancer. Only those rela-
tives with a mutant version have a high risk of being affected.
Despite the ambiguities, the different uses for the word ‘gene’
are so ingrained that any attempt to change them is futile.
LINEAR POLYMERS OF DNA ARE THE
CHEMICAL BASES FOR GENES
The chemical material that contains genetic information is
DNA. This is a linear polymer with two complementary
strands. Each strand is made up of a linear array of purine bases,
guanine (G) and adenine (A), and pyrimidine bases, cytosine (C)
and thymine (T). Each base is linked covalently to a pentose;
the combination is called a nucleoside. A single strand of
DNA has a series of the four bases coupled through these
carbohydrate moieties by phosphate bonds. The genetic
information is contained in the specific sequence of the four
bases in the 5„ to 3„ direction, where the 5„ and 3„ designations
refer to the sites on the pentose moieties where phosphate
bonds are linked. This strand is called the sense strand. The
complementary strand, or antisense strand, runs in the oppo-
site direction and invariably has nucleotides complementary to
those in the sense strand as illustrated in Figure 1.1.
DNA–RNA–PROTEIN
A gene is determined by the particular order of bases within a
specified region (locus) in a molecule of DNA. Each gene codes
for one protein. RNA is the chemical intermediate that con-
veys the base sequence in DNA to the protein-synthesizing
machinery (ribosomes) in the cytoplasm of a cell. RNA is com-
posed of the same purine and pyrimidine bases as DNA, except
that the pyrimidine base thymine (T) present in DNA is instead
uracil (U) in RNA. Another difference is that the pentose linked
to each base is ribose rather than deoxyribose. The RNA mole-
cules that transmit the DNA base sequence to the cytoplasm
of a cell are called messenger RNA molecules, or mRNA. The
synthesis of mRNA molecules from a DNA template is called
transcription. The synthesis of strands of amino acids based on
the sequence of bases in mRNA is called translation.
ORGANIZATION OF A EUKARYOTIC GENE
Eukaryotic genes, including human genes, are transcriptional
units; that is, each gene is organized for the synthesis of a
distinct mRNA sequence that codes for a distinct protein.
Transcriptional units are organized in the following manner
(Fig. 1.2). At the 5„ end is a region extending a few hundred
bases called the promoter region. This region has sequences
recognized by factors (typically proteins) that control the
expression of the gene, as well as one or more binding sites for
RNA polymerase. Besides the promoter region, other regions
within a gene or at some distance from it can also have roles in
determining the proper tissue-specific expression of a gene at
the proper time during the life of the organism.1
Downstream of the promoter region is the transcription
start site, which is a specific base at which the enzyme ‘RNA
polymerase’ initiates the synthesis of an RNA copy of the
DNA sequence. The sequence of bases in the transcribed RNA
molecule will be identical to the sequence in the sense strand of
DNA, except that the base uracil (U) will be used instead of
thymine (T), as noted earlier. Next comes the 5„ untranslated
region, or the region of sequence that is included in the RNA
1
SECTION 1 GENETICS

FIGURE 1.1. Chemical structure of DNA.

(a) Two hydrogen bonds (dotted lines) couple
the bases thymine and adenine, and three
hydrogen bonds couple guanine and cytosine.
(b) The double-helical structure of the linear
DNA strands.

2
 CHAPTER 1
Fundamentals of Genetics

a b c d

FIGURE 1.2. Functional organization of a transcriptional unit. The organization of the human blue cone opsin gene, which consists of ~4000 bp
of DNA within human chromosome 7, is shown.47 Top, Schematic representation of the position of each of the five exons. The letters (a) through
(d) indicate the four regions illustrated in more detail below, where the DNA sequence (sense strand only) at each of the four positions is shown.
(a) The 5„ end of the gene. The TATA box is the sequence TATAA, which is an important recognition sequence for the binding of a factor that
allows RNA polymerase to initiate transcription. The transcription start site is the point at which an RNA copy of the DNA sequence is begun.
The RNA sequence differs from the DNA sequence only in that a U (uridine) is used instead of a T (thymine). The first segment of transcribed
DNA is the 5„ untranslated region. Translation begins with the sequence AUG, which is called the initiation codon or the start codon. It specifies
methionine, which will be at the amino terminus (N) of the resultant amino acid sequence. (b) Intron 1. The first intron begins with the
dinucleotide sequence GT and ends with the sequence AG. These dinucleotide sequences are almost invariably present at the ends of introns
and are called the splice donor and splice acceptor sites, respectively. Notice that a codon is split by the intron. This is neither the rule nor the
exception. (c) Termination of translation. In the last exon (exon 5) a stop codon occurs – in this case the sequence TGA. Although transcription
of RNA continues beyond this codon, the remaining RNA sequence is not translated into an amino acid sequence and therefore is called the 3„
untranslated region. (d) Polyadenylation. The polyadenylation signal sequence, ATTAAA, is recognized by factors that cause the termination of
transcription 20 bases downstream. At the end of the RNA sequence, a large string of As is added. The final RNA transcript, after the excision of
the four introns and the addition of the poly-A sequence, is called a messenger RNA, or mRNA. It is transported to the cytoplasm for translation 3
by the ribosomes.
SECTION 1 GENETICS
transcript but is not used to code for a protein. The coding
region begins with the initiation codon, which is always the
triplet of bases ‘ATG’ coding for methionine. The succeeding
sequence of bases is called the coding region and is organized
into codons or triplets of bases that specify the amino acids
of the encoded protein. The coding region ends with a stop
codon (either TGA, TAG, or TAA), which is followed by the 3„
untranslated region. Finally, a polyadenylation signal sequence
registers the end of transcription by RNA polymerase.
A noteworthy feature of eukaryotic genes, but not prokaryotic
genes, is that the coding region in genomic DNA is generally
interrupted by one or more introns. After an RNA transcript is
produced from a gene, these intron sequences are excised. This
is one of the steps necessary to make mature messenger RNA
or mRNA. The term cDNA is given to any DNA fragment with
FIGURE 1.3. Translation of mRNA. A ribosome is depicted
schematically in the process of synthesizing a molecule of blue cone
opsin.
4
a sequence identical to that found in an mRNA molecule (i.e.,
a DNA sequence lacking intron sequences). cDNA molecules are
not normally produced in living cells; instead, they are produced
in research laboratories and are used as reagents helpful in
studying genes.
GENETIC CODE
The DNA sequence that specifies the sequence of amino acids
of a protein is in the form of a genetic ‘code’. In the cytoplasm
of cells, ribosomes translate the code (Fig. 1.3). Each set of three
consecutive nucleotides, called a codon, in the coding region of
an mRNA molecule specifies one amino acid.
Figure 1.4 shows the amino acid specified by each codon. The
codon ATG, which specifies the amino acid methionine, is
the only codon used by the ribosome to initiate translation.
Hence, all proteins are first synthesized with the amino acid
methionine at their amino terminus. (This amino acid may be
subsequently removed as a posttranslational modification of
the protein.) Ribosomes recognize the correct ATG sequence
present near the 5„ end of the mRNA for initiating translation;
other ATG codons nearby are customarily ignored through mech-
anisms that remain unclear. Downstream from the initiating
codon, every three bases specify one amino acid. There is no
skipping or overlapping of codons. This process continues until
one of the codons TAG, TGA, or TAA is encountered in the
same frame as the initiating codon. These three codons are
called stop or termination codons, because any one of them
serves to terminate the translation of an mRNA molecule.
HOW GENES ARE ORGANIZED IN HUMAN
CELLS
DNA molecules that carry genetic information are packaged
into chromosomes. A chromosome is thought to be composed
of a single long DNA molecule and numerous associated
proteins and perhaps other substances. The complex of DNA
and associated materials in chromosomes is called chromatin.
FIGURE 1.4. The genetic code. This wheel
gives the amino acid specified by any three-
base codon. The codon is read from the center
to the periphery of the wheel. Amino acids are
abbreviated using the standard three-letter
code. At the bottom of the figure is the one-
letter code, the three-letter code, and the full
name of each amino acid.
Adapted from Ausubel FM, Brent R, Kingston RE, et
al: Current protocols in molecular biology. New York:
Wiley; 1991.

HUMAN CHROMOSOMES
Each nucleus of a human cell has 23 pairs of chromosomes
(Fig. 1.5), corresponding to 46 molecules of DNA. The two
chromosomes in each pair typically have an identical appear-
ance and have the same complement of genetic loci in the same
order. They are distinguished because they can carry different
alleles at each locus. Each member of a pair of chromosomes is
derived from a different parent. Of the 23 pairs of chromo-
somes, 22 are called autosomes; the remaining pair embodies
the sex chromosomes. The 22 autosomes are numbered
according to their size, with chromosome 1 being the largest
chromosome, chromosome 2 the next in size, and so forth. The
only exception to this rule involves chromosomes 21 and 22,
because chromosome 21, not 22, is the smallest. The sex
chromosomes are not named by numbers but instead are called
the X and Y chromosomes.
Each chromosome has a centromere that divides it into two
arms, the short arm and the long arm (Fig. 1.6). The short arm
and long arm are called the ‘p’ arm and the ‘q’ arm, respectively.
The proximal portion of a chromosome arm is the region close
to the centromere; the distal portion is far from the centromere.
A chromosome with a very small short arm is called an
acrocentric chromosome. Acrocentric human chromosomes are
numbers 13, 14, 15, 21, and 22. The short arms of acrocentric
chromosomes contain multiple copies of the genes coding for
ribosomal RNA rather than for proteins.
Until the early 1970s, chromosomes could only be dis-
tinguished on the basis of their overall size and the relative size
of their short and long arms. Because of this, many human
chromosomes could not be uniquely distinguished, and
chromosomes of similar morphology were lumped into groups
(e.g., the ‘A’ group, ‘B’ group). As an example, the ‘D’ group
included chromosomes 13, 14, and 15; all of these are acro-
CHAPTER 1
Fundamentals of Genetics
FIGURE 1.5. A normal human karyotype.
Below the 22 pairs of autosomes are the sex
chromosomes. Since both X and Y
chromosomes are present, this karyotype is
from a male.
Courtesy of Cynthia Morton, PhD.
centric chromosomes of approximately the same size. A patient
with a deletion of any of those three chromosomes was diag-
nosed as having a ‘D-deletion’. A few cases of retinoblastoma
with a deletion of a D group chromosome were reported in
the 1960s, and this association was called ‘D-deletion
retinoblastoma’.2,3
Improved chromosome banding techniques, using dyes such
as quinacrine or Giemsa, became widely used by the early
1970s. A pattern of staining that is unique to each chromosome
arm allowed the recognition of every human chromosome.
There is now a standardized nomenclature for the set of darkly
and lightly staining bands characteristic of each human chro-
mosome arm. To continue the example of ‘D-deletion’ retino-
blastoma, after the new karyotyping techniques were developed,
it was discovered that in all cases of ‘D-deletion retino-
blastoma’, the deleted chromosome was always chromosome
13, hence the name of the association was changed to ‘13-
deletion retinoblastoma’. Furthermore, in every case, the deletion
included the band 14 on the long arm of the chromosome,4 so
that the term 13q14 deletion or 13q14– is more precise.
Another important deletion associated with ophthalmologic and
systemic abnormalities involves chromosome 11p13; deletions
of this chromosomal segment cause a syndrome including
aniridia and elevated predisposition to Wilms’ tumor.5
SIZE OF THE HUMAN GENOME
A set consisting of one of each autosome as well as both sex
chromosomes is called a human genome. It includes one copy
of every human locus. The chromosomal molecules of DNA
from one human genome, if tandemly arranged end to end,
contain a sequence of ~3.2 billion bp. The amount of informa-
tion contained within 3.2 billion bp can be instructively related 5
SECTION 1 GENETICS
FIGURE 1.6. Anatomy of a chromosome, in this case human
chromosome 7.
to the quantity of information stored on modern desktop com-
puters. At each position in DNA there is one of four possible
bases (A, T, G, and C), which is equivalent to two bits of
computer code. Since there are eight bits in a byte of computer
memory, each byte could store the equivalent of four bases of
DNA sequence. The DNA sequence of the human genome
would occupy ~800 MB. The sequence could be stored on a
1-GB hard drive (small by today’s standards) with plenty of
room to spare. Obtaining the complete sequence of the human
genome within the first decade of the twenty-first century was
one of the initial goals of the Human Genome Project. The first
draft of the complete human genome sequence was obtained
in 2001.6
In terms of the physical size of the human genome, the cor-
responding DNA would be 1 m long but only 2 nm in diameter.
The total volume of a human genome, assuming the DNA is a
cylinder, is about one hundred millionth of a microliter. Current
estimates are that there are 60 000–100 000 genes embedded in
this DNA sequence. On an average, there is one gene about
every 30 000 bp.
HAPLOIDY, DIPLOIDY, TRIPLOIDY
A set consisting of one of each autosome as well as an X or a Y
chromosome is called a haploid set of chromosomes. The
normal complement of two copies of each gene (or two copies of
each chromosome) is called diploidy. In unusual circumstances,
a cell or organism may have three copies of each chromosome;
this is called triploidy. A triploid human is not viable; however,
6 some patients have an extra chromosome or an extra segment
of a chromosome. In such a situation, the abnormality is called
trisomy for the chromosome involved. For example, patients
with Down’s syndrome have three copies of chromosome 21,
also referred to as trisomy 21. Much the same phenotype can
also result from trisomy of only the long arm of chromosome
21, or trisomy 21q.
If one copy of a pair of chromosomes is absent, the defect
is called haploidy or deletion. Haploidy for an entire human
chromosome is probably lethal, but individuals do exist who
have a deletion of a segment of a chromosome.
TRANSLOCATIONS
Occasionally, a hybrid chromosome will be observed in the
karyotype of an individual, with a mixture of material derived
from two separate chromosomes. As a hypothetical example, a
part of chromosome 1q might be fused to 3p. Depending on the
number of normal chromosomes 1 and 3, an individual who
carries a translocation (1q;3p) could be trisomic or monosomic
for these chromosome arms. A translocation is ‘balanced’ if
there is a diploid amount of each chromosome band.
SISTER CHROMATIDS
Just before a cell divides, each chromosome arm is duplicated,
so that chromosomes have two identical short arms and two
identical long arms (see Fig. 1.6). At this point, there are four
copies of each gene in a cell. Each chromosome has two short
arms and two long arms, and each arm is called a chromatid. A
pair of similar arms from the same chromosome is called a pair
of sister chromatids. When one examines the ‘karyotype’ of a
cell, the chromosomes are observed just before the cell divides.
Consequently, each chromosome has two sister chromatids cor-
responding to the short arm and two sister chromatids corre-
sponding to the long arm. Sister chromatids always share the
same alleles, whereas the two chromosome homologs in a human
cell (one derived from each parent), can have different alleles at
any locus.
ALLELES ARE VARIATIONS IN THE
NUCLEOTIDE SEQUENCE
An allele is a specific nucleotide sequence at a locus that is
associated with an observable phenotype. The most common
allele at a locus is called the wild-type allele, often abbreviated
‘+’ or ‘wt’. An allele that is different from the wild type is
customarily given an abbreviated name that is somehow related
to the phenotype or the nucleotide sequence. For example, an
allele in the rhodopsin gene causing autosomal dominant retinitis
pigmentosa could be labeled RhoPro23His or rhodopsin, Pro23His,
where Pro23His indicates that codon 23, which specifies proline
in the wild-type allele, specifies histidine in the mutant allele.7
Although a genetic locus usually corresponds with a tran-
scriptional unit, the boundaries of a locus in a DNA sequence
are often not very precise. One reason for this is that DNA
sequences many thousands of bases from the transcriptional
unit can be important for the proper expression of a gene at the
correct time during the development of a specific cell type.1 It
is conceivable that a mutation in such distant sequences can
change the expression of a transcriptional unit and produce a
phenotype associated with the locus. Hence, it is a simplifica-
tion to state that alleles are the result of variations in the
nucleotide sequence inside a transcriptional unit. In practice,
however, this is usually the case.
If an allele has a frequency of 1–2% or higher and is not
associated with a disease, it is called a polymorphism. Since
humans have two alleles at each locus, the arbitrary criterion of

a 1% allele frequency corresponds with a polymorphism for
which ~2% of unrelated individuals are carriers. An example is
the still unidentified locus on chromosome 19, where a poly-
morphism specifies the presence or absence of green iris color.8
If an allele occurs with a frequency less than 1%, it is a rare
variant. If an allele causes disease, it is customarily called a
mutation. Most mutations are rare variants. However, at least
one is at a frequency high enough to be considered a poly-
morphism: ~2% of whites carry the Phe508del mutation that
causes cystic fibrosis.9
Genetic diseases are defined clinically before the underlying
causative gene defects are known. Most clinically defined
hereditary diseases turn out to be genetically heterogeneous.
Allelic heterogeneity is the term used when different mutant
alleles at the same locus can produce the same disease. For
example, numerous mutations in the Rab escort protein gene
have been found to produce choroideremia.10 Nonallelic hetero-
geneity refers to the situation when mutations in different genes
can produce the same clinically defined disease. An example of
nonallelic heterogeneity is retinitis pigmentosa, which can be
produced as a result of defects in any of dozens of different
genes.11 Gene sharing occurs if different mutations in the same
gene can produce different phenotypes. For instance, defects in
the Norrie disease gene can produce either Norrie disease,
exudative vitreoretinopathy, or predisposition to retinopathy
of prematurity.12–14 Another example of two diseases sharing
the same genes is retinitis pigmentosa and congenital
stationary night blindness. Different defects in the rhodopsin
gene can produce these two diseases;7,15,16 so too can different
defects in the gene encoding the b subunit of rod cGMP-
phosphodiesterase.17,18
Key Features: Fundamentals of Genetics
• Genes are defined by phenotypes and are chemically
composed of DNA.
• In cells DNA is packaged into chromosomes, and a genome is
a complete set of chromosomes. The human genome contains
two copies each of 22 autosomes and two sex chromosomes.
• Alleles are variations in DNA sequence at genetic loci.
• Human disorders can be inherited as dominant, recessive,
X-linked, mitochondrial (also called maternal), digenic, and
polygenic traits.
• DNA sequence variations among human populations have
made it possible to develop a map of the human genome.
• Mutations are changes in DNA sequence that have biological
consequences.
HEREDITARY TRANSMISSION OF GENETIC
INFORMATION
SOMATIC CELLS VERSUS GERM CELLS
Most of the cells in the human body are somatic cells. Somatic
cells have a ‘diploid’ set of chromosomes (i.e., two copies of
each autosome, one derived from each parent) and two sex
chromosomes (either XX or XY). Somatic cells are produced as
a consequence of mitosis or cell division (Fig. 1.7). Before a cell
divides into two daughter cells, the entire complement of
chromosomes duplicates so that the cell has four copies of every
autosomal gene. Each daughter cell receives a complete, diploid
set of chromosomes with solitary short and long arms.
The second category of human cells involves those in the
germ line; that is, cells whose descendants are ‘germ cells’
(sperm and ova). Germ cells are haploid. The process that
creates germ cells is called meiosis. Meiosis encompasses two
cell divisions (Fig. 1.7). In the first meiotic division, each
CHAPTER 1
Fundamentals of Genetics
daughter cell receives one member of each homologous pair.
The daughter cells are therefore haploid. They, nevertheless,
have two of each chromatid. The chromosomes separate during
the second meiotic division to produce haploid germ cells with
only one of each chromatid.
RECOMBINATION
In somatic cells, it is the general rule that each chromosome
homolog has a set of alleles derived from one parent. After
meiosis, a germ cell is haploid; that is, it has only one member
of each pair of chromosomes. Hence, a germ cell could have the
maternally derived chromosomes 1, 2, 4, 7, and so on, and the
paternally derived chromosome 3, 5, 6, 8, and so forth.
This mixing of chromosomes is one source of the diversity
that is provided by sexual reproduction. However, it is only half
of the story. During the first meiotic division, chromatids from
homologous chromosomes can recombine or crossover (Fig.
1.7). During this process, the chromatids exchange linear sets
of alleles so that the daughter chromosomes have a mixture of
maternal and paternal alleles. This is the second major source
for new combinations of genes. The resultant germ cells receive
a random mixture of these hybrid chromosomes.
Roughly 30 crossovers (also called ‘recombination events’)
occur during each meiosis. Crossovers can take place anywhere
along the length of a chromosome arm, although there appear
to be regions that are especially susceptible to it (called ‘recom-
bination hot spots’). Also, there is a relatively greater likelihood
of a crossover happening in the distal portion of a chromosome
arm compared with the proximal portion. The rate of recom-
bination occurring at any particular region of a chromosome
can be different in males and females.
During oogenesis, the two X chromosomes carried by a
female can recombine anywhere along their length just as with
autosomes. In contrast, the X and Y chromosomes of a male
usually do not recombine, and if they do, crossovers occur only
within the distal short arms.
Considering that during meiosis an average of 30 crossovers
occur among the 23 pairs of human chromosomes, most
chromosomes in germ cells are recombinant. Furthermore,
because there is also a random assortment of chromosomes
during meiosis, there is the potential for a huge number of
possible combinations of alleles. In effect, each gamete has a
unique, haploid set of alleles. An individual conceived as the
union between two such gametes is likewise unique.
HOMOZYGOTES AND HETEROZYGOTES
Since an individual has two copies of each autosome, he or she
will have two copies of each autosomal locus. One copy is
derived from the mother and one from the father. How similar
are these two copies? Between any two chromosomes in a pair,
the nucleotide sequence of the DNA is very similar: more than
99 of 100 bp are identical. Most of the variations result in no
observable phenotype and are therefore ‘silent’ polymorphisms
or rare variants. The less frequent variations in DNA sequence
that correspond with a phenotype are the fundamental chemical
basis for alleles.
The two copies of a given locus in an individual can by
chance be identical, in which case the individual is homozygous
for that particular allele. On the other hand, an individual can
have two different alleles, one derived from each parent, and
the individual is then heterozygous. An individual who is
heterozygous for two different alleles, neither of which is wild-
type, is called a compound heterozygote.
Uniparental disomy or isodisomy is the term given for the
rare occasions when a locus is homozygous, but both identical 7
SECTION 1 GENETICS

FIGURE 1.7. Steps involved in mitosis and meiosis. In both processes, the first step involves the replication of DNA so that each chromosome
arm is duplicated, producing chromosomes with sister chromatids. In mitosis, the chromosomes divide so that each daughter cell receives a
short and long chromatid from each chromosome in the pair. In meiosis, there is often recombination between chromatids from homologous
chromosomes. After this, there is the first meiotic division, which segregates the chromosome pairs, followed by the second meiotic division
8 which produces gametes with one set of chromatids from only one member of each pair of chromosomes.

alleles are derived from the same parent. As an illustration,
some patients with cystic fibrosis have been found who are
homozygous for a mutant allele that is present in only one
parent.19 A patient with rod monochromatism has been
reported with isodisomy for chromosome 14q; this case possibly
indicates that a recessive gene for the disease is on that
chromosome.20 Isodisomy has also been implicated in Usher’s
syndrome21 and retinal dystrophy associated with mutations in
RPE65 and MERTK genes.22
PATTERNS OF HUMAN INHERITANCE
The major types of inheritance of human disease are: dominant,
recessive, X-linked, mitochondrial (also called maternal),
digenic, and polygenic. Of these, the first four are the most
commonly considered in ophthalmologic practice and will be
discussed in most detail. For reference, Figure 1.8 provides
schematic pedigrees illustrating each of these four inheritance
patterns.
DOMINANT (ALSO CALLED AUTOSOMAL
DOMINANT)
If a mutation is present in one of the two gene copies at an
autosomal locus, and if this heterozygous mutation produces a
disease, the mutation is called dominant. For example, a patient
FIGURE 1.8. Factitious pedigrees illustrating various hereditary patterns. Circles represent females; squares represent males. Filled-in circles or
squares represent individuals exhibiting a hypothetical hereditary trait.
CHAPTER 1
Fundamentals of Genetics
with dominant retinitis pigmentosa will have a defect in one
copy of one retinitis pigmentosa gene inherited from one parent
who, in most cases, is also affected with retinitis pigmentosa.
The other copy of that gene, the one inherited from the
unaffected parent, is normal (wild type). The term ‘dominant’
comes from the fact that the defective copy ‘dominates’ over the
wild-type gene copy to cause disease.
1. Nature of a dominant gene defect. Most dominant
mutations cause disease through one of the following three
general mechanisms.
a. Novel function. The mutant allele produces a protein
that has a new function not present in the wild-type
protein. The mutant protein might have a novel
enzyme activity, or it might be toxic.
b. Dominant-negative effect. The mutant protein forms a
complex with the wild-type protein encoded by the
homologous wild-type allele and thus inactivates the
wild-type protein. The phenotype is then a
consequence of little or no functional protein
remaining.
c. Haplo-insufficiency. The mutation produces no
functional protein. The homologous wild-type allele
produces functional protein, but because this is the
only functional allele, the target tissues have only 50%
of the normal level of the protein. This reduced level of
functional protein results in disease.
9
SECTION 1 GENETICS
2. Note on the classical definition of a dominant allele. It is
customary in human genetics to view a dominant
mutation as one that confers a disease or some other
phenotype when present heterozygously. However, in the
classic, mendelian lexicon, a dominant allele is one that
produces its designated phenotype whether it is present
homozygously or heterozygously. Proven examples of
classically defined, dominant alleles in humans are
uncommon. The Val30Met mutation in the transthyretin
gene is a true dominant, because patients who are
heterozygous for this allele have vitreous amyloidosis and
polyneuropathy comparable in severity to those who are
homozygous.23 In contrast, most ‘dominant’ human alleles
are loosely categorized as such if they are known to
produce phenotypes when present heterozygously,
regardless of the phenotype produced in a homozygote or
compound heterozygote. This definition is necessary
because individuals who are homozygotes or compound
heterozygotes for ‘dominant’ alleles causing disease may be
nonexistent. The disease alleles might be so rare that the
likelihood that two affected heterozygous carriers mating, a
precondition for the production of a homozygous offspring,
is exceedingly low. Occasionally, the disease produced by a
‘dominant’ mutation is so severe that affected
heterozygotes do not reproduce at all; again, there would be
little possibility for a homozygous individual to be
conceived and the corresponding phenotype to be
displayed. In some exceptional circumstances individuals
who are homozygotes or compound heterozygotes for
purportedly dominant ophthalmic disease alleles have been
identified. They are sometimes found to have a phenotype
that is markedly different from that found in
heterozygotes. For example, a newborn with mutations of
both copies of the aniridia gene had anophthalmia and
severe developmental defects of the central nervous system
that led to death soon after birth.24 If a homozygote for a
dominant allele has a more severe form of the same
recognizable phenotype, the mutant allele is more
appropriately called semidominant. Alleles in the PAX3
gene, causing Waardenburg’s syndrome, are semidominant,
exemplified by the report of a family in which a homozygote
had very severe disease (very exaggerated dystopic
canthorum and severely malformed upper limbs) compared
with the heterozygote relatives with more typical disease.25
3. Transmission of a dominant gene defect. A patient with a
dominant mutation at a disease locus can transmit the
normal copy or the defective copy to a child. Each copy has
an equal chance of being passed on, so that each child will
have a 50/50 chance of getting the defective gene copy.
Male and female children are equally likely to inherit the
defective copy. A dominant disease can be inherited from a
father or a mother. Unaffected individuals in a family do
not carry the defective gene copy and therefore cannot pass
a defective copy to their children.
4. Features of a family with a dominant disease. One can be
fairly confident that a disease is dominant in a family if
the following criteria are met:
a. The disease is found in three consecutive generations,
such as grandparents, parents, and children.
b. Every affected member has an affected parent.
c. There is at least one instance of transmission from an
affected father to an affected son.
Many families with a dominant disease do not meet all three
criteria. One will still be able to presume that a dominant mode
of inheritance is likely if some of the criteria are met. For exam-
10
ple, if there is transmission of the disease directly from a parent
to a child, it is likely that the gene defect is a dominant one.
There are two common sources of error in cataloguing a domi-
nant gene. First, in a family with two generations of affected
individuals, there is the possibility that the allele under study is
actually recessive, that the affected parent is homozygous for
the allele, and that the unaffected parent carries the allele
heterozygously. In this situation, offspring would invariably
inherit the recessive, disease-inducing allele from the affected
parent and would have a 50% chance of inheriting the recessive
allele from the unaffected parent. This situation is called
pseudodominance and is covered later. Pseudodominance is
very unlikely if a family exhibits three consecutive generations
of affected family members.
A second problem occurs when an X-linked allele is incor-
rectly designated as an autosomal dominant allele. Through a
process called lyonization (discussed later), it is possible for
females heterozygous for an X-linked recessive mutation to
exhibit the corresponding phenotype. If such a female had two
affected sons among four or five children in all, the pedigree
would mimic that found for autosomal dominant retinitis
pigmentosa. Suspicion of this type of mistake should be high
whenever all affected children of an affected mother are male.
This mistake is eliminated if one stipulates that a pedigree must
show father-to-son transmission of a trait before autosomal
dominant inheritance is diagnosed conclusively.
RECESSIVE (ALSO CALLED AUTOSOMAL
RECESSIVE)
A recessive disease arises if it is necessary for defects to be
present in both gene copies at an autosomal locus. One wild-
type allele together with one recessively defective allele does not
cause disease. Hence a wild-type allele always dominates over a
recessive one. The same recessive defect might affect both gene
copies, in which case the patient is said to be a homozygote.
Different recessive defects might affect the two gene copies, in
which case the patient is a compound heterozygote.
1. Nature of a recessive gene defect. Most recessive
mutations that have been functionally characterized result
in null alleles, which are defined as alleles that produce no
functional protein. It is the lack of the protein’s activity
that causes disease. For example, patients with gyrate
atrophy have recessive mutations in both copies of the
locus normally encoding the enzyme ornithine
aminotransferase. The disease is produced as a
consequence of the lack of functional enzyme.26
2. Note on the classical definition of a recessive allele.
Classically defined recessive mutations are frequently
encountered in human genetics. The heterozygote parents
of an affected child (who is either a homozygote or a
compound heterozygote) have a wild-type phenotype. In
certain cases, however, recessive mutations are loosely
defined. Consider alleles at the hemoglobin locus, where
the sickle-cell allele is called recessive. However, an
individual homozygous for a wild-type allele is not
phenotypically equivalent to the heterozygote that carries
one wild-type and one sickle allele. The latter individual,
who has the ‘sickle trait’, can become symptomatic if he or
she visits an environment with low oxygen pressure such
as the upper atmosphere.
3. Transmission of a recessive gene defect. In a family with
recessive disease, both parents are unaffected carriers, each
having one wild-type allele and one mutant allele. Each
parent has a 50% chance of transmitting the defective
allele to a child. Since a child must receive a defective

allele from both parents to be affected, each child has a
25% chance of being affected (50% µ 50% = 25%).
4. Features of a family with a recessive disease. The following
features make it likely that a family has a recessive disease.
a. The parents are unaffected, and there is no previous
family history of the disease. If the parents are blood
relatives (e.g., cousins), the disease in the offspring is
even more likely to be recessive.
b. Male and female children are affected equally severely.
On an average, one in four offspring of two carrier parents will
be a homozygote and affected. Consanguineous mates tend to
be carriers of the same rare alleles, so that children with reces-
sive disease are often the product of such marriages. If a sibship
with a presumed recessive disease has only affected males, the
possibility of X-linked inheritance should be considered.
X-LINKED (ALSO CALLED X-LINKED
RECESSIVE)
Mutations of the X chromosome produce distinctive inheri-
tance patterns, because males have only one copy of the
X chromosome whereas females have two. Almost all X-linked
gene defects are of the X-linked recessive category. Carrier
females are unaffected because they have one normal copy of
the gene in question and one defective copy. Carrier males will
be affected because their only copy is defective; that is, there is
no normal copy to ‘compensate’ for the recessive defect.
1. Nature of an X-linked recessive defect. Like recessive
mutations involving autosomal loci, most recessive
mutations of the X chromosome result in null alleles that
produce no functional protein.
2. Transmission of an X-linked recessive gene defect. First
consider the situation of a male affected with an X-linked
disease. He has only one copy of any X-linked gene, thus
he will transmit his defective X-linked gene to every
daughter. All his daughters will be carriers. All his sons
will be unaffected and will not be carriers, because fathers
do not pass any X-linked genes to sons. Note that neither
the daughters nor the sons of a male affected with an
X-linked disease will be affected.
Next consider the situation of a carrier female who
carries one defective allele at an X-chromosome locus.
Each child of the carrier female has a 50% chance of
inheriting the defective allele. If a son inherits the defective
copy, he will be affected. If a daughter inherits the defective
copy, she will be a carrier like her mother. If either a
daughter or a son inherits the mother’s normal gene copy,
the child will be unaffected and will not be a carrier.
Ordinarily, no carrier females will be affected. However,
for some X-linked diseases, female carriers can exhibit a
phenotype that is usually less severe than that found in the
affected male relatives. This could be due to the process of
lyonization. In order for males (with one X chromosome)
and females (with two X chromosomes) to have equal
levels of expression of X-linked genes, female cells express
genes from only one of the two X chromosomes that they
have. The decision as to which X chromosome is expressed
is made early in embryogenesis, and the line of cells
descending from each decision-making progenitrix cell
faithfully adheres to the choice of the active X chromosome
of the progenitrix. Hence, females are mosaics with some
of the cells in each tissue expressing the maternally derived
set of X-linked alleles and the remainder expressing the
paternally derived X-linked alleles. The proportion of cells
that express the mutant versus the wild-type alleles in
CHAPTER 1
Fundamentals of Genetics
each tissue can vary. By chance a susceptible tissue might
have a preponderance of cells expressing the mutant
X chromosome, in which case the corresponding disease
would become manifest. An example of this is offered by
some female carriers of X-linked retinitis pigmentosa who
develop symptoms, fundus signs, and electroretinographic
abnormalities of the disease. Most females affected with
X-linked retinitis pigmentosa because of lyonization have
milder disease than that found in their male relatives.27
Another explanation for a female affected with an
X-linked disease involves the unusual situation in which
the father is affected and the mother is a carrier. The
father invariably will transmit his defective copy to every
daughter. If the mother happens to transmit the defective
copy to a daughter, the daughter will be a homozygote or
compound heterozygote at the disease locus. This is the
usual explanation for females who show protan or deutan
color vision abnormalities due to defects in the genes
encoding red and green cone opsins on the X chromosome.
About 6% of X chromosomes in whites have defects in the
red and green cone opsin genes, so ~6% µ 6% = 0.36% of
females, or ~1 in 280, would be homozygotes or
compound heterozygotes. For most ophthalmic diseases,
however, the proportion of female carriers is very low. For
example, for X-linked retinitis pigmentosa, only ~1 in
every 7000 women is a carrier. In view of this low
proportion of carriers, it is very unlikely for an affected
father to marry by chance a female carrier of X-linked
retinitis pigmentosa. Hence, very few females with retinitis
pigmentosa will be homozygotes or compound
heterozygotes for mutations in an X-linked retinitis
pigmentosa gene; most will have autosomal recessive or
autosomal dominant retinitis pigmentosa instead.
3. Features of a family with an X-linked recessive disease. The
following features of a family point to an X-linked recessive
disease gene:
a. The disease is found only in males. (In unusual
circumstances, females may be affected; see the
discussion earlier.)
b. There is no instance of an affected male having an
affected child.
c. If the disease is present in more than one generation,
the affected males are related through a carrier female.
For example, an affected male might have an affected
maternal uncle or an affected maternal grandfather, but
he would not have affected relatives on his father’s side.
LESS COMMON INHERITANCE PATTERNS
1. Maternal or mitochondrial inheritance. The 23 pairs of
human chromosomes described earlier are located in the
nucleus of each cell. In addition, there is a small amount
of DNA in the cytoplasm. This DNA is from the
mitochondrial chromosome, a relatively tiny chromosome
with only 16 569 bp of DNA. Thirteen mitochondrial
proteins, 2 ribosomal RNAs, and 22 tRNAs are encoded by
this chromosome. It is a clinically important chromosome
because mutations are known to cause human disease
(examples relevant to ophthalmology are Leber hereditary
optic atrophy28,29 and Kearns–Sayre syndrome30). A
noteworthy feature of these mutations is that they are
maternally inherited, because almost all the mitochondria
of a one-cell embryo are derived from the ovum. A father
does not transmit mitochondria to his offspring.
Mitochondrially inherited diseases are inherited invariably
through the maternal lineage.
11
SECTION 1 GENETICS
One other peculiar feature of alleles in the
mitochondrial genome is that an individual is neither
homozygous nor heterozygous for them but rather is
heteroplasmic. A typical cell has numerous mitochondria,
each with ~2–10 copies of the mitochondrial genome. The
proportion of mutant mitochondrial genomes in each
mitochondrion, and the proportion of mutant
mitochondria in a cell, can vary from one cell to another in
an individual. Differences in the relative proportions of
mutant mitochondria can partly explain the observed
variable severity of mitochondrial diseases. In addition, the
proportion of mutant mitochondria can change during the
lifetime of a patient, which helps to explain the variable
age of onset of mitochondrial diseases.
Upon analysis of a pedigree with a mitochondrially
inherited disease, one may note examples of mother-to-son
and mother-to-daughter transmission, but one should
never observe father-to-child transmission. In a particular
family, the severity of disease can vary tremendously
because of heteroplasmy and perhaps other factors, and
one must be aware of possible asymptomatic carriers when
scrutinizing a pedigree. In the case of Leber optic atrophy, a
mitochondrially inherited disease, individuals with the
same mutation may have significant variations in disease
progression for unknown reasons.31
2. Pseudodominance. This is the term given to an apparent
dominant inheritance pattern due to recessive defects in a
disease gene. Consider the situation in which an affected
parent has recessive disease due to defects in both copies of
a disease gene and the spouse happens to be a carrier with
one normal gene copy and one copy that has a recessive
defect. Children from this couple will always inherit a
defective gene copy from the affected parent and will have
a 50% chance of inheriting the defective gene copy from the
unaffected carrier parent. On average, half of the children
will inherit two defective gene copies and will be affected.
The pedigree would mimic a dominant pedigree (Fig. 1.9)
because of an apparent direct transmission of the disease
from the affected parent to affected children and because
~50% of the children will be affected. Pseudodominant
transmission is uncommon, because few people are
asymptomatic carriers for any particular recessive gene.
FIGURE 1.9. An example of pseudodominance. Beneath each
schematic family member are the alleles of the disease locus under
scrutiny. ‘A’ is the dominant, wild-type allele; ‘a’ is the recessive allele
that causes the hypothetical disease. The parent-to-child transmission
of the disease occurs because the unaffected parent is actually a
12 carrier of the recessive allele.
3. Autosomal dominant with reduced penetrance. In some
pedigrees with an autosomal dominant disease, some
individuals who carry the defective gene do not get disease.
This would cause ‘skipped generations’; that is, cases
where an unaffected offspring of an affected individual
would have children with the disease. This phenomenon is
typically locus-specific. For example, many families with
dominant retinitis pigmentosa with reduced penetrance
have a defective gene on chromosome 19q13;32 those with
dominant retinitis pigmentosa with full penetrance have
mutations at other loci.
4. X-linked dominant inheritance. A few families with
retinitis pigmentosa appear to have this distinctive
inheritance pattern.33 The inheritance pattern is similar to
X-linked recessive inheritance, but all carrier females are
affected rather than unaffected. All carrier males are
affected as well. Other diseases with ophthalmic
manifestations that are loosely considered to have X-linked
dominant inheritance are Aicardi syndrome (frequent
features are agenesis of the corpus-callosum and patches
of absent retinal pigment epithelium) and incontinentia
pigmenti (irregularly pigmented atrophic scars on the
trunk and the extremities, congenital avascularity in the
peripheral retina with secondary retinal neovascularization).
Both Aicardi syndrome and incontinentia pigmenti occur
almost exclusively in females; it is likely that the X
chromosome gene defects causing these diseases are
embryonic lethals when present hemizygously in males.34,35
5. Digenic inheritance. This is another rare form of
inheritance, which till now has been found only in a few
families with retinitis pigmentosa or ocular albinism.36,37
Digenic inheritance occurs when a patient has
heterozygous defects in two different genes, and the
combination of the two gene defects causes disease.
Individuals who are heterozygous for a mutation only at
one or the other locus are wild-type. Digenic inheritance is
different from recessive inheritance, because the two
mutations involve different gene loci. Affected individuals
are called ‘double heterozygotes’ rather than compound
heterozygotes. Triallelic inheritance (three mutations
required for disease) has recently been reported in patients
with Bardet–Biedl syndrome.38
6. Polygenic and multifactorial inheritance. If the expression
of a heritable trait or predisposition is influenced by the
combination of alleles at multiple loci, it is polygenic. The
contributing loci may be ‘quantitative trait loci’ reflecting
the mathematical formulations used to calculate their
relative impacts on the phenotype or the predisposition.
If environmental factors contribute to a polygenic trait or
disease, the term multifactorial is used. Examples of
phenotypes in ophthalmology likely to be multifactorial
are myopia,39 age-related macular degeneration,40 and
adult-onset open-angle glaucoma.41
PEDIGREE ANALYSIS TO CATEGORIZE
ALLELES
The classification of a genetic disease or trait can often be made
by examining the relationships between the affected individuals
in a pedigree. The following are general guidelines for using this
method. It should be noted that in many circumstances, it is
not possible to be certain of the mode of inheritance in a
particular family because of the small size of the family or
because of uncertainties in the diagnosis of key family members
who might be too young, unavailable, or deceased.
Pedigree analysis is sometimes not necessary to determine
the inheritance pattern in a family, because for some conditions

there is only one known inheritance pattern. In those cases, the
diagnosis will immediately provide the inheritance pattern. For
example, currently, all known cases of choroideremia have an
X-linked pattern of inheritance. For other diseases, such as
hereditary cataract or hereditary retinal degeneration, many
different inheritance patterns have been observed. In those
cases, pedigree analysis can often be helpful. One constructs a
family tree indicating which members in the family have the
disease in question. It is important to make sure that the infor-
mation on the pedigree is as complete and correct as possible.
For example, if a distant relative is reported to have had ‘poor
eyesight’, one must know whether that report reflects the
ophthalmic disease in question or simply the relative’s need for
eyeglasses. Examination of the pedigree rarely ‘proves’ the type
of inheritance beyond any doubt, but it can allow one to infer
the most likely inheritance pattern.
DISEASE IS PRESENT IN ONLY ONE FAMILY
MEMBER
‘Isolate’ or ‘simplex’ cases of disease refer to families in which
two parents with no previous family history of the disease in
question have one affected child. In some cases, a simplex case
might not have a hereditary disease at all. For example,
~80–90% of unilateral, simplex cases of retinoblastoma are not
hereditary. Alternatively, simplex cases might represent
autosomal recessive disease, with both parents being carriers
and the affected child having inherited a defective gene copy
from each parent. If the affected simplex case is a male, it is
possible that he has X-linked disease, with the mother possibly
being a carrier. For some diseases such as retinitis pigmentosa,
a careful ophthalmologic evaluation including an electro-
retinogram of the mother might give clues as to her status in
this regard. Another possibility is that the simplex case has a
new gene defect not present in either parent. This is thought to
be infrequent, because so few genes become mutant from one
generation to the next.
DISEASE PRESENT IN TWO OR MORE
INDIVIDUALS IN THE SAME GENERATION
An example of this situation would be a family with two or
more siblings with a disease and no previous family history of
the disease. In such families, the inheritance pattern is usually
autosomal recessive. However, if the affected children are all
males, the possibility of X-linked disease should be considered.
Other unusual inheritance patterns, such as maternal, digenic,
or multifactorial are possible.
DISEASE PRESENT IN TWO CONSECUTIVE
GENERATIONS
The disease is most likely to be autosomal dominant. If there is
direct transmission from a father to a son, an autosomal
dominant gene is inferred with even more certainty. Uncom-
mon exceptions include pseudodominance or digenic inheritance.
If there is direct transmission from a mother to a child, an
autosomal dominant gene is still very likely, but maternal and
X-linked inheritance should be considered as well.
DISEASE PRESENT IN TWO GENERATIONS
SEPARATED BY AN UNAFFECTED
GENERATION
If the unaffected individual connecting the affected generations
is a female and if all affected individuals are male, X-linked
inheritance is likely. Alternatively, this could represent
CHAPTER 1
Fundamentals of Genetics
autosomal dominant inheritance with reduced penetrance. This
type of inheritance pattern may also result from imprinting,
where the disease is expressed only when inherited from the
mother (for some disease) or the father (for other disease).42
DISEASE PRESENT IN THREE OR MORE
CONSECUTIVE GENERATIONS
Dominant inheritance is most likely, although digenic and
X-linked dominant inheritance are also possibilities.
MAP OF THE HUMAN GENOME
LINKAGE
Because of the mixing of genes caused by meiotic crossovers and
the random assortment of chromosomes, alleles at two distinct
loci are usually inherited together ~50% of the time. In the less
common circumstance when alleles at two loci are inherited
together more than 50% of the time, the two loci are linked.
Linked loci are physically close to each other on the same
chromosome.
The distance between two linked loci can be measured two
ways: by the number of base pairs of DNA separating the loci
(physical distance) or by the frequency of meiotic crossovers
occurring between the two loci (genetic distance or recom-
bination distance). How are the two measures related? A
haploid human genome contains ~3.2 billion bp of DNA. Since
30 crossovers occur in a typical meiosis, there is an average of
one crossover per 100 million bp per meiosis. Between two loci
physically separated by a distance of 1 million bp, there would
be approximately one crossover per 100 meioses, or a 1% cross-
over rate. This distance is called 1 centimorgan (cM) and is one
of the basic units in genetics for measuring the separation
between two loci. The conversion of 1 cM/million bp is an
overall average for the human genome, since the frequency of
crossovers is not equal throughout the length of each
chromosome. The actual figure for a segment of a chromosome
can be more than 10 times greater or less. Furthermore, it can
be different in germ cells from males compared with females.
One of the major contemporary goals in the study of human
genetics is the construction of a map of the physical position of
every human gene and the correlation of that map with the
recombination distances between linked loci. This was one goal
of the ‘human genome project’ which was a formidable task,
because the human genome is so large. The physical map that
was the first step of this endeavor was started by physically
assigning many human genes to their specific locations on
chromosomes.43 These and other landmarks within the human
genome sequence led to the final determination of the DNA
sequence for each chromosome of the human genome.6
DNA POLYMORPHISMS
A major step in the human genome project was the construc-
tion of a linkage map of the human genome. This involves the
determination of which human loci are linked and the recom-
bination distances between them. This work is based on sites
in the human genome where there is variation in the DNA
sequence, called DNA polymorphisms. Most DNA poly-
morphisms are unrelated to clinically evident phenotypes,
however single nucleotide polymorphisms (SNPs), may change
the amino acid sequence of a protein causing an abnormal
function and disease phenotype.
Three major categories of DNA polymorphisms were used for
linkage maps of the human genome: RFLPs (for restriction frag-
ment length polymorphisms), VNTRs (for a variable number of 13
SECTION 1 GENETICS

tandem repeats), and microsatellites. RFLPs are the result of

occasional variations that typically affect a single base pair in
the DNA sequence. They are detectable with enzymes, called
restriction endonucleases, that are purified from bacteria. A
restriction endonuclease cleaves DNA at specific locations,
usually specified by a particular stretch of 4–6 bp called the
recognition sequence. If even a single base pair is altered at a
recognition site, a restriction endonuclease will not cleave
DNA at that site. For example, the restriction endonuclease
EcoRI cleaves DNA at the sequence GAATTC (its recognition
sequence) but would not cleave the sequence GAAGTC or
GATTTC. Restriction endonucleases allow one to trace relatively
easily the inheritance of a single-base polymorphism if a recog-
nition sequence is created or destroyed by the variation.
VNTRs are sites in the human genome where there is a
tandem repetition of a DNA sequence. The repeat unit is
~15–60 bp in length and typically has a core sequence that is
common to all VNTRs.44 The number of repeat units at a
VNTR varies from a few to dozens, and this variation is the
basis for the alleles specified by these polymorphisms. In some
cases, VNTR variation may contribute to regulation of gene
expression.45 Microsatellites are like VNTRs in that they are
tandemly repeated DNA sequences, but the repeated unit is
much smaller, typically 2–4 bp. The most frequently used
microsatellites are repeats of the dinucleotide sequence ‘CA’;
these microsatellites are also known as ‘CA repeats’. VNTRs
and microsatellites were preferred for the linkage studies that
defined the human genome because they are multiallelic. A
higher proportion of individuals are heterozygous for poly-
morphisms with numerous alleles, and therefore VNTRs and
microsatellites provide more linkage data than RFLPs, which
are biallelic.
By following the inheritance of distinct DNA polymorphisms
in human pedigrees, one can learn which are linked with each
other and at what recombination distances. To date, linkage
maps of each human chromosome are available with highly
informative polymorphic markers distributed roughly every
1–3 cM or less.46
With such a linkage map, it is possible to determine the
location of a gene causing a human disease once one has a set
of families with the disease available for study. DNA samples
from family members are first obtained. Leukocyte DNA is
typically used; DNA from 10 mL of venous blood is sufficient to
assay hundreds of DNA polymorphisms distributed throughout
the genome. The polymorphic site that most often correlates
with the disease is the one that is closest to the disease gene
(Fig. 1.10). By knowing the chromosomal location of that DNA
polymorphism, one has the approximate chromosomal location
for the disease gene. The strategies embodied in the term
‘positional cloning’ allow one to proceed from the approximate
chromosomal location of a disease gene, based on the data from
the DNA polymorphisms, to the actual isolation of the gene.
Positional cloning approaches are typically very labor-intensive,
but they have been successful in identifying a number of genes
causing ophthalmologic disease. Examples are the retinoblastoma
gene (on chromosome 13), X-linked genes for choroideremia
and one form of retinitis pigmentosa (RPGR), the aniridia gene
(chromosome 11), and a gene for Usher syndrome type I
(chromosome 11).
SNPs are single-letter variations in a DNA base sequence,
and are the most common source of genetic variation in the
human genome.47 Over 10 million SNPs are present in the
human genome with a density of one SNP approximately every
100 bases. In addition to their abundance, SNPs are useful
genetic markers because the high quality of the data makes the
automation of the analysis possible. Some SNPs (nonsynonomous
14 SNPs) change the amino acid composition of the protein and
FIGURE 1.10. An example of a linkage study using RFLPs or other
DNA markers. In this hypothetical example, a large pedigree with
autosomal dominant retinitis pigmentosa is illustrated. Filled circles
and squares indicated affected individuals. The numbers beneath
each symbol are the alleles at marker loci that have been studied. This
figure only shows the results of informative markers, i.e., for markers
where the affected members of generations I and II are heterozygotes
(1,2) and the unaffected spouses were homozygotes (2,2). (Note that
any markers that are not heterozygous in the affected members of
generations I and II would provide little useful information for this
analysis.) Beneath the symbols for the members of the generation III
are the alleles at the informative markers, as well as the chromosomal
location of each marker. At each of the marker loci, the ‘1’ allele is
defined as the allele that was transmitted from the affected male in
generation I to the affected male in generation II. (This way of naming
the ‘1’ allele is done for pedagogic purposes for this figure.) If a
marker locus is close to the disease gene, then the affected members
of generation III should usually have marker ‘1’ allele and the
unaffected members should not. The markers G and S most closely fit
this prediction. For both of these markers, nine out of the 10 members
of generation III fit the expected pattern for close linkage; the two
members who do not probably are examples of meiotic recombination
between the marker loci and the disease locus. Since both these
markers come from the long arm of chromosome 3 (bands 3q21 and
3q24, respectively), these data indicate that the locus for the disease
gene in this family is probably within or near this region. Data of this
sort led to the search for mutations of the rhodopsin gene in patients
with autosomal dominant retinitis pigmentosa, since the rhodopsin
gene was known to lie in the region 3q21–q24.
can be associated with disease. For example, the amino acid
change in the complement factor H gene recently shown to be
a risk factor for macular degeneration is a nonsynonomous
SNP.48 Although SNPs are biallelic (RFLPs are a subset of SNPs)
whole genome association studies using automated tech-
nologies are currently possible, allowing a large number of SNPs
to be evaluated in a genetic study. Screening many SNPs and
creating haplotypes, which are groups of SNPs that are inherited
together, compensates for the low information content of the
polymorphism. Another recent advance of the Human Genome
Project is the HapMap which defines haplotype blocks for four
ethnic populations to be used for disease gene identification
studies.49
 CHAPTER 1
Fundamentals of Genetics

MUTATIONS

CATEGORIES OF MUTATIONS
A new alteration in the DNA sequence of a gene is called a
mutation. The word mutant can refer to the specific sequence
abnormality (i.e., a mutant base pair), to the defective allele
(mutant gene or mutant allele), to the gene product (mutant
protein), or to the organism that is affected by the mutation
(mutant mouse). There are various ways that mutations can be
organized for didactic purposes. Mutations can be grouped
according to whether they cause a dominant or a recessive
phenotype, or no phenotype at all (silent mutations). Recessive
mutations are often loss-of-function, or null mutations because
they often interfere in some way with the production of an
active protein product. Dominant alleles can be loss-of-
function, but typically represent gain-of-function mutations.

TYPES OF LESIONS IN DNA

Another way to classify mutations is according to the type of
lesion affecting the DNA sequence. A point mutation is the
change of a single base for another. If a purine changes to
another purine, or if a pyrimidine changes to another
pyrimidine, the point mutation is called a transition. If a purine
changes to a pyrimidine or vice versa, the mutation is a trans-
version. Although there are 12 possible transversions and four
possible transitions (Fig. 1.11), transitions outnumber trans-
versions at most human loci where naturally occurring muta-
tions have been characterized. Among the transitions, the change
from a C to a T is the most frequent and most commonly
occurs if the C is part of the dinucleotide sequence CG.
A point mutation can change a codon so that it specifies a
different amino acid. This is called a missense mutation. For
example, a C-to-A transversion in codon 23 of the human
rhodopsin gene, a cause of autosomal dominant retinitis
pigmentosa, changes that codon from one that specifies proline
(CCT) to one specifying histidine (CAT).7
A nonsense mutation, also called a premature stop codon, is
one that changes a codon that normally specifies an amino acid
into a termination codon. For example, a C-to-T transition in
codon 446 of the retinoblastoma gene, found to be the cause of
hereditary retinoblastoma in one pedigree, changes the codon
from CGA (arginine) to TGA (stop). During translation of the
resultant mRNA, the encoded protein will have only the first
445 amino acid residues, whereas the normal protein product
has 928 residues. The truncated, nonfunctional, mutant protein
will not be able to prevent retinoblastoma.
A point mutation or other alteration affecting either of the
ends of an intron will interfere with the proper splicing of the
transcribed RNA. The 5„ end of an intron absolutely requires
the dinucleotide sequence GT (called the splice donor sequence),
and the 3„ end must have the dinucleotide sequence AG (the
splice acceptor sequence). If a mutation changes either the splice
acceptor or splice donor sequences, it is called a splice site
mutation. The mRNA transcript will either improperly include
sequence from the intron or will eliminate part or all of an exon.
In either case, one expects a major alteration of the translated
protein product.
Other areas of a transcriptional unit may be exquisitely sen-
sitive to single base changes. For example, the promoter region
upstream of a transcribed sequence has binding sites for factors
necessary for the proper expression of a gene. A change in the
sequence of these binding sites can bring about underexpression
or overexpression of the protein product. Additional sequences
that modulate the expression of a gene can be located in diverse
regions of a transcriptional unit, such as within introns or
FIGURE 1.11. Transitions and transversions. The black arrows
indicate base changes that would be termed transitions, because they
involve an interchange of two bases of the same type (e.g., both
purines). Transversions (gray arrows) involve the interchange of a
purine and a pyrimidine.
within the 5„ or 3„ untranslated regions, or even many thousands
of bases away from the cluster of exons and introns. Mutations
in these regions can also affect the expression of a gene and
cause an observable phenotype.
A frameshift mutation occurs when one or more bases are
inserted into or deleted from the coding region of a gene. A
frameshift mutation changes the reading frame of the encoded
message. Since the genetic code uses consecutive, nonover-
lapping triplets of DNA sequence, the number of bases that are
inserted or deleted to cause a frameshift cannot be a multiple of
three. Downstream of a frameshift mutation there is a drastic
alteration of the amino acid sequence, often with a premature
termination codon so that the encoded protein is truncated as
well. If the number of base pairs removed or inserted in the
coding region is a multiple of 3, the mutation is called an
in-frame deletion or insertion. Only the amino acids encoded by
the deleted or inserted codons will be affected.
Large deletions might remove a large portion of a tran-
scriptional unit (an internal deletion), or the 5„ or 3„ end of a
gene, or an entire transcriptional unit. Very large deletions
might remove a number of closely linked genes. To be
observable in a karyotype (i.e., to be detectable cytogenetically),
a deletion must remove at least a few million base pairs of
DNA. Since the density of genes in the human genome is ~1
per 30 000–50 000 bp, a cytogenetically detectable deletion
usually affects dozens of genes. Like deletions, insertions can
interfere with a gene if they interrupt a coding region or if they
occur in a region that is important for proper RNA splicing or
the proper expression of a gene. 15
SECTION 1 GENETICS
This general categorization of mutations is not always appli-
cable to naturally occurring defects in human DNA. Occasion-
ally a single mutational event causes many single-base
substitutions in a gene. Some deletions are complex, causing a
foreign segment of DNA to be inserted where the normal
sequence was deleted. More complex rearrangements have been
documented, such as inversions where a segment of DNA is
flipped backwards and relocated to a different region of the
gene or to another gene. Such complex mutations represent a
minority of the lesions that cause a disease.
Finally, because of our limited understanding of the molecular
control of the regulation of transcription, splicing, and tran-
slation, the precise effect of a mutation sometimes cannot be
deduced with certainty from inspection of the DNA sequence
alone. The arrangement of bases in the coding region of a gene
not only specifies the amino acid sequence of the protein
product but also has some role in the recognition of splice sites
and in maintaining the nuclear and cytoplasmic stability of the
final mRNA product. Consequently, a point mutation labeled
as a ‘missense’ mutation, since it changes the amino acid
specificity of a codon, might actually interfere with the splicing
of an RNA transcript so that a very different protein product is
produced. In some cases, considerable effort in a research labo-
ratory is necessary to establish the exact biochemical con-
sequences of a mutant allele of a known DNA sequence.
ORIGIN OF MUTATIONS
Germline mutations either arise de novo in an individual or are
inherited from a carrier parent. Actually, all mutations arise
de novo in some individuals. Sometimes that individual is a
distant ancestor who is called the founder or progenitor of the
mutation.
VARIABILITY IN THE RATE OF NEW GERMLINE
MUTATIONS
For any given genetic disease, the proportion of patients who
have a new germline mutation (as opposed to those who have
inherited a mutation) is dependent on the mutation rate and the
ability of those who carry the mutation to survive and reproduce.
In practice, the quantification of both of these factors is dif-
ficult. Mutation rates at human loci extend over many orders of
magnitude. New mutations at some loci, such as the Duchenne
muscular dystrophy locus or the retinoblastoma locus, occur in
more than one in 50 000 live births. For other diseases, such as
tritanopia (due to a defect in the gene for blue cone opsin), the
mutation rate is thought to be well below one in 10 million live
births. The explanation for the wide range of mutation rates at
different human loci is obscure. Possibilities include the size of
the transcriptional unit (the Duchenne locus and the retino-
blastoma locus are both large, encompassing 2 million and 180
thousand bp, respectively), limitations on the types of mutations
that can cause a disease (almost all mutations of the rhodopsin
gene causing dominant retinitis pigmentosa are missense muta-
tions), or inherent variation in the mutability of loci based on
their DNA sequences or their positions in the genome.
MUTATION SPECTRUM OF A GENE
An examination of mutations might provide clues to the
mechanisms that are responsible for them. A mutation spec-
trum is a compilation of the frequency of each type of mutation
at a specified locus; that is, the percentage of deletions, inser-
tions, point mutations (broken down into transitions and trans-
versions, or the specific nucleotide changes), frameshifts, and so
16 forth. Tabulating the types of mutations causing a disease can
give clues as to the functional domains of the encoded protein.
Laboratory studies suggest that each class of mutagens causes
certain types of mutations. For example, approximately half of
the mutations resulting from gamma radiation are deletions
and only ~20% are transitions. Ultraviolet light, on the other
hand, induces deletions very infrequently but appears to facili-
tate transitions (~50% of the resultant mutations). Thus, knowl-
edge of the mutation spectrum can provide evidence implicating
specific environmental mutagens as the cause of a disease.
Indeed, ultraviolet light has been implicated by such evidence in
the genesis of squamous cell carcinoma in sun-exposed skin.50
Unfortunately, the mutation spectrum of only a few genes is
known with any accuracy. The available data do not implicate
any specific environmental mutagen as the cause of most natu-
rally occurring mutations in humans.
PARENTAL ORIGIN OF NEW MUTATIONS
An individual with a new germline mutation carries that muta-
tion on the gene copy derived from either the mother or the
father (except for males with a new mutation on the X chromo-
some, a chromosome necessarily derived from a son’s mother).
The parental origin of an autosomal allele with a new mutation
can be determined in some situations. At many human loci, the
general rule is that new germline mutations preferentially arise
on a paternally derived allele. For example, ~80–90% of new
germline mutations at the retinoblastoma locus51 or the von
Recklinghausen neurofibromatosis locus52 affect the paternally
derived allele. One attractive explanation for this bias relates to
the fact that more than 300 cell divisions separate a one-cell
male embryo from his resultant sperm (produced decades later)
compared with ~20 cell divisions separating a one-cell female
embryo from her resultant ova (produced while the female is
still in utero).53 The excess of mutant sperm may pertain to the
fact that mutations chiefly arise during DNA replication.
EPIGENETIC MUTATIONS
Defects that do not alter the sequence of DNA are called
epigenetic. How such defects are transmitted through the
germline, if at all, is open to speculation. One possible basis for
epigenetic defects is that some bases of DNA are modified by
the addition of methyl groups. The classic example of this
involves the dinucleotide sequence CG. The cytosine in a CG
dinucleotide sequence is customarily methylated in human
DNA. However, in the vicinity of the promoter region at the 5„
end of a gene, cytosines are unmethylated in cells that express
the gene.54 If this region of a gene is aberrantly methylated, the
gene will not be expressed. Despite no change in the DNA
sequence, the allele will be inactive and thus equivalent to one
with a null mutation. There is evidence that epigenetic defects
in the retinoblastoma gene are one cause of retinoblastoma.55–57
IMPRINTING
Human cells have the capacity to distinguish the maternally
derived allele from the paternally derived allele at some loci.
This may be due to differences in the pattern of methylation of
the two alleles or to differences in the configuration of DNA-
binding factors that are present in chromatin. This imprinting
of DNA has clinical importance because it explains peculiar
patterns seen for some genetic diseases. For example, a deletion
of q11–q13 of human chromosome 15 causes Prader–Willi
syndrome if it affects the paternally derived chromosome 15,
but Angelman syndrome if it affects the maternally derived
chromosome homolog.58 Angelman syndrome can be associated
with oculocutaneous albinism.59

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Prader–Willi/Angelman imprinted domain
 CHAPTER
2 Molecular Mechanisms of Inherited Disease
Janey L. Wiggs
DNA mutations occurring in genes may result in the formation
of a defective gene product. If the normal protein product of a
mutated gene is necessary for a critical biologic function, then
an alteration of the normal phenotype may occur. Many changes
in phenotype are considered normal variations among humans,
for example, brown hair instead of blond hair. However, some
changes produce phenotypes that seriously affect health; these
are the major focus of study in clinical genetics laboratories.
The inheritance pattern of a disease is determined by the func-
tion of the normal and abnormal protein products of the gene as
well as the type of mutation causing the disease. For example,
mutations that create an abnormal protein that is detrimental
to cells are typically dominant, because only one mutant gene is
required to disrupt the normal functions of the cell. Mutations that
result in proteins with reduced biologic activity (loss of function)
may be inherited as dominant or recessive conditions depending
on the number of copies of normal genes (and the amount of nor-
mal protein) required. Disorders caused by mutations in mito-
chondrial DNA have a characteristic inheritance pattern, and
mutations in genes carried on the X chromosome also result in
typical inheritance patterns. Examples of the types of mutations
responsible for different inheritance patterns are described in
the following sections.
One of the goals of disease gene discovery is the development
of novel therapeutics. Disease treatment, including gene ther-
apy, cannot be developed without knowledge about the under-
lying molecular mechanisms. Diseases that are caused by a loss
of protein function could be treated by protein and gene replace-
ment therapies, while disease caused by a gain of function or
dominant negative effect would require inactivation of the
abnormal gene.
AUTOSOMAL DOMINANT DISORDERS
Disorders inherited as autosomal dominant traits result from
mutations that occur in only one copy of a gene (i.e., in hetero-
zygous individuals). Usually the parental origin of the mutation
does not matter. However, if the gene is subject to imprinting
(see further ahead), then mutations in the maternal or paternal
copy of the gene may give rise to different phenotypes.
HAPLOINSUFFICIENCY
Some cellular processes require a level of protein production that
can only be furnished if both copies of a particular gene are active.
Such proteins may be involved in a variety of biologic processes.
If one copy of a gene is mutant and the protein level is reduced
by half, a disorder may result.
Aniridia-PAX6
Mutations in the PAX6 gene cause disease through haploinsuf-
ficiency. Most of the mutations responsible for these disorders
alter the paired-box sequence within the protein product, which
is in the homeobox family of transcription factors (Fig. 2.1).1
The paired box is an important region of the protein that parti-
cipates in the regulation of expression of other genes.2 PAX6 plays
a critical role in ocular development, presumably by regulating
the expression of a set of genes that are essential for this process.3
A reduction in the amount of active PAX6 gene product changes
the level at which these other genes operate.
There is extensive variation in the range of phenotypes exhi-
bited by patients with PAX6 mutations. Patients typically have
various anterior segment abnormalities, such as aniridia,4 Peters’
anomaly,5 or autosomal dominant keratitis.6,7 This spectrum of
phenotypic abnormalities resulting from mutations in one gene
is termed variable expressivity and is a common feature of dis-
orders that result from haploinsufficiency. The variability of the
mutant phenotype possibly results from the random activation
of downstream genes that occurs when only half the required
gene product is available.
Other examples of ocular disorders caused by haploinsuffi-
ciency are: PITX2 causing Axenfeld–Rieger syndrome,8 LMX1B
causing nail patella syndrome and glaucoma,9 FOXC1 causing
anterior segment dysgenesis syndromes,10 SALL4 defects causing
Duane-radial ray syndrome,11 OPA1 causing autosomal dominant
(Kjer’s) optic atrophy,12 CRX causing cone–rod dystrophy,13 and
Waardenberg’s syndrome caused by defects in another homeo-
box gene, PAX3.14 Of interest, the majority of these genes are
regulatory proteins involved in ocular developmental processes,
suggesting that gene dosage of regulatory proteins is an important
factor in eye development.
LOSS OF FUNCTION
Autosomal dominant traits may result from mutations in one
copy of a gene that increase the likelihood, but are not sufficient
to cause the disease. For the disease to become manifest, a ‘second
hit’ that affects the remaining copy of the gene must occur. If the
second hit is a common event, the inheritance of one mutant
copy of the gene almost always results in the disease and the
trait appears to be inherited in a dominant fashion. However, at
the cellular level, the mutations appear recessive since cells
must be homozygotes or compound heterozygotes to display the
mutant phenotype.
Retinoblastoma
Tumor suppressor genes such as the retinoblastoma gene provide
good examples of loss-of-function dominant mutations. A gene
19
SECTION 1 GENETICS
FIGURE 2.1. Schematic diagram of the PAX6 gene.
responsible for retinoblastoma was identified in 1986 on chro-
mosome 13q14.15 The gene product is involved in regulating the
cell cycle.16 An absence of this protein in a sensitive embryonic
retinal cell results in uncontrolled cell growth that eventually
produces a tumor. Susceptibility to hereditary retinoblastoma is
inherited as an autosomal dominant trait. Mutations in the
retinoblastoma gene result in underproduction of the protein
product or in production of an inactive protein product.17 A
retinal cell with only one mutant copy of the retinoblastoma gene
will not become a tumor. However, inactivation of the remaining
normal copy of the retinoblastoma gene is very likely in at least
one retinal cell out of the millions present in each retina. Most
individuals who inherit a mutant copy of the gene sustain a
second hit to the remaining normal copy of the gene and
develop the disease (Fig. 2.2).18
GAIN-OF-FUNCTION DOMINANT NEGATIVE
EFFECT
Autosomal dominant disorders can be caused by mutant proteins
that have a detrimental effect on the native tissue. Under this
scenario, mutations in one copy of a gene produce a mutant
protein that may interfere with normal cellular processes or may
accumulate as a toxic product, or both. This toxicity is a func-
tion not present in the wild-type protein; hence the mutation is
termed a gain-of-function mutant. If the mutant protein inter-
feres with the function of the wild-type protein expressed by the
remaining normal copy of the gene, the mutation is described
as dominant negative.19 It is possible to have gain-of-function
mutations which can also be dominant negative because the new
FIGURE 2.2. Inheritance of retinoblastoma. Individuals inheriting a
mutation in the retinoblastoma gene are heterozygous for the mutation
in all cells of their bodies. The ‘second hit’ to the remaining normal
copy of the gene occurs in a developing retinal cell and leads to
20 tumor formation.
function of the protein also interferes with the function of the
remaining normal copy of the gene.
Corneal Dystrophies
The autosomal dominant corneal dystrophies are excellent exam-
ples of gain-of-function mutations that result in the formation
of an aberrant protein. The four most common autosomal domi-
nant corneal stromal dystrophies are: Groenouw’s (granular)
type 1,20 lattice type 1,21 Avellino’s (combined granular lattice),22,23
and Reis–Bücklers.24 Although all four corneal dystrophies
affect the anterior stroma, the clinical and pathologic features
differ. The granular dystrophies typically form discrete white
localized deposits that progressively obscure vision. Histo-
pathologically, these deposits stain bright red with Masson’s
trichrome and have been termed hyalin. In lattice dystrophy,
branching amyloid deposits gradually opacify the cornea. These
deposits exhibit a characteristic birefringence and dichroism
under polarized light after staining with Congo red. Avellino’s
dystrophy has features of both granular and lattice dystrophies.
Reis–Bücklers primarily involves Bowman’s layer and the
superficial stroma.24 All four dystrophies have been genetically
mapped to a common interval on chromosome 5q31.25–28
Mutations in a single gene, TGFB1/BIGH3, have been iden-
tified in a number of affected families.29 An abnormal protein
product of this gene, keratoepithelin, accumulates in patients
carrying mutations. The normal protein product is probably an
extracellular matrix protein that modulates cell adhesion. Four
different missense mutations occurring at two arginine codons
in the gene have been found (Fig. 2.3). Interestingly, different
mutations at the same arginine codon cause lattice dystrophy
type I or Avellino’s dystrophy, the two dystrophies characterized
by amyloid deposits. The mutations that cause Avellino’s and
lattice dystrophies abolish a putative phosphorylation site that
is probably required for the normal structure of keratoepithelin.
Destruction of this aspect of the protein structure leads to the
formation of the amyloid deposits that cause opacification of
the cornea. As a result, the mutant protein is destructive to the
normal tissue. Mutations at the other arginine codon appear to
result in either granular dystrophy or Reis–Bücklers dystrophy.
The mutation analysis of this gene demonstrates that different
mutations within a single gene can result in different phenotypes.
Of interest, pathologic deposits caused by keratoepithelin accu-
mulation have only been observed in the cornea and not in other
tissues or organs.30 Because the TGFB1/BIGH3 gene is expressed
in other tissues, these results suggest a cornea-specific mecha-
nism causing the accumulation of mutant keratoepithelin.
Retinitis Pigmentosa – Rhodopsin
Examples of gain-of-function mutations causing retinal degen-
erative disorders include: rhodopsin causing retinitis pigmen-
tosa, transthyretin mutations causing vitreous amyloidosis,31
and possibly TIMP3 mutations causing Sorsby’s dystrophy.32
Mutations in rhodopsin demonstrate how a gain-of-function
mechanism can cause a retinal degeneration. Mutations in the
gene for rhodopsin can cause retinitis pigmentosa.33 To explore the
pathogenic mechanisms relating to these mutations, transgenic
 CHAPTER 2
Molecular Mechanisms of Inherited Disease

FIGURE 2.3. Schematic diagram of the keratoepithelin gene. D1 to D4, homologous domains. Arrows point to the location of the reported
mutations.

mice were created that carried mutant copies of the gene.34 Histo-
pathologic studies of these mice showed an accumulation of
vesicles containing rhodopsin at the junction between the inner
and the outer segments of the photoreceptors. The vesicles
probably interfere with the normal regeneration of the photo-
receptors, causing photoreceptor degeneration.

Osteogenesis Imperfecta
Osteogenesis imperfecta is an example of a dominant negative-
type mutation. Osteogenesis imperfecta is a group of inherited
disorders of type I collagen that predispose a patient to easy frac-
turing of bones, and skeletal deformity. Ocular findings include
thinned sclera. The type I procollagen molecule is formed from
two proalpha-1 chains and one proalpha-2 chain. To create a
collagen molecule, the three chains form an a-helix beginning
at the carboxyl terminus. Mutations that affect the amino acid
sequence of an individual procollagen molecule disrupt the
formation of the helix, and this results in the disease.35

ANTICIPATION – TRINUCLEOTIDE REPEATS

A new class of mutations responsible for autosomal dominant
inheritance was discovered with the identification of the gene
responsible for Huntington’s disease.36 Huntington’s disease is
a neurodegenerative disorder that results in motor, cognitive,
and emotional disturbance. Huntington’s disease demonstrates
anticipation, which means that subsequent generations of affected
individuals are more severely affected and are affected at an
earlier age than their predecessors.37 The gene defect responsible
for this disease is an expanded and unstable trinucleotide repeat in
the open-reading frame of the Huntington disease gene located on
chromosome 4. The repeated DNA sequence causes the encoded
protein to have a long span of the same amino acid residue
repeated many times. A critical observation was made when the
repeat lengths were correlated with the severity and the age of
onset of the disease. Longer repeat lengths result in more severe
disease at an earlier age of onset. The number of repeats within
the gene expands with each subsequent generation and is likely to
be the cause of the increased severity of the disease (Fig. 2.4).38
Since the discovery of the Huntington gene, a number of other
disorders caused by unstable trinucleotide repeats have been recog-
nized, including myotonic dystrophy,39 spinocerebellar ataxia,40
Friedreich’s ataxia,41 and fragile X syndrome.42 Although the
specific mechanisms responsible for trinucleotide repeat disease
are not completely understood, the autosomal dominant inheri-
tance suggests that only one mutant copy of the gene is required
and that the repeat in some way has a detrimental effect on the
cell. This molecular mechanism should be considered whenever
FIGURE 2.4. Pedigree illustrating anticipation associated with
expansion of a trinucleotide repeat. Affected individuals are shown as
solid circles or squares. The age of onset of the disease is shown
beneath the pedigree symbol for each affected individual. The number
of trinucleotide repeats within the disease gene (e.g., the gene
responsible for Huntington’s disease) is schematically represented
beneath each affected individual. Successive generations have an
earlier age of onset and a higher number of repeats (compare
individual one with individual six).
pedigree analysis shows increased disease severity with each
new generation.
IMPRINTING
Some mutations give rise to autosomal dominant traits that are
transmitted by parents of either sex, but they are expressed only
when inherited from a parent of one particular sex. In families
affected with these disorders they would appear to be transmitted
in an autosomal dominant pattern from one parent (either the
mother or the father) would not be transmitted from the other
parent. Figure 2.5 provides an example of a trait that is expressed
only when transmitted from the father. Occasionally the same
mutation gives rise to a different disorder, depending on the sex 21
SECTION 1 GENETICS

Autosomal dominant Paternal imprinting

I 1 2 I 1 2

M M

II 1 2 3 4 5 6 II 1 2 3 4 5 6

M M M M

III 1 2 3 4 5 6 7 8 9 10 III 1 2 3 4 5 6 7 8 9 10

M M M M M M M M M

1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8

M M M M IV M M M M
IV

9 10 11 12 13 14 15 16

M M M M
FIGURE 2.5. Pedigree illustrating paternal imprinting compared with segregation of an autosomal dominant trait that is not imprinted. Affected
individuals are shown as solid circles or squares. Those individuals carrying a mutation are indicated by the ‘M’ beneath the pedigree figure.
Notice that in the pedigree transmitting the mutation as an autosomal dominant trait, all individuals carrying the mutation are affected, while in
the paternally imprinted pedigree, only individuals who have inherited the mutation from their father are affected. Individuals can inherit the
mutation from the mother, but in that case it is not expressed and they are phenotypically normal. These mutation carriers can, however, transmit
the mutation to their offspring, and the offspring who inherit the mutation from male mutation carriers will be affected.

of the parent transmitting the trait. These parental sex effects
are evidence of a phenomenon called ‘imprinting’. Although the
molecular mechanisms responsible for imprinting are not com-
pletely understood, it appears to be associated with DNA methyla-
tion patterns that can mark certain genes with their parental
origin.43 Prader–Willi syndrome and Angelman syndromes are
examples of imprinted conditions.44 Diseases caused by muta-
tions in imprinted genes can give rise to unusual inheritance
patterns (Fig. 2.5).
AUTOSOMAL RECESSIVE DISORDERS
Autosomal recessive disorders result from mutations present on
both the maternal and the paternal copies of a gene. Mutations
responsible for recessive disease typically cause a loss of biologic
activity, either because they create a defective protein product that
has little or no biologic activity or because they interfere with
the normal expression of the gene (regulatory mutations). Most
individuals heterozygous for autosomal recessive disorders are
clinically normal.
LOSS OF FUNCTION (Albinism)
Autosomal recessive diseases often result from defects in enzyma-
tic proteins. Albinism is the result of a series of defects in the
synthesis of melanin pigment.45 Melanin is synthesized from
the amino acid tyrosine, which is first converted to dihydroxy-
phenylalanine through the action of the copper-containing
enzyme tyrosinase. An absence of tyrosinase results in one form
of albinism. Mutations in the gene coding for tyrosinase are
responsible for this disease cluster in the binding sites for copper,
disrupting the metal ion–protein interaction necessary for enzyme
function.46 Both copies of the gene for tyrosinase must be mutated
before a significant interruption of melanin production occurs.
22 Heterozygous individuals do not have a clinically apparent pheno-
type, suggesting that one functional copy of the gene produces
sufficient active enzymes that the melanin level is phenotypically
normal.
X-LINKED RECESSIVE DISORDERS
X-linked recessive disorders, like autosomal recessive disorders,
result from a mutant gene that causes a loss of a critical biologic
activity. Because males have only one X chromosome, one mutant
copy of a gene responsible for an X-linked trait results in the
disease. Usually females are heterozygous carriers of recessive
X-linked traits. In somatic cells of females, only one X chro-
mosome is active; the second X chromosome is inactivated and
becomes a Barr body. X inactivation has been associated with the
geneticist Mary Lyon, and has been called Lyonization. Inacti-
vation of either the maternal or the paternal X chromosome
occurs early in embryonic life. In any one cell, the inactive X may
be maternal or paternal, and once the X is inactivated, it remains
inactive. Because females inherit two copies of the X chromo-
some, they can be homozygous for a disease allele at a given
locus, heterozygous, or homozygous for the normal allele at the
locus. Since only one X chromosome is active in any given
somatic cell, about half the cells of a heterozygous female express
the disease allele, and about half express the normal allele. Like
autosomal recessive traits, the female heterozygote expresses
~50% of the normal level of the protein product. For recessive
conditions, this is sufficient for a normal phenotype.
Retinoschisis
Retinoschisis is a maculopathy that is caused by intraretinal split-
ting. The defect most likely involves retinal Müller’s cells.47
Retinoschisis is inherited as an X-linked recessive trait.48 Female
carriers with one normal and one abnormal copy of the gene do
not demonstrate any clinical abnormalities. Fifty percent of the
male offspring of female carriers are affected by the disease.

Mutations in a gene located in the retinoschisis interval and
expressed in the retina have been found in a protein that is impli-
cated in cell–cell interaction and may be active in cell adhesion
processes during retinal development. Mutational analysis of
the retinoschisis gene (XLRS1) in affected individuals from nine
unrelated families showed one nonsense, one frame shift, one
splice acceptor, and six missense mutations.49 Presumably these
mutations all result in an inactive protein product.
X-LINKED DOMINANT DISORDERS
X-linked dominant mutations are less common than X-linked
recessive mutations. Clinically, X-linked dominant inheritance
is difficult to recognize because of the random inactivation of the
X chromosome in females (Lyon’s hypothesis).50 The random
inactivation of the X chromosome produces females who are X
chromosome mosaics, with ~50% of the cells expressing genes
from the paternally derived X and 50% of the cells expressing
genes from the maternally derived X. If one of the X chromosomes
has a mutant gene, these cells may display the phenotype; how-
ever, 50% of the female cells are normal, even for a ‘dominant’
mutation. As a result, for recessive and dominant X-linked traits,
the disease phenotype may not be evident in females carrying the
mutation. X-linked dominant mutations could produce a pro-
tein that has a detrimental effect on normal biologic processes
(gain-of-function or dominant negative effect). Mutations that
result in haploinsufficiency of the X chromosome could also be
X-linked dominant. X-linked dominant disorders include incon-
tinentia pigmenti and X-linked hypophosphatemia rickets. A
family with X-linked dominant retinitis pigmentosa has also
been described.51
DIGENIC INHERITANCE
Digenic inheritance describes a pattern of inheritance that is
similar to recessive inheritance, except that the trait only develops
when mutations are found in one copy of each of the two inde-
pendent genes simultaneously. In recessive disorders the muta-
tions are found in both copies of one gene. Digenic inheritance
is an example of the complex interactions that occur between mul-
tiple gene products in polygenic inheritance (see further ahead).
Retinitis Pigmentosa – Peripherin and ROM1
At least one form of retinitis pigmentosa is inherited as a digenic
trait.52 In pedigrees demonstrating digenic inheritance there is
direct parent-to-child transmission of the disease; however, affected
families have unusual features for a dominantly inherited disease:
the disease originates in the offspring of an ancestral mating
between two unaffected individuals, and the affected individuals
transmitted the disease to less than 50% of their offspring
(~25% rather than 50%). In some retinitis pigmentosa families,
mutation analysis of the peripherin gene and the ROM1 gene
showed that the affected individuals had specific mutations in
both genes. Individuals who had a mutation in one copy of either
gene were unaffected by the disease. Mutant copies of ROM1
and peripherin can also cause autosomal dominant forms of
retinitis pigmentosa.53,54 These results suggest that some mutant
forms of peripherin and ROM1 cause retinitis pigmentosa in a
digenic pattern, whereas other mutations can independently
cause autosomal dominant forms of the disease.
Bardet–Biedl Syndrome
Bardet–Biedl syndrome (BBS) is a genetically heterogeneous dis-
order characterized by multiple clinical features that include
pigmentary retinal dystrophy, polydactyly, obesity, developmental
delay, and renal defects. BBS is considered an autosomal recessive
disorder, and positional cloning efforts have identified eleven
CHAPTER 2
Molecular Mechanisms of Inherited Disease
BBS genes.55 In some BBS pedigrees, affected individuals carry
three mutations in one or two BBS genes. In these pedigrees
unaffected individuals only had two abnormal alleles.56 In some
families it has been proposed that BBS may not be a single-gene
recessive disease but a complex trait requiring at least three
mutant alleles to manifest the phenotype. This would be an
example of triallelic inheritance.57
MITOCHONDRIAL DISORDERS
Mutations in mitochondrial DNA can also result in human dis-
ease. The characteristic segregation and assortment of Mendelian
disorders depends on the meiotic division of chromosomes found
in the nucleus of cells. There are several hundred mitochondria
in a cell, and each mitochondrion contains several copies of the
mitochondrial genome. Mitochondria divide in the cellular
cytoplasm by simple fission. Not all mitochondria present in a
disease tissue carry DNA mutations. During cell division, mito-
chondria and other cytoplasmic organelles are arbitrarily dis-
tributed to the daughter cells. Because each cell contains a
population of mitochondrial DNA molecules, a single cell can
contain DNA molecules that are normal as well as DNA mole-
cules that are mutant (Fig. 2.6). This heterogeneity of DNA
composition, called heteroplasmy, is an important cause of
variable expression in mitochondrial diseases. As the diseased
mitochondria are distributed to developing tissues, some tissues
accumulate more abnormal mitochondria than others.
Disorders that result from mutations in mitochondrial DNA
demonstrate a maternal inheritance pattern (see also Chapter 1).
Maternal inheritance differs from Mendelian inheritance in that
only affected females transmit the disease to their offspring.
Unlike nuclear DNA that is equally contributed to the embryo
by the mother and the father, mitochondria and mitochondrial
DNA are derived solely from the maternal egg. A mutation
occurring in mitochondrial DNA is present in cells containing
mitochondria, including the female gametes. Sperm have few
mitochondria, and they are not transmitted to the egg. A male
FIGURE 2.6. Heteroplasmy in mitochondria. Daughter cells resulting
from the division of a cell containing mitochondria with mutant DNA
may contain unequal numbers of mutant mitochondria. Subsequent
divisions lead to a population of cells with varied numbers of normal
and abnormal mitochondria. 23
SECTION 1 GENETICS

carrying a mitochondrial DNA mutation will not transmit the
disease to his offspring.
Leber’s Hereditary Optic Neuropathy
Leber’s hereditary optic neuropathy was one of the first diseases
to be recognized as a mitochondrial DNA disorder.58 For some time
clinicians had observed maternal inheritance of this condition
in affected families, but it wasn’t until mutations in mito-
chondrial DNA of affected individuals were demonstrated that
the cause of the inheritance pattern was understood. In familial
cases of the disease, all affected individuals are related through
the maternal lineage, consistent with the inheritance of human
mitochondrial DNA.
Patients affected by Leber’s hereditary optic neuropathy
typically present with acute or subacute, painless, central vision
loss leading to a permanent central scotoma and loss of sight.
The manifestation of the disease can vary tremendously especially
with respect to the onset of loss of vision and severity of the
outcome.59 The eyes can be affected simultaneously or sequen-
tially. The vision may be lost rapidly over a period of weeks to
months, or slowly over several years. Within a family the disease
may also vary among affected family members. Several factors
contribute to the variable phenotype of this condition. Certain
mutations are associated with more severe disease. For example,
the most severely affected patients with the 11 778-bp mutation
may have no light perception,60 whereas the most severely
affected patients with the 3460-bp mutation may retain light
perception.61 Another important factor that affects the severity
of the disease in affected persons is the heteroplasmic distribu-
tion of mutant and normal mitochondria. This partially explains
why some patients develop a more severe optic neuropathy. Other
genetic or environmental factors are likely to play a role as well.
POLYGENIC INHERITANCE
Human phenotypes inherited as polygenic or ‘complex’ traits do
not follow the typical patterns of Mendelian inheritance. Gen-
erally, complex traits are commonly found in the human popu-
lation. Multiple genes are likely to contribute to the expression
of the disease phenotype. Some genes may render an individual
susceptible to a disease, and other genes or environmental con-
ditions may influence the full expression of the phenotype.
Secondary genes responsible for the modulation of the expres-
sion of a specific genetic mutation are called ‘modifier genes’;
modifier genes may be inherited completely independently from
the gene directly responsible for the disease trait. For example,
recent evidence suggests that WDR36, a gene associated with
glaucoma but not sufficient to cause glaucoma, is a modifier
gene that contributes to the severity of the glaucoma phenotype
in individuals carrying a WDR36 variant in addition to another
glaucoma gene.62 Not every individual who inherits a mutation
partly responsible for a complex trait also inherits the set of
modifier genes that is required for full expression of the disease.
The digenic inheritance of retinitis pigmentosa seen by certain
mutant alleles of peripherin and ROM1 is an example of the
simplest form of polygenic inheritance (see previous discussion).
Certain conditions may require multiple genes or a combination
of different genes and environmental conditions to be manifest.
In addition to adult-onset primary open-angle glaucoma, exam-
ples of ocular disorders that are multifactorial are age-related
macular degeneration, and myopia.63
Key Features
• Disease treatment, including gene therapy, cannot be
developed without knowledge about the underlying molecular
mechanisms that are responsible for the disease.
• Autosomal dominant disorders result from one abnormal copy
of a gene; the defect may cause a loss of protein function, or a
gain of a novel detrimental function.
• Autosomal recessive disorders are caused by abnormalities in
both copies of a gene. The defective gene copies usually result
in loss of protein function.
• Digenic inheritance describes a pattern of inheritance that is
similar to recessive inheritance except that the trait only
develops when mutations are found in one copy of each of the
two independent genes simultaneously.
• Disorders that result from mutations in mitochondrial DNA
demonstrate a maternal inheritance pattern.

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25
 CHAPTER
3 Genetic Testing
Janey L. Wiggs
The identification of genes responsible for inherited ocular dis-
orders makes it possible to perform genetic testing for disease-
associated mutations that can help determine the clinical
diagnosis and prognosis. For some diseases, genetic testing can
serve as a screening tool to identify individuals at risk before the
clinical symptoms of the disease are manifest. The amount of
information provided by a genetic test and the methods used for
testing depend on what information is known about the gene(s)
involved. If the gene has been identified then direct genetic testing
can be performed, if only the location of the gene is known then
an indirect testing approach is used.
Direct testing uses a biological sample from the patient to
prepare DNA, RNA, or protein, to test for a specific alteration.
Typically, DNA or RNA is evaluated to determine if a specific
sequence change, or genotype, causing the disease is present in
the patient’s sample. Protein samples can be analyzed for specific
amino acid changes. Direct testing requires only a biologic sample
from the patient; however, detailed knowledge about the gene,
including the gene structure and the normal gene sequence, is
required.
Indirect testing uses family analysis to detect copies of the chro-
mosome that contains the mutant form of a disease-causing gene.
In this approach, DNA samples from all family members (affected
and unaffected) are analyzed for genetic markers that are known
to be located near the disease gene. The advantage of this approach
is that specific knowledge of the disease gene is not required. The
disadvantage is that multiple family members need to be tested.
Figure 3.1 shows a flow diagram outlining the protocol for
clinical genetic testing. The evaluation begins with a patient with
phenotypic characteristics of a disease (clinical findings, imaging
studies, laboratory studies) who presents to a physician. The clini-
cal evaluation may suggest a diagnosis that could be confirmed
by genetic testing. The first step is to determine if there is a family
history of the disease that would support a Mendelian inheritance
pattern (autosomal dominant, autosomal recessive, X-linked domi-
nant, X-linked recessive). If Mendelian inheritance is supported
by the family history, the next step is to determine if a gene has
been genetically mapped to a chromosomal region (genetic locus),
and if the gene has been identified within the locus. If the gene
has been mapped but not identified, indirect testing can be
performed using all available family members. If the gene has
been identified, and the gene sequence is known, the gene can
be screened using direct testing for disease-causing mutations.
If Mendelian inheritance is not supported by the family history,
the next step is to determine if there is a maternal inheritance
pattern that would support a diagnosis of a mitochondrial DNA
disorder. If the disease affects both male and female offspring and
is only transmitted by an affected mother, then mitochondrial
DNA screening should be considered. In the absence of Mendelian
inheritance or maternal inheritance, a diagnosis of a complex
genetic trait remains a possibility and screening of genetic risk
factors associated with the trait should be considered. If genetic
risk factors have not been identified, then genetic counseling
focused on risk avoidance (environmental exposures) and risk of
familial recurrences can be provided. In all cases, genetic coun-
seling can help the physician and patient understand the genetic
risks associated with the disease.
DIRECT TESTING APPROACHES AND
METHODS
The optimal, though not always practical or possible, method of
laboratory genetic diagnosis is to test a person’s gene or gene
product directly to determine if the sequence is normal or mutant.
Direct genetic testing can only be performed if the gene(s) respon-
sible for a disease have been identified and the normal sequence
is known. Most of the methods used for direct testing are
dependent on the polymerase chain reaction (PCR) (Fig. 3.2).
This enzymatic procedure makes many copies of the DNA (or
RNA) that will be used for genetic analysis.1 For PCR short
oligonucleotide segments (usually 20–30 bp in length) are syn-
thesized to match the normal DNA sequence that flanks the
DNA region of interest (usually an exon of a gene). The patient
DNA is denatured into two single strands and the synthetic
oligonucleotides are allowed to hybridize. A thermoresistant
version of DNA polymerase is added to the reaction which adds
a new DNA strand from the end of each of the two oligonucleotides
flanking the region of interest, thus making two copies of the
DNA segment to be tested. The process is repeated 30–50 times
resulting in an exponential expansion of the number of copies
of the desired DNA segment. The copied DNA segment can be
purified and used for additional tests to detect mutations. Typi-
cally, screening an entire gene is done by selectively amplifying
each gene exon followed by further analysis after purification of
the amplification products. Since PCR is the fundamental step
for direct genetic testing, PCR artifacts or reaction failures can
result in testing errors (see further ahead).
For direct testing, a biological sample needs to be obtained
from the patient. Family members may be included, but are not
necessary for the testing. Blood samples are the most widely used
source of DNA from adults, and yield more DNA than other
sources. For children or individuals not comfortable with blood
drawing, mouthwash samples or buccal swabs can be used.2 These
samples yield sufficient DNA for initial screening of a typical
gene. If more tests are required, or if patient resampling is diffi-
cult, then whole genome amplification can be used to make many
copies of the patient DNA sample before selectively amplifying
regions of the DNA for testing.3 Occasionally, direct testing is
27
SECTION 1 GENETICS

FIGURE 3.1. Decision flow diagram for genetic

Clinical evaluation testing.
Phenotype Laboratory tests
Imaging studies

Mendelian Inheritance
Maternal Genetic Loci
Inheritance NO YES Identified

Genetic Gene
Risk Factor Identified
NO YES NO YES

Screen
NO YES Mitochondrial Recurrence NO YES
DNA Risks
Risk Genetic Indirect Direct
Avoidance Test Testing Testing

FIGURE 3.2. PCR. A DNA sample is heated to

Double-stranded DNA produce single-stranded DNA which is then
allowed to be hybridized with an excess of
Heat and denature short oligonucleotide primers. Taq DNA
Single–stranded DNA Single–stranded DNA polymerase is added and DNA synthesis
proceeds elongating the primers to full-length
+ oligonucleotides strands. The newly synthesized double-
stranded DNA is heated again, and the cycle
repeats. At the end of the second cycle, four
Taq DNA polymerase double-stranded copies have been formed.
DNA synthesis Cycles are repeated 30–50 times to generate
sufficient DNA for further studies.

Heat and denature

+ oligonucleotides

Taq DNA polymerase

DNA synthesis

performed after a patient is deceased on material obtained from
archived pathology specimens4 or from hair recovered from a
hair brush.5
Genetic testing can be performed using DNA, RNA, or protein.
Of these, DNA is the easiest to purify and analyze. RNA in the
form of an RT-PCR product may be preferable for a large gene
such as retinoblastoma (Fig. 3.3).6 However, RNA is less stable
than DNA, and samples must be processed rapidly and under
specific conditions to avoid degradation. RNA expression in
accessible tissues may be a problem, and the mutant form of the
RNA may not be stable in vivo and may not be recovered in the
sample to be assayed. Protein assays can determine if a mutation
exists and if the mutation interferes with the protein function.
Ideally, the protein function information would be useful for all
genetic tests; however, proteins are far more difficult to purify and
assay for activity than DNA. For example, mutations in myocilin,
a gene responsible for some forms of early onset glaucoma, cause
the protein to be insoluble in an in vitro assay.7 However, to
28 perform this assay on patients would require access to disease
tissue and purification of the protein product. Information about
the gene mutation can be gained from purifying the DNA from
a blood or mouthwash sample and identifying the mutation
responsible for the abnormal protein. If the mutation can be
linked to abnormal protein function (using other information
such as this in vitro assay), then the same information has been
gained. If protein function information is not available for a
specific mutation, then it would be necessary to validate the muta-
tion in other ways. Despite the attraction of accessing the pro-
tein abnormality directly, for routine screening it is more efficient
to purify a DNA sample from the patient and identify the DNA
sequence change that causes the mutation, than to purify and
characterize the abnormal protein product.
For some diseases, affected individuals may carry the same
mutation, or one of a small number of mutations associated with
the disease. For example, most patients affected by Leber’s her-
editary optic neuropathy have one of three mutations.8 Hence,
for patients who are suspected of having a maternally inherited
optic neuropathy, testing would focus first on these three

E x on 1
Gene
Intron 1
mRNA
Exon 1 Exon 2
Add reverse transcriptase
and oligonucleotide
Reverse transcriptase
Reverse transcriptase
extends oligonucleotide to
make a DNA copy of the mRNA
Reverse transcriptase
RNase H removes mRNA
leaving single–stranded DNA
DNA polymerase
Add DNA polymerase
and oligonucleotide
DNA polymerase synthesizes
second strand DNA
Amplify using PCR
mutations. Such mutation redundancy among a population of
affected individuals may be the result of a hot spot in the gene
for mutations, a dependency of the disease on a specific type of
abnormality in the protein product caused by only a few muta-
tions, or a founder effect caused by a limited number of original
mutations. For some CYP1B1 gene mutations causing congenital
glaucoma, founder chromosomes have been identified,9 and the
mutations located on these chromosomes have been found in
multiple populations indicating the ancient distributions of the
original mutations. Approximately 50% of mutations in the
BIGH3 gene responsible for dominant corneal dystrophies involve
two sites in the gene, identifying these regions as mutation hot
spots.10 Generally, for disorders that are caused by a limited
number of mutations, those mutations are tested for initially,
and if the common mutations are not found then the entire
causative gene is screened.
METHODS FOR DIRECT MUTATION
TESTING
TESTING FOR A KNOWN MUTATION
Testing for a specific mutation can be done when there are a limited
number of mutations that have been associated with a disease,
or for diagnosis within a family when screening the entire causa-
tive gene has already defined a mutation in one family member.
Methods to test for a known mutation are simpler and less time
consuming than methods used to screen the entire gene. All of
the methods used to test for known mutations depend on PCR
amplification of a portion of the causative gene, followed by a
DNA sequence-dependent assay. There are many such assays
including: testing for the presence or absence of a restriction
enzyme site,11 allele-specific oligonucleotide hybridization,12 allele-
specific PCR amplification,13 oligonucleotide ligation assay,14 and
more recently quantitative PCR approaches using TaqMan or
related fluorescer-quencher methods.15 The general principles of
these direct methods are illustrated by a more detailed descrip-
tion of the TaqMan assay (Fig. 3.4).
CHAPTER 3
Genetic Testing
FIGURE 3.3. RT-PCR. mRNA is purified from
E x on 2 the patient, and is the starting material for PCR
rather than DNA. The first step is to make a
DNA copy of the mRNA using reverse
transcriptase and an oligonucleotide primer that
matches the target sequence. After
synthesizing a DNA copy, RNase H is used to
remove the original mRNA. Next DNA
polymerase and another oligo marking the
other end of the target sequence are used to
make a double-stranded DNA. The target
double-stranded DNA can then be used for
PCR as described in Figure 3.2.
RNase H
DNA polymerase
The TaqMan assay uses quantitative PCR to identify alleles at
a selected single nucleotide variation. Single nucleotide changes
can be missense or nonsense mutations or may be polymor-
phisms called single-nucleotide polymorphisms (SNPs).16 In the
TaqMan assay, a specific probe of 20–30 bp is designed to
hybridize specifically with the DNA sequence of interest. The
TaqMan probe is labeled with both a fluorescent reporter dye and
a fluorescent quencher dye and is also altered so that it cannot
be used as a primer for extension. Two additional unlabeled pri-
mers that flank the sequence of interest including the TaqMan
probe are used for PCR after hybridization of the TaqMan probe.
During PCR, the 5„ exonuclease activity of the Taq DNA poly-
merase degrades the TaqMan probe from the 5„ end, thus
releasing the reporter dye that is now able to fluoresce because
the quencher dye is no longer in proximity. As the PCR reaction
continues the fluorescence intensity of the reporter dye increases.
To detect a specific DNA sequence variation, two TaqMan probes
are developed, one for each allele, with reporters that fluoresce
as different colors. An advantage of this approach is that it is a
closed system without the need for purification or electrophoresis
of the amplification products, thus reducing the opportunity for
sample mix-up and contamination. The procedure also allows
for relatively high throughput as 96 samples can be analyzed in
a single assay and two to three assays can be run each day. Only
a very small amount (50 ng) of template DNA is required.
The protein truncation test (PTT) is a specific test for frame
shifts, splice site, or nonsense mutations that truncate a protein
product.17 Since the PTT only detects certain classes of mutations,
it is only useful for diseases that are predominantly caused by
mutations that cause a truncated protein product. An advantage
of this method is that it only detects pathogenic mutations.
SCREENING THE ENTIRE CAUSATIVE GENE
For most diseases many different mutations can be responsible
for the disorder, and genetic testing requires a search for muta-
tions anywhere within or near the relevant gene. To comprehen-
sively screen a gene for mutations, PCR amplification of gene 29
SECTION 1 GENETICS
R Q
TaqMan probe hybridized
with reporter (R) and
quencher (Q) tags, as well
as unlabeled primers and
DNA polymerase
R extension. Two additional unlabeled primers
Primer extension with
initial degradation by DNA
polymerase exonuclease
activity
R quencher dye is no longer in proximity. As the
Further extension and
degradation releasing the
reporter and allowing fluorescence
R
Completion of extension and
release of the reporter, cycle
ready to repeat
segments (typically exons) followed by direct sequencing is usually
the method of choice. It is possible to screen gene segments with-
out sequencing using techniques such as SSCP (single-strand
conformation polymorphism)18 or DGGE (denaturing gradient
gel electrophoresis);19 however, these methods are laborious and
can miss some mutations. Mutations identified by the screening
methods are typically confirmed by sequencing. Direct sequenc-
ing is costly; however, it provides the most reliable and repro-
ducible results.
DNA microarrays or ‘chips’ have been adapted for DNA
sequence detection.20–22 Hybridization chips contain oligonu-
cleotides matching all wild-type and single-nucleotide substitution
sequences in a gene. The patient DNA to be tested is amplified
using PCR, fluorescently labeled and hybridized to the array.
Minisequencing chips use arrayed oligonucleotide primers with
a free end that will be used for extension by DNA polymerase if
the free end matches the patient DNA. If the oligonucleotide
primer is allowed to extend the sequence of the new DNA strand
can be determined. The arrays are made with primers specific
for the normal sequence as well as for all possible mutations.
MUTATION VALIDATION
Direct mutation testing frequently reveals novel DNA sequence
changes that have not been previously associated with a disease
phenotype. Such sequence variants may be causative mutations
or they may be benign polymorphisms. Before the sequence change
can be recognized as disease-causing, it is important that the asso-
ciation of the putative mutation with the disease is supported
by additional studies. Ideally it would be best to demonstrate
that the mutant protein has an abnormal function, but this is
not always practical or feasible. Creating a transgenic animal
that carries the mutation and inspecting for signs of the disease
is another approach, but this can be extremely laborious and
time consuming and could not be done for every new mutation
discovered. It is important to determine if the sequence change
30 affects a region of the gene coding for a portion of the protein
FIGURE 3.4. TaqMan Assay. A specific
TaqMan oligonucleotide of 20–30 bp is
designed to hybridize specifically with the DNA
sequence to be tested. The TaqMan probe is
labeled with both a fluorescent reporter dye
and a fluorescent quencher dye and is also
altered so that it cannot be used as a primer for
that flank the sequence of interest including the
Q TaqMan probe are used for PCR after
hybridization of the TaqMan probe. During
PCR, the 5„ exonuclease activity of the Taq
DNA polymerase degrades the TaqMan probe
from the 5„ end, thus releasing the reporter dye
that is now able to fluoresce because the
PCR reaction continues the fluorescence
Q intensity of the reporter dye increases. To
detect a specific DNA sequence variation two
TaqMan probes are developed, one for each
allele, with reporters that fluoresce as different
colors.
Q
that is critically important for its function. It is also possible to
determine if the DNA sequence change is in a part of the
protein that is evolutionarily conserved which is an indication
that the changed sequence is in a region of the protein that is
functionally important. A control group of individuals without
evidence of the disease should be screened for the mutation. To
be reasonably certain that the DNA sequence change is not a rare
polymorphism, at least 100 control patients (200 chromosomes)
should be analyzed. If the patient carrying the putative mutation
has family members (both affected and unaffected) then segre-
gation of the sequence change in the family with the disease can
be evaluated. The characteristics of a disease-causing mutation
would include location in an evolutionarily conserved region of
the protein that may have critical function, not present in at least
100 controls and evidence of segregation in affected families.
Studies that will advance the knowledge of disease gene (and
protein product) functions and development of disease-specific
mutation databases will help make this task easier in the future.
INDIRECT TESTING AND METHODS
If the causative gene is not known, but the chromosome location
of the gene is known, then it may be possible to use genetic
markers located in the same region as the gene to identify family
members at risk for the disease. This method can only be used
if the disease is inherited as a Mendelian trait, and if the chro-
mosome location of the causative gene has been previously deter-
mined using genetic linkage studies. In addition, the individual
to be tested must have affected family members and also a
sufficiently large family that the parental chromosomes and the
chromosome carrying the abnormal copy of the gene can be
identified (Fig. 3.4). Identifying the chromosome carrying the
disease gene (determining phase) is enhanced by genetic markers
that are ‘informative’ in the parents, i.e., that they carry different
alleles at the marker (heterozygous) so that both copies of their
chromosomes can be identified. Microsatellite repeat markers
are highly informative because they have on average six to eight

alleles. With the completion of the human genome, over 10 000
microsatellite markers have been mapped across the human
genome, making it almost always possible to find an infor-
mative marker that maps close to the disease locus.23
Because indirect testing is looking for a DNA marker located
near the gene and not the gene itself, there is a risk that a recom-
bination event will occur between the marker and the gene which
can cause the disease chromosome to be inaccurately identified.
The closer the marker is to the true location of the gene, the less
the risk of a recombination event occurring between the marker
and the disease gene. Thus, indirect testing is most accurate for
disease genes that have been tightly linked to a small chromo-
some region, and with multiple highly polymorphic markers
located on opposite sides of the disease locus so that recom-
bination events can be visualized. The actual genetic risk can be
calculated using several methods including Bayesian calculations
and linkage programs.24–26
POPULATION SCREENING
Screening a population for a disease-related risk factor may iden-
tify a group of individuals who are at high risk for the disease.
If this knowledge enables actions that can modify the risk, then
the screening test has merit. For example, patients with higher
than normal intraocular pressure are at increased risk for optic
nerve disease related to glaucoma. Knowing that their pressure
is high, patients can initiate treatment to reduce their pressure
and lower their risk.27 A genetic risk factor could identify a popu-
lation of individuals at increased risk for developing a disease,
and if the knowledge of this increased risk makes it possible to
pursue treatment or behavior modification to reduce the risk
then the genetic testing is useful. Ideally the useful outcome is
treatment, but for many diseases this is not currently possible.
Other outcomes that may be useful are to avoid environmental
exposures that increase the risk and increase disease surveil-
lance. Emerging evidence may suggest that screening macular
degeneration patients for the complement factor H risk allele
and the LOC387715 risk allele may help identify groups of
patients that should avoid smoking.28–30
SPECIFICITY AND SENSITIVITY OF
GENETIC TESTING
An ideal test should be both specific and sensitive. Specificity is
the number of unaffected individuals that are negative for the
test compared with the total number of unaffected individuals
tested (including those that tested positive for the test). Sensi-
tivity is the number of affected individuals that are positive for
a test compared with the total number of affected individuals
(including those that tested negative for the test) (Fig. 3.5). In
general, false positives (individuals without the disease who test
positively) and false negatives (individuals with the disease who
test negatively) are serious failures of a diagnostic test. For gene-
tic tests, false positives are rare. The most likely causes of false
positives in DNA testing are laboratory or clerical errors. False-
negative tests are much more common in DNA testing. False-
negative tests can arise for a number of reasons including:
genetic heterogeneity (more than one gene is responsible for the
condition), PCR artifacts caused by primer binding site poly-
morphisms and deletions/insertions of the PCR primer sites,
deletion/insertion of an entire exon or the entire gene that inter-
feres with PCR amplification, preferential amplification of the
smaller allele in a large insertion, and tissue mosaicism. Because
a negative result cannot completely eliminate the possibility that
a person carries a mutation in a causative gene, genetic counseling
and patient and physician education are important components
of genetic testing.
CHAPTER 3
Genetic Testing
Specificity and sensitivity
Affected Unaffected
individuals individuals
Individuals
A B
positive for test
Individuals
C D
negative for test
A
Sensitivity
A+C
D
Specificity
B+D
FIGURE 3.5. Definition of sensitivity and specificity for a laboratory
test. Sensitivity is defined as the number of affected individuals
positive for the test (A) divided by the total number of affected
individuals tested (A + C). Specificity is defined as the number of
unaffected individuals negative for the test (D) divided by the total
number of unaffected individuals tested (B + D).
CLIA LABORATORIES
Laboratories offering genetic testing must comply with regula-
tions under the Clinical Laboratory Improvement Amendments
of 1988 (CLIA). CLIA, administered by the Centers for Medicare
and Medicaid Services, requires that laboratories meet certain
standards related to personnel qualifications, quality control
procedures, and proficiency testing programs in order to receive
certification. This regulatory system was put in place to encourage
safe, accurate, and accessible genetic tests. In addition to ensuring
that consumers have access to genetic tests that are safe, accu-
rate, and informative, these policies encourage the development
of genetic tests, genetic technologies, and the industry that pro-
duces these products. A number of CLIA-certified laboratories
performing genetic testing for eye diseases exist in the United
States. For a list of CLIA-certified laboratories participating in
the National Eye Institute sponsored eyeGENE network, see the
NEI website at: http://www.nei.nih.gov.
Key Features
• Genetic testing uses information about the gene(s) responsible
for a disease to identify individuals who carry abnormal forms
of a gene that may increase their risk of disease, alter the
progression of a disease, or identify them as carriers of a
disease.
• The type of genetic testing depends on the available
information about the genetic disease. If the disease gene is
known then direct testing can be performed, if only the
chromosomal location of the gene is known then indirect
testing is performed.
• Direct testing evaluates the DNA or RNA from a patient for a
specific sequence change, or genotype that causes the
disease. In some cases, protein samples can be analyzed for
specific amino acid changes.
• Indirect testing uses family analysis to detect copies of the
chromosome that contains the mutant form of a disease-
causing gene.
• Laboratories offering genetic testing must comply with
regulations under the CLIA of 1988, and genetic counseling
and patient and physician education are important
components of genetic testing.
31
SECTION 1 GENETICS
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1. Saiki RK, Bugawan TL, Horn GT, et al:
Analysis of enzymatically amplified beta-
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2. Mulot C, Stucker I, Clavel J, et al:
Collection of human genomic DNA from
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3. Barker DL, Hansen MS, Faruqi AF, et al:
Two methods of whole-genome
amplification enable accurate genotyping
across a 2320-SNP linkage panel. Genome
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4. Onadim Z, Cowell JK: Application of PCR
amplification of DNA from paraffin
embedded tissue sections to linkage
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Genet 1991; 28:312–316.
5. Suenaga E, Nakamura H: Evaluation of
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6. Chuang EY, Chen X, Tsai MH, et al:
Abnormal gene expression profiles in
unaffected parents of patients with
hereditary-type retinoblastoma. Cancer Res
2006; 66:3428–3433.
7. Zhou Z, Vollrath D: A cellular assay
distinguishes normal and mutant
TIGR/myocilin protein. Hum Mol Genet
1999; 8:2221–2228.
8. Spruijt L, Kolbach DN, de Coo RF, et al:
Influence of mutation type on clinical
expression of Leber hereditary optic
neuropathy. Am J Ophthalmol 2006;
141:676–682.
9. Sena DF, Finzi S, Rodgers K, et al: Founder
mutations of CYP1B1 gene in patients with
congenital glaucoma from the United
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11. Sieving PA, Bingham EL, Kemp J, et al:
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Mutation analysis of the KIF21A gene in an
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TNF-857T, a genetic risk marker for acute
anterior uveitis. Invest Ophthalmol Vis Sci
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method for point mutation detection using
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15. Hantash FM, Olson SC, Anderson B, et al:
Rapid one-step carrier detection assay of
mucolipidosis IV mutations in the
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2006; 8:282–287.
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throughput genotyping with single
nucleotide polymorphisms. Genome Res
2001; 11:1262–1268.
17. Tsai T, Fulton L, Smith BJ, et al: Rapid
identification of germline mutations in
retinoblastoma by protein truncation
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19. Mashima Y, Shiono T, Inana G: Rapid and
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20. Mandal MN, Heckenlively JR, Burch T, et
al: Sequencing arrays for screening
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26. Wiggs JL, Dryja TP: Predicting the risk of
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29. Schmidt S, Hauser MA, Scott WK, et al:
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 CHAPTER
4 Principles of Genetic Counseling
Gretchen Schneider and Pamela Hawley
The rapid advance in knowledge about genetic diseases and the
genetic contribution to common disorders, the improvements
in diagnostic testing, and the availability of some therapeutic
options have greatly enhanced the usefulness of genetic coun-
seling to families. The principles of genetic counseling can be
readily appreciated from the definition recommended by an ad
hoc committee of the American Society of Human Genetics.1
This defines genetic counseling as a communication process
aimed at helping families or individuals understand the impli-
cations of a definitive diagnosis or a risk for a disease, and the
hereditary implications for the patient, parents, and, when indi-
cated, other family members. Properly trained professionals
must be prepared to help the individual and the family com-
prehend available options for dealing with risk and to appro-
priately guide and support them in choosing the best course
of action.
Although the committee published this definition in 1974, these
goals of genetic counseling still remain widely accepted and
disseminated.2 What are changing rapidly are the diagnostic
tools available to meet these goals as well as the use of the
principles of genetic counseling as they apply to an increasingly
broadened scope of clinical scenarios. Because accurate genetic
counseling is predicated on a precise risk or accurate diagnosis,
knowledge of these new diagnostic tools and a consistent
approach to clinical evaluation are essential to the process.
WHO PROVIDES GENETIC COUNSELING
The providers of genetic counseling have changed greatly in the
past few decades. In the 1970s, when genetic counseling was
growing in recognition, many counselors were MDs and PhDs
who had no formal training. Physicians, nurses, and social workers
have continued to provide genetic counseling, mostly by learning
from experience. As genetic counseling became better defined,
the need was recognized for persons trained specifically to deal
with this process and its integration with medical science and
psychology.
Master’s level genetic counseling programs are designed to
train medical professionals, called genetic counselors, who pro-
vide such a service. These 2-year programs have combined
molecular and clinical genetics with counseling psychology in
settings that emphasize clinical rotations to gain experience.
More than 1500 genetic counselors have been trained at over 30
2-year programs. Genetic counselors often work with other health
professionals, including board-certified geneticists, obstetricians,
genetic fellows, nurses, social workers, and laboratory personnel.
This team approach allows comprehensive genetic services in
prenatal, pediatric, adult, cancer, specialty clinic, and commercial
settings.
WHY REFER PATIENTS FOR A GENETIC
EVALUATION
Accurate genetic counseling starts with a thorough genetic
evaluation. It is important for both families and physicians to
realize what is involved in the process and its value to the
patient and immediate relatives. The genetic evaluation is
important in a number of major ways:
1. It may help in understanding a patient’s problems by
providing a unifying diagnosis. When the diagnosis is a
well-described entity, it can sometimes provide prognostic
information. It may also change the clinical management
of a patient.
2. It may establish an increased risk of developing a disease
based on genetic markers, for example, breast or colon
cancer. This, too, can provide insight into options for
increased surveillance, or changes in management based
on this risk.
3. A specific diagnosis or the presence of a genetic risk factor
may have implications for other family members. Relatives
may also be at risk or become similarly affected. In many
instances, these relatives should be encouraged to receive
genetic counseling. Future children in the family may be at
risk. This risk is called the recurrence risk, and it
sometimes can be mathematically quantified.
INDICATIONS FOR REFERRAL TO A
GENETICS SPECIALIST
Although the need for a genetic evaluation or genetic counseling
often is obvious, this is not always the case. A child born with mul-
tiple anomalies may have no clearly identifiable diagnosis until
pedigree analysis reveals a pattern diagnostic of a genetic syn-
drome. This is particularly important whenever parents are
planning additional children and are justifiably concerned about
those children having similar problems. Even when a clinical
diagnosis and the relevant genetic counseling may seem straight-
forward, unanticipated beneficial information might be gained
from a visit to a genetics specialist.
ESTABLISHED GENETIC CONDITION
For a child or adult with an established diagnosis, the focus of a
genetics visit might be to understand the hereditary implications
of the diagnosis and the recurrence risks. For example, in a child
with retinoblastoma and a positive family history, the diagnosis
is clear. These families may be referred for genetic counseling to
review recurrence risks in a setting separate from the ophthal-
mologist’s office. An ophthalmologist may not feel well versed
33
SECTION 1 GENETICS
in the details of molecular testing and its use in testing other
family members and in prenatal diagnosis. A genetics specialist
can also discuss alternative reproductive options for those who
may not want prenatal testing.
Genetic evaluation sometimes suggests a clinical diagnosis of
a disorder that displays genetic heterogeneity. An example is oculo-
cutaneous albinism. There are several types of albinism due to
various mutations in any of several genes. A genetic evaluation
might uncover relatives who clearly have albinism; this infor-
mation might allow diagnosis with a mildly affected index patient.
Confirmation of that diagnosis might require biochemical or
molecular tests.
EYE FINDINGS WITH OTHER CONGENITAL
ANOMALIES
A child is sometimes born with a number of malformations
including ophthalmologic abnormalities. Some cases obviously
fit a particular syndrome, but others do not. For example, a child
might have microphthalmia, congenital heart disease, and delays
in development, with no syndrome diagnosis immediately recog-
nizable. Yet these multiple medical problems suggest a unifying
explanation for these findings. This constellation of findings could
be the syndrome of coloboma, heart defects, choanal atresia,
retarded growth and development, genital hypoplasia in males,
and ear anomalies – the CHARGE syndrome – or it could be
caused by a chromosome anomaly such as 13q–. In these situa-
tions, the experience of a geneticist in recognizing malformation
patterns and understanding the variability of genetic conditions
can aid in diagnosis. A genetics professional is also more likely
to be aware of the latest testing available, which may also be an
important component of the evaluation and diagnostic process.
If an underlying cause is identified, relatives can then undergo
genetic counseling.
EYE FINDINGS WITH OTHER MINOR
ANOMALIES
Some patients referred to the ophthalmology clinic may have no
obvious extraocular medical problems. During their visit, how-
ever, one may observe dysmorphic features or other seemingly
unrelated minor medical signs or symptoms. For example, reti-
nitis pigmentosa is a feature of a number of syndromes whose
other signs and symptoms may be subtle. A child with retinitis
pigmentosa, obesity, and polydactyly may have Bardet–Biedl
syndrome, whereas one with prominent central incisors and
slender hands and feet may have Cohen’s syndrome. Similarly,
a child referred for myopia who has micrognathia could have
Stickler’s syndrome. One with ectopia lentis due to Marfan’s
syndrome might be tall and lanky. Physical features that may
not be classified as medical problems, when combined with eye
findings, may lead to a syndrome diagnosis which is more easily
recognizable by a genetics professional.
SPECIFIC EYE DISEASES
A genetic evaluation may be important for patients with a purely
ocular disease for a number of reasons. A family history might
reveal similar eye disease or other findings that, when compared,
may lead to a genetic diagnosis in the family. A comprehensive
pedigree analysis sometimes reveals a genetic basis for such
diseases. Many frequently encountered ophthalmologic diseases,
such as cataracts or glaucoma, have a well-documented Mendelian
inheritance pattern. Others may not be purely Mendelian, but
the presence of multiple affected family members would indicate
increased risk for other relatives. Identifying the inheritance
34
pattern might lead to the identification of affected relatives who
could be diagnosed and treated early in the course of disease.
This is especially important in families with such conditions as
dominantly inherited juvenile glaucoma.
INCIDENTAL EYE FINDINGS
Eye findings with important genetic implications are sometimes
observed incidentally during ophthalmologic evaluation. For
example, a child may undergo ophthalmologic evaluation because
of a failed eye test at school but be found to have Lisch nodules,
which suggests neurofibromatosis type 1. Another child might
have the stellate iris pattern of Williams’ syndrome. Hetero-
chromia irides indicate an examination for the possibility of
Waardenburg’s syndrome. Although such findings may not have
any clinical implications, in some patients their strong association
with genetic conditions is an indication for a genetic evaluation.
Despite the numerous situations in which it is important to
explore the possibility of a genetic etiology, an identifiable gene-
tic condition is often not found. This does not exclude the
possibility of an underlying genetic cause for the individual’s
problems. Family members need to be aware of the possibility
of recurrence risk even if no specific diagnosis is made.
WHAT IS INVOLVED IN A GENETIC
EVALUATION
A genetic counselor begins a visit by ascertaining the client’s
understanding of the reason for the referral. The components of
a genetics evaluation are described and, when appropriate, the
client is cautioned that the evaluation does not always result in
a definite diagnosis or establish a specific genetic etiology.
FAMILY HISTORY
A detailed pregnancy, medical, and developmental history is
obtained, as is a three-generation family health history that
includes the ethnic origins of the ancestors. The possibility of con-
sanguinity should be explored. The family history is obtained
not only to establish a hereditary pattern for the referring diag-
nosis but also to identify other conditions that could have here-
ditary implications. For example, if the parents of the patient
are of Eastern European Jewish ancestry, their children are at
increased risk for Tay–Sachs disease, a recessive neurodegen-
erative condition for which carrier testing is available. If the
family history reveals developmental delay in a pattern
suggestive of fragile X syndrome, carrier testing could be offered.
Several modes of inquiry ascertain whether families could be at
risk for certain conditions unrelated to the referring diagnosis
(Table 4.1).
PHYSICAL EXAMINATION
A complete physical examination is performed with attention to
growth parameters, developmental milestones and subtle physi-
cal findings that can be important for establishing a syndrome
diagnosis. Careful anthropometric measurements (e.g., inner
canthal, outer canthal, and interpupillary distances; mid-
finger/total hand length; and upper body to lower body ratios) may
be obtained. Photographs also can be used to record nonmea-
surable dysmorphic features.
Examination of other family members may be indicated to
determine if a particular finding is hereditary. Sometimes this is
incidental to the reason for referral. Findings such as fifth-finger
clinodactyly, although a part of many syndromes, may also be
an isolated hereditary trait without other medical implications.

TABLE 4–1. Family History Considerations Regardless of Reason for Referral
Family History Positive for:
Ancestry
Eastern European Jewish*
French Canadian
Caucasian
African American
Mediterranean
Southeast Asian
More than two miscarriages
Birth defects in near relatives
Developmental delay
Maternal age over 35
Neonatal/childhood deaths in
first-degree relative
Known genetic disease
* The extent of screening for individuals of Ashkenazi descent varies by institution and laboratory and may include fewer, or more, tests than those listed.
COMPUTER-ASSISTED DIAGNOSTICS
Many databases can be accessed as part of the genetics evalua-
tion (Table 4.2). Pregnancy exposures may be assessed through
REPROTOX, a computerized database of potential teratogens
(available at many institutions free of charge through
MicroMedex). Standard computer literature searches are per-
formed. If findings are multiple and the patient’s history and
clinical findings do not suggest an obvious syndrome, the patient’s
information may be entered into genetic syndrome databases
such as POSSUM or London Dymorphology (these are available
by purchase) in an effort to diagnose a syndrome. If a specific
syndrome is being considered or an isolated finding has been
established, On-Line Mendelian Inheritance in Man (OMIM) is
often useful. OMIM is a frequently updated catalog of more
than 8400 human genetic conditions that is available to the
public through the NIH. It contains a historical summary of the
condition, current information regarding available diagnostic and
treatment options, details of genetic etiology, and references.
GENETESTS is another database of up-to-date clinical and
research diagnostic testing for specific conditions as well as a
library of comprehensive reviews written by genetic experts on
many genetic diseases. When circumstances and time permit,
computer searches such as these are conducted prior to or
during the initial visit. While there are many additional sources of
information on the Internet, it is advisable to select well-known
databases or websites with accurate and up to date information
when using it for patient assessment.
CHAPTER 4
Principles of Genetic Counseling
Consider:
Tay–Sachs disease carrier testing
Canavan’s disease carrier testing
Cystic fibrosis carrier testing
Fanconi anemia type C
Gaucher disease
Niemann–Pick type A
Tay–Sachs disease carrier testing
Cystic fibrosis carrier testing
Cystic fibrosis carrier testing
Sickle cell anemia carrier testing
b-Thalassemia carrier testing
a and b-thalassemia carrier testing
Parental chromosome studies to rule out translocation
Chromosome studies in parent
Fragile X testing if family history indicates pattern
Because of the possibility of asymptomatic transmitting males and
affected females, the inheritance is not the typical X-linked
recessive pattern
Prenatal chromosome studies
Review of records, particularly autopsy
Possible carrier testing (i.e., cystic fibrosis, Duchenne’s muscular
dystrophy)
ASSESSMENT
The initial assessment of an individual may include recommend-
ing testing or specialty consultations based on the history,
examination, or computer searches. Ophthalmologic examina-
tions for relatives may be indicated to detect relevant eye find-
ings. These examinations can be helpful in establishing familial
patterns when autosomal dominant or X-linked conditions are
being considered. For example, Best’s disease is an autosomal
dominant form of macular degeneration that causes a distinc-
tive macular lesion in its early stage. Scarring at the site of the
lesion can lead to decreased central vision. Macular lesions are
not present in all affected patients, but all affected patients have
abnormal electrooculogram findings. Ophthalmologic exam-
inations of the parents of an affected child can help provide
them with a recurrence risk assessment as well as identify
which side of the family may have affected relatives. Another
example is Lowe syndrome, an X-linked condition with findings
that include congenital cataracts, neurologic impairment, and
renal tubular dysfunction. Female carriers typically show no
neurologic or renal defects as detected by physical examination
or laboratory testing. However, slit-lamp examination reveals
specific lenticular changes in up to 94% of carriers.3 Although
molecular diagnostic testing is clinically available, careful
ophthalmologic examination is also valuable in assessing the
carrier status and therefore the recurrence risk for this con-
dition, particularly in families in which diagnostic testing was
negative. 35
SECTION 1 GENETICS
TABLE 4–2. Computer-Assisted Diagnostics
Program
REPROTOX
Reprotoxicology Center
Columbia Hospital For Women, Washington, DC
London Dysmorphology
Oxford University Press
POSSUM
Murdoch Institute for Research into Birth Defects
Royal Children’s Hospital, Melbourne, Australia
OMIM
http://www3.ncbi.nlm.nih.gov/omim/
GENETESTS
http://www.genetests.org/
PubMed
http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed
It may be necessary to obtain documentation of previous
testing including chromosome analysis, DNA testing or other
types of diagnostic tests and to review the studies (such as a
karyotype) to confirm the adequacy of the study. Obtaining records
to document a condition reported in a family member may also
be indicated. Because of these numerous steps involved in the
assessment process, review of the final assessment sometimes
requires a follow-up visit.
At the completion of the genetic evaluation of a patient
referred with a specific ocular finding, assessments can fall into
one of three general areas:
1. Isolated ocular disease or anomaly.
2. Nonocular findings with a pattern that fits no
recognized genetic syndrome.
3. Nonocular findings with a pattern that fits a
recognizable syndrome or association.
In the latter two situations, the ophthalmologist may not recog-
nize other clinical implications and the family may benefit from
discussion of these with a genetics professional. In any of these
three situations, a genetic component may be at work that
influences the risk of disease in the patient’s offspring, parents,
and other family members.
EXPLANATION OF CONCLUSIONS
Genetic counseling involves explaining the assessment process
and its conclusions to the family, including what is known about
the genetics of the patient’s condition and any possible medical
and developmental implications.
MEDICAL AND DEVELOPMENTAL
IMPLICATIONS
A genetic evaluation that results in a specific diagnosis may
provide information regarding previously obscure medical or
developmental implications. It is important to discuss clinical
variability in syndromes and to note that individuals do not
36
Database
Teratogens
Syndrome identification
Syndrome identification
Human genetic conditions
Availability of clinical and research
diagnostic testing
Expert Written Disease Reviews
Literature search
usually develop all the findings associated with a given condition.
Even if genetic testing has confirmed a diagnosis, it seldom
provides information regarding the likelihood or severity of spe-
cific features of a genetic disease. However, for some syndromes,
empirical data exist regarding the probability of the associated
findings. A genetic specialist can explain the indications for
medical monitoring or evaluations and can make appropriate
referrals. The importance of age-appropriate developmental
assessment and intervention programs in helping patients reach
their maximum potential is also emphasized. An established
diagnosis may have no additional medical or developmental impli-
cations, or no definitive diagnosis may be reached. In these
cases, the focus is primarily on the genetic implications of the
diagnosis.
GENETIC IMPLICATIONS
PRECISION
The extent to which the genetic component of a disorder is
understood can vary a great deal. This understanding affects the
precision of risk assessment and the options available for
modifying the risk. Some diseases have a definite inheritance
pattern that permits risks to be calculated according to the laws
of Mendelian genetics. For example, in a patient with Marfan’s
syndrome, an autosomal dominant condition, there is high
confidence in declaring a risk of 50% for offspring. Similarly, in
a family with a child with an autosomal recessive disease such
as Bardet–Biedl syndrome, the risk of recurrence in siblings is
one in four.
In contrast, in other diseases there is genetic heterogeneity, and
various inheritance patterns are possible. This can complicate
the prediction of risk. Instructive examples are nonsyndromic
retinitis pigmentosa or congenital cataracts. The inheritance
pattern can be autosomal recessive, autosomal dominant, or
X-linked recessive. For an isolated male case of retinitis pig-
mentosa, empirical data suggest that his offspring have a 12%
risk of recurrence.4 In fact, the recurrence risk ranges from less

than 1%, if it can be established that the patient has recessive
retinitis pigmentosa, and up to 50% if he has dominant retinitis
pigmentosa. In other scenarios, the risk differs from case to case.
One example is when a syndrome whose genetic etiology is not
well defined has been diagnosed in a child, but a recurrence risk
of 2% has been reported. Another is when a child has a constel-
lation of findings that has not previously been recognized. The
actual recurrence in siblings could be negligible if the etiology is
nongenetic, 25% if it is autosomal recessive, or ~50% if a
parent carries the mutant gene but does not express it clinically
(i.e., nonpenetrant).
Counselors must be cautious in providing recurrence risk in
a family with a child who has a well-established dominant syn-
drome if neither parent shows evidence of the disease. At first
glance, we might assume that the affected child represents a new
dominant mutation, in which case the parents are genetically
normal and the recurrence risk for siblings is vanishingly small.
However, two possibilities by which recurrence risk could be much
higher need to be considered. One, nonpenetrance, is defined as
the absence of phenotypic features in a person who has the
mutant genotype. If one of the parents is a nonpenetrant carrier,
the recurrence risk for subsequent children approaches 50%.
Another possibility is gonadal mosaicism, in which the mutation
has occurred during the growth and development in a parent, so
that it is present in a proportion of that parent’s germ cells.
Although genetic testing or empirical data may be available to
determine if a parent is a nonpenetrant carrier, testing is often
not available to evaluate gonadal mosaicism, and empirical data
on the frequency of gonadal mosaicism for specific conditions
are rare.
PATIENT’S UNDERSTANDING OF RISKS AND
OPTIONS
It is important to explain inheritance patterns and risks in ways
that patients will understand. A patient’s understanding of the
risks can be aided by presenting the risk estimates in more than
one way. Risk can be given as a fraction and as a percentage, and
risks can be given for both affected and unaffected offspring. For
example, one might explain that there is a 25%, or one in four,
chance that a disease would occur in the next child and a 75%,
or three in four, chance that it will not. The risk of recurrence
can also be put into context by providing the general population
risk for the particular condition, when available, as well as the
general population risk for a newborn child to have a serious
birth defect (3–4%).
A person’s interpretation of a recurrence risk is affected by a
number of factors, including personality (e.g., risk-taker versus
risk-averse), family goals and beliefs, and perceived physical,
emotional, and financial consequences of having a child with a
particular condition. In addition, a patient’s actual experience with
the condition in question can significantly affect the perception
of risk. The woman at risk for sons with Lowe syndrome might
feel differently about this condition if her uncle experienced the
renal failure associated with this disorder and died before she
was born than if her yet mildly affected son had been recently
diagnosed.
It is not surprising, therefore, that a risk considered high by
some will be viewed as low by others. Reviewing how these dif-
ferent factors affect interpretation of information and the choices
that are made can help clients. The counselor also needs to be
aware of his or her own perceptions of risk and burden. To the
greatest extent possible, the information provided to a patient
should emphasize the objective nature of risk figures and avoid
the subjective nature of how people perceive risk and the con-
sequences of a disease. There is no cutoff as to whether a given
CHAPTER 4
Principles of Genetic Counseling
risk figure is high or low or whether a specific disease-given con-
sequence is severe or minor. Clients also need to hear whether
a specific disease is severe or mild. Patients should be told that
decisions regarding having (more) children, seeking prenatal
testing, or considering alternative ways to have families are
their own decisions and are not based on perceived ‘orders’ of
their doctor or genetic counselor. Patients choose their future based
on their own goals, beliefs, and values.
RISK MODIFICATION FOR FUTURE
OFFSPRING
PRENATAL DIAGNOSIS
One means of risk modification for future children is prenatal
diagnosis. For conditions in which a diagnosis can be confirmed
with chromosome, biochemical, or molecular studies, three
procedures can usually be offered:
1. Routine amniocentesis at 15–16 weeks’ gestation.
2. Early amniocentesis at 12 weeks’ gestation.
3. Chorionic villus sampling at 10–12 weeks’ gestation.
If diagnostic testing is not available for a condition that includes
major congenital malformations, serial ultrasound examinations
may be performed as a means of prenatal diagnosis. The exami-
nations need to be performed by an ultrasonographer expert at
detecting fetal malformations; even then, the rate of detection
is not 100%.
If prenatal diagnosis is an option, a separate session should
be arranged to discuss the information more thoroughly. The risks,
benefits, and limitations of the procedures can be reviewed in
detail. Couples need to be reminded that many conditions cannot
be detected prenatally and that normal results from prenatal
diagnostic evaluation do not guarantee a healthy child. All
couples, regardless of their ages or family history, have a 3–4%
risk of having a child with a birth defect. Also, many inherited
conditions display considerable clinical variability. Couples need
to be aware that prenatal diagnosis usually does not predict the
severity of a condition.
In counseling for prenatal diagnosis, it is important to stress
to parents that they are not committed in advance to any parti-
cular course of action in the event of an abnormal finding.
Although termination of an affected pregnancy is available, this
is clearly not an acceptable alternative for all couples. Some may
wish to know in advance if the baby will be affected because this
may affect delivery site and neonatal management. For others,
early knowledge can help their families prepare and adjust for the
baby. Many couples consider prenatal testing for the reassurance
associated with the more likely event that the results are normal.
Thus, prenatal diagnosis should not be summarily dismissed
for those couples who indicate that they will not consider elec-
tive pregnancy termination.
A relatively recent option for some conditions is preimplan-
tation diagnosis with in vitro fertilization (IVF). Following IVF,
typically at the 8–16-cell blastomere stage, genetic material from
single cells is analyzed for DNA or chromosomal abnormalities.
Only embryos with a normal genetic complement (for the dis-
order testing for) are then implanted into the mother’s uterus.
Though this procedure is very accurate, follow-up prenatal diag-
nosis is recommended to confirm the findings discovered by PGD.
The procedure is also expensive and may not be covered by
insurance. Although some states require third-party payers to
cover IVF, this is usually mandated for infertile couples, and those
seeking preimplantation diagnosis are not infertile. Finally,
because relatively few facilities offer the procedure, logistics can
preclude its availability.
37
SECTION 1 GENETICS
ASSISTED REPRODUCTIVE TECHNOLOGIES
AND ADOPTION
Some risk revision options do not involve prenatal testing. Assisted
reproductive technologies offer a means for reducing risk,
particularly for Mendelian disorders or familial chromosome
changes. IVF with donor egg when the mother has an autosomal
dominant condition or is a carrier for an X-linked condition
reduces the risk to the level of population incidence. Risk is
similarly reduced with artificial insemination by donor if the
father has an autosomal dominant condition. With recessive
conditions, artificial insemination by donor usually reduces the
risk to less than 1%. Adoption can be an alternative for couples
who perceive the recurrence risk or consequences to be too high
but whose personal goals include a (larger) family. Therefore, for
families faced with risks to future children, alternative options
should be discussed as well.
CARRIER TESTING
For some conditions, carrier testing is available to revise risk. If
the concern is regarding future children, this means that pre-
natal diagnosis is available as well. However, assessment of
carrier status sometimes helps a couple decide if they wish to
pursue another pregnancy even if prenatal diagnosis is available.
It could also have implications for other family members.
For example, if a child has microphthalmia and other conge-
nital anomalies related to a translocation trisomy 13 and both
parents have normal chromosomes, the risk of recurrence for their
offspring and those born to other relatives is extremely low. In
contrast, if one parent carries a balanced arrangement involving
chromosome 13, the empirical recurrence risk data would be
known for both parents and any sibling of the parent who
carries the rearrangement. Fabry’s disease is another example in
which carrier testing is useful. This is an X-linked condition in
which affected patients accumulate glycolipid as a result of an
a-galactosidase deficiency. Onset is typically in childhood or ado-
lescence and includes episodes of severe extremity pain, angio-
keratomas, and characteristic corneal and lenticular opacities.
Cardiac, renal, and cerebrovascular complications can occur later
in life. Carrier assessment includes ophthalmologic examina-
tion. Corneal opacities detectable only by slit-lamp examination
are present in ~80% of carriers.5 Assaying a-galactosidase levels
is another carrier testing option for this disorder. Both eye
examination results and enzyme level can be normal in carriers,
however, because of X-chromosome inactivation. Therefore, mole-
cular testing may offer more definitive results to identify
females in a family who are at risk of having affected sons.
MOLECULAR TESTING: DISTINCTIONS AND
LIMITATIONS
Molecular testing often is used for prenatal testing and carrier
detection. When newly developed technology is being considered,
it is important that families be aware of whether the testing is
provided on a clinical or research basis. Clinical testing implies
well-established protocols with quality control measures and
available data regarding sensitivity and specificity. The time
required for testing is predictable, and a charge is often involved.
Research testing is performed in an unpredictable time frame,
and usually there is no charge.
Progress toward understanding the genetic basis of disease can
be expected to affect diagnostic capabilities first. Treatment or
management of a genetic disease generally lags behind consi-
derably, although considerable progress has been made for inborn
errors of metabolism such as Gaucher disease and Fabry disease
38
for which enzyme replacement therapy now exists. Although
‘gene therapy’ receives a great deal of media attention, clinical
application is so far limited. Genetic counselors must explain
this distinction between diagnostic and therapeutic interventions.
If a gene is mapped and DNA markers linked to the gene are
available, linkage analysis in some families may be used to
predict affected status in at-risk individuals. This, however, may
not always be informative because of the limited size of the
family. Studies should be performed on several family members
before it is known whether linkage studies will be useful for carrier
or prenatal assessment in that family. For those families in
which study results are informative, the studies will provide a
revised risk rather than a definitive answer, because with link-
age studies, recombination is always possible. The degree of risk
revision varies from family to family, depending on which markers
are used. Accuracy is highest for families with informative
flanking markers. Another limitation of linkage studies is the
possibility that an altered gene at a location unlinked to the
markers could cause a similar clinical condition. If the gene
mutation or product is not testable, this potential heterogeneity
remains a concern.
When direct analysis of an actual gene mutation or gene pro-
duct is possible, issues need to be discussed with families to help
them understand how the information is useful to them. For
example, if all possible mutations causing a condition cannot be
identified, testing will not be definitive in all cases. Although
blood is an easily accessible source of genetic material and use-
ful for linkage and mutation analysis, it may not be a good source
for gene product testing. In this situation, additional tissue may
be necessary, and the appropriateness of a more invasive test
needs to be discussed with family members. The invasiveness
of a test should be weighed against the additional information
that will likely be obtained.
DOCUMENTATION AND FOLLOW-UP
Clients who are counseled should receive a detailed written sum-
mary of the evaluation. Although writing clear and informative
summaries can be extremely time consuming, it is necessary for
several reasons. It is unlikely that all the verbal information
provided during the visit will be remembered, and what is
remembered may be difficult for an individual to explain to
others. A summary serves as an extension of the communication
process that allows for review by the recipient.
Genetic counselors are available to clients on an ongoing basis
to reexamine and clarify the issues covered during the visit(s)
and in the written summary. They provide reassurance that the
clients’ responses to a situation are expected and appropriate;
this can be reinforced by providing families with information about
support organizations. In addition, families need to be informed
that genetics is a rapidly advancing area of medicine. Even if an
evaluation has failed to identify a specific diagnosis, families who
have received genetic counseling are encouraged to reestablish
contact whenever planning a pregnancy to take advantage of
any pertinent new developments.
ETHICAL CONSIDERATIONS IN GENETIC
COUNSELING
The increased understanding of genetic disease and the genetic
components of common disease as well as the availability of test-
ing bring many challenges to genetic assessment and counseling
and raise a number of ethical issues. Although most genetic
counseling situations do not give rise to these dilemmas, it is
important for health professionals to be aware of these
possibilities.

CONFIDENTIALITY
Issues of genetic privacy are much discussed in the genetics com-
munity and society as a whole. There is debate over who should
have access to genetic information and how it can be used. Of
particular concern is the potential for discrimination by insurance
companies or employers. There is fear that insurance companies
may use test results to deny coverage, claiming that a genetic
disease is a preexisting condition. Alternatively, they may con-
sider an affected individual to be an insurance risk if his or her
condition could cause medical problems in the future. Others
are concerned that employers may try to use genetic informa-
tion to make hiring decisions, basing their assessment on risk for
medical complications or disability. Currently, numerous states
have genetic privacy legislation which protects patients from
discrimination, and national legislation has been presented but
is currently under review.
Regardless of protections in place, these issues often lead fami-
lies or individuals to be wary of genetic testing. Some decide to
decline testing even if a positive test result could alter medical
management. Others choose to pay for testing themselves to
prevent the insurance company from having access to this infor-
mation. Still others request that test results not be put in their
medical record. Families may desire to have total control over
the information to help minimize the risk of the information
being used against them.
Genetic professionals support the patients’ right to privacy
with regard to results of genetic testing. Those arranging testing
should discuss the issues of confidentiality prior to the initia-
tion of testing so there is consensus on how results are reported,
who receives results, and where the information is documented.
CONTROVERSIAL USES OF GENETIC
TESTING
A number of situations may arise where patients want to use
genetic testing for less traditional purposes. Because many patients
have access to different types of genetic testing, particularly if
they pay for it themselves, genetic counselors may be asked to
arrange testing for reasons with which they do not necessarily
agree. It is important for medical professionals to be aware of
these scenarios, recognize their own opinions, and be able to
refer patients to others if they do not feel that they can support
such patients’ wishes. Some specific examples of these situa-
tions are discussed below.
SEX SELECTION
A couple might wish to choose the sex of their child by testing
during a pregnancy, or through PGD with implantation of only
embryos of the desired sex. Having a child of a particular gender
has strong roots in some cultures, justifying these measures for
some couples. Other couples may simply wish to ensure that they
have children of both sexes in their family, a concept known as
‘family balancing’. Although this is not illegal, and is offered at
some institutions, it can make those providing the testing
uncomfortable.
PRESYMPTOMATIC TESTING OF CHILDREN
Because testing is available for a number of disorders with later
onset, such as Huntington disease or breast cancer, it is possible
to test children or even fetuses for conditions that may not affect
their lives for many years. Although parents may feel that this
is in the best interest of their children, some fear it may cause
stigmatization. Others argue that undergoing testing should be
CHAPTER 4
Principles of Genetic Counseling
the decision of the individual, once he or she reaches adulthood,
particularly if it would not affect medical management. Current
recommendations discourage testing in children for disorders for
which the results would not warrant a change in their immediate
medical management. However, parents with strong desires to
pursue such testing may be able to find someone willing to do it.
TESTING FOR SELECTION OF AFFECTED
PERSONS
Patients with certain conditions or physical limitations may desire
to have similarly affected children. Patients with achondroplasia,
for example, have wanted to have children with achondroplasia
because this is what they have come to consider normal. This
could lead them to choose prenatal diagnosis to ‘rule in’ achon-
droplasia, possibly resulting in the termination of an unaffected
pregnancy. The same might be true of a couple in which both are
deaf. Such testing is theoretically available, if a genetics center
feels comfortable performing it.
DISCOVERY OF UNANTICIPATED OR
HARMFUL INFORMATION
Because genetic testing can involve looking for a broad array of
abnormalities (e.g., when looking at chromosomes) or studying
a number of persons in a family (via linkage analysis), it can some-
times uncover information that family members did not anti-
cipate or do not want to know. Prior to the initiation of testing,
it is important to discuss not only the possible benefits of
genetic testing but also the potential for unanticipated results.
NONPATERNITY
Genetic testing can lead to the discovery of nonpaternity. Raising
this as a possible outcome prior to testing may help to avoid an
awkward situation when test results become available.
DISCLOSURE OF DISEASE STATUS
In large families studied by linkage analysis, a number of persons
may learn a family member’s disease status. Some such persons
may have no relationship with the physician or genetic counselor
that organized the testing. If possible, these persons should be
referred to a qualified physician or local genetics center where
they can learn about their disease status and discuss the impli-
cations of their test results. It is also best to determine which
family members do not want to know their results before testing
begins. Care must be taken to avoid divulging their status to other
family members. Those not requesting information should have
the option of obtaining it later, should they change their minds.
NONDISCLOSING PRENATAL DIAGNOSIS
A special situation surrounding genetic testing involves prenatal
diagnosis for an autosomal dominant condition in which a parent
is at risk but does not want to know his or her disease status.
Prenatal diagnosis using linkage analysis is most accurate in
families with affected individuals in more than one generation.
In this scenario, if a fetus is found to be unaffected, the parent’s
status would not need to be conveyed (Fig. 4.1). However, the
diagnosis of an affected fetus would indicate that the parent is
also affected. This would necessarily prompt a couple to come to
terms with the diagnosis in the parent.
Alternatively, testing to determine which grand-parental allele
a fetus received without establishing linkage of the disease gene
to a particular allele can be used when only one affected family
39
SECTION 1 GENETICS

affected. Another situation might be during prenatal diagnosis

for advanced maternal age, where the couple is told the fetus is
being tested for Down syndrome, but turns out to have a dif-
ferent chromosomal abnormality. In situations where genetic
testing is performed, all possible testing outcomes should be
discussed prior to testing.

a b
FIGURE 4.1. Linkage analysis with letters (A–E) represents specific
RFLPs (see Chapter 1). The fetus is unaffected in both scenarios. The
father’s disease state is determined but need not be disclosed. (a) The
fetus and father both have the nondisease allele of the affected
grandmother. (b) The fetus receives the allele of the unaffected
grandfather, but the father has the disease allele from the affected
grandmother.
DUTY TO RECONTACT
In the era of rapid scientific discovery, particularly in molecular
diagnostics, the question arises as to how to keep families informed
of new information. Parents of a child with albinism seen years
ago might now benefit from molecular testing. Carriers of a fragile
X pre-mutation who had been told in the past that this has no
medical implications may need to be informed of the now-
recognized risk of premature ovarian failure or tremor-ataxia
syndrome. Therefore, what a family is told at a genetic counseling
session could eventually become outdated. At the same time, it
is not generally possible for medical professionals to contact
previous patients when new knowledge or testing becomes
available.
As discussed previously (see section on Documentation and
Follow-Up), genetic counselors must remain available to families.
In addition, the importance of genetic counseling for affected
children when they reach child-bearing age should be stressed.
This allows for a review of the genetic implications as well as an
update on the possibilities for diagnostic testing. Finally,
periodic follow-up visits may be suggested to help families keep
up-to-date on both clinical and molecular developments.

FIGURE 4.2. The risk of the fetus being affected is 50%. The father’s
risk remains unchanged. The fetus receives the grand-maternal allele,
but testing cannot determine whether it is the disease allele.
CONCLUSION
Genetic counseling involves the transfer of technical and con-
ceptual information that is complex and sometimes different
from information the family may have previously encountered.
This information is often conveyed to persons who are feeling
anxious, guilty, depressed, or overwhelmed. By recognizing and
exploring the psychological impact of genetic counseling issues,
counselors can better integrate medical and genetic information
so that families feel competent in making informed decisions.
Such autonomy can reestablish their sense of control and aid in
their psychological adjustment.

member is available for testing and when parents want to
guarantee that their status is not determined by testing. This
could exclude (within the limits of recombination) a fetus being
affected if it received an allele from the unaffected grandparent.
If the fetus received the allele of the affected grandparent, this
would not prove that the fetus is affected but would increase the
risk from 25% to 50% (Fig. 4.2).
DIAGNOSIS OF AN UNANTICIPATED
DISORDER
At times, a genetic test may provide unanticipated results.
Examples include performing hemochromatosis or CF carrier
testing on an individual only to determine they are actually
Key Features
• Genetic counselors often work with other health professionals,
including board-certified geneticists, obstetricians, genetic
fellows, nurses, social workers, and laboratory personnel to
provide genetic counseling.
• A genetic evaluation includes family history, physical
examination, and assessment of laboratory and ancillary
testing.
• Genetic counseling involves explaining the assessment
process and its conclusions to the family, including what is
known about the genetics of the patient’s condition, any
possible medical and developmental implications, and risk of
recurrence to other family members.

REFERENCES
1. Fraser FC: Genetic counseling. Am J Hum
Genet 1974; 26:636–659.
2. Marks JH: 2003 ASHG award for
excellence in human genetics education.
The importance of genetic counseling. Am
J Hum Genet 2004; 74:395–396.
40
3. Nussbaum RL, Suchy SF: The
oculocerebrorenal syndrome of Lowe (Lowe
syndrome). In: Scriver CR, Beaudet AL,
Sly WS, Valle D, eds. The metabolic and
molecular bases of inherited disease. 8th edn.
New York: McGraw Hill; 2001:6257–6266.
4. Hartong DT, Berson EL, Dryja TP: Retinitis
pigmentosa. Lancet 2006; 368:1795–1809.
5. Metabolic disorders. In: Gorlin RJ, Cohen
MM Jr, Hennekam RCM, eds. Syndromes
of the head and neck. 4th edn. New York:
Oxford University; 2001.

6. Baker DL, Schuette JL, Uhlmann WR eds:
A guide to genetic counseling. New York:
Wiley-Liss; 1998.
7. Bernhardt BA: Empirical evidence that
genetic counseling is directive: where do
we go from here? Am J Hum Genet 1997;
60:17–20.
8. Furu T, Kaarianinen H, Sankilla EM, et al:
Attitudes towards prenatal diagnosis and
Principles of Genetic Counseling
selective abortion among patients with
retinitis pigmentosa or choroideremia as
well as among their relatives. Clin Genet
1993; 43:160–165.
9. Harper PS: Practical genetic counseling.
6th edn. Oxford: Butterworth-Heinemann;
2004.
10. Raz AE, Atar M: Nondirectiveness and its
lay interpretations: the effect of counseling
CHAPTER 4
style, ethnicity and culture on attitudes
towards genetic counseling among Jewish
and Bedouin respondents in Israel. J Genet
Couns 2003; 12:313–332.
11. Weil J: Psychosocial genetic counseling in
the post-nondirective era: a point of view.
J Genet Couns 2003; 12:199–211.
12. Weil J: Psychosocial genetic counseling.
Oxford: Oxford University Press; 2000.
41
 SECTION 2 IMMUNOLOGY
Edited by C. Stephen Foster and M. Reza Dana
CHAPTER
5 Immunology – An Overview
Reza Dana and C. Stephen Foster
All organisms live under the threat of attack from other living
organisms that express foreign, potentially immunogenic,
antigens. Additionally, a wide array of ‘non-pathologic’ cellular
exposures (ultraviolet radiation from sun exposure, injury, etc.)
and responses (e.g., cell death, stress, and oxidation) can lead to
activation of immune responses to autoantigens. Among primi-
tive single-celled eukaryotes, defense depends on physico-
chemical barriers at the cell surface and the capacity to engulf,
phagocytize, and digest the attacking pathogen. As multicellular
organisms evolved, and individual cells assumed differentiated
functions important to the well-being of the host, defense
against invading pathogens and complex immunoregulatory
pathways that ensure a ‘measured’ response to immunogenic
insults, became the responsibility of specialized cells and mol-
ecules. The multifaceted array of sophisticated cells and mol-
ecules of the mammalian immune system is the evolutionary
descendant of these early forms of defense mechanisms.
The immune system found in mammals and higher
vertebrates is divided into two functionally distinct, but also
overlapping and interregulated, components termed ‘innate’
and ‘adaptive’ immunity. Innate immunity is evolutionarily
more ancient and provides the host organism with an imme-
diate protective response that does not require gene arrange-
ment and is not antigen-specific. Adaptive immunity, by
contrast, provides protection that takes time to develop, is
antigen-specific, but is remembered through time (involves
‘memory’), thereby allowing for efficient responses to be
generated in case of chronic or recurrent challenge by the
inciting antigen. Whereas innate immunity has the capacity to
recognize and respond to invading pathogens, the capacity to
accurately distinguish between self-molecules and molecules
of the pathogen (non-self) is much more highly developed in
the adaptive immune system (Table 5.1).
INNATE IMMUNITY
Innate, or ‘natural’, immunity consists of physicochemical
barriers, erected at interfaces between the host and the
environment, and a distinctive array of cells and molecules.1–3
Intact body surfaces, such as the skin and mucous membranes
with tight junctions among adjacent epithelial lining cells,
provide physical barriers to the entry of pathogens. In the case
of the eye, mechanical phenomena such as the wiping action of
eyelids, coverage of much of the epithelia with mucinous
glycoproteins, and the bulk flow of tears across the ocular
surface, all provide natural protection against pathogens. The
chemical components of body fluids (such as the tears)
including fatty acids, lysozyme, and complement components,
also make essential contributions to innate immunity. Finally,
cellular effectors of innate immunity include bone marrow-
TABLE 5.1. Characteristics of Innate vs Adaptive Immunity
Innate Immunity Adaptive Immunity
Specificity Not antigen-specific Antigen-specific
Efficiency Rapid Primary responses
slower
Memory Absent Present
Chief Effectors Neutrophils, macrophages, Lymphocytes
natural-killer (NK) T cells
derived cells, including neutrophils, macrophages, and natural
killer cells, that are mobilized in the natural defenses against
invading pathogens.
Innate immunity is activated, for example, when an invading
bacterium, perhaps by releasing endotoxins or other bacterial
products, elicits a stereotypic inflammatory response by inter-
acting with toll-like receptors on host cells, inducing micro-
vascular dilatation, leukocyte infiltration, and participation of
serum complement proteins. Innate immunity is also revealed
when a virus penetrates through the skin and evokes within the
draining lymph node an accumulation of natural killer cells
with the capacity to lyse virus-infected cells directly. In both of
these examples, the cells and molecules responsible for innate
immunity recognize and respond to the pathogen, but in neither
case is the recognition specific for the particular organism.
Moreover, if and when the attacker has been eliminated, the host
is not protected against a second invasion from the same agent
any more than it was the first time, since there is no memory.
ADAPTIVE IMMUNITY
Adaptive, or acquired, immunity depends on a highly devel-
oped, sophisticated set of lymphoid organs (thymus, spleen,
lymph nodes, bone marrow, mucosa-associated lymphoid
tissues), cells (T and B lymphocytes, antigen-presenting cells
including dendritic and Langerhans cells, and macrophages),
and molecules (antibodies, cytokines, growth factors, and cell-
adhesion molecules).1 The interactions between and among
these elements allow the adaptive immune system to meet four
important challenges as listed in Table 5.2.
FEATURES OF ADAPTIVE IMMUNITY
Certain features of the adaptive immune response set it apart
from all other ways in which an organism can respond to its
environment: 43
 IMMUNOLOGY
TABLE 5.2. Critical Functions of the Immune System
1. To create a repertoire of recognition structures (antibodies by
B cells, T cell receptors for antigen) that recognize biologically
important molecules in our universe
2. To eliminate or suppress lymphocytes whose recognition
structures bind to self-molecules and therefore threaten
autoimmunity and autoimmune disease
SECTION 2
3. To create a diversity of effector mechanisms designed to
counter the diverse virulence strategies used by the many
different potential pathogens
4. To fashion immune responses in individual organs and tissues
such that protection is provided without interfering with the
tissue’s differentiated function
1. Adaptive immunity is acquired. Exposure of an adult
individual to a foreign antigen for the first time leads to an
immune response that is first detected (e.g., as antibody in
the blood) within 5–7 days. During the ‘silent’ interval
after initial exposure, the adaptive immune system is
‘learning’ about the presence of the antigen. Thus, adaptive
immunity is ‘acquired’.
2. The immune response is specific for the eliciting antigen.
The antibodies that form within 5–7 days react with the
eliciting antigen alone and not with any other molecule
(unless there are shared structural residues between the
antigen that elicited the response and another antigen to
which the immune response is reacting). Exposure of the
same individual to a second (different) antigen elicits
another antibody response that is equally specific for the
second antigen and nonreactive with the first antigen.
Thus, adaptive immunity is molecularly ‘specific’.
3. Reexposure of an individual to an antigen for a second
time elicits a response that is accelerated in ‘onset’ and
exaggerated in ‘amount’. This means that what was
‘learned’ by the immune system during its first exposure to
an antigen is ‘remembered’ through time, and the
secondary response is the manifestation of that memory.
Thus, adaptive immunity is ‘remembered’.
4. Adaptive immunity can be transferred from an individual
who has it to another individual, thus conferring an
identical immunity on the recipient. Both antibodies and
specifically sensitized lymphocytes are capable of
transferring adaptive immunity. Thus, adaptive immunity
is ‘transferable’.
5. Adaptive immunity can be specifically prevented by
administering antigen under highly specialized, often
experimental, conditions. Individuals treated with antigen
in this manner may be rendered unable subsequently to
acquire immunity to the same antigen if administered in a
conventional fashion. Individuals rendered specifically
unable to respond to a particular antigen are said to be
immunologically ‘tolerant’. Thus, tolerance is a
manifestation of adaptive immunity.
BENEFITS OF IMMUNITY
In mature mammals and higher vertebrates, both innate and
adaptive immune systems exist. Virtually every immune
response represents the summation of both innate and adaptive
responses, and the two systems are inextricably entwined.4 To
describe briefly the interplay between innate and adaptive
immunity, the following examples are given. Infection of the
lung with Streptococcus pneumoniae is prevented from
44 proceeding to consolidating pneumonia primarily by the innate
immune response. Neutrophils and, to a lesser extent, macro-
phages form the primary defense system, aided by acute-phase
reactants (e.g., C reactive protein) and members of the
complement cascade of proteins. The innate response in this
setting is activated to phagocytose and neutralize the invading
pathogen before large numbers of cells are infected. Adaptive
immunity, in which S. pneumoniae-specific antibodies are
produced, comes into play well after the primary infection has
already been contained, providing additional protection for that
infection, but most importantly also for protection once the
host gets reexposed to S. pneumoniae. In influenza virus infec-
tions of the lung, natural killer cells act early to limit virus
spread, but the infection appears to be terminated by virus-
specific cytotoxic T cells that eliminate all virus-infected cells.
In parasitic infections, where clearance and elimination of the
organism may never be achieved, adaptive immunity plays the
key role in containing the organism in situ.
While the importance of immunity in infectious disease is
obvious, immunity is also believed to play a key role in the
control of neoplasms.5,6 Because tumors arise from host tissues,
the antigenic differences between tumors (‘non-self ’) and ‘self ’
tissues are necessarily narrower. On the one hand, this makes
it more difficult for the immune system to detect neoplastic
cells, and, on the other hand, raises the possibility that
immunity directed at antigens on tumor cells may spill over
onto normal tissues because of shared antigenic moieties. Still,
the immunity generated against neoplastic tissues is important,
manifested by the enhanced propensity of chronically
immunosuppressed individuals to a variety of malignancies.
HAZARDS OF IMMUNITY
There are two important ways in which immunity can harm
the host. First, most (if not all) immune responses that lead to
elimination of a pathogen require the participation of non-
specific host defense (innate immune) mechanisms. Because
they lack the high specificity of antibodies, T lymphocytes,
neutrophils, macrophages, and natural killer cells are incapable
of confining their destructive forces to pathogenic organisms.
Similarly, activated proteases of the complement system are
indiscriminate in their choice of substrates at the site of
infection. Thus, host tissues adjacent to an infection are usually
damaged, sometimes irreparably, by the intense inflammation
taking place in their midst. This penchant for innate immunity
to cause unwanted tissue damage is further enhanced by cells
and molecules of the adaptive immune system.1 For example,
the T cells that mediate delayed hypersensitivity responses
secrete cytokines that can serve as powerful attractants and
stimulants of macrophages and other leukocytes. As a
consequence, tissue injury and death is almost an invariant
outcome of delayed hypersensitivity responses directed at
infecting pathogens. Similarly, complement-fixing antibodies
recruit and amplify the participation of neutrophils and
macrophages at the site where they bind target pathogens, lead-
ing to exaggerated inflammation and necrosis. Thus, immunity
can inadvertently produce injury to otherwise healthy host
tissues, and immunopathogenic mechanisms are important
causes of disease in many different organs and tissues.
Second, the adaptive immune response must meet the
challenge of eliminating or suppressing T and B cells with
recognition structures (e.g., T cell receptors) specific for self-
antigens, so-called ‘autoreactive’ lymphocytes.7 This is one of
the central tenets of central tolerance that allows the thymus
to delete such autoreactive cells from circulation. When this
challenge is not met, autoimmunity can arise. In truth, not all
‘autoimmunity’ is deleterious. For example, there is evidence
suggesting that immunity against certain self-components may
 Immunology – An Overview

be a necessary part of the healing response to injury and
infection. However, certain types of autoimmunity are destruc-
tive, and these can give rise to tissue-restricted inflammatory
diseases. Examples abound, including rheumatoid arthritis,
Sjögren’s syndrome, uveitis, inflammatory bowel disease, and
others. A hierarchy of self-antigens exists, dictated by the extent
to which the antigens are accessible to lymphocytes of the
systemic immune apparatus. For instance, circulating plasma
ment, including microbial pathogens, and needs the protection
afforded by the immune system. And yet, on the other hand,
immunity is necessarily mediated in part by nonspecific host
defense mechanisms that carry the threat of innocent bystander
injury. To resolve this dilemma evolutionarily, the eye and the
immune system have arranged a compromise in which certain
forms of immunity are permitted, whereas others are sup-
pressed. This compromise is expressed experimentally in the

proteins have an extremely low potential for evoking an auto-
immune response. By contrast, proteins expressed on cells found
only in the eye (e.g., photoreceptors) or testis (spermatozoa)
have a high potential for eliciting an autoimmune response. In
addition, tissue-restricted factors (e.g., blood–tissue barriers)
influence whether a response that is autoimmune becomes
immunopathogenic and therefore causes disease.
SPECIAL CASE OF THE EYE: IMMUNE
PRIVILEGE
Most organs of the body can sustain substantial amounts of
permanent damage from immune and inflammatory reactions
without losing appreciable function. For example, inflammation
in the skin, heart, liver, kidney, and bone can be associated
with the typical consequences of inflammation-damage to the
normal cells of the organ and scarring from the compensatory
reparative processes associated with injury. These organs, how-
ever, are very forgiving, in that they can each sustain substantial
amounts of inflammation (provided that it is temporary) and
still retain sufficient viability after the reparative processes to
carry on the normal functions required for normal living
activities. The same is not true for the eye.
Inflammation that in other tissues would be trivial is not
tolerated well by the eye and visual system. The vulnerability of
the eye to even small amounts of inflammation derives from
the need to preserve the anatomic integrity of the visual axis.
Very slight alterations in components of the visual axis prevent
light images from landing precisely on the retina, causing sig-
nificant visual impairment. Thus, innocent bystander damage
to ocular tissues during the course of inflammation can be
associated with a profound loss of function (i.e., blindness or
substantial impairment of useful vision). For example, even
slight temporary inflammation in the central part of the cornea
can have substantial, long-term effects on functional visual
acuity after resolution of the inflammation, simply because the
reparative processes result in disorganization of the normally
ordered arrangement of collagen fibrils within the corneal
stroma, an organization that is critical to continued clarity in
the cornea. Similarly, inflammation involving the retina
(especially the macula), the vitreous, and the uveal tract can
also produce significant loss in visual function.
Thus, the eye is confronted with a dilemma. On the one
hand, the eye is covered by a mucosal surface that leaves it
largely exposed to the myriad noxious stimuli of the environ-
CHAPTER 5
phenomenon of ‘immune privilege’.8 It has been known for
more than a century that foreign tissues implanted in the
anterior chamber of the eye enjoyed prolonged survival com-
pared with the fate of foreign tissues implanted at conventional
body sites. In the 1950s, Medawar correctly inferred that the
ability of foreign grafts to survive in the eye was due to a failure
of immunologic rejection.9 At the time, Medawar proposed that
immune privilege resulted from sequestration of intraocular
antigenic material from the systemic immune apparatus. The
term ‘immunologic ignorance’ has been used to identify this
situation. However, in recent years, it has become clear that
ocular immune privilege is a state that is actively maintained by
a variety of immunoregulatory mechanisms, rather than simply
antigenic sequestration afforded by physical and tight junction
barriers.
Immune privilege is an actively acquired and maintained
state in which ocular factors, acting on cells of the immune
system, suppress both the induction and expression of
immunity within the eye, and alter the induction of systemic
immunity to ocular antigens, leading to a stereotypic systemic
immune response called anterior chamber associated immune
deviation (ACAID).10 As a consequence, systemic immune
responses to eye-derived antigens are deficient in T cells that
mediate delayed hypersensitivity and in antibodies that activate
complement components. Thus, systemic immunity engen-
dered by eye-derived antigens lacks the two effector modalities
most closely linked to intense inflammation and innocent
bystander injury-delayed hypersensitivity and complement-
fixing antibodies.
It is important to emphasize that immune privilege in the eye
is not simply the consequence of a ‘failed’ immune response;
rather, it results from modifications in the immune response
that afford immune protection for the eye that carries a mini-
mal threat to nonspecific injury. The importance of this
understanding lies in the implications that it holds for the
diagnosis and treatment of ocular inflammatory and infectious
disorders. The sections and chapters that follow are designed to
provide more specific information to ophthalmologists and
vision scientists about the cells and molecules that affect and
regulate inflammation and immunity in the eye.
ACKNOWLEDGMENT
The authors would like to acknowledge the significant material contribution
of Dr J Wayne Streilein to the previous edition of this chapter.

REFERENCES
1. Janeway CA Jr, Travers P, eds:
Immunobiology. 6th edn. New York:
Garland Publishing Inc; 2004.
2. Akira S, Uematsu S, Takeuchi O: Pathogen
recognition and innate immunity. Cell 2006;
124:783–801.
3. Koehn B, Gangappa S, Miller JD, et al:
Patients, pathogens, and protective
immunity: the relevance of
virus-induced alloreactivity in
transplantation. J Immunol 2006;
176:2691–2696.
4. Pulendran B, Ahmed R: Translating innate
immunity into immunological memory:
implications for vaccine development.
Cell 2006; 124:849–863.
5. Moller G: Tumor immunology. Immunol Rev
1995; 145:1–12.
6. Karin M, Lawrence T, Nizet V: Innate
immunity gone awry: linking microbial
infections to chronic inflammation and
cancer. Cell 2006; 124:823–835.
7. Hogquist KA, Baldwin TA, Jameson SC:
Central tolerance: learning self-control in
the thymus. Nat Rev Immunol 2005;
5:772–782.
8. Streilein JW: Perspective: unraveling
immune privilege. Science 1995; 270:1158.
9. Medawar PB: Immunity to homologous
grafted skin. III. The fate of skin homografts
transplanted to the brain, to subcutaneous
tissue, and to the anterior chamber of the
eye. Br J Exp Pathol 1948; 29:58.
10. Streilein JW: Ocular immune privilege and
the Faustian dilemma. Invest Ophthalmol
Vis Sci 1996; 37:1940–1950.
45
 CHAPTER
A Cast of Thousands: The Cells of the Immune
6 System
C. Stephen Foster
The cellular components of the immune system include lym-
phocytes, macrophages, Langerhans’ cells, neutrophils, eosi-
nophils, basophils, and mast cells. Many of these cell types can
be further subdivided by subtypes and subsets. For example,
lymphocytes include T lymphocytes, B lymphocytes, and non-
T, non-B (null) lymphocytes. Each type can be further subcate-
gorized, both by functional differences and by differences in
cell-surface glycoprotein specializations and uniqueness. The
latter differentiating aspect of cell types and cell-type subsets
has been made possible through the development of hybridoma-
monoclonal antibody technology. This phenomenon of cell-
surface glycoprotein specialization and uniqueness among cell
types, and the technology for identifying those unique dif-
ferences among cell types, are so important that a synopsis of
the evolution and current understanding of this phenomenon
follows.
Jeorges Kohler and Cesar Milstein, at Cambridge University,
succeeded in immortalizing antibody-producing cells in 1975
by fusing them with myeloma tumor cells using a myeloma cell
line with a selective deficiency of hypoxanthine phosphoribo-
syltransferase.1 These researchers developed a technique for
successfully recovering only the cells that had successfully fused
to the myeloma cells (i.e., the hybridomas). Only the hybridoma
cells survived in a tissue culture medium containing hypo-
xanthine, aminopterin, and thymidine, because the antibody-
forming cell component of the hybridoma contributed enough
hypoxanthine phosphoribosyltransferase to ensure survival of
the hybrid. Selecting individual hybrids that produce the desired
antibody against a particular immunogen (antigen or antigenic-
determinant or epitope) and then allowing that hybrid cell
(hybridoma) to proliferate generated an immortal monoclonal
cell population (i.e., a hybrid cell population derived from a
single original cell) and thus produced a never-ending supply of
a highly specific antibody (monoclonal antibody) directed
against the original immunogen of interest. For this innovative
and important work, these researchers were awarded the Nobel
Prize for Medicine in 1984.
Reinherz and Schlossman2 exploited the monoclonal
antibody technology in the late 1970s, first taking advantage of
the fact that T lymphocytes possess well-known, unique cell-
surface determinants (e.g., a binding receptor for sheep
erythrocytes), which made it possible to separate T lymphocytes
into pure preparations from peripheral blood lymphocytes.
Immunization of mice with such a purified preparation of T
cells, with subsequent preparation of hybridomas from spleen
cell populations harvested from those immunized mice, was
followed by screening and selection of hybridomas that
synthesize antibodies that would stick to the cell surface of T
cells and by cloning of these hybridomas. This same strategy or
similar strategies based on functional assays (e.g., beginning
with cells that were efficient at helping an immune response to
develop or beginning with cells that efficiently suppressed an
immune response) resulted in the additional development of
monoclonal antibody reagents that were specific for and
identified the two major T lymphocyte subsets, helper-inducer
T cells, and suppressor-cytotoxic T cells.
Because the original work was performed in collaboration
with Ortho Pharmaceuticals, the original designation of the
cell-surface determinants for T cells was OKT 3, the de-
signation for helper-inducer T cells was OKT 4, and that for
suppressor-cytotoxic T cells OKT 8. As additional companies
began to develop their reagents using the same technology,
additional naming schemes developed, and the name game for
cell-surface determinants became extremely complicated.
Investigator workshops have now generated a universal no-
menclature system for cell-surface glycoproteins, or ‘antigens’,
and this system is based on the so-called clusters of
differentiation designation. Hence, the proper designation for
the cell-surface glycoprotein unique to T cells is now CD3, and
the designation for the cell-surface glycoprotein unique to
helper/inducer T cells is CD4. Table 6.1 presents a partial list of
current clusters of differentiation designations and the cell
types that express these CD antigens.
LYMPHOCYTES
Lymphocytes are mononuclear cells, round, 7–8 mm in
diameter, found in lymphoid tissue (lymph node, spleen,
thymus, gut-associated lymphoid tissue, mammary-associated
lymphoid tissue, and conjunctiva-associated lymphoid tissue)
and in blood. They ordinarily constitute ~30% of the total peri-
pheral white blood cell count. The lymphocyte is the premier
character in the immune drama; it is the primary recognition
unit for foreign material, the principal specific effector cell type
in immune reactions, and the cell exclusively responsible for
immune memory.
T lymphocytes, or thymus-derived cells, compose 65–80% of
the peripheral blood lymphocyte population, 30–50% of the
splenocyte population, and 70–85% of the lymph node cell
population. B lymphocytes compose 5–15% of peripheral blood
lymphocytes, 20–30% of splenocytes, and 10–20% of lymph
node cells.
T cells possess cell-surface receptors for sheep erythrocytes
and for the plant-derived mitogens concanavalin A and phyto-
hemagglutinin. They do not possess surface immunoglobulin or
surface membrane receptors for the Fc portion of antibody-two
notable cell-surface differences from B lymphocytes, which do
possess these two entities. B cells also exhibit cell-surface
receptors for the third component of complement, for the
Epstein–Barr virus and the plant mitogen known as pokeweed 47
 IMMUNOLOGY

TABLE 6.1. Clusters of Differentiation (CD) Designations

Clusters Cell Specificity Function

CD1a b c d Thymocytes, Langerhans’ cells dendritic cells, MHC class I-like molecule, associated with b
B cells (CD1c), intestinal epithelium, smooth 2-microglobulin. Role in presentation of lipid antigens
muscle, blood vessels (CD1d)
CD2 T cells, NK subset Receptor/sheep erythrocyte receptor; adhesion molecule
SECTION 2

— binds to LFA-3 (CD58), binds Lck intracellularly and

activates T cells
CD3 T cells T-cell antigen-complex receptor
CD4 Helperinducer T cells, TH1 and TH2T cells MHC class II immune recognition; HIV receptor (HIV-1 and
HIV-2 gp120)
CD5 T cells, B-cell subset Scavenger receptor
CD6 T cell, subset, B cells in chronic lymphatic leukemia Binds CD166 (scavenger receptor)
CD7 T cells, NK cells, platelets Binds PI 3-kinase. Marker for T cell acute lymphatic
leukemia and pluripotential stem cell leukemias
CD8 Cytotoxic suppressor T cells MHC class I immune recognition, binds Lck kinase
CD9 Pre-B cells, monocytes, eosinophils, basophils, Mediates platelet aggregation and activation via FcgRIIa,
platelets, activated T cells, brain and peripheral may play a role in cell migration
nerves, vascular smooth muscle
CD10 Pre-B cells, neutrophils Neutrophil endopeptidase.
Common acute Zinc metalloproteinase, marker for pre-B acute lymphatic
lymphocytic leukemia (ALL)
leukemia
antigen (CALLA)
CD11a Leukocytes Adhesion molecule (LFA-1) binds to CD54 (ICAM-1), CD102
(ICAM-2), and CD50 (ICAM-3)
CD11b ( Mac-1) Monocytes, granulocytes, NK cells a-Chain of complement receptor CR3;, binds CD54,
complement component iC3b, and extracellular matrix
proteins
CD11c Monocytes, granulocytes, NK cells Adhesion (aX subunit of integrin CR4 (associated with
CD18), binds fibrinogen)
CD11d Leukocytes aD subunits of integrin; associated with CD18; binds to
CD50
CDw12 Monocytes, granulocytes, platelets Unknown
CD13 Monocytes, granulocytes, Aminopeptidase N (Zinc metalloproteinase)
CD14 Macrophages Lipopolysaccharide receptor
CD15 Neutrophils, activated T cells, eosinophils Terminal trisaccharide expressed on glycolipids and many
cell-surface glycoproteins
CD15s Leukocytes, endothelium Ligand for CD62E, P
CD15u Sulphated CD15 Terminal trisaccharide expressed on glycolipids and many
cell-surface glycoproteins
CD16 Granulocytes, macrophages, NK cells Fc receptor IgG (Fc-g RIII); activation of NK cells
CDw17 Neutrophils, monocytes, platelets Lactosyl ceramide, a cell-surface glycosphingolipid
CD18 Leukocytes Intergrin b2 subunit; associates with CD11a, b, c, and d
CD19 B cells B-cell activation (binds tyrosine kinases and PI 3-kinase)
CD20 B cells B-cell activation (oligomers from a calcium channel)
CD21 B cells Complement receptor CR2 (C3d) — Epstein–Barr virus
receptor
CD22 B cells Adhesion; B-cell activation
CD23 Activated B cells, macrophages, activated Low-affinity Fc-e receptor, induced by IL-4
macrophages, eosinophils, follicular dendritic
cells, platelets
CD24 B cells, granulocytes Unknown
CD25 Activated T cells, B cells IL-2 receptor

Continued
48
 A Cast of Thousands: The Cells of the Immune System

TABLE 6.1. Clusters of Differentiation (CD) Designations—Cont’d

Clusters Cell Specificity Function

CD26 Activated B and T cells, macrophages Exopeptidase, cleaves N terminal X-Pro or X-Ala dipeptides
from polypeptides
CD27 Medullary thymocytes, T cells, NK cells, some B cells TNF receptor, Binds CD70; can function as a co-stimulator
for T and B cells

CHAPTER 6
CD28 T cells Receptor for co-stimulator molecules B7.1 (CD80) and B7.2
(CD86)
CD29 Leukocytes Integrin b1 subunit, associates with CD49a in VLA-1 integrin
CD30 Activated B and T cells Binds CD30L (CD153); cross-linking CD30 enhances
proliferation of B and T cells
CD31 Platelets, monocytes, and B cells Role in leukocyte–endothelial adhesion (PECAM-1 mediated
leukocyte-endothelial and endothelial-endothelial
interactions)
CD32 B lymphocytes, granulocytes, macrophages, Fc receptor IgG (Fc-gRIII) ADCC
eosinophils
CD33 Myeloid progenitor cells, monocytes Binds sialoconjugates
CD34 Hematopoietic precursors, capillary endothelium Ligand for CD62L (L-selectin)
CD35 B cells, erythrocytes, neutrophils, mononuclear cells Complement receptor CR1 (binds C3b and C4b, mediates
phagocytosis)
CD36 Platelets, monocytes, endothelial cells Platelet adhesion molecule, (GPIV, GPIIIb) involved in
recognition and phagocytosis of apoptosed cells
CD37 B cells Unknown, may be involved in signal transduction
CD38 Activated T and plasma cells, early B and T cells NAD glycohydrolase, augments B cell proliferation
CD39 Activated B cells, activated NK cells, macrophages, Unknown, may mediate adhesion of B cells
dendritic cells
CD40 B cells Co-stimulatory molecule for B-cell activation by T-cell
contact binds CD154 (CD40L), promotes growth,
differentiation, and isotype switching of B cells
CD41 Megakaryocytes, platelets Associates with CD61 to form GPIIb; binds fibrinogen,
fibronectin, von Willebrand factor, and thrombospondin;
Fn receptor,
CD42 a,b,c,d Megakaryocytes, platelets GpIb —platelet adhesion; binds von Willebrand factor,
thrombin
CD43 Leukocytes T-cell activation
CD44 Leukocytes Pgp1 (Hermes) receptor; homing receptor for matrix
components (e.g., hyaluronate)
CD45 All leukocytes Leukocyte common antigen —signal transduction
(tyrosine phosphatase)
CD45RA Naive cells
CD45RO Activated/memory T cells
CD45RB B cells, T-cell subsets, monocytes, macrophages,
granulocytes
CD46 Hematopoietic and nonhematopoietic nucleated cells Membrane co-factor protein; binds to C3b and C4b to
permit their degradation by Factor I
CD47 All cells Adhesion molecule; thrombospondin receptor
CD48 Leuckocytes Putative ligand for CD244
CD49a (VLA-1) Activated T cells, monocytes, neuronal cells, smooth a1 integrin; associates with CD29; binds collagen, laminin-1
muscle
CD49b (VLA-2) B cells, monocytes, platelets, megakaryocytes, a2 integrin; associates with CD29; binds collagen, laminin
neuronal, epithelial and endothelial cells, osteoclasts
CD49c (VLA-3) B cells, many adherent cells a3 integrin; associates with CD29; binds laminin-5,
fibronectin, collagen, entactin, invasin

Continued

49
 IMMUNOLOGY

TABLE 6.1. Clusters of Differentiation (CD) Designations—Cont’d

Clusters Cell Specificity Function

CD49d (VLA-4) Broad distribution includes B cells, thymocytes, a4 integrin; associates with CD29; binds fibronectin,
monocytes, granulocytes, dendritic cells MAdCAM-1, VCAM-1
CD49e (VLA-5) Broad distribution includes memory T cells, monocytes, a5 integrin; associates with CD29; binds fibronectin,
platelets invasin
SECTION 2

CD49f (VLA-6) T lymphocytes, monocytes, platelets, megakaryocytes, a6 integrin; associates with CD29; binds laminin, invasin,
trophoblasts merosine
CD50 (ICAM3) Thymocytes, T cells, B cells, monocytes, granulocytes Binds integrin CD11a/CD18
CD51 Platelets, megakaryocytes aV integrin; associates with CD61; binds vitronectin, von
Willebrand factor, fibrinogen, and thrombospondin; may
be receptor for apoptotic cells
CD52 Thymocytes, T cells, B cells (not plasma cells), Unknown
(CAMPATH 1) monocytes, granulocytes, spermatozoa
CD53 Leukocytes Unknown
CD54 (ICAM-1) Activated cells Adhesion to LFA-1 (CD11a/CD18 integrin) and MAC
1(CD11b/CD18); rhinovirus receptor
CD55 Hematopoietic and nonhematopoietic cells Decay accelerating factor (DAF); binds C3b; disassembles
C3/C5 convertase
CD56 NK NCAM (neural cell adhesion molecule) —adhesion
CD 57 NK cells, subsets of T cells, B cells, and monocytes Oligosaccharide, found on many cell-surface glycoproteins
CD58 (LFA-3) B cells, antigen-presenting cells Binds to CD2
CD59 Hematopoietic and nonhematopoietic cells Binds complement components C8 and C9; blocks
assembly of membrane attack complex
CD60a,b,c
CD61 Platelets, megakaryocytes, macrophages Intergrin b3 subunit; associates with CD41 (GPIIb/IIIa) or
CD51 (vitronectin receptor)
CD62E (E-selectin, Endothelial cells Adhesion (binds CD34, GlyCAM, mediates
ELAM-1) rolling interactions with endothelium)
CD62L T cells, B cells Adhesion (binds CD34, GlyCAM, mediates rolling
(L-selectin, interactions with endothelium)
LAM-1)
CD62P (P-selectin) Platelets, endothelial cells, megakaryocytes Adhesion (binds CD162 (PSGL-1), mediates interaction
PADGEM of platelets with endothelial cells, monocytes, and rolling
leukocytes on endothelium)
CD63 Activated platelets, monocytes, macrophages Unknown
CD64 Monocytes, macrophages Adhesion, FC-g receptor; antibody-dependent, cell
mediated cytotoxicity
CD65 Myeloid cells Oligosaccharide component of a ceramide
dodecasaccharide
CD66a Neutrophils Unknown
CD66b Granulocytes Unknown
CD66c Neutrophils Unknown
CD66d Neutrophils Unknown
CD66e Adult colon epithelium, colon carcinoma Unknown
CD66f Unknown
CD68 Monocytes, macrophages, neutrophils, basophils, Unknown
large lymphocytes
CD69 Activated lymphocytes Unknown
CD70 Activated T and B cells, and macrophages Ligand for CD27
CD71 Proliferating cells Transferrin receptor
CD72 B cells Ligand for CD5; B cell – T cell interactions
CD73 B and T cells Ecto-5„-nucleotidase; dephosphorylates nucleotides to
allow nucleoside uptake
50
Continued
 A Cast of Thousands: The Cells of the Immune System

TABLE 6.1. Clusters of Differentiation (CD) Designations—Cont’d

Clusters Cell Specificity Function

CD74 B cells, macrophages, monocytes, MHC class II MHC class II-associated invariant chain
positive cells
CD75 Mature B cells, T-cells subsets Lactosamines; ligand for CD22; mediates B-cell-B-cell
adhesion

CHAPTER 6
CD75s Mature B cells, T-cells subsets a-2,6-sialylated lactosamines
CD77 Germinal center B cells Neutral glycosphingolipid; binds Shiga toxin; cross-linking
induces apoptosis
CD79 B cells Components of B-cell antigen receptor analogous to CD3;
required for cell-surface expression and signal transduction
CD80 (B7-1) B cells, dendritic cells, macrophages Ligand for CD28 and CTLA4; co-stimulator for T-cell
activation
CD81 Lymphocytes Associates with CD19, CD21 to form B cell co-receptor
CD82 Leukocytes Unknown
CD83 Leukocytes Unknown
CDw84 Monocytes, platelets, circulating B cells Unknown
CD85 Dendritic cells ILT/LIR family
CD86 Monocytes, activated B cells, dendritic cells Ligand for CD28 and CTLA4
CD87 Granulocytes, monocytes, macrophages, T cells, Receptor for urokinase plasminogen activator
NK cells, wide variety of nonhematopoietic cell types
CD88 Polymorphonuclear leukocytes, macrophages, mast cells Receptor for complement component C5a
CD89 Neutrophils, monocytes IgA-dependent cytotoxicity
(Fc-a receptor)
CD90 CD34 + prothymocytes (human), thymocytes Unknown
CD91 Monocytes, many nonhematopoietic cells a2-macroglobulin receptor
CD92 Neutrophils, monocytes, platelets, endothelium Unknown
CD93 Neutrophils, monocytes, endothelium Unknown
CD94 T-cell subsets, NK cells Unknown
CD95 (Fas) Multiple cell types Role in programmed cell death (Bbinds TNF-like Fas ligand)
CD96 Activated T cells, NK cells Unknown
CD97 Activated B and T cells, monocytes, granulocytes Binds CD55
CD98 T cells, B cells, natural killer cells, granulocytes, all Unknown
human cell lines
CD99 Peripheral blood lymphocytes, thymocytes Unknown
CD100 Hematopoietic cells Unknown
CD101 Monocytes, granulocytes, dendritic cells, activated Unknown
T cells
CD102 (ICAM-2) Endothelial cells, monocytes Ligand for LFA-1 integrin (CD11a/CD18)
CD103 (HML-1) T cells Role in T-cell homing to mucosae
CD104 CD4 – CD8 – thymocytes, neuronal, epithelial, and some Integrin b4 associates with CD49f;, binds laminins
endothelial cells, Schwann cells, trophoblasts
CD105 Endothelial cells, activated monocytes and Binds TGF-b
macrophages, bone marrow cell subsets
CD106 (VCAM-1) Endothelial cells, macrophages Receptor for VLA-4 integrin; adhesion
CD107a,b Activated platelets, activated T cells, activated Unknown
neutrophils, activated endothelium
CD108 Erythrocytes, circulating lymphocytes, lymphoblasts Unknown
CD109 Activated T cells, activated platelets, vascular Unknown
endothelium
CD110 Platelets MPL, TPO R

Continued 51
 IMMUNOLOGY

TABLE 6.1. Clusters of Differentiation (CD) Designations—Cont’d

Clusters Cell Specificity Function

CD111 Myeloid cells PPR1/Nectin1

CD112 Myeloid cells PPR2
CD114 Granulocytes, monocytes Granulocytes colony-stimulating factor (G-CSF) receptor
SECTION 2

CD115 Monocytes, macrophages Macrophage colony-stimulating factor (M-CSF) receptor

CD116 Monocytes, neutrophils, eosinophils, endothelium Granulocyte-macrophage colony-stimulating factor
(GMCSF) receptor a chain
CD117 Hematopoietic progenitors Stem-cell factor (SCF) receptor
CD118 Many cell types Interferon-a, b receptor
CD119 Macrophages, monocytes, B cells, endothelium Interferon-g receptor
CD120a,b Hematopoietic and nonhematopoietic cells TNF receptor; binds both TNF-a and TNF-b
CD121a Thymocytes, T cells Type I interleukin-1 receptor; binds IL-1a and IL-b
CDw121b B cells, macrophages, monocytes Type II interleukin-1 receptor; binds IL-1a and IL-1b
CD122 NK cells, resting T-cell subsets, some B-cell lines IL-2 receptor b chain
CD123 Bone marrow stem cells, granulocytes, monocytes, IL-3 receptor a chain
megakaryocytes
CD124 Mature B and T cells, hematopoietic precursor cells IL-4 receptor
CD125 Eosinophils, basophils, activated B cells IL-5 receptor
CD126 Activated B cells and plasma cells (strong), most IL-6 receptor a subunit
leukocytes (weak)
CD127 Bone marrow lymphoid precursors, pro-B cells, mature IL-7 receptor
T cells, monocytes
CDw128 Neutrophils, basophils, T-cell subsets IL-8 receptor
CD129 Unknown
CD130 Most cell types, especially activated B cells and plasma Common subunit of IL-6, IL-11, oncostain-M (OSM)
cells and leukemia inhibitory factor (LIF) receptors
CDw131 Myeloid progenitors, granulocytes Common b subunit of IL-3, IL-5, and GM-CSF receptors
CD132 B cells, T cells, NK cells, mast cells, neutrophils IL-2 receptor g chain; common subunit of IL-2, IL-4, IL-7,
IL-9, and IL-15 receptors
CD133 Stem/progenitor cells AC133
CD134 Activated T cells May acts as adhesion molecule co-stimulator
CD135 Multipotential precursors, myelomonocytic and B-cell Growth factor receptor
progenitors
CDw136 Monocytes, epithelial cells, central and peripheral Chemotaxis, phagocytosis, cell growth, and differentiation
nervous system
CDw137 T and B lymphocytes, monocytes, some epithelial cells Co-stimulator of T-cell proliferation
CD138 B cells Heparan sulphate proteoglycan binds collagen type I
CD139 B cells Unknown
CD140a.b Stromal cells, some endothelial cells Platelet-derived growth factor (PDGF) receptor a and b
chains
CD141 Vascular endothelial cells Anticoagulant; binds thrombin, the complex then activates
protein C
CD142 Epidermal keratinocytes, various epithelial cells, Inducible by inflammatory mediators Binds Factor VIIa;
astrocytes, Schwann cells this complex activates Factors VII, IX, and X in blood
clotting
CD143 Endothelial cells, except large blood vessels and kidney, Soluble form in plasma Zn 2+ metallopeptidase dipeptidyl
epithelial cells of brush borders of kidney and small peptidase; cleaves angiotensin I and bradykinin from
intestine, neuronal cells, activated macrophages and precursor forms
some T cells.
CD144 Endothelial cells Organizes adherens junction in endothelial cells (cadherin)
CD145 Endothelial cells, some stromal cells Unknown
CD146 Endothelium Potential adhesion molecule, localized at cell-cell junctions
52
Continued
 A Cast of Thousands: The Cells of the Immune System

TABLE 6.1. Clusters of Differentiation (CD) Designations—Cont’d

Clusters Cell Specificity Function

CD147 Leukocytes, red blood cells, platelets, endothelial cells Potential adhesion molecule
CD148 Granulocytes, monocytes, dendritic cells, T cells, Contact inhibition of cell growth
fibroblasts, nerve cells
CD150 Thymocytes, activated lymphocytes Unknown

CHAPTER 6
CD151 Platelets, megakaryocytes, epithelial cells, endothelial Associates with b integrins
cells
CD152 (CTLA 4) Activated T cells Receptor for B7.1 (CD80), B7.2 (CD86); negative regulator
of T-cell activation
CD153 Activated T cells, activated macrophages, neutrophils, Ligand for CD30, may co-stimulate T cells
B cells
CD154 Activated CD4 T cells Ligand for CD40; inducer of B-cell proliferation and
activation
CD155 Monocytes, macrophages, thymocytes, CNS neurons Normal function unknown; receptor for polio virus
CD156a.b Neutrophils, monocytes Unknown
CD157 Granulocytes, monocytes, bone marrow stromal cells, ADP-ribosyl cyclase; cyclic
vascular endothelial cells, follicular dendritic cells ADP-ribose hydrolase
CD158a,b NK cells Inhibits NK cell cytotoxicity
CD159a NK cells Binds CD94 to form NK receptor; inhibits NK cell
cytotoxicity on binding MHC class I molecules
CD160 T cells Unknown
CD161 NK cells, T cells Regulates NK cytotoxicity
CD162 Neutrophils, lymphocytes, monocytes Ligand for CD62P
CD162R NK cells Unknown
CD163 Monocytes, macrophages Unknown
CD164 Epithelial cells, monocytes, bone marrow stromal cells Unknown
CD165 Thymocytes, thymic epithelial cells, CNS neurons, Adhesion between thymocytes and thymic epithelium
pancreatic islets, Bowman’s capsule
CD166 Activated T cells, thymic epithelium, fibroblasts, neurons Ligand for CD6; involved integrin neurite extension
CD167a Normal and transformed epithelial cells Binds collagen
CD168 Breast cancer cells Adhesion molecule.
CD169 Some macrophages Adhesion molecule.
CD170 Neutrophils Adhesion molecule
CD171 Neurons, Schwann cells, lymphoid and myelomonocytic Adhesion molecule, binds CD9, CD24, CD56
cells, B cells, CD4 T cells
CD172a Unknown Adhesion molecule; is a substrate of activated receptor
tyrosine kinases and binds to SH2 domains
CD173 All cells Blood group H type 2; carbohydrate moiety
CD174 All cells Lewis y blood group; carbohydrate moiety
CD175 All cells Tn blood group; carbohydrate moiety
CD175s All cells Sialyl-Tn blood group; carbohydrate moiety
CD176 All cells TF blood group; carbohydrate moiety
CD177 Myeloid cells Unknown
CD178 Activated T cells Fas ligand; binds to Fas to induce apoptosis
CD179a Early B cells Associates noncovalently with immunoglobulin l-like
polypeptide 1 to form a surrogate light chain that is
selectively expressed at the early stages of B-cell
development. Mutations in the CD179b gene have been
shown to result in impairment of B-cell development and
agammaglobulinemia in humans
CD179b Associates noncovalently with immunoglobulin iota chain to
form a surrogate light chain (a component of the pre-B-
cell receptor which plays a critical role in early B-cell
differentiation)
53
Continued
 IMMUNOLOGY

TABLE 6.1. Clusters of Differentiation (CD) Designations—Cont’d

Clusters Cell Specificity Function

CD180 B cells Membrane protein consisting of extracellular leucine-rich

repeats
CD183 Malignant B cells from chronic lymphoproliferative CXC chemokine receptor involved in chemotaxis of
disorders malignant B lymphocytes
SECTION 2

CD184 Immature CD34 + haematopoietic stem cells 1 Binding to SDF-1 (LESTR/fusin); acts as a co-factor for
fusion and entry of T-cell line; trophic strains of HIV-
CD195 Promyelocytic cells Receptor for a CC type chemokine; binds to MIP-1a,
MIP-1b and RANTES; may play a role in the control of
granulocytic lineage proliferation or differentiation; acts as
co-receptor with CD4 for HIV-1
CDw197 Activated B and T lymphocytes, strongly upregulated in Receptor for the MIP-3b chemokine; probable mediator
B cells infected with EBV and T cells infected with of EBV effects on B lymphocytes or of normal lymphocyte
HHV6 or 7 functions
CD200 Normal brain and B-cell lines Unknown
CD201 Endothelial cells Endothelial cell-surface receptor that binds with high-affinity
to protein C and activated protein C; downregulated by
exposure of endothelium to tumor necrosis factor
CD202b Endothelial cells Receptor tyrosine kinase, binds angiopoietin-1; important in
angiogenesis, particularly for vascular network formation
in endothelial cells; defects in TEK are associated with
inherited venous malformations; the TEK signaling
pathway appears to be critical for endothelial cell-smooth
muscle cell communication in venous morphogenesis
CD203c Myeloid cells Ectoenzymes that are involved in hydrolysis of extracellular
nucleotides. They catalyze the cleavage of phosphodiester
and phosphosulfate bonds of a variety of molecules,
including deoxynucleotides, NAD, and nucleotide sugars
CD204 Myeloid cells Mediate the binding, internalization, and processing of a
wide range of negatively charged macromolecules;.
Iimplicated in the pathologic deposition of cholesterol in
arterial walls during atherogenesis
CD205 Dendritic cells Lymphocyte antigen 75; putative antigen-uptake receptor on
dendritic cells
CD206 Macrophages, endothelial cells Type I membrane glycoprotein; only known example of a
C-type lectin that contains multiple C-type CRDs
(carbohydrate-recognition domains); it binds high-
mannose structures on the surface of potentially
pathogenic viruses, bacteria, and fungi
CD207 Langerhans’ cells Type II transmembrane protein; Langerhans’ cell specific
C-type lectin; potent inducer of membrane superimposition
and zippering leading to BG (Birbeck granules) formation
CD208 Interdigitating dendritic cells in lymphoid organs Homologous to CD68, DC-LAMP is a lysosomal protein
involved in remodeling of specialized antigen-processing
compartments and in MHC class II-restricted antigen
presentation; upregulated in mature DCs induced by
CD40L, TNF-a and LPS.
CD209 Dendritic cells C-type lectin; binds ICAM3 and HIV-1 envelope glycoprotein
gp120 enables T-cell receptor engagement by
stabilization of the DC/T-cell contact zone, promotes
efficient infection in trans cells that express CD4 and
chemokine receptors; type II transmembrane protein
CDw210 B cells, T-helper cells Interleukin 10 receptor a and b
CD212 Activated CD4, CD8, and NK cells IL-12 receptor b chain; a type I transmembrane protein
involved in IL-12 signal transduction.
CD213a1 B cells, monocytes, fibroblasts, endothelial cells Receptor which binds IL-13 (low affinity); together with IL
4Ra can form a functional receptor for IL-13, also serves
as an alternate accessory protein to the common
cytokine receptor gamma chain for IL-4 signaling
CD213a2 B cells, monocytes, fibroblasts, endothelial cells IL-13 receptor which binds as a monomer to interleukin-13
(high affinity), but not to IL-4; human cells expressing
IL-13RA2 show specific IL-13 binding with high affinity
54
Continued
 A Cast of Thousands: The Cells of the Immune System

TABLE 6.1. Clusters of Differentiation (CD) Designations—Cont’d

Clusters Cell Specificity Function

CDw217 Activated memory T cells Interleukin 17 receptor homodimer

CD220 Nonlineage molecules Insulin receptor; integral transmembrane glycoprotein
comprised of two a and two b subunits; this receptor
binds insulin and has a tyrosine- protein kinase activity

CHAPTER 6
– autophosphorylation activates the kinase activity
CD221 Nonlineage molecules Insulin-like growth factor I receptor binds insulin-like growth
factor with a high affinity. It has tyrosine kinase activity
and plays a critical role in transformation events.
Cleavage of the precursor generates a and b subunits
CD222 Nonlineage molecules Transmembrane protein. Its main functions include
internalization of IGF-II, internalization or sorting of
lysosomal enzymes, and other M6P-containing proteins
CD223 Activated T and NK cells Involved in lymphocyte activation; binds to HLA class-II
antigens; role in downregulating antigen-specific response
CD224 Nonlineage molecules Predominantly a membrane-bound enzyme; plays a key role
in the g-glutamyl cycle, a pathway for the synthesis and
degradation of glutathione. This enzyme consists of two
polypeptide chains, which are synthesized in precursor
form from a single polypeptide
CD225 Leukocytes and endothelial cells Interferon-induced transmembrane protein 1 is implicated
in the control of cell growth.
CD226 NK cells, platelets, monocytes, and a subset of T cells Adhesion glycoprotein; mediates cellular adhesion to other
cells bearing an unidentified ligand and cross-linking
CD226 with antibodies causes cellular activation
CD227 Human epithelial tumors, such as breast cancer Epithelial mucin containing a variable number of repeats
with a length of twenty amino acids, resulting in many
different alleles. Direct or indirect interaction with actin
cytoskeleton
CD228 Predominantly in human melanomas Tumor-associated antigen (melanoma) identified by
monoclonal antibodies 133.2 and 96.5; involved in cellular
iron uptake.
CD229 Lymphocytes May participate in adhesion reactions between T
lymphocytes and accessory cells by homophilic interaction
CD230 Expressed both in normal and infected cells Unknown
CD231 T-cell acute lymphoblastic leukemia, neuroblastoma Unknown
cells, and normal brain neuron
CD232 Nonlineage molecules Receptor for an immunologically active semaphorin (virus
encoded semaphorin protein receptor)
CD233 Erythroid cells Band 3 is the major integral glycoprotein of the erythrocyte
membrane. It has two functional domains. Its integral
domain mediates a 1:1 exchange of inorganic anions
across the membrane, whereas its cytoplasmic domain
provides binding sites for cytoskeletal proteins, glycolytic
enzymes, and hemoglobin. Multifunctional transport protein
CD234 Erythroid cells and nonerythroid cells Fy-glycoprotein; Duffy blood group antigen; nonspecific
receptor for many chemokines such as IL-8, GRO,
RANTES, MCP-1, and TARC. It is also the receptor for the
human malaria parasites Plasmodium vivax and
Plasmodium knowlesi
CD235a Erythroid cells Major carbohydrate-rich sialoglycoprotein of human
erythrocyte membrane which bears the antigenic
determinants for the MN and Ss blood groups. Also binds
influenza virus
CD235b Erythroid cells This protein is a minor sialoglycoprotein in human
erythrocyte membranes. Along with GYPA, GYPB is
responsible for the MNS blood group system.
CD236 Erythroid cells Glycophorin C (GPC) and glycophorin D (GPD) are closely
related sialoglycoproteins in the human red blood cell
membrane. GPD is a ubiquitous shortened isoform of
GPC, produced by alternative splicing of the same gene.
The Webb and Duch antigens, also known as glycophorin D,
result from single point mutations of the glycophorin C gene
55
Continued
 IMMUNOLOGY

TABLE 6.1. Clusters of Differentiation (CD) Designations—Cont’d

Clusters Cell Specificity Function

CD236R Erythroid cells Glycophorin C (GPC) is associated with the Gerbich (Ge)
blood group deficiency. It plays an important role in
regulating the mechanical stability of red cells and is a
putative receptor for the merozoites of Plasmodium
falciparum
SECTION 2

CD238 Erythroid cells KELL blood group antigen; homology to a family of zinc
metalloglycoproteins with neutral endopeptidase activity,
type II transmembrane glycoprotein
CD239 Erythroid cells A type I membrane protein.The human F8/G253 antigen,
B-CAM, is a cell-surface glycoprotein that is expressed
with restricted distribution pattern in normal fetal and
adult tissues, and is upregulated following malignant
transformation in some cell types.
CD240CE Erythroid cells Rhesus blood group, CcEe antigens.
CD240D Erythroid cells Rhesus blood group, D antigen. May be part of an
oligomeric complex which is likely to have a transport or
channel function in the erythrocyte membrane.
CD241 Erythroid cells Rhesus blood group-associated glycoprotein RH50,
component of the RH antigen multisubunit complex;
required for transport and assembly of the Rh membrane
complex to the red blood cell surface. Defects in RhAg are
a cause of a form of chronic hemolytic anemia associated
with stomatocytosis, and spherocytosis, reduced osmotic
fragility, and increased cation permeability
CD242 Erythroid cells Intercellular adhesion molecule 4, Landsteiner–Wiener
blood group. LW molecules may contribute to the
vasoocclusive events associated with episodes of acute
pain in sickle cell disease
CD243 Stem/progenitor cells Multidrug resistance protein 1 (P-glycoprotein). P-gp has
been shown to utilizese ATP to pump hydrophobic drugs
out of cells, thus increasing their intracellular concentration
and hence their toxicity.
CD244 NK cells 2B4 is a cell-surface glycoprotein related to CD2 and
implicated in the regulation of natural killer and T
lymphocyte function.
CD245 T cells Cyclin E/Cdk2 interacting protein p220. NPAT is involved in
a key S phase event and links cyclical cyclin E/Cdk2 kinase
activity to replication-dependent histone gene transcription
CD246 Expressed in the small intestine, testis, and brain but Anaplastic (CD30+ large cell) lymphoma kinase; plays an
not in normal lymphoid cells important role in brain development; involved in anaplastic
nodal non-Hodgkin’s lymphoma or Hodgkin’s disease
with translocation t(2;5)(p23;q35) or inv2(23;q35).
CD247 T cells, NK cells T-cell receptor z; has a probable role in assembly and
expression of the TCR complex as well as signal
transduction upon antigen triggering. TCR z together with
TCRa:b and g:b heterodimers and CD3-g, -d, and -e, forms
the TCR-CD3 complex. The z chain plays an important
role in coupling antigen recognition to several intracellular
signal-transduction pathways. Low expression of
the antigen results in impaired immune response
(Adapted in part from Janeway CA, Travers P, Walport M, Shlomchik M: Immunobiology 6: the immune system in health and disease, 6th Edition, New York, Garland
Science 2004.)
ELAM, endothelial leukocyte adhesion molecule; LAM, leukocyte adhesion molecule; MAC, macrophage; HIV, human immunodeficiency virus; ICAM, intercellular
adhesion molecule; IL, interleukin; LPS, lipopolysaccharide; NCAM, neutrophil cellular adhesion molecule; NK, natural killer; MHC, major histocompatibility complex; LFA,
a2‚b2-integrins; VCAM, vascular cellular adhesion molecule; VLA, a2‚b1-integrins.

mitogen, as well as for the purified protein derivative of Myco-
bacterium tuberculosis and lipopolysaccharide.
Null cells are lymphocytes that possess none of the afore-
mentioned cell-surface antigens characteristic of T cells or B
cells. This cell population is heterogeneous, and some author-
ities include natural killer (NK) cells among the null cell
population even though the origin of NK cells appears to be
56 in monocyte/macrophage precursor lines rather than the
lymphocyte lineage. Nonetheless, the morphologic charac-
teristics and behaviors of NK cells, along with the ambiguity of
their origin, allow one license to include them under the null
cell rubric. NK cells are nonadherent (unlike macrophages, they
do not stick to the surface of plastic tissue culture dishes)
mononuclear cells present in peripheral blood, spleen, and
lymph node. The most notable function of these cells is killing
of transformed (malignant) cells and virus-infected cells.
 A Cast of Thousands: The Cells of the Immune System
Because they do this without prior sensitization, they are an
important component of the early natural response in the
immune system. The cytotoxicity of NK cells is not major
histocompatibility complex (MHC)-restricted, a dramatic
contrast with cytotoxic T cells. (More about the MHC and the
products of those gene loci later.) The large granules present in
NK cells (the cells are sometimes called large granular lym-
phocytes) contain perforin and perhaps other cell membrane-
lysing enzymes, and it is the enzymes in these granules that are
responsible for the lethal-hit cytolysis for which NK cells are
famous.
Killer cells or LAK cells (lymphocyte-activated killer cells) are
the other notable null cell subpopulation. These cells do have
receptors for the Fc portion of immunoglobulin G (IgG) and
thus can attach themselves to the Fc portion of IgG molecules.
Through this receptor, they are a primary cell responsible for
the cytolysis in the so-called antibody-dependent, cell-mediated
cytotoxicity reaction. These cells probably participate in type II
Gell and Coombs hypersensitivity reactions and are involved in
immune removal of cellular antigens when the target cell is too
large to be phagocytosed.
It is clear that both B cells and T cells can be further divided
into specialized subsets. B cells, for example, are subdivided
into the B cells that synthesize the five separate classes of im-
munoglobulin (IgG, IgA, IgM, IgD, and IgE). All B cells initially
produce IgM specific for an antigenic determinant (epitope) to
which it has responded, but some subsequently switch from
synthesis of IgM to synthesis of other immunoglobulin classes.
The details of the control of antibody synthesis and class-
switching are covered in Chapter 8. Less known is the fact that
functionally distinct subsets of B cells exist, in addition to the
different B cells in terms of antibody class synthesis. The field
of B-cell diversity analysis is embryonic, but it is clear that the
exploitation of monoclonal antibody technology will dis-
tinguish, with increasingly fine specificity, differences in B-cell
subpopulations. It is clear, for example, that a subpopulation of
B lymphocytes possesses the CD5 glycoprotein on the cell
surface plasma membrane (a CD glycoprotein not ordinarily
present on B lymphocytes but rather on the cell surface of T
cells).3 These cells appear to be associated with autoantibody
production.4
It is also clear now that B cells are functionally important as
antigen-presenting cells (APCs), a fact that startles most
physicians who studied immunology before 1991. T-cell
receptors (TCRs) cannot react with native antigen; rather they
respond to processed antigenic determinants of that antigen.
APCs phagocytose the antigen, process it, and display
denatured, limited peptide sequences of the native antigen on
the cell surface of the APC in association with cell surface class
II MHC glycoproteins. B cells, as well as classic APCs, such as
macrophages and Langerhans’ cells, can perform this function.
The antigen is endocytosed by the B cell and processed in the B-
cell endosome (possibly through involvement of cathepsin D) to
generate short, denatured peptide fragments, which are then
transported to the B-cell surface bound to class II glycoprotein
peptides, where the antigenic peptides are ‘presented’ to CD4
helper T lymphocytes, along with the delivery of a co-
stimulatory signal via its B7–1 and –2 molecules’ (CD80 and
CD 86) interaction with T-cell stimulatory molecules, CD 28
and CTLA 4.
Finally, regarding B-cell heterogeneity, it is becoming
apparent that some B lymphocytes also have suppressor or
regulatory activity. The emerging data on B-cell functional and
cell surface heterogeneity will be exciting to follow in the
coming years.
Much more widely recognized, of course, is that subsets of T
lymphocytes exist. Helper (CD4) T cells ‘help’ in the induction
of an immune response, in the generation of an antibody
response, and in the generation of other, more specialized
components of the immune response. Cytotoxic (CD8) T cells,
as the name implies, are involved in cell killing or cytotoxic
reactions. Delayed-type hypersensitivity (CD4) T cells are
the classic participants in the chronic inflammatory responses
characteristic of certain antigens such as mycobacteria. Re-
gulatory T cells, Treg, are responsible for modulating immune
CHAPTER 6
responses, preventing uncontrolled, host-damaging inflam-
matory responses. There are at least 2 subsets of Treg, cells:
CD4+ CD25+ and CD8+ CD25+ cells. It is even likely that
there are sub-subsets of these T cells. Excellent evidence exists,
for example, that there are at least three subsets of regulatory T
cells and at least two subsets of helper T cells.
Mosmann and Coffman5 described two types of helper (CD4)
T cells with differential cytokine production profiles. TH1 cells
secrete interleukin-2 (IL-2) and interferon-g (IFN-g) but do not
secrete IL-4 or IL-5, whereas TH2 cells secrete IL-4, IL-5, IL-10,
and IL-13, but not IL-2 or IFN-g. Furthermore, TH1 cells can be
cytolytic and can assist B cells with IgG, IgM, and IgA synthesis
but not IgE synthesis. TH2 cells are not cytolytic but can help B
cells with IgE synthesis as well as with IgG, IgM, and IgA
production.6 It is becoming clear that CD4 TH1 or CD4 TH2
cells are selected in infection and autoimmune diseases. Thus,
TH1 cells accumulate in the thyroid of patients with auto-
immune thyroiditis,7 whereas TH2 cells accumulate in the
conjunctiva of patients with vernal conjunctivitis.8 The T cells
that respond to M. tuberculosis protein are primarily TH1 cells,
whereas those that respond to Toxocara canis antigens are TH2
cells. Romagnani has proposed that TH1 cells are preferentially
‘selected’ as participants in inflammatory reactions associated
with delayed-type hypersensitivity reactions and low antibody
production (as in contact dermatitis or tuberculosis), and TH2
cells are preferentially selected in inflammatory reactions
associated with persistent antibody production, including
allergic responses in which IgE production is prominent.9
Further, it is now clear that these two major CD4 T-lymphocyte
subsets regulate each other through their cytokines. Thus, TH2
CD4 lymphocyte cytokines (notably IL-10) inhibit TH1 CD4
lymphocyte proliferation and cytokine secretion, and TH1 CD4
lymphocyte cytokines (notably IFN-g) inhibit TH2 CD4 lympho-
cyte proliferation and cytokine production.
MACROPHAGES
The macrophage ( ‘large eater’) is the preeminent professional
APC. These cells are 12–15 mm in diameter, the largest of the
lymphoid cells. They possess a high density of class II MHC
glycoproteins on their cell surface, along with receptors for
complement components, the Fc portion of Ig molecules, re-
ceptors for fibronectin, interferons-a, -b, and -g, IL-1, tumor
necrosis factor, and macrophage colony-stimulating factor.
These cells are widely distributed throughout various tissues
(when found in tissue, they are called histiocytes), and the
microenvironment of the tissue profoundly influences the
extent of expression of the various cell surface glycoproteins as
well as the intracellular metabolic characteristics. It is clear that
further compartmentalization of macrophage subtypes occurs
in the spleen. Macrophages that express a high density of class
II MHC glycoproteins are present in red pulp, and macrophages
with significantly less surface expression are in the marginal
zone, where intimate contact with B cells exists. It is likely that
just as in the murine system,10 in humans one subclass
of macrophage preferentially presents antigen to one particular
subset of helper T cell responsible for induction of regulatory
T-cell activation, whereas a different subset of macrophage
preferentially presents antigen to a different helper T-cell subset 57
 IMMUNOLOGY

responsible for cytotoxic or delayed-type hypersensitivity

TABLE 6.2. Neutrophil Granules and Their Contents
effector functions.
Macrophages also participate more generally in inflammatory Azurophil Granules Specific Granules Other Granules
reactions. They are members of the natural (early defense)
immune system and are incredibly potent in their capacity to Myeloperoxidase Alkaline Acid
phosphatase phosphatase
synthesize and secrete a variety of powerful biologic molecules,
including proteases, collagenase, angiotensin-converting Acid phosphatase Histaminase Heparinase
enzyme, lysozyme, IFN-a, IFN-b, IL-6, tumor necrosis factor-a, 5„-Nucleotidase Collagenase b-Glucosaminidase
SECTION 2

fibronectin, transforming growth factor-b, platelet-derived

growth factor, macrophage colony-stimulating factor, Lysozyme Lysozyme a-Mannosidase
granulocyte-stimulating factor, granulocyte-macrophage colony- Elastase Vitamin B12-binding Acid proteinase
stimulating factor, platelet-activating factor, arachidonic acid proteins
derivatives (prostaglandins and leukotrienes), and oxygen Cathepsins B, D, G Plasminogen
metabolites (oxygen free radicals, peroxide anion, and hydrogen activator
peroxide). These cells are extremely important, even pivotal,
participants in inflammatory reactions and are especially Lactoferrin Elastase gelatinase
important in chronic inflammation. The epithelioid cell typical Proteinase 3 Glycosamino-
of so-called granulomatous inflammatory reactions evolves glycans
from the tissue histiocyte, and multinucleated giant cells form b-Glycerophosphatase
through fusion of many epithelioid cells.
Specialized macrophages exist in certain tissues and organs, b-Glucuronidase
including the Kupffer cell of the liver, dendritic histiocytes in N-acetyl- Cytochrome
lymphoid organs, interdigitating reticulum cells in lymphoid b-glucosaminidase
organs, and Langerhans’ cells in skin, lymph nodes, conjunc- a-Mannosidase
tiva, and cornea.
Langerhans’ cells are particularly important to the ophthal- Arylsulfatase ·
mologist. They probably are the premier APC for the external a-Fucosidase
eye. Derived from bone marrow macrophage precursors, like
Esterase
macrophages, their function is basically identical to that of the
macrophage in antigen presentation. They are rich in cell- Histonase
surface class II MHC glycoproteins and have cell-surface Cationic proteins
receptors for the third component of complement and the Fc
portion of IgG. Langerhans’ cells are abundant in the mucosal Defensins
epithelium of the mouth, esophagus, vagina, and conjunctiva. Bactericidal
They are also abundant at the corneoscleral limbus, less so in permeability-
the peripheral cornea; they are normally absent from the central increasing
third of the cornea.11 If the center of the cornea is provoked protein (BPI)
through trauma or infection, the peripheral cornea Langerhans’ Glycosaminoglycans
cells quickly ‘stream’ into the center of the cornea.12 These
CD1-positive dendritic cells possess a characteristic racket-
shaped granule on ultrastructural analysis, the Birbeck granule. fibrinolytic and kinin system components, and products from
Birbeck granules are subdomains of the endosomal recycling other leukocytes, platelets, and certain bacteria), neutrophils
compartment that are rich in Langerin (CD 207), a protein move from blood to tissues through margination (adhesion
specific to Langerhans’ cells, and a type II membrane-associated to receptors or adhesion molecules on vascular endothelial cells)
C-type lectin which recognizes mannose residues and may and diapedesis (movement through the capillary wall).
serve with CD 1 to present lipid antigens by Langerhans’ cells Neutrophils release the contents of their primary (azurophilic)
after endocytosis and processing.13,14 granules (lysosomes) and secondary (specific) granules (Table
6.2) into an endocytic vacuole, resulting in:1 phagocytosis of a
POLYMORPHONUCLEAR LEUKOCYTES microorganism or tissue injury;2 type II antibody-dependent,
cell-mediated cytotoxicity; or3 type III hypersensitivity reactions
Polymorphonuclear leukocytes (PMNs) are part of the natural (immune complex-mediated disease). Secondary granules
immune system. They are central to host defense through release collagenase, which mediates collagen degradation. Aside
phagocytosis, but if they accumulate in excessive numbers, from the products secreted by the granules, neutrophils produce
persist, and are activated in an uncontrolled manner, the result arachidonic acid metabolites (prostaglandins and leukotrienes)
may be deleterious to host tissues. As the name suggests, they as well as oxygen free radical derivatives.
contain a multilobed nucleus and many granules. PMNs are
subcategorized as neutrophils, basophils, or eosinophils, depen-
ding on the differential staining of their granules. EOSINOPHILS
Eosinophils constitute 3–5% of the circulating PMNs. They
possess surface receptors for the Fc portion of IgE (low affinity)
NEUTROPHILS and IgG (CD16) and for complement components, including
Neutrophils account for more than 90% of the circulating C5a, CR1 (CD35), and CR3 (CD11b). Eosinophils play a
granulocytes. They possess surface receptors for the Fc portion special role in allergic conditions and parasitoses. They also par-
of IgG (CD16) and for complement components, including C5a ticipate in type III hypersensitivity reactions or immune
(important in chemotaxis), CR1 (CD35), and CR3 (CD11b) complex-mediated disease following attraction to the inflam-
(important in adhesion and phagocytosis). When appropriately matory area by products from mast cells (eosinophil chemotactic
58 stimulated by chemotactic agents (complement components, factor of anaphylaxis), complement, and other cytokines from
 A Cast of Thousands: The Cells of the Immune System

TABLE 6.3. Granular Contents of Eosinophils TABLE 6.4. Mast Cell Types and Characteristics

Lysosomal hydrolases Characteristic Mucosal Mast Cell Connective Tissue

Mast (MC-T, MMC) Mast Cell (MC-TC,
Arylsulfatase CTMC)
b-Glucuronidase
Morphology
Acid phosphatase
Size Small, pleomorphic Large, uniform

CHAPTER 6
b-Glycerophosphatase
Nucleus Unilobed or bi-lobed Unilobed
Ribonuclease
Granules Few Many
Proteinases
Location Gut Peritoneum, skin
Collagenase Histochemistry
Cathepsin Protease Tryptase Tryptase and
chymase
Histaminase
Proteoglycans Chondroitin sulfate Heparin
Peroxisomes
Histamine <1 pg/cell 15 pg/cell
Major basic proteins
IgE Surface and Surface
Eosinophil cationic protein cytoplasmic
Eosinophil peroxidases Formalin sensitive Yes No
Phospholipases In Vitro Effect of:
Lysophospholipases Compound 48/80 Proliferation Degranulation
Polymyxin Proliferation Degranulation
Life Span ≤40 days >40 days
other inflammatory cells. Eosinophils release the contents of
Proliferation Thymus-dependent Thymus-independent
their granules to the outside of the cell after fusion of the
intracellular granules with the plasma membrane (degranulation). Secretagogues
Table 6.3 shows the known secretory products of eosinophils; Antigen Yes Yes
the role these products of inflammation play, even in nonallergic
diseases (such as Wegener’s granulomatosis), is underappreciated. Anti-IgE Yes Yes
Compound 48/80 No Yes
BASOPHILS Bee venom No Yes
Basophils account for less than 0.2% of the circulating Con A Yes Yes
granulocytes. They possess surface receptors for the Fc portion Staining
of IgE (high affinity) and IgG (CD16) and for complement
components, including C5a, CR1 (CD35), and CR3 (CD11b). Alcian blue Yes Yes
Their role, other than perhaps as tissue mast cells, is unclear. Safranin No Yes
Berberine sulfate No Yes
MAST CELLS
Antiallergic
The mast cell is indistinguishable from the basophil in many Compounds No Yes
respects, particularly its contents. There are at least two classes
of mast cells based on their neutral protease composition, Cromoglycate No Yes
T-lymphocyte dependence, ultrastructural characteristics, and Theophylline Yes Yes
predominant arachidonic acid metabolites (Table 6.4). Mucosa-
Doxantrile
associated mast cells (MMC or MC-T) contain primarily
tryptase as the major protease (hence, some authors designate Enhancement of No Yes
these MC-T, or mast cells-tryptase) and prostaglandin D2 as Secretion
the primary product of arachidonic acid metabolism. MMCs are Phosphatidyl serine Yes Yes
T-cell-dependent for growth and development (specifically IL-3-
Adenosine
dependent), and are located predominantly in mucosal stroma
(e.g., gut). MMCs are small and short-lived (< 40 days). They Predominant Prostaglandin D2 Leukotrienes B4,
contain chondroitin sulfate but not heparin, and their Arachidonic Acid C4, D4
histamine content is modest (Table 6.5). MMCs degranulate in Metabolite
response to antigen-IgE triggering but not to exposure to Ultrastructural Lattice Scroll
compound 48/80, and are not stabilized by disodium cro- Features of Granules
moglycate. They are formalin-sensitive, so formalin-fixation of
tissue eliminates or greatly reduces our ability to find these cells
by staining technique. With special fixation techniques, MMC arachidonic acid metabolism. CTMCs are T-cell-independent.
granules stain with alcian blue but not with safranin. They are larger than MMCs and are located principally in skin
Connective tissue mast cells (CTMCs) contain both tryptase and at mucosal interfaces with the environment. They contain
and chymase (so some authors designate them MC-TC), as well heparin and large amounts of histamine, and degranulate in
as leukotrienes B4, C4, and D4, as the primary products of response to compound 48/80 in addition to antigen-IgE 59
 IMMUNOLOGY

interactions. CTMCs are stabilized by disodium cromoglycate.

TABLE 6.5. Mast Cell Contents
They stain with alkaline Giemsa, toluidine blue, alcian blue,
safranin, and berberine sulfate. Histamine
The ultrastructural characteristics of MMCs and CTMCs are
Serotonin
also different. Electron microscopy shows that the granules of
MMCs contain lattice-like structures; the granules of CTMCs Rat mast-cell protease I and II
contain scroll-like structures. Mast cells play a special role in Heparin
allergic reactions – they are the preeminent cell in the allergy
SECTION 2

drama. They also can participate in type II, III, and IV hyper- Chondroitin sulfate
sensitivity reactions, however. Their role in these reactions, b-Hexosaminidase
aside from notable vascular effects, is not well understood. Non-
b-Glucuronidase
IgE-mediated mechanisms (e.g., C5a) can trigger mast cells to
release histamine, platelet-activating factor, and other biologic b-4DGalactosidase
molecules when antigen binds to two adjacent IgE molecules Arylsulfatase
on the mast cell surface. Histamine and other vasoactive
amines cause increased vascular permeability, allowing Eosinophil chemotactic factor for anaphylaxis (ECF-A)
immune complexes to become trapped in the vessel wall. Slow reactive substance of anaphylaxis (SRS-A)
High molecular weight neutrophil chemotactic factor
PLATELETS
Arachidonic acid derivatives
Blood platelets, cells well adapted for blood clotting, also are Platelet-activating factor
involved in the immune response to injury, a reflection of their
evolutionary heritage as myeloid (inflammatory) cells. They
possess surface receptors for the Fc portion of IgG (CD16) and
IgE (low affinity), for class I histocompatibility glycoproteins
(human leukocyte antigen-A, -B, or -C), and for factor VIII. TABLE 6.6. Platelet Granules and Their Contents
They also carry molecules such as GpII b/ III a (CDw41), which a-Granules
bind fibrinogen, and Gp1b (CDw42), which binds von
Fibronectin
Willebrand’s factor.
After endothelial injury, platelets adhere to and aggregate at Fibrinogen
the endothelial surface, releasing permeability-increasing Plasminogen
molecules from their granules (Table 6.6). Endothelial injury
may be caused by type III hypersensitivity. Platelet-activating Thrombospondin
factor released by mast cells after antigen-IgE antibody complex von Willebrand factor
formation induces platelets to aggregate and release their
a2-Plasmin inhibitor
vasoactive amines. These amines separate endothelial cell tight
junctions and allow the immune complexes to enter the vessel Platelet-derived growth factor (PDGF)
wall. Once the immune complexes are deposited, they initiate Platelet factor 4 (PF4)
an inflammatory reaction through activation of complement
components and neutrophil lysosomal enzyme release. Transforming growth factor (TGF) a and b
Thrombospondin
ONTOGENY OF THE IMMUNE SYSTEM b-Lysin
Cells of the hematologic system are derived from primordial Permeability factor
stem cell precursors of the bone marrow. Embryonically, these Factors D and H
cells originate in the blood islands of the yolk sac.13 These cells
populate embryonic liver and bone marrow.14 All the blood Decay-accelerating factor
elements are derived from these primordial stem cells: Dense granules
erythrocytes, platelets, PMNs, monocytes, and lymphocytes.
Serotonin
These primordial stem cells are pluripotential, and the exact
details of the influences that are responsible for a particular Adenosine diphosphate (ADP)
pluripotential primordial stem cell’s evolving along one Others
differentiation pathway (e.g., into a monocyte) as opposed to
some other differentiation pathway (e.g., into a lymphocyte) are Arachidonic acid derivatives
incompletely understood. It appears, however, that special
characteristics of the microenvironment in the bone marrow,
particularly with respect to the association with other resident so-called bursal equivalent that is responsible for B-cell
cells in the bone marrow, contribute to or are responsible for the differentiation in humans were proposed for many years before
different pathways of maturation and differentiation. For the role of the bone marrow itself for this function became
example, specific cells in the bone marrow in the endosteal evident. Extra-bone marrow tissues that had been proposed as
region promote the differentiation of hematopoietic stem cells bursal equivalent candidates included the appendix, tonsils,
into B lymphocytes.15–21 In birds, primordial pluripotential stem liver, and Peyer’s patch.
cells that migrate to a gland near the cloaca of the chicken T-cell development results from pluripotential hematopoietic
known as the bursa of Fabricius (for reasons of probable stimuli stem cell migration (stimulus unknown) from the bone marrow
in the bone marrow as yet not understood) are influenced by the to the thymus. Thymic hormones (at least 20 have been
epithelial cells in that gland to terminally differentiate into preliminarily described) produced by the thymic epithelium
60 B lymphocytes.22,23 Interestingly, various candidates for the initiate the complex series of events that result not only in
 A Cast of Thousands: The Cells of the Immune System
TABLE 6.7. Thymic Hormones
Hormone No. of Amino Acids
Thymosin 28
Thymopoietin 49
Thymic humoral factor 31
Facteur thymique serique 9 The thymus consists of a medulla, containing thymic epi-
differentiation of the hematopoietic stem cells into T
lymphocytes but also in subdifferentiation of T lymphocytes
into their various functional subsets; helper function, killer
function, and suppressor function are acquired while the T cells
are still in the thymus. Table 6.7 lists the four thymic hormones
most rigorously studied to date. Note that all are involved in T-
cell differentiation and in the development of helper T-cell
function and that three of the four can be involved or are
involved in the acquisition of suppressor T-cell activity. Clearly,
the story is considerably more complex than the part we
currently understand, and additional factors are undoubtedly
responsible for the final differentiation of T lymphocytes into
their functionally distinct subsets.
These various hormones are also undoubtedly responsible for
the induction of cell surface glycoprotein expression on the
surface of T cells. The cell-surface expression of the various
glycoproteins changes during T-cell maturation in the thymus.
For example, the CD2 glycoprotein is the first that can be
identified on the differentiating T cell, but this is eventually
joined by CD5; these are both eventually replaced (CD2
completely and CD5 partially) by CD1 glycoprotein, which in
turn is lost and replaced by the mature CD3 marker. CD4 and
CD8 glycoproteins are acquired prior to emigration from the
thymus of helper and cytotoxic-regulatory T cells, respectively.
Monocytes, NK cells, and killer cells evolve from pluri-
potential hematopoietic stem cells through influences that are
incompletely understood. All three types of cells do arise from a
common monocyte precursor and later subdifferentiate under
unknown influences.
a b
c
PRIMARY (CENTRAL) LYMPHOID ORGANS
The primary or central lymphoid organs are the bone marrow,
thymus, and liver. The peripheral lymphoid organs include
lymph nodes, spleen, gut-associated lymphoid tissue, bronchus-
associated lymphoid tissue, and conjunctiva-associated
lymphoid tissue. The anatomic characteristics of the thymus,
lymph node, and spleen are described briefly.
CHAPTER 6
thelial tissue and lymphocytes, and a surrounding cortex densely
packed with small, proliferating T lymphocytes (Fig. 6.1).
The cells in the cortex emigrate from the thymus: The cell
population turns over completely every 3 days. Only ~1% of the
cells produced in the thymus, however, actually emigrate from
it; 99% are destroyed locally, probably in a process designed to
prevent autoreactive T lymphocytes from gaining access to the
extrathymic regions of the organism. Thymic nurse cells, epi-
thelial cells in the cortical region, may be responsible in part for
some of the later events in T-lymphocyte differentiation (e.g.,
into helper and regulatory T cells).
Lymph nodes (Fig. 6.2) are also composed of medulla and
cortex. The medulla, rich in the arterial and venous compo-
nents of the lymph node, contains reticular cells that drain into
the efferent lymphatic vessels. The cortex contains the primary
lymphoid follicles, containing mature, resting B cells, secondary
lymphoid follicles with their germinal centers (full of antigen-
stimulated B cells and dendritic cells) and mantle, and lym-
phocytes. The paracortical region close to the medulla is rich in
T cells, particularly CD4+ T cells.
The arrangement of the spleen is similar to that of the
thymus and lymph node, though lymph node-type follicles are
not so clearly distinguished (Fig. 6.3). The lymphoid follicles
and surrounding lymphocytes are called the white pulp of the
spleen. The red pulp of the spleen is composed of the sinusoidal
channels that typically contain a relatively large number of red
blood cells. Popiernik has described the white pulp as being or-
ganized as a lumpy cylindrical sheath surrounding central
arterioles. The arterioles curve back on the white pulp to
develop it as the marginal sinus, which separates the white pulp
from the red.24 B cells predominate in the marginal zone, but
FIGURE 6.1. (a) and (b) Human thymus. Note
the organization into individual lobules
separated by connective tissue trabeculae, with
dense collections of tightly packed, deeply
stained immature thymocytes in the cortex
and more mature lymphocytes in the medulla.
(c) Hassall’s corpuscles, probably composed
of degenerated epithelial cells, are found
scattered throughout the medulla.
61
 IMMUNOLOGY
SECTION 2
a b
a b
CD4+ T cells are present as well. T cells are clustered tightly
around the central arteriole, where ~70% of the T cells are
CD4+. B cells also predominate in the lumpy eccentric follicle
of white pulp. Table 6.8 outlines some of the characteristics of
these three lymphoid organs and their organization. The spleen
is the primary site of immune responses to intravenous and
anterior chamber-introduced antigens.
LYMPHOID TRAFFIC
Lymphatic vessels and blood vessels connect these lymphatic
organs to each other and the other organs of the body. Lym-
phatic vessels drain every organ except the nonconjunctival
parts of the eye, internal ear, bone marrow, spleen, cartilage, and
some parts of the central nervous system. The interstitial fluid
and cells entering this system are propelled (predominantly by
skeletal muscle contraction) to regional lymph nodes. Efferent
lymphatics draining these regional nodes converge to form large
lymph vessels that culminate in the thoracic duct and in the
right lymphatic duct. The thoracic duct empties into the left
subclavian vein, carrying approximately three-quarters of the
lymph, whereas the right lymphatic duct empties into the right
subclavian vein.
The subject of lymphocyte traffic, like so many areas of
immunology, has undergone intensive reexamination since the
1980s; since then, discoveries relating to homing receptors,
addressins, and other adhesion molecules have revolutionized
TABLE 6.8. Lymphoid Organs
Primary Secondary
Thymus Lymph nodes
Bone marrow Spleen
Mucosa-associated lymphoid tissue
62
FIGURE 6.2. (a) Human lymph node. Note the
organization, in some respects similar to that of
the thymus, into two predominant areas – the
cortex and the medulla. The cortex is rich in B
cells; the medulla contains cords of lymphoid
tissue that contain both B and T cells; and an
intermediate zone called the paracortex is rich
in T cells. The paracortex, in addition to being
rich in T cells, contains APCs. (b) The medulla
contains macrophages and plasma cells as well
as B and T cells. The cortex contains the
primary and secondary follicles, the distinction
between the two being the germinal center (site
of actively proliferating B cells) in the secondary
follicles.
FIGURE 6.3. (a) Human spleen. Note the red
pulp, primarily involved in destruction of old red
blood cells and red blood cells containing
immune complexes, and white pulp, organized
primarily around central arterioles and hence
forming a ‘follicle’ or a periarteriolar lymphoid
sheath (PALS). (b) T cells are particularly rich
around the central arteriole of the PALS And B
cells in the periphery of the PALS. The far
periphery of the PALS, adjoining the red pulp,
contains macrophages as well as B cells.
our understanding of how lymphoid cells migrate into and out
of specific areas. For example, it is clear that one or more
homing receptors is present on the surface of all lymphoid cells.
These receptors can be regulated, induced, and suppressed.
Furthermore, induction and suppression of other cell-surface
moieties that may regulate lymphoid cell exit from one location
or another occurs. For example, cortical thymocytes rich in
peanut agglutinin on their surface have a paucity of homing
receptors, a fact that might ordinarily allow them to migrate out
of the thymus to some other location. Butcher and Weissman
have hypothesized that “terminal sialidation could release
formerly peanut agglutinin-positive thymocytes from hypo-
thetical peanut agglutinin-like lectins in the thymus, providing
‘exit visas’ for their release from the thymus.”25 In any event,
one thing is clear: mature T cells emerging from the thymus
cortex toward the medulla are rich in cell surface or plasma
membrane-homing receptors, or adhesion molecules or
‘adhesomes’, which are ligands for various addressins or
adhesion molecules at other, remote loci. In the mouse, homing
receptors on the surface of mature T cells have been identified
for the lymph node (MEL-14 or L-selectin (LFA-1)) and for
Peyer ’s patch (LPAM-1 a4b7 integrin, CD44). Equivalent
homing receptors exist in humans.26 The Hermes glycoprotein
on the surface of T and B lymphocytes has been shown to be
identical to the CD44 molecule.27 Antibodies to this
glycoprotein prevent binding of lymphocytes to mucosal lymph
node high endothelial venules.28 Other cell-surface homing and
adhesion molecules, along with their homing receptor ligands,
are shown in Table 6.9.
IMMUNE RESPONSE
Professional APCs phagocytose foreign material (antigens),
process it through protease endosomal-lysosomal degradation,
‘package’ it with MHC molecules, and transport the peptide-
MHC complex to the cell surface. B cells and dendritic cells
(including Langerhans’ cells) perform this function too, but
 A Cast of Thousands: The Cells of the Immune System

sites that bind proteins (enzymes), like phosphatidylinositol

TABLE 6.9. Adhesion Molecules
phospholipase C-g1 (PI-PLC-g1) with SH2 binding domain. PI-
LFA-1a (CD11a) PLC-g1 in turn is phosphorylated (and thereby activated), and it
catalyzes hydrolysis of plasma membrane phosphatidylinositol
MAC-1 (CD11b)
4,5 bisphosphate into inositol 1,4,5 triphosphate (ID3) and
GP150,95 (CD11c) diacylglycerol. IP3 then provokes the release of calcium from
LFA-1b (CD18) its endoplasmic reticulum storage sites. The increased
intracellular calcium concentration that results from the release

Integrin a4 (CD49d)
TCRab
TCRg/d
LFA-2 (CD2)
CD 22
NCAM (CD56)
ICAM-1 (CD54)
LFA-3 (CD58)
LECAM-1
CD5
HCAM (CD44)
HPCA-2 (CD34)
CD28
88-1
PECAM (CD31)
GMP140 (CD62)
HNK-1 (CD57)
differences in protease types and class II MHC molecules among
these APCs may influence the type of T cell activated by an
antigen. It is this unit of antigenic peptide determinant and self-
MHC glycoproteins, along with the aid of adhesion molecules
(ICAM-1([CD54) and LFA-3 (CD58)) and co-stimulatory
molecules (B7 (CD80)), that forms the recognition unit for the
TCRs specific for the antigenic epitope of the foreign material.
The TCR is composed of recognition units for the epitope and
for the autologous MHC glycoprotein. Endogenous antigens,
such as endogenously manufactured viral protein, typically
result in cytoplasm, associate with class I MHC molecules, and
are transported to the surface of the APC, where the class I
MHC-peptide complex preferentially associates with the TCR
of CD8+ cells. Exogenous antigens that are phagocytized
typically associate, as described earlier, in the endosomal,
endoxytic, exocytic pathways with class II MHC molecules, and
this type of complex preferentially associates with CD4+ TCRs.
The ab heterodimer of the TCR is associated with CD3 and
zh proteins and (for CD4 cells) the CD4 molecule, forming the
TCR complex. Antigen presentation can then occur as the TCR
complex interacts with the antigenic determinant/MHC
complex on the macrophage, with simultaneous CD28-CD80
interaction. Macrophage secretion of IL-1 during this cognitive
‘presentation’ phase of the acquired immune response to CD4
T cells completes the requirements for successful antigen
presentation to the helper T cell (see Fig. 6.1).
The CD3 and zh proteins are the signal-transducing
components of the TCR complex; transmembrane signaling via
this pathway results in activation of several phosphotyrosine
kinases, including those of the tyk/jak family and other signal
transduction and activation of transcription molecules and
phosphorylation of tyrosine residues in the cytoplasmic tails of
the CD3 and zh proteins, resulting in the creation of multiple
CHAPTER 6
from storage in turn results in increased binding of calcium to
calmodulin; this then activates the phosphatase, calcineurin.
Calcineurin catalyzes the conversion of phosphorylated nuclear
factor of activated T cells, cytoplasmic component (NFATc), to
free NFATc. This protein (and probably others) then enters the
cell nucleus, where gene transcription of cellular proto-
oncogenes/transcription factor genes, cytokine receptor genes,
and cytokine genes is then activated and regulated by it (or
them). For example, NFATc translocates to the nucleus, where
it combines with AP-1 proteins; this complex then binds to the
NFATc-binding site of the IL-2 promoter. This, coupled with
NFkB binding by proteins possibly induced by the events
stimulated by CD28-CD80 signal transduction, results in IL-2
gene transcription typical of T-cell activation (see Fig. 6.2).
Thus, this activation phase of the acquired immune response is
characterized by lymphocyte proliferation and cytokine
production.
EXPRESSION OF IMMUNITY
The emigration of hematopoietic cells from the vascular system
typically occurs at the region of postcapillary high endothelial
venule cells. These cells are rich in the constitutive expression
of so-called addressins, which are tissue- or organ-specific
endothelial cell molecules involved in lymphocyte homing.
These adhesion molecules are lymphocyte-binding molecules
for the homing receptors on lymphocytes. Thus, the mucosal
addressin27 specifically binds to the Hermes 90-kDa
glycoprotein. In the murine system, a 90-kDa glycoprotein
(designated MECA-79) is a peripheral lymph-node addressin
specifically expressed by high endothelial venules.30 In
peripheral lymph nodes.29 MECA-367 and MECA-89 are
additional addressin glycoproteins in the murine system that
are specific for mucosal vascular high endothelial venules. In
addition to the constitutive expression of addressins or adhesion
molecules, expression of additional adhesion molecules is induced
by a panoply of proinflammatory cytokines. It is this directed
trafficking of inflammatory cells via adhesion molecules that
gives the expression of an immune response its focus, its
specifically directed, targeted expression.
Lymphocytes, monocytes, and neutrophils preferentially
migrate or ‘home’ to sites of inflammation because of this up-
regulation of cytokines and the induction of adhesion molecules
they promote. Thus, L-selectin (CD62L) on the neutrophil cell-
surface membrane does not adhere to normal vascular
endothelium, but intercellular adhesion molecule (ICAM) and
endothelial leukocyte adhesion molecule (ELAM) (CD62E)
expression on the vascular endothelial cell surface induced by
IFN-a, IFN-g, IL-1, IL-17, or a combination thereof results in
low-affinity binding of CD62L, with resultant slowing of
neutrophil transit through the vessel, neutrophil ‘rolling’ on the
endothelial surface, and (with complement split product and IL-
8-driven chemotaxis of increasing numbers of neutrophils)
neutrophil margination in the vessels of inflamed tissue.31
Neutrophil LFA-1 (CD11a, CD18) activated expression
(stimulated by IL-6 and IL-8) then results in stronger adhesion
of the neutrophil to endothelial cell ICAM molecules, with
resultant neutrophil spreading and diapedesis into the sub-
endothelial spaces and the surrounding tissue. 63
 IMMUNOLOGY

IMMUNOLOGIC MEMORY TABLE 6.10. Cytokines and Target Cells

The anamnestic capacity of the acquired immune response Cytokine Source Target Cell
system is one of its most extraordinary properties. Indeed, it is
this remarkable property that was the first to be recognized by IL-1 Mj, TH, FB, NK, Pluripotent stem cells, or not
B, Nj, EC TCTH, B, Mj, FB, Nj
the Chinese ancients and (later) by Jenner. We take it as
axiomatic that our immunization in childhood with killed or IL-2 TH1 TCTH, B, NK
attenuated smallpox and polio virus provoked not only a IL-3 BM, TH, MC TCTH, B, MC, stem cells
SECTION 2

primary immune response but also the development of long-

lived ‘memory’ cells that immediately produce a rapid, vigo- IL-4 TH2, MC TH1, B, Mj, MC, TH2, NK,
FC
rous secondary immune response whenever we might
encounter smallpox or polio virus, thereby resulting in specific IL-5 TH2, MC, Ej TCTH, B, Ej
antibody and lymphocyte-mediated killing of the microbe and IL-6 BM, Mj, MC, EC, Pluripotent stem
defending us from the harm the virus would otherwise have B, TH2, FB cells, or not TCTH, B, FB, Nj
done. But just what do we know about the cells responsible for
this phenomenon? What special characteristics enable memory IL-7 FB, BM Subcapsular and thymocytes,
TCTH, F, FB
cells to live for prolonged periods in the absence of continued or
repeated antigen exposure? IL-8 BM, FB, EC, Mj, TCTH, Mj, Nj
Neils Jerne first hypothesized a clonal selection theory to Nj, Ej
explain at once the specificity and diversity of the acquired IL-9 TH2 Pluripotent stem cells, or not
immune response, and Macfarlene Burnet expanded on Jerne’s TCTH, MC
original hypothesis, clearly predicting the necessary features IL-10 TH2, B, Mj TCD2, TC, TH1, MC, Mj
that would prove the theory; many subsequent studies have
done so. Clones are derived from the development of antigen- IL-11 BM Pluripotent stem cells, or not
specific clones of lymphocytes arising from single precursors TCTH, B
prior to and independent from exposure to antigen. Appro- IL-12 Mj, Nj, B NK, TH–TH1
ximately 109 such clones have been estimated to exist in an
IL-13 TH2 TH1, Mj, B
individual, allowing him or her to respond to all currently
known or future antigens. Antigen contact results in pre- IL-14 T B
ferential activation of the preexisting clone with the cell-surface IL-15 Mj, FB, BM T, NK, B
receptors specific for it, with resultant proliferation of the clone
and differentiation into effector and memory cells. The IL-16 T, Ej, MC T, Ej
secondary or anamnestic immune response is greater and more IL-17 TH FB, T
rapid in onset than is the primary immune response because of IL-18 Mj T, NK
the large number of lymphocytes derived from the original clone
of cells stimulated by the primary contact with antigen, as well TNF-a Mj, NK,T TCTH, B, Mj,
as the long-lived nature of many of the cells (memory cells). FB
The memory cells can survive for very long periods, even TNF-b TC, TH1, B EC, Nj
decades. They express certain cell-surface proteins not ex-
GMCSF TH, Mj, MC
pressed by nonmemory cells (CD45RO). In memory cells, the
level of cell-surface expression of peripheral lymph node Null cells, FB TCTH, Ej, Nj
homing receptors is low compared with the population of such GCSF BM, Mj, FB TCTH, FB, Nj
receptors on the surface of nonmemory cells; in contrast, the
population of other adhesion molecules on the surface of MCSF BM, Mj, FB
memory cells is much greater than that of the surface of LIF BM, fibroblasts Myeloid progenitor
nonmemory cells. These adhesion molecules include CD11a,
SCF BM Myeloid progenitor
CD18 (LFA-1), CD44, and VLA molecules. Because of the Cortical thymocytes
constitutive expression of the cell-surface adhesion molecules,
memory T cells rapidly home to sites of inflammation, ‘looking’ IFN-g NK, TH1 NK, TC, TH2, B, FB, MC
for antigen to which they might respond. IFN-a Mf TCTH, B
IFN-b FB TCTH
SUMMARY
TGF-b Mf,T, TCTH, B, Mf, FB
The evolutionary advantage of the immune system is obvious. chondrocytes
The complexity of the system that has evolved to protect us, B, B cell; BM, bone marrow; CSF, colony-stimulating factor; Ej, eosinophil; EC,
however, is extraordinary, and our understanding of the endothelial cell; FB, fibroblast; GM, granulocyte, macrophage; IFN, interferon;
IL, interleukin; LIF, leukocyte inhibitory factor; Mj, macrophage; MC, mast cell;
immune system is far from complete. The major cell types of Nj, neutrophil; NK, natural killer cell; SCF, stem cell factor; TC, cytotoxic T cell;
the system are well known, but subtypes and sub-subtypes are TGF, transforming growth factor; TH, helper T cell; TNF, tumor necrosis factor.
still being identified. The primary products of one of the major
cell types, the B lymphocytes, have been well characterized
(antibody), but additional cellular products or cytokines from
these cells, which in the 1980s were believed to secrete only at once as fascinating as any Shakespeare play and as frustrating
immunoglobulins in their mature (plasma cell) state, are being as attempting to understand the universe and the meaning of
discovered. Thus, the 18 interleukins and other cytokines listed life. Each year, a chapter brings new knowledge and new ques-
in Table 6.10 will be an incomplete list of the known cytokines tions, and the wise physician will realize that schooling never
of the immune system by the time this edition is published. ends in immunology as in so many other biologic sciences. Stay
64 The seemingly never-ending story of immunologic discovery is tuned.

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65
 CHAPTER
7 T-Lymphocyte Responses
Reza Dana and J. W. Streilein
T lymphocytes, or T cells, stand at the center of the adaptive
immune response (see Chapter 5 for discussion of innate vs
adaptive immunity).1 T cells are absolutely critical for antigen-
specific cell-mediated immunity, as well as for tolerance. In the
absence of T cells, only primitive antibody responses and no
cell-mediated immune responses can be made; even there,
the repertoire of antibodies generated suffers in the absence
of T cell help since CD4+ T cells play an important role in
supporting B-cell responses. The majority of T cells undergo
differentiation in the thymus gland and, upon reaching
maturity, disseminate via the blood to populate secondary
lymphoid organs and to circulate among virtually all tissues of
the body. A second population of T cells undergoes differen-
tiation extra-thymically and has a somewhat different set of
functional properties. T cells are exquisitely antigen-specific, a
property conferred on them by unique surface receptors that
recognize antigenic material in a highly distinctive manner.
Once activated, T cells initiate or participate in the various
forms of cell-mediated immunity, humoral (antibody-mediated)
immunity, and tolerance.
T-LYMPHOCYTE DEVELOPMENT
The ontogeny of the various lymphocyte populations is complex
and incompletely understood. In essence, it is believed that a
‘pluripotent’ hematopoietic stem cell leads to a lineage of
cells that becomes the ‘oligopotent’ lymphocyte progenitor.2
During fetal life, this lineage of cells is observed first in the
liver, but as the fetus matures, the lymphocyte progenitors
shift to the bone marrow. According to developmental signals
not completely understood, lymphocyte progenitors in the
marrow differentiate into (at least) three distinct lineages of
committed precursor cells: pre-thymocytes, pre-B lymphocytes,
and pre-natural killer (NK) lymphocytes. Pre-thymocytes,
which give rise eventually to T lymphocytes, escape from
the bone marrow (or fetal liver) and migrate via the blood
primarily to the thymus, where cell-adhesion molecules on
microvascular endothelial cells direct them into the cortex.
The differentiation process that thymocytes experience within
the thymus accomplishes several critical goals: (1) each cell
acquires a unique surface receptor for an antigen; (2) cells with
receptors that recognize non-self antigenic molecules in the
context of self class I or class II molecules (encoded by genes
within the major histocompatibility complex (MHC)) are
positively selected;3 (3) cells with receptors that recognize
self-antigenic molecules in the context of self-MHC molecules
are negatively selected (i.e., deleted);4 and (4) each mature cell
acquires unique effector functions – the capacity to respond to
antigen by secreting cytokines or by delivering a ‘lethal hit’ to a
target cell.
DIFFERENTIATION IN THE THYMIC
CORTEX
Within the thymus cortex, pre-thymocytes receive differen-
tiation signals from resident thymic epithelial cells and thus
initiate the process of maturation.2 A unique set of genes is
activated, including: (1) genes that commit the cells to pro-
liferation, (2) genes that encode the T-cell receptors for antigen,
and (3) genes that code accessory molecules that developing and
mature T cells use for antigen recognition and signal
transduction. The genes that make it possible for T cells to
create surface receptors for antigen are the structural genes that
encode the four distinct polypeptide chains (a, b, g, d) from
which the T-cell receptor (Tcr) for antigen is composed, as well
as the genes that create genetic rearrangements that confer an
extremely high degree of diversity on Tcr molecules. The
portion of the Tcr that is involved in antigen recognition resides
at the ends of the peptide chains distal to the cell surface and is
called the ‘combining site’. It is thus that within the thymus
cortex, individual pre-thymocytes proliferate, come to express a
unique Tcr for an antigen, and simultaneously express CD3,
CD4, and CD8 on the cell surface. Each day, a very large
number of thymocytes is generated and, therefore, an enormous
diversity of Tcr is generated. Conservative estimates place
the number of novel Tcr produced each day in excess of 109, or
one billion!
NATURE OF ANTIGEN RECOGNITION BY
T CELLS
Understanding the nature of the antigenic determinants
detected by individual T-cell receptors for antigen is central to
understanding the differentiation process that occurs among
thymocytes in the thymus gland. Thymocytes acquire one of
two types of T-cell receptors: ab-Tcr are heterodimers composed
of polypeptides encoded by the Tcr-a and Tcr-b chain genes;
gd-Tcr are heterodimers composed of polypeptides encoded by
the Tcr-g and Tcr-d chain genes.5 Because much is known about
ab-Tcr, whereas much remains to be learned about gd-Tcr, this
discussion is limited to the former.
The ab-T-cell receptor for antigen does not recognize a
protein antigen in its native configuration. Rather, the Tcr
recognizes peptides (ranging in size from 7 to 22 amino acids in
length) derived from limited proteolysis of the antigen, and it
recognizes these peptides when they are bound noncovalently
to highly specialized regions of antigen-presenting molecules.6
Two types of antigen-presenting molecules exist, and both are
encoded within the MHC.7 Class I molecules are trans-
membrane proteins expressed on antigen-presenting cells
(APC). These molecules possess on their most distal domains a 67
 IMMUNOLOGY
groove that accommodates peptides (generated by regulated
proteolysis of antigenic proteins) ranging from seven to nine
amino acids in length. Class II molecules are also trans-
membrane proteins expressed on APC, and the platforms on
their distal domains contain similar grooves that accept
peptides of 15–22 amino acids in length. Thus, the conditions
that must be met for successful recognition of antigen by Tcr
are: (1) a class I or class II molecule must be available on an
SECTION 2
APC, and (2) a peptide must occupy the groove of the presenting
MHC molecule.
Within the thymus cortex, epithelial cells express class I and
class II molecules encoded by the individual’s own MHC genes.2
When Tcr-bearing thymocytes are generated in the cortex, cells
with Tcr that recognize peptide-containing self-class I or self-
class II molecules are induced to undergo successive rounds of
proliferation, leading to clonal expansion. By contrast, Tcr-
bearing thymocytes that fail to recognize peptide-containing
self-class I or self-class II molecules are not activated within the
cortex. In the absence of this cognate signal, all such cells enter
a default pathway, which ends inevitably in cell death (apoptosis).
This process is called positive selection, because thymocytes
with Tcr that have an affinity for self-MHC molecules (plus
peptide) are being selected for further clonal expansion.
Unselected cells simply die by apoptosis. At the completion of
their sojourn in the thymus cortex, large numbers of positively
selected Tcr+, CD3+, CD4+, and CD8+ thymocytes migrate into
the thymus medulla.
DIFFERENTIATION IN THE THYMIC
MEDULLA AND MATURATION OF T CELLS
In addition to epithelial cells, the thymic medulla contains a
unique population of bone marrow-derived cells called dendritic
cells.8,9 These cells express large amounts of class I and class II
molecules and actively endocytose proteins in their environ-
ment. Peptides derived from these proteins by proteolysis are
loaded onto the grooves of MHC-encoded antigen presentation
platforms. Within the thymic medulla, the vast majority of such
endocytosed proteins are self proteins. As thymocytes enter the
medulla from the cortex, a subpopulation expresses Tcr that
recognize peptides of self proteins expressed on self-class I or
self-class II molecules. When these cells engage self-derived
peptides plus MHC molecules on the medullary dendritic cells,
a death (apoptotic) signal is generated to the T cells, and all
such cells undergo apoptosis. This process is called negative
selection because thymocytes with Tcr that have an affinity
for self-peptides in self-MHC molecules are being eliminated so
as to prevent these autoreactive cells from reacting to self
antigens – a process that could lead to autoimmune disease.
Many other thymocytes that enter the medulla express Tcr
that are unable to engage self-class I or self-class II molecules on
dendritic cells, because the relevant peptide does not occupy the
antigen-presenting groove. T cells of this type proceed to
downregulate expression of either CD4 or CD8 and acquire the
properties of mature T cells. The mature T cells that are ready
at this point to leave the thymus are Tcr+, CD3+, and either
CD4+ or CD8+ (but not both). Moreover, they are in G0 of the
cell cycle, and hence resting. The number of such cells exported
from the thymus per day is very large; in humans, it is
estimated that more than 108 new mature T cells are produced
daily. These cells are fully immunocompetent and are prepared
to recognize and respond to a large diversity of foreign antigens,
but because they are antigen-inexperienced, they are called
naive. It is estimated that the number of different antigenic
specificities that can be recognized by mature T cells (i.e., the
T cell repertoire for antigens) exceeds 109, that is, far more than
68 the number of proteins expressed by the genome.
PROPERTIES AND FUNCTIONS OF
MATURE T LYMPHOCYTES
Mature, resting T cells migrate from the thymus to all tissues
of the body, but there are vascular specializations (postcapillary
venules) in secondary lymphoid organs (lymph nodes, Peyer’s
patches, tonsils) that promote the selective entry of T cells into
these tissues. More than 99% of T cells in blood that traverse a
lymph node are extracted into the parafollicular region of the
cortex. This region of the nodal cortex is designed to encourage
the interaction of T cells with APC, since this region is also the
preferential site where a majority of antigen-bearing APCs that
drain from peripheral tissues, also home. Because the encounter
of any single, antigen-specific T cell with its antigen of interest
on an APC is a relatively rare event, most T cells that enter a
secondary lymphoid organ fail to find their antigen of interest –
that is, the antigen for which they express the specific Tcr. In
this case, the T cells migrate into the effluent of the node,
passing through lymph ducts back into the general blood circu-
lation. An individual unstimulated T cell may make journeys
such as this numerous times during a single day, and countless
journeys are accomplished during its lifetime. Remarkably, this
monotonous behavior changes dramatically if and when a
mature T cell encounters its specific antigen loaded on an APC
in a secondary lymphoid organ. It is this critical encounter that
initiates T cell-dependent antigen-specific immune responses.
T-CELL ACTIVATION BY ANTIGEN
There is a general rule regarding the requirements for activation
of lymphocytes, including T cells, which are normally in a
resting state: two different surface signals received simul-
taneously are required to arouse the cell out of G0.8 One signal
(referred to as ‘signal 1’) is triggered by successful engagement
of the Tcr with its peptide in association with an MHC
molecule. The other signal (referred to as ‘signal 2’) is delivered
through numerous cell surface molecules other than the Tcr.
Signals of this type are also referred to as co-stimulatory signals
and are the result of receptor/ligand interactions in which the
receptor is on the T cell and the ligand is expressed on the APC.
For example, B7.1 (CD80) and B7.2 (CD86) are surface
molecules expressed on APC; these molecules engage the
receptor CD28 on T cells, thus delivering an activation signal
to the recipient cells that also promotes their survival through
upregulation of signals that oppose apoptosis.10 Similarly,
CD40 ligand on T cells and CD40 on APC function in a co-
stimulatory manner. When both conditions are met – signal 1
(Tcr binds to peptide plus MHC molecule) and signal 2
(e.g., B7.1 binds to CD28) – the T cell receives coordinated
signals across the plasma membrane, and these signals initiate
a cascade of intracytoplasmic events that lead to dramatic
changes in the genetic and functional programs of the T cells.
ANTIGEN-ACTIVATED T-CELL RESPONSES
When a T cell encounters its antigen of interest along with a
satisfactory signal 2, it escapes from G0. Under these circum-
stances, the genetic program of the cell shifts in a direction that
makes it possible for the cell to proliferate and to undergo
further differentiation. Proliferation results in emergence of a
‘clone’ of cells, all of the identical phenotype, including the Tcr.
This process is called clonal expansion, and results from the
elaboration of growth factors (e.g., IL-2), and represents a hall-
mark of the process of immunization or sensitization, that is,
the process by which the lymphocytes that are specific to an
antigen expand. The signal that triggers proliferation arises first
from the APC, but sustained T-cell proliferation takes place

because the responding T cell activates its own IL-2 and IL-2
receptor genes.11,12 IL-2 is a potent growth factor for T cells, and
T cells expressing the IL-2R respond to IL-2 by undergoing repe-
titive rounds of replication. IL-2 is not the only growth factor for
T cells; another important growth factor is IL-4, which is also
made by T cells. Thus, once activated, T cells have the capacity
to autocrine stimulate their own proliferation, so long as their
Tcr remains engaged with the antigen (plus MHC) of interest.
In addition to proliferation, antigen-activated T cells proceed
down pathways of further differentiation. This is an important
concept, since not all antigen-specific T cells, even when acti-
vated, share the same functional properties. For example, CD4+
T cells can differentiate down distinct paths that allow them to
contribute differentially to the type of immune response
(T helper-1 vs T helper-2 type) generated.13 Additionally, CD8+
T cells can acquire the capacity for cytotoxicity, that is the
ability to lyse target cells.14 These functional properties are
often called the ‘functional phenotype’ of the T-cell response,
and are largely determined by the pattern of cytokines produced
by the T cell(s). The list of lymphokines that an activated
mature T cell can make is long: IL-2, IL-3, IL-4, GM-CSF, IL-5,
IL-6, IL-10, interferon-gamma, etc. Similarly, the range of
biologic activities attributable to these cytokines is extremely
broad, and no single T cell produces all of these factors simul-
taneously, but in general, the specific immune response gener-
ated to an antigen (e.g., microbial, transplant, allergen, etc.) is
dominated by a specific T-cell response phenotype.
The ability of cytotoxic T cells to lyse antigen-bearing target
cells is embodied in specializations of the cells’ cytoplasm and
cell surface, including possession of granules that contain a
molecule, perforin, that can polymerize and insert into the
plasma membrane of a target cell, creating large pores. The
granules also contain a series of lytic enzymes (granzymes) that
enter the target cell, perhaps through the perforin-created pores,
and trigger apoptosis. There is a second mechanism by which T
cells can cause death of neighboring cells. Activated T cells
express high levels of Fas (also known as CD95), a cell-surface
glycoprotein that binds Fas ligand (CD95 ligand). It is a
member of the TNF receptor superfamily and its cytoplasmic
tail contains a ‘death domain’. After sustained activation,
T cells also express Fas ligand; when Fas interacts with Fas
ligand, the cell bearing Fas undergoes programmed cell death.
Thus, Fas ligand+ T cells can trigger apoptotic death in adjacent
cells that are Fas+, including other T cells. In fact, the ability of
antigen-activated T cells to elicit apoptosis among neighboring,
similarly activated, T cells serves as an important mechanism
for downregulating the immune response.
T-CELL ANERGY
On occasion, T cells may encounter their antigen of interest
(in association with an MHC molecule) under circumstances
where an appropriate signal 2 does not exist. In this case,
delivery of signal 1 alone fails to activate the T cells. However,
if these same T cells are re-exposed subsequently to the same
antigen/MHC signal 1 on viable APC capable of delivering a
functional signal 2, activation of the T cells still fails. The
inability of T cells first activated by signal 1 in the absence of
signal 2 to respond subsequently to functional signal 1 and
signal 2 is referred to as anergy (discussed in more detail in
Chapter 10).
T-CELL HETEROGENEITY AND
REGULATORY T CELLS
The adaptive immune response is separable into a cell-mediated
immune arm and an antibody or humoral immune arm (see
T-Lymphocyte Responses
Chapter 5). T cells initiate and mediate cell-mediated immunity,
and also play a critical role in promoting antibody-mediated
responses.
CELL-MEDIATED IMMUNITY
Cell-mediated immunity arises when effector T cells are
generated within secondary lymphoid organs in response to
CHAPTER 7
antigen-induced activation. Effector cells can be broadly divided
into two types: (1) for the most part CD4+ T cells that elicit
delayed-type hypersensitivity (DTH), and (2) CD8+ T cells that
are cytotoxic for antigen-bearing target cells. T cells that elicit
DTH recognize their antigen of interest on cells in peripheral
tissues and upon activation secrete proinflammatory cytokines
such as IFN-g and TNF-a, and thereby can cause significant
‘bystander’ damage to neighboring cells. These cytokines act on
microvascular endothelium, promoting edema formation and
recruitment of monocytes, neutrophils, and other leukocytes to
the site. In addition, monocytes and tissue macrophages exposed
to these cytokines are activated to acquire phagocytic and
cytotoxic functions. Since it takes hours for these inflammatory
reactions to emerge, they are called ‘delayed’. It is generally
believed that the T cells that elicit delayed hypersensitivity
reactions are CD4+ and recognize antigen of interest in asso-
ciation with class II MHC molecules. However, ample evidence
exists to also implicate CD8+ T cells in this process (especially
in reactions within the central nervous system). Although the
elicitation of delayed hypersensitivity reactions is antigen-
specific, the inflammation that attends the response is itself
nonspecific since there the cytokines secreted by DTH effector
T cells have profound paracrine effects on other nearby cells. In
contrast, effector responses elicited by cytotoxic T cells possess
much less nonspecific inflammation. Cytotoxic T cells interact
directly with antigen-bearing target cells and deliver a ‘lethal
hit’ that is ‘clean’ and highly cell-specific; there is virtually no
innocent bystander injury in this response.
HUMORAL IMMUNITY
Humoral immunity arises when B cells produce antibodies in
response to antigenic challenge. Although antigen alone may be
sufficient to activate B cells to produce IgM antibodies, this
response is amplified in the presence of helper CD4+ T cells.
Significant research since the 1990s has focused on how the
patterns of cytokines secreted by T cells can regulate B-cell
responses and the type of immunity generated.13 For example,
one polar form of helper T cell – called Th1 – responds to antigen
stimulation by producing IL-2, IFN-g, and TNF-a. In turn,
these cytokines influence B-cell differentiation in the direction
of producing complement-fixing IgG antibodies. Th1 cells are
also responsible for generating DTH (as discussed earlier), and
hence are relevant to both humoral and cell-mediated
immunity. By contrast, Th2 cells (the other polar form of helper
T cell) respond to antigen stimulation by producing IL-4, IL-5,
IL-6, and IL-10. In turn, these cytokines influence B-cell
differentiation in the directions of producing non-complement-
fixing IgG antibodies or IgA and IgE antibodies. The discovery
of these two polar forms of helper T cells (as well as numerous
intermediate forms) has had a profound impact on our under-
standing of the immune response and its regulation.
REGULATORY T CELLS
It is important to appreciate that the ‘default’ setting of the
immune system is unresponsiveness, or more precisely having
a measured response. Were it not for this feature of immunity,
unchecked clonal expansion of lymphocytes would result in 69
 IMMUNOLOGY

lymphomatous growths, and unregulated inflammatory T CELLS IN DISEASE: INFECTIOUS,

responses in peripheral tissues in response to antigenic
challenges would lead to relentless tissue destruction. Since
these responses are not compatible with normal organ/tissue
function, or indeed life in some cases, the immune system has
generated protean mechanisms for tightly regulating how it
responds to challenges and how quickly these responses are
quenched (see Chapter 10 for details). Immune regulation at the
SECTION 2
IMMUNOPATHOGENIC, AUTOIMMUNE
It is generally believed that T cells were developed in response
to evolutionary pressure to respond to microbial, in particular
intracellular, pathogens, a belief based on the ability of T cells
to detect peptides derived from degradation of intracellular
or phagocytosed pathogens. This property is most obviously

level of T cells is effected by numerous mechanisms: anergy,
clonal deletion, tolerance, regulation of APC maturity and
migration to lymphoid compartments, and cell death. These
mechanisms are tightly controlled and work in concert to
regulate both the induction and expression of immunity. Of
critical importance are ‘natural’ T-regulatory cells that actively
promote immunologic quiescence in an antigen-dependent
fashion.15,16 In this way, immunity generated to foreign
(e.g., transplant) or self-antigens can be quenched in a timely
manner; thus, reexposure to the antigen will lead to a measured
response. Significant research is currently underway to use
these T-regulatory cells in a manner that provides therapeutic
potential in autoimmune diseases.
T-CELL-DEPENDENT INFLAMMATION
Primarily by virtue of the lymphokines they produce, T cells
can cause immunogenic inflammation if they encounter their
antigen of interest in a peripheral tissue (see Chapter 9). As
noted above, CD4+ T cells are particularly capable of causing
tissue injury. In the case of Th1 type CD4+ T cells, these cells
produce IFN-g and other proinflammatory molecules. IFN-g
is a potent activator of microvascular endothelial cells and
macrophages. Activated endothelial cells become ‘leaky’,
permitting edema fluid and plasma proteins to accumulate at
the site. Activated endothelial cells also promote the immi-
gration of blood-borne leukocytes, including monocytes, into
the site, and it is the activated macrophages that provide
much of the ‘toxicity’ at the inflammatory site. These cells
respond to IFN-g by upregulating the genes responsible for nitric
oxide (NO) synthesis. NO, together with newly generated
reactive oxygen intermediates, creates much of the local necro-
sis associated with immunogenic inflammation. Because Th2
cells do not make IFN-g in response to antigenic stimulation,
one might expect that Th2 cells would not promote inflam-
matory injury, but this does not appear to be the case.13 Th2
cells have been directly implicated in immune inflammation,
including that found in the eye. One responsible Th2 cytokine
in this setting known to be capable of causing inflammation
is IL-4.
revealed in viral infections where CD8+ T cells detect peptides
on virus-infected cells derived from viral proteins in association
with self class I molecules (so called ‘altered self ’ recognition).
Once recognition has occurred, a ‘lethal hit’ is delivered to the
target cell, and lysis aborts the viral infection. T-cell immunity
is also conferred when CD4+ T cells detect peptides derived
from bacteria (or other pathogens) phagocytosed by macro-
phages or other antigen-presenting cells. Recognition in this
case does not result in delivery of a ‘lethal hit’; instead,
proinflammatory cytokines released by the activated T cells
cause the macrophages to acquire phagocytic and cytotoxic
functions that lead to the death of the offending pathogen.
To a limited extent with CD8+ cells, but to a greater extent
with CD4+ cells, the inflammation associated with the immune
attack on the invading pathogen can lead to injury of surround-
ing tissues. If the extent of this injury is of sufficient magnitude,
disease may result from the inflammation itself, quite apart
from the ‘toxicity’ of the pathogen. This is the basis of the
concept of T-cell-dependent immunopathogenic disease. As
previously mentioned (see chapters on Overview of Immuno-
logy and Immune regulation), certain organs and tissues, espe-
cially the eye, are particularly vulnerable to immunopathogenic
injury. In tissues of this type, the immune response may prove
to be more problematic than the triggering infection itself!
In some pathologic circumstances, T cells mistakenly
identify self molecules as ‘foreign’, thus mediating an auto-
immune response that can eventuate in disease. Although this
idea is conceptually sound, it is often difficult to identify the
offending self-antigen. Because of this difficulty, it is frequently
impossible to determine whether a particular inflammatory
condition, initiated by T cells, is immunopathogenic in origin
(and, therefore, triggered by an unidentified pathogen) or auto-
immune in origin. This is a particularly common problem in
the eye. To make matters more complicated, the increasing
appreciation for regulatory T cells makes it clear that not all
T lymphocytes are pathogenic, and that certain populations of
these cells may actually aid in terminating or attenuating
the immunoinflammatory response, providing yet one more
untoward complication of nonspecific immunosuppressive
medicines, in particular those that cause lymphopenia.

REFERENCES
1. Janeway CA Jr, Travers P, eds.
Immunobiology. 6th edn. New York:
Garland Publishing Inc; 2004.
2. Wu L: T lineage progenitors: the earliest
steps en route to T lymphocytes. Curr Opin
Immunol 2006; 18:121–126.
3. Ladi E, Yin X, Chtanova T, Robey EA:
Thymic microenvironments for T cell
differentiation and selection. Nat Immunol
2006; 7:338–343.
4. Siggs OM, Makaroff LE, Liston A: The why
and how of thymocyte negative selection.
Curr Opin Immunol 2006; 18:175–183.
5. Krogsgaard M, Davis MM: How T cells
‘see’ antigen. Nat Immunol 2005;
6:239–245.
6. Germain RN: MHC-dependent antigen
70 processing and peptide presentation:
providing ligands for T lymphocyte
activation. Cell 1994; 76:287.
7. Germain RN, Jenkins MK: In vivo antigen
presentation. Curr Opin Immunol 2004;
16:120–125.
8. Janeway CA, Bottomly K: Signals and
signs for lymphocyte responses. Cell 1994;
76:275.
9. Sprent J, Webb SR: Intrathymic and
extrathymic clonal deletion of
T cells. Curr Opin Immunol 1995; 7:196.
10. Kroczek RA, Mages HW, Hutloff A:
Emerging paradigms of T-cell
co-stimulation. Curr Opin Immunol 2004;
16:321–327.
11. Jain J, Loh C, Rao A: Transcription
regulation of the IL-2 gene. Curr Opin
Immunol 1995; 7:333.
12. Minami Y, Kono T, Miyazaki T, Taniguchi T:
The IL-2 receptor complex: its structure,
function, and target genes. Annu Rev
Immunol 1993; 11:245.
13. Gor DO, Rose NR, Greenspan NS: Th1-
Th2: a procrustean paradigm. Nat Immunol
2003; 4:503–505.
14. Catalfamo M, Henkart PA: Perforin and the
granule exocytosis cytotoxicity pathway.
Curr Opin Immunol 2003; 15:522–527.
15. Randolph DA, Fathman CG: CD4+CD25+
regulatory T cells and their therapeutic
potential. Annu Rev Med 2006;
57:381–402.
16. Picca CC, Caton AJ: The role of self-
peptides in the development of
CD4+CD25+ regulatory T cells. Curr Opin
Immunol 2005; 17:131–136.
 CHAPTER
8 B-Lymphocyte Responses
C. Stephen Foster and Fahd Anzaar
B-lymphocyte development from pluripotential bone marrow
stem cells influenced by endosteal region bone marrow
interstitial cells is introduced in Chapter 6. The first stage to
develop in the bone marrow is designated the pro-B lym-
phocyte, which represents the earliest committed B-cell pre-
cursor. CD 19 expression is first seen in this cell type, and
continues to be expressed in all subsequent (‘downstream’)
B-cell lineages (including plasma cells) earning its designation
as the ‘pan-B cell’ marker. However, it does not express CD 20,
whose expression is first seen in the next stage of development,
the pre-B lymphocyte. Pro-B cells express the recombination
activating genes (RAG1 and 2), terminal deoxynucleotidyl
transferase (TdT) as well as genes that encode the surrogate
light chains, and the pro-B cell receptor, which has an unknown
function. Expression of the pre-B-cell receptor allows deve-
lopment and further maturation of the pre-B cells, which
contain cytoplasmic, but not membrane, immunoglobulin M
(IgM) heavy chains that associate with ‘surrogate light chains’
devoid of variable regions. These primitive immunoglobulin
molecules in pre-B cells, composed of complete, mature heavy
chains and surrogate light chains, are critical to the further
development of the B cell into the immature B lymphocyte
containing complete k or l light chains with suitable variable
regions. IgM is then expressed on the immature B-cell surface.
Interleukin-7, BAFF (B-cell activating factor of the TNF family)
and APRIL (a proliferation-inducing ligand) are important in the
process of B-cell development (acting by phosphorylating and
thus activating STAT5)1 as is tyrosine kinase in bone marrow
stromal cells and stem cells. Several B-cell transcription factors
(e.g., the E box proteins (E2A, HEB, E2–2) and early B-cell factor
(EBF)) are involved in this process, activating the B-cell
commitment factor Pax5, which in turn activates B-cell specific
genes (such as CD 19 and BLNK) and simultaneously represses
genes for other cell lines (through a TLE4 Groucho protein).2
Inhibition of Pax 5 is so detrimental to the development of B
cells that it has been shown to ‘reprogram’ them to become
macrophages.3 When an antigen encounters cell-surface IgM
that has binding specificities for the antigen (e.g., self-antigens),
tolerance to the antigen is the typical result if such an encounter
precedes emigration of the B cell from the bone marrow.
Once the immature B cell has acquired its ‘exit visa’ (com-
plete surface IgM), it leaves the bone marrow, residing primarily
in the peripheral lymphoid organs (and blood), where it further
matures to express both IgM and IgD on its cell surface. It is
now a mature B cell, responsive to antigen with proliferation
and antibody synthesis.
CD 20 expression is limited to pre-B, immature, and mature
B cells. It is not seen in plasma cells or memory cells. This
forms the basis of therapy with Rituxan® (rituximab), a chimeric
monoclonal antibody against CD 20, which induces lympho-
cyte death by activating apoptotic pathways (phospholipase Cg,
c-myc, bax, STAT3). The United States Food and Drug
Administration has approved it for treating B-cell non-
Hodgkin’s lymphomas, but is has also been used successfully
for treating autoimmune thrombocytopenia, systemic lupus
erythematosus, and rheumatoid arthritis.4 A major advantage
of Rituxan® is that it does not affect stem cells or plasma cells,
and so has no effect on immunoglobulin levels, and does
not subject patients to the risk of developing opportunistic
infections. Conversely, the presence of long-lived plasma cells
may lead to continued production of pathogenic autoantibodies,
necessitating indefinite treatment. CD 19 monoclonal anti-
bodies have been tested in animal models of autoimmune
disease, and show a more durable depletion of B cells than does
anti-CD 20 therapy, affecting pre-B and immature B cells
(present, for example, in early lymphoblastic leukemias
unresponsive to Rituxan®), eliminating them before antigen-
receptor selection (and production of other pathogenic anti-
bodies responsible for other disease states) occurs.5
The hallmark of the vertebrate immune system is its ability
to mount a highly specific response against virtually any foreign
antigen, even those never before encountered. The ability to
generate a diverse immune response depends on the assembly
of discontinuous genes that encode the antigen-binding sites of
immunoglobulin and T-cell receptors during lymphocyte
development. Diversity is generated through the recombination
of various germline gene segments, imprecise joining of
segments with insertion of additional nucleotides at the junc-
tions, and somatic mutations occurring within the recombining
gene segments. Other factors, such as chromosomal position of
the recombining gene segments and the number of homologous
gene segments, may play a role in determining the specificities
of the antigen-recognizing proteins produced by a maturing
lymphocyte.
ANTIBODY DIVERSITY
The paradox of an individual possessing a limited number of
genes but the capability to generate an almost infinite number
of different antibodies remained an enigma to immunologists
for a considerable time. The discovery of distinct variable (V)
and constant (C) regions in the light and heavy chains of
immunoglobulin molecules (Fig. 8.1) raised the possibility that
immunoglobulin genes possess an unusual architecture. In
1965, Dreyer and Bennett proposed that the V and C regions of
an immunoglobulin chain are encoded by two separate genes in
embryonic (germline) cells (germline gene diversity).6 According
to this model, one of several V genes becomes joined to the C
gene during lymphocyte development. In 1976, Hozumi and
Tonegawa discovered that variable and constant regions are 71
 IMMUNOLOGY

several J (joining)-segment genes, which encodes part of the last

hypervariable segment (Fig. 8.4).9–11 Additional diversity is
generated by allowing V and J genes to become spliced in
different joining frames (junctional diversity) (Fig. 8.5).10 There
are at least three frames for the joining of V and J. Two forms
SECTION 2

FIGURE 8.1. Structure of IgG showing the regions of similar

sequence (domains).

FIGURE 8.3. Hypervariable or CDRs on the antigen-binding site of

encoded by separate, multiple genes far apart in germline DNA the variable regions of IgG.
that join to form a complete immunoglobulin gene active in B
lymphocytes.7 Immunoglobulin genes are thus translocated
during the differentiation of antibody-producing cells (somatic
recombination) (Fig. 8.2).

STRUCTURE AND ORGANIZATION OF

IMMUNOGLOBULIN GENES
The V regions of immunoglobulins contain three hypervariable
segments that determine antibody specificity (Fig. 8.3).8 Hyper-
variable segments of both the light (L) and heavy (H) chains
form the antigen-binding site. Hypervariable regions are also
called complementarity-determining regions (CDRs). The V
regions of L and H chains have several hundred gene segments
in germline DNA; the exact number of segments is still being
FIGURE 8.4. A V gene is translocated near a J gene in forming a
debated but is estimated to range between 250 and 1000
light-chain V region gene.
segments.

LIGHT-CHAIN GENES
A complete gene for the V region of a light chain is formed by
the splicing of an incomplete V-segment gene with one of

FIGURE 8.2. Translocation of a V-segment gene to a C gene in the FIGURE 8.5. Imprecision in the site of splicing of a V gene to a J gene
72 differentiation of an antibody-producing B cell. (junctional diversity).

of light chains exist: (k) and (l). For kl chains, assume that
there are ~250 V-segment genes and four J-segment genes.
Therefore, a total of 250 µ 4 µ 3 (for junctional diversity), or
3000, kinds of complete VK genes can be formed by com-
binations of V and J.
HEAVY-CHAIN GENES
Heavy-chain V-region genes are formed by the somatic
recombination of V, an additional segment called D (diversity),
and J-segment genes (Fig. 8.6). The third CDR of the heavy
chain is encoded mainly by a D segment. Approximately 15 D
segments lie between hundreds of VH and at least four JH gene
segments. A D segment joins a JH segment; a VH segment then
becomes joined to the DJH to form the complete VH gene. The
D to J rearrangements occur in pro-B cells, when the recom-
bination activating genes (RAG 1 and 2) introduce a single-
stranded nick on either side of the segments, assisted by
DNA-bending high mobility group proteins (HMGB1 and 2).
The V to DJ joining occurs in pre-B cells, and a pre-B-cell
receptor is expressed. The light chain gene rearragements now
take place, forming an immature B cell with a complete immu-
noglobulin molecule that is then expressed on the cell’s surface.
To further diversify the third CDR of the heavy chain, extra
nucleotides are inserted between V and D and between D and J
(N-region addition) by the action of terminal deoxyribonucleo-
tidyl transferase.12 Introns, which are noncoding intervening
sequences, are removed from the primary RNA transcript.
The site-specific recombination of V, D, and J genes is
mediated by enzymes (immunoglobulin recombinase) that
recognize conserved nonamer and palindromic heptamer
sequences flanking these gene segments.13,14 The nonamer and
heptamer sequences are separated by either 12-base pair (bp) or
23-bp spacers (Fig. 8.7). Recombination can occur only between
the 12- and 23-bp types but not between two 12-bp types or two
23-bp types (called the 12/23 rule of V-gene-segment
recombination). For example, VH segments and JH segments are
flanked by 23-bp types on both their 5„ and 3„ ends.
Consequently, they cannot recombine with each other or
among themselves. Instead, they recombine with D segments,
FIGURE 8.6. The variable region of the heavy chain is encoded by
V-, D-, and J-segment genes.
B-Lymphocyte Responses
which are flanked on both 5„ and 3„ ends by recognition
sequences of the 12-bp type.
SOURCES OF IMMUNOGLOBULIN GENE
DIVERSITY
For 250 VH, 15 DH, and 4 JH gene segments that can be joined
in three frames, at least 45 000 complete VH genes can be
CHAPTER 8
formed. Therefore, more than 108 different specificities can be
generated by combining different V, D, and J gene segments and
by combining more than 3000 L and 45 000 H chains. If the
effects of N-region addition are included, more than 1011
different combinations can be formed. This number is large
enough to account for the immense range of antibodies that can
be synthesized by an individual.
Far fewer V genes than VK genes encode light chains.
However, many more V amino-acid sequences are known.15–17
It is therefore likely that mutations introduced somatically give
rise to much of the diversity of l light chains (somatic hyper-
mutation).10 Likewise, somatic hypermutation further amplifies
the diversity of heavy chains. To summarize, four sources of
diversity are used to form the almost limitless array of anti-
bodies that protect a host from foreign invasion: germline gene
diversity, somatic recombination, junctional diversity, and
somatic hypermutation.
REGULATION OF IMMUNOGLOBULIN
GENE EXPRESSION
Immunoglobin gene rearrangements are separated in time
(as discussed earlier) and also restricted to one locus. An in-
complete V gene becomes paired to a J gene on only one of a pair
of homologous chromosomes. Successful rearrangement of one
heavy-chain V region prevents the process from occurring on
FIGURE 8.7. Recognition sites for the recombination of V-, D-, and
J-segment genes. V and J genes are flanked by sites containing
23-bp spacers, whereas D-segment genes possess 12-bp spacers.
Recombination can occur only between sites with different classes of
spacers. 73
 IMMUNOLOGY
the other heavy-chain allele. Only the properly recombined
immunoglobulin gene is expressed. Therefore, all of the V
regions of immunoglobulins produced by a single lymphocyte
are the same. This is called allelic exclusion.18,19
There are five classes of immunoglobulins. An antibody-
producing cell first synthesizes IgM and then IgG, IgA, IgE, or
IgD of the same specificity. Different classes of antibodies are
formed by the translocation of a complete VH (VHDH) gene from
SECTION 2
the CH gene of one class to that of another.20 Only the constant
region of the heavy chain changes; the variable region of the
heavy chain remains the same (Fig. 8.8). The light chain
remains the same in this switch. This step in the differentiation
of an antibody-producing cell is called class switching and is
mediated by another DNA rearrangement called SS recom-
bination (Fig. 8.9).21 This process is regulated by cytokines
produced by helper T cells, and also by BAFF10,22 For example,
switching to IgE class immunoglobulin production is provoked
by the CD4 TH2 cytokine, IL-4. Repetitive DNA sequences
called switch regions are located upstream of each CH gene;
double-stranded breaks in these regions precede the deve-
lopment of stem-and-loop structures, and a CSR recombinase
enzyme (aided by AID (activation-induced cytidine deaminase))
then combines the new variable and heavy chain segments.
New evidence indicates that in addition to the cytokine milieu,
the type of antibody produced is also biased towards those heavy
chain gene segments that are in closest proximity to the pre-
switch heavy chain gene.22 The number of cells that have
undergone class switching depends on the number of divisions
the cell has performed rather than on the time since
stimulation by cytokines.22
DETERMINATION OF B-CELL REPERTOIRE
V-segment genes can be grouped into families based on their
DNA sequence homologies. In general, variable genes sharing
greater than 80% nucleotide similarity are defined as a family.23
There are 11 VH gene families currently known in the
mouse23–26 and 6 in humans.27–30 At least 29 families are known
for the V of murine light-chain genes.31,32 In fetal pre-B cells,
FIGURE 8.8. The VH region is first associated with Cm and then with
another C region to form an H chain of a different class in the
74 synthesis of different classes of immunoglobulins.
chromosomal position is a major determinant of VH rearrange-
ment frequency, resulting in a nonrandom repertoire that is
biased toward use of VH families closest to the JH segments.33–36
In contrast, random use of VH families based on the number of
members in each family occurs in mature B cells without bias
toward JH proximal families.37–39 The preferential VH gene re-
arrangement frequency seen in pre-B cells presumably becomes
normalized when contact of the organism with a foreign antigen
selects for the expression of the entire VH gene repertoire. One
can speculate that members of VH families preferentially used in
the pre-B cell encode antibody specificities that are needed in
the early development of the immune system.40
Immunoglobulins are serum proteins that migrate with the
globulin fractions by electrophoresis.7 Although they are glyco-
proteins, the molecules’ primary functions are determined by
their polypeptide sequence.8 At one end of the immunoglobulin,
the amino terminus, is a region that binds a site (epitope) on an
antigen with great specificity. At the other end, the carboxyl
terminus, is a non-antigen-binding region responsible for
various functions, including complement fixation and cellular
stimulation via binding to cell-surface Ig receptors. The general-
ized structure of immunoglobulin is best understood initially by
examining its most common class, IgG (see Fig. 8.1).
IgG is composed of four polypeptide chains: two identical
heavy chains and two identical light chains. Heavy chains
weigh about twice as much as light chains. The identical heavy
chains are covalently linked by two disulfide bonds. One light
chain is associated with each of the heavy chains by a disulfide
bond and noncovalent forces. The two light chains are not
linked. Asparagine residues on the heavy chains contain carbo-
hydrate groups. The amino terminals of one light chain and its
linked heavy chain compose the region for specific epitope-
binding. The carboxyl termini of the two heavy chains con-
stitute the non-antigen-binding region.
Each polypeptide chain, whether light or heavy, is composed
of regions that are called constant (C) or variable (V). A variable
FIGURE 8.9. The VHDJH gene moves from its position near Cm to one
near Cg1 by SS recombination.
 B-Lymphocyte Responses

region on a light chain is called VL, the constant region of a

heavy chain is called CH, and so forth. If the amino acid
sequence of multiple light or heavy chains is compared, the
constant regions will vary little, whereas the variable regions
differ greatly. The light chains are divided approximately equally
into a constant (CL) and variable (VL) region at the carboxyl and
amino terminals, respectively. The heavy chains also contain a
similar length of variable region (VH) at the amino terminals,

CHAPTER 8
but the constant region (CH) is three times the length of the
variable region (VH). The variable regions are responsible for
antigen-binding, and it is this variability that accounts for the
ability to bind to millions of potential and real epitopes.9 Be-
cause each antibody molecule has two antigen-binding sites
with variable regions, cross-linking of two identical antigens
may be performed by one antibody. The constant regions carry
out effector functions that are common to all antibodies of a
a
given class (e.g., IgG) without the requirement of unique
binding sites.
The function of various regions of the immunoglobulin
molecule was determined in part by the use of proteolytic
enzymes that digest these molecules at specific locations. These
enzymes have also been exploited for the development of
laboratory reagents. The enzyme papain splits the molecule on
the amino terminal side of the disulfide bonds that link the
heavy chains, resulting in three fragments: two identical Fab
fragments (each composed of the one entire light chain and a
portion of the associated heavy chain) and one Fc fragment
composed of the linked carboxyl terminal ends of the two
heavy chains. In contrast, treatment with the enzyme pepsin b
results in one molecule composed of two linked Fab fragments
known as F(ab„).7 The Fc fragment is degraded by pepsin
treatment.
Within some classes of immunoglobulins, whole molecules
may combine with other molecules of the same class to form
polymers with additional functional capabilities. J chains
facilitate the association of two or more immunoglobulins
(Fig. 8.10), most notably IgA and IgM. Secretory component is
a polypeptide synthesized by nonmotile epithelium found near
mucosal surfaces. This polypeptide may bind noncovalently to
IgA molecules, allowing their transport across mucosal surfaces
to be elaborated in secretions.
Five immunoglobulin classes are recognized in humans: IgG,
IgM, IgA, IgE, and IgD (Table 8.1). Some classes are composed
c
of subclasses as well. The class or subclass is determined by the
structure of the heavy-chain constant region (CH).10 The heavy FIGURE 8.10. Schematic diagram of polymeric human
chains g, m, a, e, and d are found in IgG, IgM, IgA, IgE, and IgD, immunoglobulins. (a) IgM. (b) Secretory IgA. (c) Serum IgA.
respectively. Four subclasses of IgG and two subclasses of both
IgA and IgM exist (Table 8.2). The two light chains on any
immunoglobulin are identical and, depending on the structure
of their constant regions, may be designated k or l. Kappa
chains tend to predominate in human immunoglobulins fixation. IgG is the only immunoglobulin class to cross the
regardless of the heavy chain-determined class. Whether an placenta, an important aspect in fetal defense. Via their Fc
immunoglobulin is composed of two k or two l chains does not portion, IgG molecules bind Fc receptors found on a host of
determine its functional capabilities. Heavy chain-determined inflammatory cells. Such binding activates cells such as macro-
class does dictate important capacities.11 phages and natural killer cells, enhancing cytotoxic activities
important in the immune response.
IMMUNOGLOBULIN G
The most abundant of the human classes in serum, immuno- IMMUNOGLOBULIN M
globulin G (IgG) constitutes about three-quarters of the total Less abundant in the serum than IgG, IgM typically exists as a
serum immunoglobulins. Respectively, IgG1 and IgG2 make up pentameric form, stabilized by J chains, theoretically allowing
~60% and 20% of the total IgG. IgG3 and IgG4 are relatively the binding of 10 epitopes. (In vivo, this is usually limited by
minor components. IgGs are the primary immunoglobulin steric considerations.) IgM appears early in the immune
providing immune protection in the extravascular compart- response to antigen and is especially efficient at initiating agglu-
ments of the body. IgG is able to fix complement in the serum, tination, complement fixation, and cytolysis. IgM probably
an important function in inducing inflammation and con- preceded IgG in the evolution of the immune response and is
trolling infection. IgG3 and IgG1 are most adept at complement the most important antibody class in defending the circulation. 75
 IMMUNOLOGY

IMMUNOGLOBULIN A IMMUNOGLOBULIN INTRACLASS

Immunoglobulin A (IgA) is found in secretions of mucosal
surfaces as well as in the serum. In secretions, it exists as a
dimer coupled by J chains and stabilized by secretory com-
ponent. IgA protects mucosal surfaces from infections but may
also be responsible for immunologic surveillance at the site of
first contact with antigen. IgA in secretion is hardy, able to
SECTION 2
DIFFERENCES
Differences among the immunoglobulin classes are known as
isotypes, because all of the normal individuals in a species
possess all of the classes. Allotype refers to antigenic structures
on immunoglobulins that may differ from one individual to
another within a species. Idiotype refers to differences among

withstand the ravages of proteolytic degradation.
IMMUNOGLOBULIN D
Immunoglobulin D (IgD) is present in minute amounts in the
serum and is the least stable of the immunoglobulins. Its
function is not known, but it probably serves as a differentiation
marker. IgD is found on the surface of B lymphocytes (along
with IgM) and may have a role in class switching and tolerance.
IMMUNOGLOBULIN E
Immunoglobulin E (IgE) is notable for its ability to bind to mast
cells; when cross-linked by antigen, it causes a variety of
changes in the mast cell, including release of granule contents
and membrane-derived mediators. Although recognized as a
component of the allergic response, the role of IgE in protective
immunity is speculative.
individual antibodies and is determined by the variable domain.
Just as the variable domain allows for antibodies to recognize
many antigens (epitopes), these differences also allow individual
antibodies to be recognized on the basis of their idiotype. In
fact, antibodies directed against antibodies exist and are called
anti-idiotypic antibodies. They are crucial to the regulation of
the antibody response and constitute the basis for Jerne’s
idiotype network.
COMPLEMENT
The complement system functions in the immune response by
allowing animals to recognize foreign substances and defend
themselves against infection.29 The pathways of complement
activation are complex (Fig. 8.11).30 Activation begins with
the formation of antigen-antibody complexes and the ensuing
generation of peptides that lead to a cascade of proteolytic
events. The particle that activates the system accumulates a

TABLE 8.1. Diversity in TCR and Immunoglobulin Genes

Immunoglobulin TCR

H k a b g d

Germline Variable (V) 250–1000 250 100 25 7 10

Segments Diversity (D) 15 0 0 2 0 2
Joining (J) 4 4 50 12 3 2
Variable region combinations 62 500–250 000 2500 50
Use of different D and Yes Yes Yes Yes – Yes
J segments
Junctional Variability in 3„ Rarely Rarely Yes No Yes Yes
Diversity Joining of V and J
D joining in all three Rarely – – Often – Often
reading frames
N-region diversity V-D, D-J None V-J V-D, D-J V-J V-D, D1-D2
Junctional combinations 108 1015 1018
Total repertoire 1011 1017 1019
The numbers of the V, D, and J gene segments in the murine genome are shown. Total repertoire produced by the various mechanisms for generating diversity was
estimated.

TABLE 8.2. Human Immunoglobulin Subclasses

Immunoglobulin Subclasses Predominant Subclass Unique Characteristics

IgG 1, 2, 3, and 4 1 (65%) and 2 (25%) IgG2 – crosses placenta poorly

IgG3 – aggregates spontaneously
IgG4 – blocks IgE binding; poor classic complement fixation
IgA 1 and 2 1
IgM 1 and 2 1
76

FIGURE 8.11. Simplified schematic of steps in classic and alternate
complement cascades.
protein complex on its surface that often leads to cellular
destruction via disruption of membranes.
Two independent pathways of complement activation are
known. The classic pathway is initiated by IgG- and IgM-
containing immune complexes.31 The alternative pathway is
activated by aggragated IgA or complex polysaccharides from
microbial cell walls.32 One component, C3, is crucial to both
pathways and in its proactive form can be found circulating in
plasma in large concentrations. Deficiency or absence of C3
results in increased susceptibility to infection.33 Cleavage of C3
may result in at least seven products (lettered a through g), each
with biologic properties related to cellular activation and
immune and nonimmune responses.34 C3a, for instance,
causes the release of histamine from mast cells, neutrophil
enzyme release, smooth muscle contraction, suppressor T-cell
induction, and secretion of macrophage IL-1, prostaglandin,
and leukotriene.35 C3e enhances vascular permeability. C3b
binds to target cell surfaces and allows opsonization of biologic
particles.
The alternative pathway probably is a first line of defense,
because unlike the classic pathway, it may neutralize foreign
material in the absence of antibody. The initiating enzyme of
this pathway, factor D, circulates in an active form and may
protect bystander cells from inadvertent destruction following
activation of the pathway.
The final step of both pathways is membrane damage leading
to cytolysis. Both pathways require the assembly of five
precursor proteins to effect this damage: C5, C6, C7, C8, and
C9. The mechanism of complement-mediated cell lysis is
B-Lymphocyte Responses
similar to that of cell-mediated cytotoxicity (as with natural
killer cells). Membrane lesions result from insertion of tubular
complexes into the membranes, leading to uptake of water with
ion-exchange disruption and eventual osmotic lysis.
The complement system interfaces with a variety of immune
responses, as outlined earlier, and with the intrinsic coagulation
pathways.36 Complement activity is usually measured by
assessing the ability of serum to lyse sensitized sheep red blood
CHAPTER 8
cells.37 Values are expressed as 50% hemolytic complement
units per millimeter. The function of an individual component
may be studied by supplying excess quantities of all the other
components in a sheep red blood cell lysis assay.38 Components
are quantitated by radial diffusion or immunoassay. Comple-
ment may be demonstrated in tissue sections by immuno-
fluorescence or enzymatic techniques.
Complement plays a role in a number of human diseases.
Complement-mediated cell lysis is the final common pathologic
event in type III hypersensitivity reactions. Deficiencies of
complement exist in the following human disorders: systemic
lupus erythematosus, glomerulonephritis, Raynaud’s pheno-
menon, recurrent gonococcal and meningococcal infections,
hereditary angioedema, rheumatoid disease, and others.33
B-CELL RESPONSE TO ANTIGEN
PRIMARY RESPONSE
Naive B cells respond to protein antigen in much the same way
that T cells do, through the help of antigen-presenting cells and
‘helper’ T cells. An antigen-presenting cell (usually a macro-
phage or dendritic cell) processes the antigen and presents it to
an antigen-specific helper (CD4) T cell, generally in the T-cell-
rich zones of the required lymph node. The T cell is thus
activated, expresses the membrane protein gp39, secretes
cytokines (e.g., IL-2 and IL-6), and binds to similarly activated
antigen-specific B cells (activated by the binding cross-linking of
antigen to surface IgM- and IgD-binding sites). The T-cell/B-cell
proliferation and a cascade of intracellular protein phosphoryl-
ation events, together with T-cell cytokine signals, result in
production of transcription factors that induce transcription of
various B-cell genes, including those responsible for production
of IgM light and heavy chains with paratopes specific to the
antigen epitopes that initiated this primary B-cell response. The
proliferating B cells form germinal centers in the lymph node
follicles, and somatic hypermutation of the IgV genes in some
of these cells results in the evolution of a collection of B cells in
the germinal center with surface IgM of even higher antigen-
binding affinity. This phenomenon is called affinity maturation
of the primary antibody response. Those cells with the greatest
antigen-binding affinity survive as this primary B-cell response
subsides, persisting as long-lived memory cells responsible for
the classic distinguishing characteristics of the secondary
humoral immune response.
SECONDARY RESPONSE
The development of the secondary humoral immune response
is markedly accelerated compared with the primary response,
and it is greatly amplified in terms of magnitude of antibody
production (Fig. 8.12). The secondary response differs from the
primary one in the isotype or isotypes of antibody produced, as
well as in the avidity of the paratopes for the epitopes on the
elicited antigen. IgG, IgA, and IgE isotypes may now be seen
in the effector phase of this secondary humoral immune
response, and the binding affinities of these antibodies are
usually greater than that of the IgM elicited in the primary
response. 77
 IMMUNOLOGY

The cellular and molecular events of the secondary B-cell

response are considerably different from those of the primary
response. Memory B cells themselves become the preeminent
antigen-binding, processing, and presenting cells, presenting
peptide fragments (antigenic determinants) to CD4 helper T
cells in the typical major histocompatibility complex-restricted
fashion, with ‘processed’ peptide/human leukocyte antigen/DR
motifs interacting with the appropriate elements of the T-cell
SECTION 2

FIGURE 8.12. Relative synthesis of IgG and IgM following initial and
subsequent antigen injection.
receptor for antigen at the same time that B-cell CD40 and T-
cell gp39 signaling occurs. Additionally, various T-cell cytokines
induce the memory B cells to divide, proliferate, produce
antibody, and switch the class of antibody being produced,
depending on the sum-total message being received by the B
cell: the nature of the antigenic stimulus, the amount and the
site of stimulation, and the site of the cells involved in the
cognitive and activation phases of the secondary response.
Memory cells of each immunoglobulin isotype involved in the
secondary response will, of course, persist after devolution of
the response.

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12. Alt FW, Baltimore D: Joining of
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13. Early P, Huang H, Davis M, et al: An
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DNA: VH, D and JH. Cell 1980; 12:981.
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Tonegawa S: Sequences at the somatic
recombination sites of immunoglobulin
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15. Weigert MG, Cesari IM, Yondovich SJ,
Cohn M: Variability in the lambda light
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16. Brack C, Hirama M, Lenhard-Schuller R,
Tonegawa S: A complete immunoglobulin
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17. Bernard O, Hozumi N, Tonegawa S:
Sequences of mouse immunoglobulin light
chain genes before and after somatic
changes. Cell 1978; 15:1133.
18. Pernis BG, Chiappino G, Kelus AS,
Gell PGH: Cellular localization of
immunoglobulins with different allotypic
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19. Cebra J, Colberg JE, Dray S: Rabbit
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alpha-, gamma-, and mu-heavy
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20. Kataoka T, Kawakami T, Takahasi N,
Honjo T: Rearrangement of immunoglobulin
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21. Gritzmacher CA: Molecular aspects of
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CHAPTER 8
79
 CHAPTER

9 Immune-Mediated Tissue Injury

C. Stephen Foster, Miguel C. Coma, and J. Wayne Streilein

The immune response of an organism to an antigen may be
either helpful or harmful. If the response is excessive or
inappropriate, the host may incur tissue damage. The term
‘hypersensitivity reactions’ has been applied to such excessive
or inappropriate immune responses. Four major types of
hypersensitivity reactions are described, and all can occur in the
eye (Table 9.1). The necessary constituents for these reactions
are already present in or can be readily recruited into ocular
tissues. Immunoglobulins, complement components, inflam-
matory cells, and inflammatory mediators can, under certain
circumstances, be found in ocular fluids (i.e., tears, aqueous
humor, vitreous) and in the ocular tissues, adnexa, and orbit.
Unfortunately, these tissues (especially the ocular tissues) can
be rapidly damaged by inflammatory reactions that produce
irreversible alterations in structure and function. Some authors
have described a fifth type of hypersensitivity reaction, but this
adds little to our real understanding of disease mechanisms and
is unimportant to us as ophthalmologists in the study and care
of patients with destructive ocular inflammatory diseases. For
this reason, this discussion is confined to the classic four types
of hypersensitivity reactions that were originally proposed by
Gell, Coombs, and Lackmann.
Multiple theories about the etiology of these autoimmune
diseases have been postulated. Infections play a crucial role in
the induction and exacerbation, but sometimes also in
inhibition of these entities. The protection, induced by
infection, against some autoimmune and atopic disorders could
be related to immunoregulation that normally keeps the
immune system balanced, generated by production of
immunosuppressive cytokines, such as IL-10 or TGF-ß.1 On
the other hand, there is also good evidence supporting infection
as a possible cause of exacerbation or even generation of
autoimmune and allergic disorders (as in postinfectious
encephalitis disseminata or rheumatic fever).2
INJURY MEDIATED BY ANTIBODY
TYPE I HYPERSENSITIVITY
The antigens typically responsible for type I (immediate)
hypersensitivity reactions are ubiquitous environmental
allergens such as dust, pollen, dander, microbes, and drugs.
Under ordinary circumstances, exposure of an individual to
such materials is associated with no harmful inflammatory
response. The occurrence of such a response is considered,
therefore, out of place (Greek, a topos) or inappropriate, and it
is for this reason that Cocoa and Cooke coined the word ‘atopy’
in 1923 to describe individuals who develop such inappropriate
inflammatory or immune responses to ubiquitous environmental
agents.3 The antibodies responsible for type I hypersensitivity
reactions are homocytotropic antibodies, principally immuno-
globulin E (IgE) but sometimes IgG4 as well. The mediators of
the clinical manifestations of type I reactions include
histamine, serotonin, leukotrienes (including slow-reacting

TABLE 9.1. Gell, Coombs, and Lackmann Hypersensitivity Reactions

Type Participating Elements Systemic Examples Ocular Examples

Type I Allergen, IgE, mast cells
Type II Antigen, IgG, IgG3, or
IgM, complement,
neutrophils (enzymes),
macrophages (enzymes)
Type III Antigen, IgG, IgG3, or IgM,
complement-immune
complex, neutrophils
(enzymes), macrophages
(enzymes)
Type IV Antigen, T cells, neutrophils,
macrophages
Allergic rhintis, allergic asthma,
anaphylaxis
Goodpasture’s syndrome, myasthenia
gravis
Stevens–Johnson syndrome,
rheumatoid arthritis, systemic
lupus erythematosus, polyarteritis
nodosa, Behçet’s disease,
relapsing polychondritis
Transplant rejection, tuberculosis,
sarcoidosis,Wegener’s
granulomatosis
Seasonal allergic conjunctivitis, vernal
keratoconjunctivitis, atopic keratoconjunctivitis,
giant papillary conjunctivitis
Ocular cicatricial pemphigoid, pemphigus vulgaris
dermatitis herpetiformis
Ocular manifestations of diseases are Systemic
Examples
Contact hypersensitivity (drug allergy), herpes
disciform keratitis, phlyctenulosis, corneal
transplant rejection, tuberculosis, sarcoidosis,
Wegener’s granulomatosis, uveitis, herpes
simplex virus, stromal keratitis, river blindness
81
 IMMUNOLOGY
SECTION 2
a b
c d
FIGURE 9.1. Type I hypersensitivity reaction mechanism. (a) Mast cell
Fc receptors have antigen-specific IgE affixed to them by virtue of the
patient’s being exposed to the antigen and mounting an inappropriate
(atopic) immune response to that antigen, with resultant production of
large amounts of antigen-specific IgE antibodies. The antibodies have
found their way to the MMC, have bound to the mast cells, but have
not provoked allergic symptoms because the patient is no longer
exposed to the antigen. (b) Second (or subsequent) exposure to the
sensitizing antigen or allergen results in a ‘bridging’ binding reaction of
antigen to two adjacent IgE antibodies affixed to the mast cell plasma
membrane. (c) The antigen–antibody bridging reaction shown in (b)
results in profound changes in the mast cell membrane, with
alterations in membrane-bound adenyl cyclase, calcium influx, tubulin
aggregation into microtubules, and the beginning of the degranulation
of the preformed mast cell mediators from their storage granules.
(d) The degranulation reaction proceeds, and newly synthesized
mediators, particularly those generated by the catabolism of
membrane-associated arachidonic acid, begin to work. The array of
liberated and synthesized proinflammatory and inflammatory
mediators is impressive.
substance of anaphylaxis (SRS-A)), kinins, and other vasoactive
amines. Examples of type I hypersensitivity reactions include
anaphylactic reactions to insect bites or to penicillin injections,
allergic asthma, hay fever, and seasonal allergic conjunctivitis.
It should be emphasized that in real life the four types of
hypersensitivity reactions are rarely observed in pure form, in
isolation from each other, and it is typical for hypersensitivity
reactions to have more than one of the classic Gell and
Coombs’ responses as participants in the inflammatory
problem. For example, eczema, atopic blepharokeratoconjunc-
tivitis, and vernal keratoconjunctivitis have hypersensitivity
reaction mechanisms of both type I and type IV. The atopic
individuals who develop such abnormal reactions to environ-
mental materials are genetically predisposed to such responses.
82 The details of the events responsible for allergy (a term coined
TABLE 9.2. Mast Cell Mediators
Preformed in Granules Newly Synthesized
Histamine LTB4
Heparin LTC4
Tryptase LTD4
Chymase Prostaglandins
Kinins Thromboxanes
Eosinophil chemotactic factor Platelet-activating factor
Neutrophil chemotactic factor
Serotonin
Chondroitin sulfate
Arylsulfatase
in 1906 by von Pirquet, in Vienna, meaning ‘changed reactivity’)
are clearer now than they were even a decade ago.4
Genetically predisposed allergic individuals have defects in
the population of suppressor T lymphocytes responsible for
modulating IgE responses to antigens. After the initial contact
of an allergen with the mucosa of such an individual, abnormal
amounts of allergen-specific IgE antibody are produced at the
mucosal surface and at the regional lymph nodes. This IgE has
high avidity, through its Fc portion, to Fc receptors on the
surface of mast cells in the mucosa. The antigen-specific IgE
antibodies, therefore, stick to the receptors on the surface of the
tissue mast cells and remain there for unusually long periods.
Excess locally produced IgE enters the circulation and binds to
mast cells at other tissue locations as well as to circulating
basophils. A subsequent encounter of the allergic individual
with the antigen to which he or she has become sensitized
results in antigen-binding by the antigen-specific IgE molecules
affixed to the surface of the tissue mast cells.
The simultaneous binding of the antigen to adjacent IgE
molecules on the mast cell surface results in a change in the
mast cell membrane and particularly in membrane-bound
adenyl cyclase (Fig. 9.1). The feature common to all known
mechanisms that trigger mast cell degranulation (including
degranulation stimulated by pharmacologic agents or
anaphylatoxins like C3a and C5a and antigen-specific IgE-
mediated degranulation) is calcium influx with subsequent
aggregation of tubulin into microtubules, which then
participate in the degranulation of vasoactive amines (see Fig.
9.1). In addition to the degranulation of the preformed
mediators such as histamine, induction of synthesis of newly
formed mediators from arachidonic acid also occurs with
triggering of mast cell degranulation (Table 9.2). The preformed
and newly synthesized mediators then produce the classic
clinical signs of a type I hypersensitivity reaction: wheal
(edema), flare (erythema), itch, and in many cases the sub-
sequent, delayed appearance of the so-called late-phase reaction
characterized by subacute signs of inflammation.
Type I hypersensitivity has been postulated as a strategy to
avoid and remove multicellular parasite infections affecting the
respiratory and gastrointestinal systems.5 The consequence of
mast-cell degranulation is not only vasodilatation and increase
in production and release of exudative fluid, but also goblet cell
hyperplasia, synthesis of mucin of augmented viscosity and
increased peristaltic movement, which are demonstrated
successful mechanisms to eliminate parasitic nematodes.
Indeed one of the main symptoms in asthmatic patients, the
viscous and obstructive mucus secreted by the respiratory

FIGURE 9.2. Diagrammatic display of IgE synthesis. Glycosylation-
enhancing factor, glycosylation-inhibiting factor, IgE-promoting factor,
IgE suppressor factor, and the helper and suppressor T lymphocytes
specific for regulation of IgE synthesis are shown.
epithelium, is thought to play a protective role in parasitic
infections (the parasite, because of the mucus, cannot
effectively penetrate the epithelial cells, which is essential to its
development). Type I hypersenstivity reactions would be host-
destructive only when they occur more intensely, improperly, or
as a result of a mistake in the perception of the existence of an
intruder, even though there is no true threat.
Control of IgE Synthesis
The Th2 subset of helper T cells bearing Fce receptors produce,
in addition to interleukin-4 (IL-4), IgE-binding factors after
stimulation by interleukins produced by antigen-specific helper
T cells activated by antigen-presenting cells and antigen. The
two known types of IgE-binding factor that can be produced are
IgE-potentiating factor and IgE-suppressor factor; both are
encoded by the same codon, and the functional differences are
created by posttranslational glycosylation. The glycosylation is
either enhanced or suppressed by cytokines derived from other
T cells. For example, glycosylation-inhibiting factor (identical to
migration inhibitory factor) is produced by antigen-specific
suppressor T cells. Glycosylation-enhancing factor is produced
by an Fc receptor helper T cell (Fig. 9.2). The relative levels of
Immune-Mediated Tissue Injury
these factors control the production of IgE-potentiating factor
and IgE-suppressor factor by the central helper T cell and, thus,
ultimately control the amount of IgE produced (see Fig. 9.2).
They probably do so through regulation of IgE B lymphocyte
proliferation and synthesis of IgE by these cells.
Mast Cell Subpopulations
It has become increasingly clear that at least two subpopu-
CHAPTER 9
lations of mast cells exist. Connective tissue mast cells
(CTMCs) contain heparin as the major proteoglycan, produce
large amounts of prostaglandin D2 in response to stimulation,
and are independent of T cell-derived interleukins for their
maturation, development, and function. These cells stain
brilliantly with toluidine blue in formalin-fixed tissue sections.
Mucosal mast cells (MMCs) do not stain well with toluidine
blue. They are found primarily in the subepithelial mucosa in
gut and lung, contain chondroitin sulfate as the major
proteoglycan, manufacture leukotriene C4 as the predominant
arachidonic acid metabolite after stimulation, and are
dependent on IL-3 (and IL-4) for their maturation and pro-
liferation. Interestingly, MMCs placed in culture with
fibroblasts rather than T cells transform to cells with the
characteristics of CTMCs. Disodium cromoglycate inhibits
histamine release from CTMCs but not from MMCs. Steroids
suppress the proliferation of MMCs, probably through
inhibition of IL-3 production.
Atopy Genetics and Immunology the Role of the
Environment
Both genetic and environmental components are clearly
involved in the allergic response. Offspring of marriages in
which one parent is allergic have ~30% risk of being allergic,
and if both parents are allergic the risk to each child is greater
than 50%. At least three genetically linked mechanisms govern
the development of atopy1: general hyperresponsiveness,2
regulation of serum IgE levels,3 and sensitivity to specific
antigens. General hyperresponsiveness, defined as positive skin
reactions to a broad range of environmental allergens, is
associated HLA-B8/HLA-DW3 phenotype, and this general
hyperresponsiveness appears not to be IgE class specific. Total
serum IgE levels are also controlled genetically, and family
studies indicate that total IgE production is under genetic
control. Finally, experimental studies using low molecular
weight allergenic determinants disclose a strong association
between IgE responsiveness to such allergens and HLA-
DR/DW2 type, whereas for at least some larger molecular
weight allergens, responsiveness is linked to HLA-DR/DW3. In
mice at least, gene regulation of IgE production occurs at several
levels, including regulation of antigen-specific,1 IgE-specific
suppressor T cells,2 manufacture of glycosylation-inhibiting
factor or of glycosylation-enhancing factor by helper T cells,3 at
the level of IL-4 regulation of class switching to IgE synthesis,
and at the level of IgE-binding factors such as IgE-potentiating
factor and IgE-suppressor factor.4
It is likely that the genetic architecture of the clinical
conditions of asthma or atopic keratoconjunctivitis differs.
However there are many common genes and pathways which
contribute to the onset, course, or severity of these related
entities. Certainly, well-known phenotypes associated with
them, such as bronchial hyperresponsiveness or the amount of
total and specific IgE, are influenced by the same genes. In 1996
the first genome-wide search for asthma and atopy
susceptibility loci was completed, and there have been multiple
publications on the genetic basis of these complex phenotypes.6
The most frequent loci reported as associated with asthma or
atopy phenotypes are the following genes: IL4, IL13, ADRB2,
TNF, HLA-DRB1, FCER1B, IL4RA, CD14, HLA-DQB1, and 83
 IMMUNOLOGY
ADAM33.7 However no one gene will be the ‘atopy’ gene in all
populations, which reflects the tremendous complexity of these
pathologies in terms of genetic predisposition and the modest
effects of these genes on risk.
The environment plays a major role in whether or not a
genetically predisposed individual expresses major clinical
manifestations of atopy. The ‘dose’ of allergens to which the
individual is exposed is a critical determinant of whether or not
SECTION 2
clinical expression of an allergic response develops. Less well
recognized, however, is the fact that the general overall quality
of the air in an individual’s environment plays a major role in
whether clinical expression of allergic responses to allergens to
which the individual is sensitive does or does not develop. It has
become unmistakably clear that as the general quality of the air
in urban environments has deteriorated and as the air has
become more polluted, the prevalence in the population of overt
atopic clinical manifestations has increased dramatically. On a
global level, the immediate environment in which an individual
finds himself much of the time, the home, plays an important
part in the expression of allergic disease. Allergically predis-
posed persons, at least one member of whose household smokes
cigarettes, have enhanced sensitivity to allergens such as house
dust, mites, and molds, among others. It is probably also true
that the overall health and nutritional status of an individual
influence the likelihood of that person developing a clinically
obvious allergy.
Evidence linking stress to the expression of conditions such
as atopy is still growing. The reported influence of stress on
neuroimmunoregulation and oxidative stress pathways may
interact with the hypersensitivity to environmental conditions
as previously described, playing a crucial role in the genesis
of the characteristic clinical manifestations.8 Both roles,
gene–gene and gene–enviroment interactions, are important in
determining susceptibility. Further studies to determine risk for
specific patients will have to consider the influence of the genes
under a certain environmental context, as much as possible, to
clarify the degree of responsibility of each factor.
Diagnosis of Type I Reactions
The definite diagnosis of type I hypersensitivity reactions
requires the passive transfer of the reaction via a method known
as the Prausnitz–Kustner reaction. Intradermal injection of the
serum of a patient suspected of having a type I hypersensitivity-
mediated problem into the skin of a volunteer is followed by
injection of varying dilutions of the presumed offending antigen
at the same intradermal sites as the patient’s serum injection.
A positive Prausnitz–Kustner reaction occurs when local flare
and wheal formation follows the injection of the antigen. This
method for proving type I reactions is not used clinically;
therefore, diagnosis of type I mechanisms contributing to a
patient’s inflammatory disorder is always based on a collection
of circumstantial evidence that strongly supports the hypothesis
of a type I reaction. A typical history (e.g., of a family history of
allergy or personal history of eczema, hay fever, asthma, or
urticaria) elicitation of allergic symptoms following exposure to
suspected allergens involves itching as a prominent symptom,
elevated IgE levels in serum or other body fluids, and blood
or tissue eosinophilia. Chapter 11 covers these points in
general, as well as the importance of the histopathologic
characteristics of conjunctival biopsy tissue, in particular in the
evaluation of patients with chronic cicatrizing conjunctivitis.
Therapy for Type I Reactions
Therapy for type I reactions must include scrupulous avoidance
of the offending antigen. This is not easy, and it is a component
of proper treatment that is often neglected by the patient and
84 the physician alike. It is crucial, however, for a patient with an
incurable disease such as atopy to recognize that throughout a
lifetime he or she will slowly sustain cumulative permanent
damage to structures affected by atopic responses (e.g., lung,
eye) if he or she is subjected to repetitive triggering of the
allergic response. Pharmacologic approaches to this disorder can
never truly succeed for careless patients who neglect their
responsibility to avoid allergens. A careful environmental
history is, therefore, a critical ingredient in history-taking, and
convincing education of the patient and family alike is an
essential and central ingredient in the care plan.
A careful environmental history and meticulous attention to
environmental details can make the difference between relative
stability and progressive inflammatory attacks that ultimately
produce blindness. Elimination of pets, carpeting, feather pillows,
quilts, and wool blankets and installation of air-conditioning
and air-filtering systems are therapeutic strategies that should
not be overlooked.9
One of the most important advances in the care of patients
with type I disease during the past two decades has been the
development of mast cell-stabilizing agents. Disodium
cromoglycate, sodium nedocromil, and lodoxamide are three
such agents. Topical administration is both safe and effective in
the care of patients with allergic eye disease.10,11 This
therapeutic approach is to be strongly recommended and is very
much favored over the use of competitive H1 antihistamines.
Clearly, if the mast cells can be prevented from degranulating,
the therapeutic effect of such degranulation-inhibiting agents
would be expected to be vastly superior to that of anti-
histamines simply by virtue of preventing liberation of an entire
panoply of mediators from the mast cell rather than competitive
inhibition of one such mediator, histamine.
Histamine action-inhibition by H1 antihistamines can be
effective in patients with ocular allergy provided the drugs are
administered systemically. The efficacy of such agents when
given topically is marginal at best, and long-term use can
result in the development of sensitivity to ingredients in the
preparations. The consistent use of systemic antihistamines,
however, can contribute significantly to long-term stability,
particularly of the newer noncompetitive antihistamines
such as astemizole. Additionally, slow escalation of the
amount of hydroxyzine used in the care of atopic patients
can help to interrupt the itch–scratch–itch psychoneurotic
component that often accompanies eczema and atopic
blepharokeratoconjunctivitis.
Generalized suppression of inflammation, through use of
topical corticosteroids, is commonly used for treatment of type
I ocular hypersensitivity reactions, and this is appropriate for
acute breakthrough attacks of inflammation. It is, however,
completely inappropriate for long-term care. Corticosteroids
have a direct effect on all inflammatory cells, including eosi-
nophils, mast cells, and basophils. They are extremely effective,
but the risks of chronic topical steroid use are considerable and
unavoidable, thus chronic use is discouraged.
Although desensitization immunotherapy can be an impor-
tant additional component to the therapeutic plan for a patient
with type I hypersensitivity, it is difficult to perform properly.
The first task, of course, is to document to which allergens the
patient is sensitive. The second task is to construct a ‘serum’
containing ideal proportions of the allergens that induce the
production of IgG-blocking antibody and stimulate the
generation of antigen-specific suppressor T cells. For reasons
that are not clear, the initial concentration of allergens in such
a preparation for use in a patient with ocular manifestations of
atopy must often be considerably lower than the initial
concentrations usually used when caring for a person with
extraocular allergic problems. If the typical starting concen-
trations for nonocular allergies are employed frequently, a

TABLE 9.3. Therapy of the Atopic Patient
Environmental control
Mast cell stabilizers
Systemic antihistamines
Topical steroids (for acute intervention only)
Desensitization immunotherapy
Plasmapheresis
Intravenous gamma globulin
Cyclosporine (systemic and topical)
Psychiatric intervention for the patient and family
dramatic exacerbation of ocular inflammation immediately
follows the first injection of the desensitizing preparation.
Plasmapheresis is an adjunctive therapeutic maneuver that
can make a substantial difference in the care of patients
with atopy, high levels of serum IgE, and documented
Staphlyococcus-binding antibodies.9 This therapeutic technique
is expensive, is not curative, and must be performed at highly
specialized centers, approximately three times each week,
indefinitely. It is also clear, from our experience, that the
aggressiveness of the plasmapheresis must be greater than that
typically employed by many pheresis centers. Three to four
plasma exchanges per pheresis session typically are required to
achieve therapeutic effect for an atopic person.
Intravenous or intramuscular gamma globulin injections
may also benefit selected atopic patients. It has been recognized
that, through mechanisms that are not yet clear, gamma
globulin therapy involves much more than simple passive
‘immunization’ through adoptive transfer of antibody
molecules. In fact, immunoglobulin therapy has a pronounced
immunomodulatory effect, and it is because of this action that
such therapy is now recognized and approved as effective
therapy for idiopathic thrombocytopenic purpura.12 The use of
gamma globulin therapy is also being explored for other
autoimmune diseases, including systemic lupus erythematosus
and atopic disease.
Cyclosporine is being tested in patients with certain atopic
diseases. Preliminary evidence suggests that topical cyclo-
sporine can have some beneficial effect on patients with atopic
keratoconjunctivitis and vernal keratoconjunctivitis.13
Furthermore, in selected desperate cases of blinding atopic
keratoconjunctivitis, we have demonstrated that systemic
cyclosporine can be a pivotal component of the multimodality
approach to the care of these complex problems.9
The calcineurin-inhibitors, such as Pimecrolimus or
Tacrolimus, were introduced in the early 2000s as alternative
topical treatments, acting more selectively and providing
certain advantages over corticosteroids. These agents have
demonstrated efficacy in the management of patients with
atopy-related diseases, such as atopic dermatitis or severe atopic
keratoconjunctivitis.14,15 These agents appear to offer the
potency of a corticosteroid without its adverse side effects.
Tacrolimus, also known as FK506, is a potent immuno-
suppressive agent (close ‘relative’ of cyclosporine in terms of
action mechanism, but up to 100 times more potent) that
has been used orally since 1994 to prevent allograft rejection in
liver and kidney transplant recipients. Its systemic use may
also be considered in selected patients with severe atopic
keratoconjunctivitis.
Finally, appropriate psychiatric care may be (and usually is)
indicated in patients with severe atopy (and family members). It
Immune-Mediated Tissue Injury
CHAPTER 9
a b
c d
FIGURE 9-3. Type II hypersensitivity. (a) A ‘sensitized’ cell with two
antibodies specific for antigenic determinants on the cell surface has
attached to the target cell. C1q, C1r, and C1s complement
components have begun the sequence that will result in the classical
cascade of complement-factor binding. (b) The complement cascade
has progressed to the point of C5 binding. Note that two
anaphylatoxin and chemotactic split products, C3a and C5a, have
been generated, and a neutrophil is being attracted to the site by
virtue of the generation of these two chemotactic moieties. (c) The
complement cascade is complete, with the result that a pore has been
opened in the target cell membrane, and osmotic lysis is the nearly
instantaneous result. (d) A variant of the type II hypersensitivity
reaction is the antibody-dependent cellular cytotoxicity (ADCC)
reaction. Target-specific antibody has attached to the target cell
membrane, and the Fc receptor on a neutrophil, a macrophage, or a
killer (K) cell is attaching to that membrane-affixed antibody. The result
will be lysis of the target cell.
is not hyperbole to state that in most cases, patients with severe
atopic disease and the family members with whom they live
demonstrate substantial psychopathology and destructive
patterns of interpersonal behavior. The degree to which these
families exhibit self-destructive, passive–aggressive, and
sabotaging behaviors is often astonishing. Productive engage-
ment in psychiatric care is often difficult to achieve, but it
can be extremely rewarding when accomplished successfully.
Table 9.3 summarizes the components of a multifactorial
approach to the care of atopic patients.
TYPE II HYPERSENSITIVITY REACTIONS
Type II reactions require the participation of complement-fixing
antibodies (IgG1, IgG3, or IgM) and complement. The
antibodies are directed against antigens on the surface of
specific cells (i.e., endogenous antigens). The damage caused by
type II hypersensitivity reactions, therefore, is localized to the
particular target cell or tissue. The mediators of the tissue
damage in type II reactions include complement as well as
recruited macrophages and other leukocytes that liberate their
enzymes. The mechanism of tissue damage involves antibody-
binding to the cell membrane with resultant cell membrane
lysis or facilitation of phagocytosis, macrophage and neutrophil
cell-mediated damage (Fig. 9.3a–c), and killer cell damage to
target tissue through antibody-dependent cell-mediated
cytotoxicity (ADCC) reaction (see Fig. 9.3d). It is important to
remember (particularly in the case of type II hypersensitivity
reactions that do not result in specific target cell lysis through
the complement cascade with eventual osmotic lysis) that
neutrophils are prominent effectors of target cell damage. 85
 IMMUNOLOGY

Neutrophil adherence, oxygen metabolism, lysosomal enzyme

release, and phagocytosis are tremendously ‘upregulated’ by
IgG–C3 complexes and by the activated split product of C5a. As
mentioned in the description of type I hypersensitivity
reactions, mast cells also participate in nonallergic inflam-
matory reactions, and type II hypersensitivity reactions provide
an excellent example of this. The complement split products
C3a and C5a both produce mast cell activation and
SECTION 2

degranulation. The result is the liberation of preformed

vasoactive amines and upregulation of membrane synthesis of
leukotriene B4, the most potent (and also other cytokines (e.g.,
TNF-a)) known chemoattractant for neutrophils, even more
potent than IL-8/RANTES, eosinophil chemotactic factor,
and other arachidonic acid metabolites. Neutrophils and
macrophages attracted to this site of complement-fixing IgG or
IgM in a type II hypersensitivity reaction cannot phagocytose
entire cells and target tissues, and thus liberate their proteolytic
and collagenolytic enzymes and cytokines in ‘frustrated
phagocytosis’. It is through this liberation of tissue-digestive
enzymes that the target tissue is damaged. Direct target cell
damage (as opposed to ‘innocent bystander’ damage caused by
liberation of neutrophil and macrophage enzymes) in type II
hypersensitivity reactions may be mediated by killer (K) cells
through the antibody-dependent cytotoxicity reaction. In fact,
definitive diagnosis of type II reactions requires the demon-
stration of fixed antitissue antibodies at the disease site as well
as a demonstration of killer cell activity in vitro against the
tissue. No ocular disease has been definitively proved to
represent a type II reaction, but several candidates, including
ocular cicatricial pemphigoid, exist.
This type II hypersensitivity reaction has been postulated as
a tactic to deal with small extracellular organisms.5 The first
step, the interaction between antibodies and antigens, results in
opsonization of extracellular microbes resistant to phagocytosis.
The second step, the liberation of neutrophil chemoattractants,
is designed to be a magnet for PMNs to the site of the
inflammation. As in the type I reaction, this would be
pathologic only if it occurs in other circumstances different
from a response to such kind of infections.
The classic human autoimmune type II hypersensitivity
disease is Goodpasture’s syndrome. Many believe ocular
cicatricial pemphigoid is analogous (in mechanism at least) to
Goodpasture’s syndrome, in which complement-fixing antibody
directed against a glycoprotein of the glomerular basement
membrane fixes to the glomerular basement membrane. This
action causes subsequent damage to the membrane by
proteolytic and collagenolytic enzymes liberated by phagocytic
cells, including macrophages and neutrophils.
Therapy for Type II Reactions
Therapy for type II reactions is extremely difficult, and
immunosuppressive chemotherapy has, in general, been the
mainstay of treatment. Experience with ocular cicatricial
pemphigoid has been especially gratifying in this regard.16–18
Progressive cicatricial pemphigoid affecting the conjunctiva
was, eventually, almost universally blinding before the advent of
systemic immunosuppressive chemotherapy for this condition.
With such therapy now, however, 90% of cases of the disease are
arrested and vision is preserved.19
TYPE III HYPERSENSITIVITY REACTIONS
Type III reactions, or immune complex diseases, require, like
type II hypersensitivity reactions, participation of complement-
fixing antibodies (IgG1, IgG3, or IgM). The antigens
participating in such reactions may be soluble diffusible
86 antigens, microbes, drugs, or autologous antigens. Microbes
FIGURE 9.4. Type III hypersensitivity reaction. Circulating immune
complexes (shown here as triangle-shaped moieties in the vascular
lumen) percolate between vascular endothelial cells but become
trapped at the vascular endothelial basement membrane. Neutrophils
and other phagocytic cells are attracted to this site of immune
complex deposition. These phagocytic cells liberate their proteolytic
and collagenolytic enzymes and damage not only the vessel but also
the surrounding tissue.
that cause such diseases are usually those that cause persistent
infections in which not only the infected organ but also the
kidneys are affected by the immune complex-stimulated
inflammation. Autoimmune–immune complex diseases are the
best known of these hypersensitivity reactions: the classic
collagen vascular diseases and Stevens–Johnson syndrome.
Kidney, skin, joints, arteries, and eyes are frequently affected in
these disorders. Mediators of the tissue damage include
antigen–antibody–complement complexes and the proteolytic
and collagenolytic enzymes from phagocytes such as
macrophages and neutrophils. As with type II reactions, the
C3a and C5a split products of complement exert potent
chemotactic activity for the phagocytes and also activate mast
cells, which through degranulation of their vasoactive amines,
TNF-a increase vascular permeability and enhance emigration
of such phagocytic cells. It is again through frustrated
phagocytosis that the neutrophils and macrophages liberate
their tissue-damaging enzymes (Fig. 9.4).
Arthus’ reaction, a special form of type III hypersensitivity, is
mentioned for completeness. Antigen injected into the skin of
an animal or individual previously sensitized with the same
antigen, and with circulating antibodies against that antibody,
results in an edematous, hemorrhagic, and eventually necrotic
lesion of the skin. A passive Arthus’ reaction can also be created
if intravenous injection of antibody into a normal host recipient
is followed by intradermal injection of the antigen. An
accumulation of neutrophils develops in the capillaries and
venule walls after deposition of antigen, antibody, and
complement in the vessel walls.
Immune complexes form in all of us as a normal conse-
quence of our ‘immunologic housekeeping’. Usually, however,

these immune complexes are continually removed from the
circulation. In humans, the preeminent immune complex-
scavenging system is the red blood cells, which have a receptor
(CR1) for the C3b and C4b components of complement. This
receptor binds immune complexes that contain complement,
and the membrane-bound complexes are removed by fixed-
tissue macrophages and Kupffer cells as the red blood cells pass
through the liver. Other components of the reticuloendothelial
system, including the spleen and the lung, also remove
circulating immune complexes. Small immune complexes may
escape binding and removal, and not surprisingly, smaller
immune complexes are principally responsible for immune
complex-mediated hypersensitivity reactions. It is also true that
IgA complexes (as opposed to IgG or IgM complexes) do not
bind well to red blood cells. They are found in the lung, brain,
and kidney rather than in the reticuloendothelial system.
The factors that govern whether or not immune complexes
are deposited into tissue (and if so, where) are complex and
rather incompletely understood. It is clear that the size of the
immune complex plays a role in tissue deposition. It is also
clear that increased vascular permeability at a site of immune
system activity or inflammation is a major governor of whether
or not immune complexes are deposited in that tissue.
Additionally, it is clear that immune complex deposition is
more likely to occur at sites of vascular trauma; this includes
trauma associated with the normal hemodynamics of a
particular site, such as the relatively high pressure inside
capillaries and kidneys, the turbulence associated with
bifurcations of vessels, and obviously at sites of artificial trauma
as well. Excellent examples of the latter include the areas of
trauma in the fingers, toes, and elbows of patients with
rheumatoid arthritis, where subsequently vasculitic lesions and
rheumatoid nodules form, and in the surgically traumatized
eyes of patients with rheumatoid arthritis or Wegener ’s
granulomatosis, where immune complexes are deposited
subsequently and necrotizing scleritis develops.20 It is likely
that addressing or other attachment factors in a local tissue play
a role in the ‘homing’ of a particular immune complex.
Antibody class and immune complex size are also important
determinants of immune complex localization at a particular
site, as is the type of the basement membrane itself.
Type III hypersensitivity reactions have been postulated as a
strategy to prevent further injury in the viremic phase of viral
infections.5 The potential harmful effect of this reaction would
be the one described by Gell and Coombs. But under more
physiological conditions, the results are probably beneficial to
the host. In fact, the binding of excess complement to
preformed antigen–antibody complexes seems to result in their
disaggregation into smaller entities that no longer bind more
complement. Furthermore, these complexes do not trigger the
lytic components of complement and do not liberate
anaphylotoxins, and can be ingested and later eliminated by the
reticuloendothelial system. This reaction may have a host-
protective response and is possibly the best one to eliminate
circulating viral particles. However, when C3 falls under critical
levels, this mechanism fails, obstructing this degradation of
antigen–antibody complexes into smaller and soluble fragments
which then deposit in certain areas of the host: this is why, for
example, renal disease in systemic lupus erythematosus (SLE) is
inversely related to complement levels.
Therapy for Type III Reactions
Therapy for type III reactions consists predominantly of large
doses of corticosteroids, of immunosuppressive chemo-
therapeutic agents, or both. Cytotoxic immunosuppressive
chemotherapy may or may not be necessary to save both the
sight and the life of a patient with Behçet’s disease, but it is
Immune-Mediated Tissue Injury
TABLE 9.4. Types of Delayed Hypersensitivity Reactions
Reaction Type Example Peak Reaction
Tuberculin contact Tuberculin skin test 48–72 h
Contact Drug contact 48–72 h
hypersensitivity
Granulomatous Leprosy 14 days
CHAPTER 9
Jones–Mote Cutaneous basophil 24 h
hypersensitivity
categorically required to save the life of a patient with either
polyarteritis nodosa21 or Wegener’s granulomatosis.22 In the
case of rheumatoid arthritis-associated vasculitis affecting the
eye, it is likely that systemic immunosuppression will also be
required if death from a lethal extraarticular, extraocular,
vasculitic event is to be prevented.23
INJURY MEDIATED BY CELLS
TYPE IV HYPERSENSITIVITY REACTIONS:
IMMUNE-MEDIATED INJURY DUE TO
EFFECTOR T CELLS
The original classification of immunopathogenic mechanisms
arose in an era when considerably more was known about
antibody molecules and serology than about T cells and cellular
immunity. Out of this lack of knowledge, T cell-mediated
mechanisms were relegated to the ‘type IV’ category, and all
manner of responses were unwittingly grouped together (Table
9.4).24 We now know that T cells capable of causing immune-
based injury exist in at least three functionally distinct
phenotypes: cytotoxic T cells (typically CD8+) and two
populations of helper T cells (typically CD4+) (Fig. 9.5). Since
cytotoxic T lymphocytes (CTLs) were discovered well after the
original Gell and Coombs classification, they were, therefore,
never anticipated in that classification system. As mentioned
previously, CD4+ T cells can adopt one of two polar positions
with regard to their lymphokine secretions (IL-12 induces Th1
cells, and IL-10 induces Th2 cells).25 Th1 cells secrete IL-2,
IFN-g, and lymphotoxin, whereas Th2 cells were identified in
the 1940s and 1950s as the initiators of delayed hyper-
sensitivity reaction by secretion of cytokines such as IL-4, IL-5,
and IL-6. The latter cells, in addition to providing helper factors
that promote IgE production, also mediate tissue inflammation,
albeit of a somewhat different type than Th1 cells.
Immunopathogenic T Cells
CTLs exhibit exquisite antigen specificity in their recognition of
target cells, and the extent of injury that CTLs effect is usually
limited to target cells bearing the relevant instigating antigens.
Therefore, if a CTL causes tissue injury, it is because host cells
express an antigen encoded by an invading pathogen, an antigen
for which the Tcr on the CTL is highly specific. Delivery of a
cytolytic signal eliminates hapless host cells, and in so doing
aborts the intracellular infection. Assuming that the infected
host cell is one of many and can thus be spared (e.g., epidermal
keratinocytes), there may be little or no physiologic conse-
quence of this CTL-mediated loss of host cells. However, if the
infected cell is strategic, limited in number, or cannot be replaced
by regeneration (e.g., neurons, corneal endothelial cells), then
the immunopathogenic consequences may be severe.
CD4+ effector cells also exhibit exquisite specificity in
recognition of target antigens. However, the extent of injury 87
 IMMUNOLOGY
SECTION 2
FIGURE 9.5. Type IV hypersensitivity reaction. DTH (CD4) T
lymphocytes and cytotoxic (CD8 and CD4) T lymphocytes directly
attack the target cell or the organism that is the target of the type IV
hypersensitivity reaction. Surrogate effector cells are also recruited
through the liberation of cytokines. The most notable surrogate or
additional effector cell is the macrophage or tissue histiocyte. If the
reaction becomes chronic, certain cytokines or signals from
mononuclear cells result in the typical transformation of some
histiocytes into epithelioid cells, and the fusion of multiple epithelioid
cells produces the classic multinucleated giant cell.
that these cells can effect is diffuse and is not limited to cells
bearing the target antigen. CD4+ effector cells secrete cytokines
that possess no antigen specificity in their own right. Instead,
these molecules indiscriminately recruit and activate macro-
phages, natural killer cells, eosinophils, and other mobile cells
that form the nonspecific host defense network. It is this
defense mechanism that leads to eradication and elimination of
the offending pathogen. In other words, CD4+ effector cells
protect by identifying the pathogen antigenically, but they cause
the elimination of the pathogen by enlisting the aid of other
cells. The ability of CD4+ effector cells to orchestrate this
multicellular response rests with the capacity of these cells to
secrete proinflammatory cytokines to arm inflammatory cells
with the ability to ‘kill’. Once armed, these ‘mindless assassins’
mediate inflammation in a nonspecific manner that leads often,
if not inevitably, to ‘innocent bystander’ injury to surrounding
tissues. For an organ that can scarcely tolerate inflammation of
even the lowest amount, such as the eye, ‘innocent bystander’
injury is a formidable threat to vision.
Autoimmune T Cells
The foregoing discussion addresses immunopathogenic injury
due to T cells that develops among host tissues invaded by
pathogenic organisms. However, there is another dimension to
immunopathology. T cells can sometimes make a mistake and
mount an immune attack on host tissues simply because those
tissue cells express self molecules (i.e., autoantigens). Although
an enormous amount of experimental and clinical literature is
devoted to autoimmunity and autoimmune diseases, very little
is known in a ‘factual’ sense that enables us to understand this
curious phenomenon. What seems clear is that T cells with
receptors that recognize ‘self ’ antigens, as well as B cells bearing
surface antibody receptors that recognize ‘self ’ antigens, exist
under normal conditions.24 Moreover, there are examples of T
88 and B cells with ‘self ’-recognizing receptors that become
activated in putatively normal individuals. Thus, immuno-
logists have learned to distinguish an autoimmune response
(not necessarily pathologic) from an autoimmune disease.
Whereas all autoimmune diseases arise in a setting where an
autoimmune response has been initiated, we understand little
about what causes the latter to evolve into the former. Whatever
the pathogenesis, autoimmune disease results when effector T
cells (or antibodies) recognize autoantigens in a fashion that
triggers a destructive immune response.26,27
The pathogenesis of autoimmunity is probably related to a
complex phenomenon called cripticity.28 This is directly
connected with the hierarchy of antigenic determinants within
self-antigens and is a product of the extent of proper presen-
tation of the antigen and the affinity of the T-cell receptor. The
well-processed and -presented determinants constitute a
‘dominant self ’, whereas the inadequately processed and/or
presented determinants will be invisible to T cells and comprise
a ‘cryptic self ’, which plays a crucial role in the genesis of
autoimmunity. A similar hierarchy is established in the thymus
with both positive and negative selections. This would explain
why experimental model systems show that T cells against
dominant self-determinants get positive tolerance, whereas
those potentially directed against cryptic epitopes escape
tolerance induction. Under normal physiological conditions,
the cryptic epitopes of a native antigen are unproductively
processed and presented and there is no threat of initiation of
an anti-self immune response by such epitopes. However, under
inflammatory and other specific conditions, upregulation of
antigen-processing events can lead to improved presentation of
the previously cryptic epitopes by the antigen-presenting cells,
that can lead to priming cryptic-epitope specific T cells.
The eye consists of unique cells bearing unique molecules.
Moreover, the internal compartments of the eye exist behind a
blood–tissue barrier. The very uniqueness of ocular molecules,
and their presumed sequestration from the systemic immune
system, has provoked immunologists to speculate that ocular
autoimmunity arises when, via trauma or infection, eye-specific
antigens are ‘revealed’ to the immune system. Sympathetic
ophthalmia is a disease that almost fits this scenario perfectly.
Trauma to one eye, with attendant disruption of the
blood–ocular barrier and spillage of ocular tissues and
molecules, leads to a systemic immune response that is specific
to the eye. This response is directed not only at the traumatized
eye but also at its putatively normal fellow eye. However, even
in sympathetic ophthalmia, not every case of ocular trauma
leads to this outcome; in fact, only in a few cases does this type
of injury produce inflammation in the undamaged eye.
Suspicion is high that polymorphic genetic factors may be
responsible for determining who will, and who will not, develop
sympathetic ophthalmia following ocular injury. However,
environmental factors may also participate.
Range of Hypersensitivity Reactions Mediated by
T Cells
Because a wealth of new information about T cell-mediated
immunopathology has accrued within the past decade, our
ideas about the range of hypersensitivity reactions that can be
mediated by T cells have expanded. But, as yet, any attempt to
classify these reactions must necessarily be incomplete. In the
past, four types of delayed hypersensitivity reactions were
described:1 tuberculin,2 contact hypersensitivity,3 granulo-
matous, and Jones–Mote.4 Delayed hypersensitivity reactions of
these types were believed to be caused by IFN-g-producing CD4+
T cells and to participate in numerous ocular inflammatory
disorders, ranging from allergic keratoconjunctivitis, through
Wegener’s granulomatosis, to drug contact hypersensitivity.
Based on recent knowledge concerning other types of effector T
 Immune-Mediated Tissue Injury

cells, this list must be expanded to include cytotoxic T cells,
and proinflammatory, but not IFN-g-secreting, Th2 type cells,
such as the cells that are believed to cause corneal clouding in
river blindness.29
Additionally, graft versus host disease is a result of cellular
immunity and is an example of a delayed T-helper cell response.
A rejected allograft has a similar histological appearance to a
tuberculin reaction, and rejection is mediated by T cells with an
with HSK in a yet different strain of mice. Fourth, T cells have
been found in association with HSK that recognize an antigen
uniquely expressed in the cornea. The evidence suggests that
this corneal antigen is unmasked during a corneal infection
with HSV, and an autoimmune response is evoked in which the
cornea becomes the target of the attack.
Only time will tell whether similar immunopathogenic
mechanisms will prove to be responsible for HSK in humans,

important role for the NK cells.30 The histopathological
findings are mononuclear cell infiltration and tissue
destruction. The CD8+ T cells are the primary cells inducing
the lesions, although a minor role for CD4+ has been described.
As in the other hypersensitivity reactions, this one is a clear
example of an anomaly in a well-organized cellular response to
pathogens. T cells represent the best choice against intracellular
infections, usually viral, in order to prevent further damage and
offspring of the infective agent.5 There appears to be a
connection between antecedent viral infection, susceptible
MHC class II alleles, and the inception of certain diseases
included in this range. The protective mechanisms to the host
(such as control of cell proliferation by cytokines or induction of
apoptosis of target cells by different ways) are the same as those
which cause injurious effects to the host.
Herpes Simplex Keratitis as an Example of T Cell-
Mediated Ocular Inflammatory Disease
Infections of the eye with herpes simplex virus are significant
causes of morbidity and vision loss in developed countries.
Although direct viral toxicity is damaging to the eye, the
majority of intractable herpes infections appear to be
immunopathogenic in origin. That is, the immune response to
antigens expressed during a herpes infection leads to tissue
injury and decompensation, even though the virus itself is
responsible for little pathology directly. Herpes stromal keratitis
(HSK) is representative of this type of disorder.31
Numerous experimental model systems have been developed
in an effort to understand the pathogenesis of HSK. Perhaps the
most informative studies have been conducted in laboratory
mice. Evidence from these model systems indicates that T cells
are central to the corneal pathology observed in HSK.31 At least
four different pathogenic mechanisms have been discovered,
each of which alone can generate stromal keratitis. Genetic
factors of the host seem to play a crucial role in dictating which
mechanism will predominate. First, HSV-specific cytotoxic T
cells can cause HSK and do so in several strains of mice.
Second, HSV-specific T cells of the Th1 type, which secrete
IFN-g and mediate delayed hypersensitivity, also cause HSK,
but in genetically different strains of mice. Third, HSV-specific
T cells of the Th2 type, that secrete IL-4 and IL-10, correlate
CHAPTER 9
but the likelihood is very great that this will be the case.
Furthermore, it is instructive to emphasize that quite different
pathologic T cells can be involved in ocular pathology, which
implies that it will be necessary to devise different therapies in
order to meet the challenge of preventing immunopathogenic
injury from proceeding to blindness.
SUMMARY
Faced with a patient who is experiencing extraocular or
intraocular inflammation, the thoughtful ophthalmologist will
try, to the best of his or her ability, to diagnose the specific cause
of the inflammation, or at the very least to investigate the
problem so that the mechanisms responsible for the
inflammation are understood as completely as possible. Armed
with this knowledge, the ophthalmologist is then prepared to
formulate an appropriate therapeutic plan rather than to
indiscriminately prescribe corticosteroids. It is clear as we move
into the twenty-first century that the past four decades of
relative neglect of ocular immunology by mainstream
ophthalmic practitioners is coming to an end. Most
ophthalmologists are no longer satisfied to cultivate practices
devoted exclusively to the ‘tissue carpentry’ of cataract surgery
or even to a broad-based ophthalmic practice that includes
‘medical ophthalmology’ but is restricted to problems related
exclusively to the eye (e.g., glaucoma) and divorced from the eye
as an organ in which systemic disease is often manifested. More
ophthalmologists than ever before are demanding the
continuing education they need to satisfy intellectual curiosity
and to prepare for modern care of the total patient when a
patient presents with an ocular manifestation of a systemic
disease. It is to these doctors that this chapter is directed.
The eye can be affected by any of the immune hypersensitivity
reactions, and understanding the mechanism of a particular
patient’s inflammatory problem lays the ground-work for
correct treatment. In the course of the average ophthal-
mologist’s working life, the diagnostic pursuit of mechanistic
understanding will also result in a substantial number of
instances when the ophthalmologist has been responsible for
diagnosing a disease that, if left undiagnosed, would have
been fatal.

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2. Bach JF: The effect of infections on
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10. Foster CS, Duncan J: Randomized clinical
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11. Foster CS: Evaluation of topical cromolyn
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15. Mark J, Kaufman HE, Insler M: Topical
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20. Sainz de la Maza M, Foster CS: Necrotizing
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21. Leib ES, Restivo C, Paulus AT:
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22. Wolf SM, Fauci AS, Horn RG, Dale DC:
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23. Foster CS, Forstot SL, Wilson LA: Mortality
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peripheral ulcerative keratitis.
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 CHAPTER
10 Regulation of Immune Responses
Reza Dana and C. Stephen Foster
Immunization with an antigen leads, under normal circum-
stances, to a robust immune response in which effector T cells
and antibodies are produced with specificity for the initiating
antigen (see Chapter 5). The purpose of these effectors is to
recognize and combine with antigen (e.g., on an invading
pathogen) in such a manner that the antigen (pathogen) and/or
infected cell are eliminated. Once the antigen has been elimi-
nated, there is little need for the persistence of high levels of
effector cells and antibodies, and what is regularly observed is
that levels of these effectors in blood and peripheral tissues fall
dramatically. Only the T cells and B cells that embody antigen-
specific memory are retained.
The ability of the immune system to respond to an antigenic
challenge in a sufficient, and yet measured, manner is a dramatic
expression of the ability of the system to regulate itself. If it
were not for this capacity, uncontrolled expansion of immune
cells against an antigen would wreak havoc in the host and
cause significant morbidity, or even lymphomatous spread of
these cells. It is therefore critical to have an understanding of
how immunity regulates itself so that its response is checked
tightly in both time and space. Table 10.1 lists several of the
key methods by which immunity is regulated locally and
systemically.
REGULATION BY ANTIGEN
Antigen itself is a critical factor in regulating an immune
response. When nonreplicating (e.g., nonviral) antigens have
been studied, it has been found that the high concentration of
antigen required for initial sensitization begins to fall through
time. In part, this occurs because antibodies produced by
immunization interact with the antigen and cause its elimi-
nation. As the antigen concentration falls, the efficiency with
which specific T and B cells are stimulated to proliferate and
TABLE 10.1. Levels of Immune Regulation
Regulation by antigen
Phenotype of the T-cell response (T-helper (Th)-1 and Th-2)
Suppressor/regulatory T cells
Induction of tolerance
Anergy
Clonal deletion
Suppression
Immune deviation
differentiate also falls, and eventually, when antigen concen-
tration slips below a critical threshold, further activation of
specific lymphocytes stops. The use of anti-Rh antibodies
(RhoGAM) to prevent sensitization of Rh-negative women
bearing Rh-positive fetuses is a clear, clinical example of the
ability of antibodies to terminate (and in this particular case,
even prevent) a specific (unwanted) immune response.
REGULATION BY TH1 AND TH2 CELLS
More than 20 years ago, experimentalists discovered that certain
antigen-specific T lymphocytes are capable of suppressing
immune responses,1 and the mechanism of suppression was
found to be unrelated to the simple act of ‘clearing the antigen’
from the system. It is now understood that CD4+ T cells primed
against an antigen can differentiate into two largely distinct
‘phenotypes’, called T-helper 1 (Th1) and Th2, based on the
cytokine products they secrete, which in turn have a significant
effect in the character of the secondary responses generated
against that antigen.
Helper T cells are so called since they facilitate other lympho-
cytes to differentiate into effector and antibody-producing cells.
Any particular immunizing event does not necessarily lead to
the production of the entire array of effector modalities, and one
of the reasons for this is that helper T cells tend to polarize into
one or other of two largely distinct phenotypes.2 Th1 cells
provide a type of help that leads to the generation of T-cell
effectors that mediate delayed hypersensitivity, and B cells that
secrete complement-fixing antibodies, and they perform this
function chiefly through expression of specific cytokines such as
interferon (INF)-g, tumor necrosis factor (TNF)-a, and inter-
leukin (IL)-2. By contrast, Th2 cells provide a type of help that
leads to the generation of B cells that secrete non-complement-
fixing IgG antibodies, as well as IgA and IgE, critical in many
humoral antibody-mediated responses in many conditions
including allergy and immunity against parasitic infection. In
turn, the ability of Th2 cells to promote these types of antibody
responses rests with their capacity to secrete a different set of
cytokines-IL-4, IL-5, IL-6, and IL-10.
As it turns out, Th1 and Th2 cells can crossregulate each
other. Thus, Th1 cells with specificity for a particular antigen
secrete IFN-g, and in the presence of this cytokine, Th2 cells
with specificity for the same antigen fail to become activated.
Similarly, if Th2 cells respond to a particular antigen by
secreting their unique set of cytokines (e.g., IL-10), Th1 cells in
the same microenvironment are prevented from responding to
the same antigen. Thus, precocious activation of Th1 cells to an
antigen, such as ragweed pollen, may prevent the activation
of ragweed-specific Th2 cells and therefore prevent the produc-
tion of ragweed-specific IgE antibodies. Alternatively, precocious 91
 IMMUNOLOGY
activation of Th2 cells to an antigen (e.g., urushiol, the agent
responsible for poison ivy dermatitis) may prevent the
activation of urushiol-specific Th1 cells and thus eliminate the
threat of dermatitis when the skin is exposed to the leaf of the
poison ivy plant.
As it turns out, there is more to regulation and differentiation
of T cells than the neat dichotomy afforded by the Th1/Th2
paradigm. Nevertheless, the discovery of Th1 and Th2 cell
SECTION 2
diversity has led to a profound rethinking of immune regulation.
However, it is still too early to know precisely the extent to
which the ability to influence an immune response toward the
Th1 or Th2 phenotypes will have therapeutic value in humans.
REGULATION BY SUPPRESSOR
(‘REGULATORY’) T CELLS
‘Suppressor’ T cells are defined operationally as cells that
suppress an antigen-specific immune response.3 Cells of this
functional property were actually described before the discovery
of Th1 and Th2 cells. While it is now apparent that some of the
phenomena attributed to suppressor T cells initially are actually
explained by the crossregulating abilities of Th1 and Th2 cells,
there are distinct examples of immune suppression that cannot
be explained by either Th1 or Th2 cells.
The designation ‘suppressor’ T cell has evolved over the past
decade in favor of ‘regulatory’ T cells. Various experimental
maneuvers have been described that lead to the generation of
these T cells. These include1 injection of soluble protein antigen
intravenously,2 application of a hapten to skin previously exposed
to ultraviolet B radiation,4 ingestion of antigen by mouth,3
injection of allogeneic hematopoietic cells into neonatal mice,5
injection of antigen-pulsed antigen-presenting cells (APCs) that
have been treated in vitro with transforming growth factor
(TGF)-b or with fluids replete with immunosuppressive
cytokines (e.g., aqueous humor, cerebrospinal fluid, or amniotic
fluid),6 and engraftment of a solid tissue (e.g., heart, kidney)
under cover of immunosuppressive agents. In each of these
examples, T cells harvested from the lymphoid organs of these
experimentally manipulated animals induce antigen-specific
unresponsiveness when injected into immunologically com-
petent but naive (antigen-inexperienced) animals.5
What is key, however, is that the suppressor function of
regulatory T cells is now understood not to be simply a con-
sequence of experimental manipulation of laboratory animals,
but also an important part of normal physiology that is critical
in preventing autoimmunity.6,7 Whether experimentally induced,
or normally present, the cast of regulatory T cells that induce
unresponsiveness to self or foreign antigens is highly hetero-
geneous; these cells can be CD4+ or CD8+ or even natural killer
(NK) T cells.8 Some of the CD8 cells (the classically defined
‘suppressor’ T cells) inhibit the activation of CD4+ helper or
CD8+ cytotoxic T cells, whereas others interfere with B-cell
function. There are even suppressor cells that inhibit the
activation and effector functions of macrophages and other
APCs. The mechanisms that mediate the suppressor function
of regulatory T cells are the subject of intense current investi-
gation. Certain T cells secrete immunosuppressive cytokines,
such as TGF-b or IL-10, whereas other regulatory cells inhibit
the function of other cells only when they make direct cell-
surface contact with target cells; example of the latter include
CD4+CD25+ cells.6,9
TOLERANCE AS AN EXPRESSION OF
IMMUNE REGULATION
Classic immunologic tolerance is defined as the state in which
92 immunization with a specific antigen fails to lead to a detec-
table immune response. In this sense, tolerance represents the
ultimate expression of the effectiveness of immune regulation
since the unresponsiveness is maintained. Originally described
experimentally in the 1950s,10 but accurately predicted by
Ehrlich and other immunologists at the end of the nineteenth
century, immunologic tolerance has been the subject of
considerable study during the past 50 years. It has been learned
that several distinct mechanisms contribute singly, or in
unison, to creation of the state of tolerance. These mechanisms
include clonal deletion, clonal anergy, suppression, and
immune deviation.
MECHANISMS INVOLVED IN TOLERANCE
The term ‘clonal’ refers to a group of lymphocytes all of which
have identical receptors for a particular antigen. During regular
immunization, a clone of antigen-specific lymphocytes responds
by proliferating and undergoing differentiation. ‘Clonal deletion’
refers to an aberration of this process in which a clone of
antigen-specific lymphocytes responds to antigen exposure by
undergoing apoptosis (programmed cell death). Deletion of a
clone of cells in this manner eliminates the ability of the
immune system to respond to that antigen, hence rendering the
immune system tolerant to that antigen. Subsequent exposures
to the same antigen fail to produce the expected immune
response (sensitized T cells and antibodies) because the relevant
antigen-specific T and B cells are missing.
‘Clonal anergy’ resembles clonal deletion in that a particular
clone of antigen-specific lymphocytes fails to respond to antigen
exposure by proliferating and undergoing differentiation.
However, in clonal anergy, the lymphocytes within the clone
are not triggered to undergo apoptosis. Rather, due to inade-
quate co-stimulation of the T cells by specific molecules, they
fail to become adequately activated to expand, but rather enter
an altered state in which their ability to respond is suspended,
even though these cells survive this encounter with antigen.
Still, subsequent encounters continue to fail to cause their
expected activation, rendering the immune system tolerant of
that antigen.
Antigen-specific immune suppression or regulation, as
described earlier, is another mechanism that has been shown to
cause immunologic tolerance. As in clonal deletion and anergy,
immune suppression creates a situation in which subsequent
encounters with the antigen in question fail to lead to signs of
sensitization. However, in suppression, the failure to respond is
actively maintained.
Immune deviation is a special form of immune suppression.11
Originally described in the 1960s, immune deviation refers to
the situation where administration of an antigen in a particular
manner leads to a response, but fails to elicit the expected
response. In the first such experiments, soluble antigens
injected intravenously into naive experimental animals failed to
induce delayed hypersensitivity responses. With respect to
delayed hypersensitivity, one could say that the animals were
tolerant. However, the sera of these animals contained unex-
pectedly large amounts of antibody to the same antigen,
indicating that the so-called tolerance was not global, but rather
‘deviant’. In other words, the immune response is deviated from
the expected pattern.
FACTORS THAT PROMOTE TOLERANCE
RATHER THAN IMMUNITY
Experimentalists have defined various factors that influence
or promote the development of immunologic tolerance. The
earliest description of tolerance occurred when antigenic mat-
erial was injected into newborn (and therefore developmentally

immature) mice. This indicates that exposure of the developing
immune system to antigens before the system has reached
maturity leads to antigen-specific unresponsiveness. However,
tolerance can also be induced when the immune system is
developmentally mature. The factors that are known to
promote tolerance under these conditions include the physical
structure of the antigen, the dose of antigen, and the route of
antigen administration. More specifically, soluble antigens are
more readily able to induce tolerance than particulate or
insoluble antigens. Very large doses as well as extremely small
quantities of antigens are also likely to induce tolerance. This
indicates that the immune system is disposed normally to
respond to antigens within a relatively broad, but defined,
range of concentrations or amounts. Injection of antigen
intravenously, or its ingestion,12 favors tolerance induction,
whereas injection of antigen cutaneously favors conventional
sensitization.
Additional factors influencing whether tolerance is induced
concern the status of the immune system itself. For example,
antigen X may readily induce tolerance when injected intra-
venously into a normal, immunologically naive individual.
However, if the same antigen is injected into an individual
previously immunized to antigen X, then tolerance will not
occur. Thus, a prior state of sensitization mitigates against
tolerance induction. Alternatively, if a mature immune system
has been assaulted by immunosuppressive drugs, either by debi-
litating systemic diseases, or by particular types of pathogens
(the human immunodeficiency virus is a good example), it may
display increased susceptibility to tolerance. Thus, when an
antigen is introduced into an individual with a compromised
immune response, tolerance may develop and be maintained,
even if the immune system recovers.
REGIONAL IMMUNITY AND THE EYE
In the Overview of Immunology chapter, we discussed how
evolution had to meet the challenge of ‘designing’ an immune
system that is at once capable of responding to pathogenic
antigens with a response that is effective in eliminating the
threat, while at the same time not damaging the tissue itself.
Because pathogens with different virulence strategies threaten
different types of tissues, the immune system consists of a
diversity of immune effectors. The diversity includes different
populations (e.g., CD4, CD8) of effector T cells and different
types of antibody molecules (IgM, IgG1, IgG2, IgG3, IgG4, IgA,
and IgE). Thus, different tissues and organs display markedly
different susceptibilities to immune-mediated tissue injury. The
regional specificity of an immune response is nowhere better
manifested than in the eye.13 Because integrity of the micro-
anatomy of the visual axis is absolutely required for accurate
vision, the eye can tolerate inflammation to only a very limited
degree. Vigorous immunogenic inflammation, such as that found
in a typical delayed hypersensitivity reaction in the skin, wreaks
havoc with vision, and it has been argued that the threat of
blindness has dictated an evolutionary adaptation in the eye
that limits the expression of inflammation. Therefore, certain
aspects of immunity in the eye are considered ‘deviant’ or
‘privileged’, a description of which follows.
OCULAR SURFACE IMMUNITY –
CONJUNCTIVA, LACRIMAL GLAND, TEAR
FILM, CORNEA, AND SCLERA
The human conjunctiva is an active participant in immune
defense of the ocular surface against invasion by exogenous sub-
stances. The presence of blood vessels and lymphatic channels
fosters transit of immune cells that can participate in the
Regulation of Immune Responses
afferent and efferent arms of the immune response. The
marginal and peripheral palpebral arteries and anterior ciliary
arteries are the main blood suppliers of the conjunctiva.
Lymphatics of the palpebral conjunctiva on the lateral side
drain into the preauricular and parotid lymph nodes, whereas
the lymphatics draining the palpebral conjunctiva on the medial
side drain into the submandibular lymph nodes. Major immune
cells found in normal human conjunctiva are dendritic cells,
CHAPTER 10
T and B lymphocytes, mast cells, and neutrophils. Dendritic
cells, Langerhans’ cells, and macrophages have been detected
in different regions of the conjunctiva and cornea, but the
normal cornea is devoid of T cells.14,15 Dendritic cells act as
APCs to stimulate antigen-specific T lymphocyte responses.15
T lymphocytes, the predominant lymphocyte subpopulation in
conjunctiva, are represented in the epithelium and the sub-
stantia propria. T lymphocytes are the main effector cells in
immune reactions such as delayed hypersensitivity or cytotoxic
responses. B lymphocytes are fewer, and mostly scattered in the
substantia propria of the fornices. Plasma cells are detected only
in the conjunctival accessory lacrimal glands of Krause or minor
lacrimal glands.16 Plasma cells from major and minor lacrimal
glands synthesize Igs, mainly IgA.17,18 IgA is a dimer that is
transported across the mucosal epithelium bound to a receptor
complex. IgA dimers are released to the luminal surface of the
ducts associated with a secretory component after cleavage of
the receptor and are excreted with the tear film. Secretory IgA is
a protectant of mucosal surfaces. Although secretory IgA does
not seem to be bacteriostatic or bactericidal, it may modulate
the normal flora of the ocular surface.19 Foreign substances can
be processed locally by the mucosal immune defense system.
After exposure to antigen, specific IgA helper T lymphocytes
stimulate B lymphocytes to differentiate into IgA-secreting
plasma cells. Dispersed T and B lymphocytes and IgA-secreting
plasma cells of the conjunctiva and lacrimal gland are referred
to as the conjunctival and lacrimal gland-associated lymphoid
tissue (CALT).17 CALT is considered part of a widespread
mucosa-associated lymphoid tissue (MALT) system, which
includes the oral mucosa and salivary gland-associated
lymphoid tissue, the gut-associated lymphoid tissue (GALT),
and the bronchus-associated lymphoid tissue (BALT). CALT
drains to the regional lymph nodes in an afferent arc; effector
cells may in turn return to the eye via an efferent arc comprised
of blood vessels; in this the lymph and blood vessels contribute
to different aspects (induction and expression, respectively) of
the immune system on the ocular surface.20
Mast cells are located mainly perilimbally, although they can
also be found in bulbar conjunctiva. Their degranulation in
response to an allergen or an injury results in the release of
vasoactive substances such as histamine, heparin, platelet-
activating factor, and leukotrienes, which can cause blood vessel
dilatation and increased vascular permeability. The tears contain
several substances known to have antimicrobial properties.
Lysozyme, immunoglobulins, and lactoferrin may be synthe-
sized by the lacrimal gland. Lysozyme is an enzyme capable of
lysing bacteria cell walls of certain Gram-positive organisms.
Lysozyme may also facilitate secretory IgA bacteriolysis in the
presence of complement. The tear IgG has been shown to
neutralize virus, lyse bacteria, and form immune complexes
that bind complement and enhance bacterial opsonization and
chemotaxis of phagocytes. Lactoferrin, an iron-binding protein,
has both bacteriostatic and bactericidal properties.21 Lactoferrin
may also interact with a specific antibody to produce an
antibacterial effect more powerful than that of either lactoferrin
or antibody alone.22
The unique anatomic and physiologic characteristics of the
human cornea explain, on the one hand, its predilection for
involvement in various immune disorders and, on the other 93
 IMMUNOLOGY
hand, its ability to express immune privilege.23 The peripheral
cornea differs from the central cornea in several ways. The
former is closer to the vascularized and lymphatic-rich con-
junctiva, rendering the peripheral cornea much more immuno-
reactive. The limbal vasculature allows diffusion of some
molecules, such as immunoglobulins and complement com-
ponents, into the cornea; moreover, it significantly facilitates
the recruitment of a wide variety of leukocyte populations into
SECTION 2
the peripheral corneal matrix since the intravascular compart-
ment is the chief source of these bone marrow-derived cells.24,25
IgG and IgA are found in similar concentrations in the peri-
pheral and central cornea; however, more IgM is found in the
periphery, probably because its high molecular weight restricts
diffusion into the central area.25,26 Both classic and alternative
pathway components of complement and its inhibitors have
been demonstrated in normal human corneas. However
although most of the complement components have a
peripheral-to-central cornea ratio of >1, C1 is denser in the
periphery by a factor of five. The higher concentration of
antibodies, complement components, APCs, and inflammatory
leukocytes in the corneal periphery and perilimbal area make
the peripheral cornea far more susceptible to involvement in a
wide variety of autoimmune and hypersensitivity disorders,
such as Mooren’s ulcer and collagen vascular diseases.27
The sclera consists almost entirely of collagen and proteo-
glycans. It is traversed by the anterior and posterior ciliary
vessels but retains a scanty vascular supply for its own use. Its
nutrition is derived from the overlying episclera and underlying
choroid;28 similarly, both classic and alternative pathway
components of complement are derived from these sources.29
Normal human sclera has few, if any, lymphocytes, macro-
phages, Langerhans’ cells, or neutrophils. In response to an
inflammatory stimulus in the sclera, the cells pass readily from
blood vessels of the episclera and choroid. Because of the
collagenous nature of the sclera, many systemic autoimmune
disorders, such as the collagen vascular diseases, may affect it.30
INTRAOCULAR IMMUNITY AND OCULAR
IMMUNE PRIVILEGE
For more than 100 years, it has been known that foreign tissue
grafts placed within the anterior chamber of an animal’s eye can
be accepted indefinitely.31 The designation of this phenomenon
as immune privilege had to await the seminal work of Medawar
and colleagues, who discovered the principles of transplantation
immunology in the 1940s and 1950s. These investigators
studied immune privileged sites – the anterior chamber of the
eye, the brain – as a method of exploring the possible ways to
thwart immune rejection of solid tissue allografts.32–34 It had
been learned that transplantation antigens on grafts were
carried to the immune system via regional lymphatic vessels
and that immunization leading to graft rejection took place
within draining lymph nodes. Because the eye and brain were
regarded at the time as having no lymphatic drainage (a concept
that has since been shown to be fallacious), and because both
tissues resided behind a blood–tissue barrier, Medawar and
associates postulated that immune privilege resulted from
immunologic ignorance. What these investigators meant was
that foreign tissues placed in immune-privileged sites were
isolated by physical vascular barriers ( ‘antigenic sequestration’)
from the immune system and that they never alerted the
immune system to their existence. During the past quarter
century and more, immunologists who have studied immune
privilege at various sites in the body have learned that this
original postulate is basically untrue.35–39 First, some privileged
sites possess robust lymphatic drainage pathways – the testis is
94 a good example. Second, antigens placed in privileged sites,
including the cornea,40 are known to escape and drain to distant
sites, including lymphoid organs such as the lymph nodes and
spleen. Third, antigens in privileged sites evoke antigen-
specific, systemic immune responses, albeit of a unique nature.
Thus, the modern view of immune privilege states that privilege
is an actively acquired, dynamic state in which the immune
system conspires with the privileged tissue or site in generating
a response that is protective, rather than destructive.
IMMUNE-PRIVILEGED TISSUES AND SITES
Immune-privileged sites (Table 10.2) are regions of the body
where allografts survive for extended, even indefinite, periods of
time, compared with nonprivileged, or conventional sites where
these same allografts are readily rejected. The eye contains
examples of both privileged tissues and sites, of which the best-
studied site is the anterior chamber, and the best-studied tissue
is the cornea. Much has been learned about the phenomenon of
immune privilege since the 1990s. The forces that confer
immune privilege have been shown to act during both induction
and expression of the immune response to antigens placed
within, or expressed on, privileged sites and tissues.
The forces that shape immune-privileged sites and tissues
include an ever-expanding list of microanatomic, biochemical,
and immunoregulatory features. A short list of privilege-
promoting features is displayed in Table 10.3. The eye expresses
virtually every one of these features. Although passive physical
features such as the blood–ocular barrier, lack of lymphatics,
and low expression of major histocompatibility complex (MHC)
class I and II molecules are important, experimental attention
has focused on immunomodulatory molecules expressed on
ocular tissues and present in ocular fluids.
REGULATION OF IMMUNE EXPRESSION IN
THE EYE
There are many levels at which immune privilege is maintained
in the eye, covering virtually every step of the induction and
expression of immunogenic inflammation.14,23,34,38 APCs are
kept at an immature state, rendering them highly capable of
picking up foreign antigen but poor in stimulating T cells; lack
of lymphatics in the cornea reduces the efficiency with which
antigen-laden APCs can gain access to lymphoid tissues; lack of
TABLE 10.2. Immune Privileged Sites
Eye
Cornea
Vitreous cavity
Subretinal space
Lens
Brain
Cartilage
Placenta/fetus
Testis
Ovary
Adrenal cortex
Liver
Hair follicles
Tumors

TABLE 10.3. Features of Immune Privileged Sites
Passive
Blood–tissue barriers
Deficient efferent lymphatics
Tissue fluid that drains into blood vasculature
Reduced expression of major histocompatibility complex class I
and II molecules
Active
Constitutive expression of inhibitory cell surface molecules: Fas
ligand, DAF, CD59, CD46
Immunosuppressive microenvironment: TGF-b, a-MSH, VIP, CGRP,
MIF, free cortisol
MIF, melanocyte-inhibiting factor; MSH, melanocyte-stimulating hormone; VIP,
vasoinhibitory peptide; CGRP, calcitonin gene-related peptide.
blood vessels and maintenance of the blood–ocular barrier
reduces the efficiency by which effector T cells can gain access
to ocular tissues; and immunosuppressive and proapoptotic
signals in the eye actively suppress or delete lymphocytes that
have gained access to ocular compartments.38 Herein, we shall
focus on a few of the mechanisms that regulated T-cell activa-
tion in the eye.
It is know that activated T cells upregulate expression of the
death receptor, Fas (CD95), on their surface, and by doing so
become vulnerable to programmed cell death if they encounter
other cells that express Fas ligand (CD95L).41 Constitutive
expression of Fas ligand on cells that surround the anterior
chamber has been shown to induce apoptosis among T cells and
other Fas+ leukocytes exposed to this anterior chamber.42 More
important, Fas ligand expressed by cells of the cornea play a key
role in rendering the cornea resistant to immune attack and
rejection.43 Similarly, constitutive expression on corneal endo-
thelial cells, as well as iris and ciliary body epithelium, of
several membrane-bound inhibitors of complement activation
are strategically located to prevent complement-dependent intra-
ocular inflammation and injury.44 More recently, another factor,
which is a member of the B7 costimulatory superfamily, known
as programmed death ligand-1 (PD-L1) has been shown to be
constitutively expressed at very high levels by the cornea, impli-
cating this factor in the active deletion of PD-1+ T cells from the
anterior segment.
Cells that are not deleted/killed in this microenvironment are
rendered less hostile by a highly immunosuppressive milieu.
For example, transforming growth factor-beta 2 (TGF-b2), a
normal constituent of aqueous humor,45 is a powerful immuno-
suppressant that inhibits various aspects of T cell and macro-
phage activation. Other relevant factors in the aqueous humor
include alpha-melanocyte-stimulating hormone,46 vasoactive
intestinal peptide,47 calcitonin gene-related peptide,48 and
macrophage migration inhibitory factor,49 among others. It is
important to emphasize, however, that aqueous humor does not
inhibit all immune reactivity. For example, antibody neutral-
ization of virus infection of target cells is not prevented in the
presence of aqueous humor.50
REGULATION OF INDUCTION OF IMMUNITY
TO EYE-DERIVED ANTIGENS
Another dimension to immune privilege is the ability of the eye
to regulate the nature of the systemic immune response to
antigens placed within it, an issue of paramount importance
Regulation of Immune Responses
as it is the systemic immune response that plays a critical role
in sustaining immunity in peripheral tissues including the
eye. It has been known from the 1980s that injection of allo-
antigenic cells into the anterior chamber of rodent eyes evokes
a distinctive type of immune deviation, now called anterior
chamber-associated immune deviation (ACAID).51,52 In
ACAID, eye-derived antigens elicit an immune response that is
selectively deficient in T cells that mediate delayed hyper-
CHAPTER 10
sensitivity, and B cells that secrete complement-fixing anti-
bodies. There is not, however, a global lack of response, because
animals with ACAID display a high level of antigen-specific
serum antibodies of the non-complement-fixing varieties and
primed cytotoxic T cells.37 In ACAID, regulatory T cells are also
generated which, in an antigen-specific manner, suppress both
the induction and expression of delayed hypersensitivity to the
antigen in question.53,54 ACAID can be elicited by diverse types
of antigens, ranging from soluble protein to histocompatibility
to virus-encoded antigens.
Induction of ACAID by intraocular injection of antigen
begins within the eye itself.55–57 After injection of antigen into
the eye, local APCs capture the antigen, migrate across the
trabecular meshwork into the canal of Schlemm, and then
traffic via the blood to the spleen. In the splenic white pulp,
the antigen is presented in a unique manner to T and
B lymphocytes, resulting in the spectrum of functionally
distinct antigen-specific T cells and antibodies found in
ACAID. The ocular microenvironment sets the stage for this
sequence of events by virtue of the immunoregulatory
properties of the aqueous humor described earlier. This ocular
fluid, or more precisely, TGF-b2, confers upon conventional
APCs the capacity to induce ACAID. Thus, the ocular micro-
environment not only regulates the expression of immunity
within the eye, but also the functions of eye-derived APCs and
thus promotes a systemic immune response that is deficient in
those immune effector modalities most capable of inducing
immunogenic inflammation-delayed hypersensitivity T cells
and complement-fixing antibodies.
IMMUNE PRIVILEGE AND INTRAOCULAR
INFLAMMATORY DISEASES
Ocular immune privilege has been implicated in1 the extra-
ordinary success of corneal allografts,58–62 progressive growth of
intraocular tumors,63 resistance to herpes stromal keratitis,64
and4 suppression of autoimmune uveoretinitis.65,66 When
immune privilege prevails within the eye, corneal allografts
succeed; trauma to the eye heals without incident; and ocular
infections are cleared without inflammation. However, the price
of this compromise is that ocular tumors may then grow relent-
lessly, and uveal tract infections may persist and recur.34,37 In
contrast, the consequences of failed immune privilege are
protean. For example, ocular trauma may result in sympathetic
ophthalmia, ocular infections may produce sight-threatening
inflammation, and corneal allografts may undergo irreversible
rejection.
CORNEAL TRANSPLANTATION
IMMUNOLOGY
Our objective here is not to provide a thorough review of the
immunobiology of corneal transplantation, which has been
extensively reviewed elsewhere.61–67 Rather, we shall focus on
the mechanisms of ocular immune privilege as they affect the
fate of corneal allografts, and demonstrate how abrogation of
such privilege can lead to immunogenic graft failure.
The cornea is an immune privileged tissue and, in part, this
accounts for the extraordinary success of corneal transplants in 95
 IMMUNOLOGY
both experimental animals and humans. However, despite the
many advances that have been made in corneal tissue pre-
servation and surgical techniques, a significant proportion of
grafts eventually fail,68 and this is nowhere as significant a
problem as when grafts are placed onto inflamed and
neovascularized host beds. Regardless of host bed parameters,
or the indication for transplantation, the main cause of corneal
graft failure is immune-mediated graft rejection, the rate
SECTION 2
ranging from as low as 10% in grafts performed for keratoconus
and bullous keratopathy, to well over 50% in grafts performed
for corneal burns and other conditions associated with surface
disease and stromal vascularization.61 Corneal vascularization,
either preoperative from recipient herpetic, interstitial, or
traumatic keratitis, or stimulated by silk or loose sutures,
contact lenses, infections, persistent epithelial defects, and
other disorders associated with inflammation, has been widely
recognized as a clear risk factor for decreased graft survival.
Other factors that increase the risk of allograft rejection include
a history of previous graft loss, eccentric and large grafts, and
glaucoma.69–71
TRANSPLANT ANTIGENS ON CORNEAL
TISSUE
In outbred species, such as humans, where genotypic variation
is high, transplants of solid tissue grafts usually fail unless the
recipient is immunosuppressed. The reason for this is
development of an immune response directed at so-called
transplantation antigens displayed on cells of the graft.
Immunologists have separated transplantation antigens into
two categories: ‘major’ and ‘minor’, primarily because of purely
empirical evidence that major antigens induce more vigorous
alloimmunity than do minor antigens. The genes that encode
the major transplantation antigens in humans are located
within the MHC, called human leukocyte antigen (HLA).
Minor histocompatibility antigens are encoded at numerous
loci spread throughout the genome. The HLA complex, which
is a large genetic region, is situated on the short arm of the sixth
human chromosome. HLA genes that encode class I and class
II antigens are extremely polymorphic. Similarly, minor
histocompatibility loci contain highly polymorphic genes. In
the aggregate, polymorphisms at the major and minor
histocompatibility loci account for the observation that solid
tissue grafts exchanged between any two individuals selected at
random within a species are acutely rejected.
The expression of HLA antigens on corneal cells is somewhat
atypical.72–74 Class I MHC antigens are expressed strongly on
the epithelial cells of the cornea, comparable in intensity to
the expression of epidermal cells of skin. Keratocytes express
less class I than conventional fibroblasts, and corneal
endothelial cells express small amounts of class I antigens
under normal circumstances. Additionally, class II MHC
(e.g., HLA D/DR) antigen expression is essentially absent in
the normal corneal tissue. However, corneal cells respond to
specific cytokines, such as INF-g, by upregulating MHC antigen
expression.
If the normal cornea exhibits little MHC expression, but can
acquire high-level expression when inflamed, what is the
benefit of tissue matching? The evidence for HLA tissue typing
in corneal transplantation is conflicting.75–81 There seems to
be little controversy regarding the influence of tissue typing
on grafts placed in eyes of low-risk patients. In the low-risk
situation, with a few exceptions,81 virtually no studies suggest a
positive typing effect. Most likely, the rate of graft success is
so high in low-risk transplants under cover of topical steroids
that there is little opportunity for a matching effect to be seen.
96 However, in high-risk situations, the literature contains many
disparate reports with conflicting conclusions regarding the
utility of HLA matching. On balance, however, notwithstanding
the results of the Collaborative Corneal Transplantation
Studies (CCTS), a multicenter study completed in the United
States in the early 1990s that failed to demonstrate any
protection from HLA matching,79 the majority of large
studies have supported the concept of antigen-matching for
corneal transplants conducted in hosts at high risk for graft
rejection.
One of the unexpected outcomes of the CCTS was the
finding that ABO blood type matching was significantly
protective of corneal transplants.79 This was difficult to explain
in the early 1990s, until studies on corneal transplantation
performed in rodents reported that minor transplantation
antigens offer a significant barrier to graft success.82,83 ‘Minor’
antigens are thus called since in conventional solid tissue
(e.g., skin) grafts, they are not as determining of graft success
as compared to MHC antigens. However, as described earlier,
there is significantly reduced expression of MHC antigens by
corneal grafts. Hence, in the cornea, minor transplantation
antigens are potentially quantitatively more numerous than
MHC antigens, and ABO antigens may well represent possible
minor antigens.
CORNEAL TRANSPLANT SURVIVAL – AN
EXAMPLE OF THE SUCCESS OF IMMUNE
PRIVILEGE
The normal cornea is an immune-privileged tissue, and several
features are known to contribute to the privileged status. First,
as mentioned earlier, the expression of MHC class I and class II
molecules is reduced and impaired, especially on the corneal
endothelium. The net antigenic load of corneal tissue is thus
reduced compared with other tissues, which has a mitigating
effect on both the induction and expression of alloimmunity.
Second, the cornea lacks blood and lymph vessels. The absence
of these vascular structures provides relative isolation for
corneal antigens in a manner that reduces, though does not
prevent, antigenic information from escaping from the tissue
while at the same time suppressing immune effectors from
gaining access to the tissue. Third, the cornea is deficient in
activated APCs that exhibit high levels of MHC class II and
requisite co-stimulatory molecules (e.g., CD40, CD80, CD86)
for priming T cells. Indeed, the bone marrow-derived cells of the
cornea are of a highly immature phenotype and uniformly
MHC class II-negative.38 Fourth, as detailed above, there is
considerable expression of a variety of immunosuppressive
factors by various tissues in the anterior segment of the eye that
impair induction and expression of conventional immunity.43–50
These immunosuppressive molecules have powerful immuno-
modulatory effects on APCs, T cells, B cells, NK cells, and
macrophages, and can suppress many forms of immunity
including alloreactive responses. Fifth, cells of the cornea
constitutively express surface molecules, including DAF, CD59,
CD46, PD-L1, and others that can inhibit numerous comple-
ment and T cell effector functions.
The dramatic expression of immune privilege is mirrored by
the success of keratoplasties performed in low-risk situations in
humans. Modest amounts of topical steroids in the early
postoperative period, even followed by cessation of all therapy
later, is still associated, in the vast majority of cases, with
indefinite survival of most corneal transplants. However, not all
grafts are successful. In high-risk transplantation, performed
in inflamed host beds, the prognosis is worse than many forms
of solid organ transplants. What are the mechanisms that
lead to graft rejection, and how does immune privilege fail in
some circumstances?
 Regulation of Immune Responses

graft, and migrate to regional lymph nodes where recipient

CORNEAL TRANSPLANT REJECTION – THE
EROSION OF IMMUNE PRIVILEGE
The immunopathogenic mechanisms that lead to corneal
transplant rejection have been reviewed elsewhere.61–67 Basic
investigations into the mechanisms responsible for allo-
immunity in the high-risk setting have shown how the prin-
cipal modalities that dictate immune privilege in the healthy/
T cells are initially activated.40 Fourth, the significant over-
expression of proinflammatory cytokines generated in inflamed
eyes in the postoperative period can effectively counteract the
function of many of the immunosuppressive cytokines that
normally downmodulate immunity in the healthy eye under
the physiologic state. Hence, under conditions of intense
inflammation, as may occur after transplant surgery, and parti-

physiologic setting can erode after sustained inflammation,
setting the stage for transplant rejection.
It is instructive to place these events in the context of
immune privilege reviewed in the earlier section. First, surgery
itself leads to expression of MHC molecules by the cornea.40
Second, inflammation leads to induction of angiogenic pro-
cesses, prompting growth of both blood and lymph vessels into
the corneal matrix, thereby affecting the relative sequestration
and protection of the cornea from the immune system.84,85
Third, profound changes occur in relation to corneal APCs; the
first is that there is massive mobilization of these cells into
the graft;38,61 the second is that under conditions of intense
inflammation the APCs change their phenotype and mature
(become activated) by acquisition of MHC class II and co-
stimulatory molecules that render them highly capable of
sensitizing host T cells.86 These changes are reflected in the fact
that in both animal models and the clinical setting, high-risk
graft rejection occurs at an accelerated rate, reflecting the
efficiency by which the host has become sensitized to graft
antigens. For example, sensitization develops in recipient
animals with surprising rapidity when grafts are placed in high-
risk eyes. Within 7 days of engraftment, immune donor-specific
T cells can be detected in lymphoid tissues. Similar grafts placed
in low-risk mouse eyes do not achieve T-cell sensitization until
at least 3 weeks after engraftment. It is very likely that the
vulnerability to rejection of grafts placed in high-risk eyes is
dictated by the efficiency with which APCs are mobilized in the
CHAPTER 10
cularly in the high-risk host, the inherent immune privileged
status of the graft is clearly insufficient to overcome the
fact that the graft site can no longer act as an immune-
privileged site.
SUMMARY AND CONCLUSION
The eye is defended against pathogens, just as is every other
part of the body. Components of both the natural and the
acquired immune systems respond to pathogens in the eye, but
the responses are different from those following antigen
encounter in most other places in the body, perhaps as a result
of evolutionary pressures resulting in the survival of those
species and species’ members in which a blinding, exuberant
inflammatory response was prevented by regulation of the
response. In any event, we are left for the moment with an
organ (the eye) in which special immunologic responsiveness
allows us to enjoy a degree of ‘privilege’ tolerance to trans-
planted tissue not experienced by other organs. It is clear now
that this tolerance is an active process, not simply a passive one
derived from the ‘invisibility’ of the transplant from the
recipient’s immune system.
ACKNOWLEDGMENT
The authors would like to acknowledge the significant material contribution
of Dr J Wayne Streilein to the previous edition of this chapter.

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deviation. J Immunol 1977; 118:809–814.
52. Kaplan HJ, Streilein JW: Immune response
to immunization via the anterior chamber of
the eye. II. An analysis of F1 lymphocyte
induced immune deviation. J Immunol
1978; 120:689–693.
53. Streilein JW, Niederkorn JY:
Characterization of the suppressor cell(s)
responsible for anterior chamber
associated immune deviation (ACAID)
induced in BALB/c mice by P815 cells.
J Immunol 1985; 134:1381–1387.
54. Ferguson TA, Kaplan HJ: The immune
response and the eye. II. The nature of
T suppressor cell induction of anterior
chamber-associated immune deviation
(ACADI). J Immunol 1987; 139:352–357.
55. Wilbanks GA, Streilein JW: Characterization
of suppressor cells in anterior
chamber-associated immune deviation
(ACAID) induced by soluble antigen:
evidence of two functionally and
phenotypically distinct T-suppressor cell
populations. Immunology 1990;
71:383–389.
56. Wilbanks GA, Streilein JW: Studies on the
induction of anterior chamber associated
immune deviation (ACAID). I. Evidence that
an antigen-specific, ACAID-inducing, cell-
associated signal exists in the peripheral
blood. J Immunol 1991; 146:2610–2617.
57. Wilbanks GA, Mammolenti MM, Streilein
JW: Studies on the induction of anterior
chamber associated immune deviation
(ACAID). II. Eye-derived cells participate in
generating blood borne signals that induce
ACAID. J Immunol 1991; 146:3018–3024.
58. Maumanee AE: The influence of donor-
recipient sensitization on corneal grafts.
Am J Ophthalmol 1951; 34:142–152.
59. Sonoda Y, Streilein JW: Orthotopic corneal
transplantation in mice: evidence that the
immunogenetic rules of rejection do not
apply. Transplantation 1992; 54:694–703.
60. Qian Y, Boisgerault F, Benichou G, Dana
MR: Blockade of CD40–CD40L
costimulatory pathway promotes survival of
allogeneic corneal transplants. Invest
Ophthalmol Vis Sci 2001; 42:987–994.
61. Qian Y, Dana MR: Molecular mechanisms
of immunity in corneal allotransplantation
and xenotransplantation. Expert Rev Mol
Med 2001; 3:1–21.
62. Boisgerault F, Liu Y, Anosova N, et al: Role
of CD4+ and CD8+ T cells in
allorecognition. Lessons from corneal
transplantation. J Immunol 2001;
167:1891–1899.
63. Niederkorn J, Streilein JW, Shadduck JA:
Deviant immune responses to allogeneic
tumors injected intracamerally and
subcutaneously in mice. Invest Ophthalmol
Vis Sci 1980; 20:355–363.
64. McLeish W, Rubsamen P, Atherton SS, et
al: Immunobiology of Langerhans cells on
the ocular surface. II. Role of central
corneal Langerhans cells in stromal keratitis
following experimental HSV-1 infection in
mice. Reg Immunol 1989; 2:236–243.
65. Mizuno K, Clark AF, Streilein JW: Induction
of anterior chamber associated immune
deviation in rats receiving intracameral
injections of retinal S antigen. Curr Eye Res
1988; 7:627–632.
66. Hara Y, Caspi RR, Wiggert B, et al:
Suppression of experimental autoimmune
uveitis in mice by induction of anterior
chamber associated immune deviation with
interphotoreceptor retinoid binding protein.
J Immunol 1992; 148:1685–1692.
67. Niederkorn JY: The immune privilege of
corneal grafts. J Leukoc Biol 2003;
74:167–171.
68. Wilson SE, Kaufman HE: Graft failure after
penetrating keratoplasty. Surv Ophthalmol
1990; 34:325–356.
69. Vlker-Dieben HJM, D’Amaro J, Kok-van
Alphen CC: Hierarchy of prognostic factors
for corneal allograft survival. Aust N Z J
Ophthalmol 1987; 15:11–18.
70. Boisjoly HM, Bernard P-M, Dube I, et al:
Effect of factors unrelated to tissue etching
on corneal transplant endothelial rejection.
Am J Ophthalmol 1989; 107:647–654.
71. Dana MR, Streilein JW: Loss and
restoration of immune privilege in eyes with
corneal neovascularization. Invest
Ophthalmol Vis Sci 1996; 37:2485–2494.
72. Fujikawa LS, Colvin RB, Bhan AK, et al:
Expression of HLA-A/B/C and -DR locus
antigens on epithelial, stromal and
endothelial cells of the human cornea.
Cornea 1982; 1:213.
73. Whitsett CF, Stulting RD: The distribution
of HLA antigens on human corneal
tissue. Invest Ophthalmol Vis Sci 1984;
25:519–524.
74. Treseler PA, Foulks GN, Sanfilippo F: The
expression of HLA antigens by cells in the
human cornea. Am J Ophthalmol 1984;
98:763–772.
75. Batchelor JR, Casey TA, Gibbs DC, et al:
HLA matching and corneal grafting. Lancet
1976; 1:551–554.
76. Foulks GN, Sanfilippo FP, Locascio JA,
et al: Histocompatibility testing for
keratoplasty in high-risk patients.
Ophthalmology 1983; 90:239–244.

77. Sanfilippo F, MacQueen JM, Vaughn WK,
et al: Reduced graft rejection with good
HLA-A and -B matching in high-risk
corneal transplantation. N Engl J Med
1986; 315:29–35.
78. Boisjoly HM, Bernard P-M, et al:
Association between corneal allograft
reactions and HLA compatibility.
Ophthalmology 1990; 97:1689–1698.
79. Stark W, Stulting D, Maguire M, et al: The
collaborative corneal transplantation
studies (CCTS): effectiveness of
histocompatibility matching of donors and
recipients in high risk corneal
transplantation. Arch Ophthalmol 1992;
110:1392–1403.
Regulation of Immune Responses
80. Gore SM, Vail A, Bradley BA, et al:
HLA-DR matching in corneal
transplantation. Transplantation 1995;
60:1033–1039.
81. Sundmacher R: A clinician’s outlook on
HLA matching for keratoplasty. Dev
Ophthalmol 2003; 36:89–97.
82. Sonoda Y, Streilein JW: Impaired cell
mediated immunity in mice bearing healthy
orthotopic corneal allografts. J Immunol
1993; 150:1727–1734.
83. Sonoda Y, Sano Y, Ksander B, et al:
Characterization of cell mediated immune
responses elicited by orthotopic corneal
allografts in mice. Invest Ophthalmol Vis
Sci 1995; 36:427–434.
84. Dana MR: Angiogenesis and
lymphangiogenesis: novel implications for
corneal immunity. Sem Ophthalmol 2006;
21:19–22.
85. Yamagami S, Hamrah P, Zhang Q, et al:
Early ocular chemokine gene expression
and leukocyte infiltration after high-risk
corneal transplantation. Mol Vision 2005:
11:632–640.
CHAPTER 10
86. Huq S, Liu Y, Benichou G, Dana MR:
Relevance of the direct pathway of
sensitization in corneal transplantation is
dictated by the graft bed
microenvironment. J Immunol 2004:
173:4464–4469.
99
SECTION 3 MICROBIOLOGY
Edited by Michael S. Gilmore
CHAPTER
11 Ocular Bacteriology
Christopher N. Ta, Robert W. Bowman, and James P. McCulley
Overview
Bacteria are ubiquitous in the environment and are part of the
normal flora of humans. The balance between the virulence of
the bacteria and the strength of the immune system plays a role
in whether or not an infection will occur. In order to initiate an
infection, bacteria must be able to adhere to the surface,
multiply, colonize, and evade the immune system, and finally,
invade the tissue. In contrast, the host defense system includes
mechanical removal of bacteria, such as the tear film and
blinking reflexes. The immune system, both humoral and cellular
response, is important in preventing and eliminating a bacterial
infection. Once an infection has occurred, the treating physician
must attempt to identify the etiology of the infection. The most
common classification of bacteria is based on the Gram stain
characteristics. The available tests include the traditional stains
and culture media, and more recently, the tests such as
polymerase chain reaction. The mainstay for treatment of
bacterial infections are antibiotics, although recent evidence
suggests that resistance to many commonly prescribed
antibiotics is on the rise. Finally, it is critical to consider
prophylaxis against infections in patients undergoing ocular
surgery with the use of antiseptic agents, most commonly with
povidone-iodine.
Bacterial infections comprise a complex and constantly
changing group of ocular diseases. Various bacteriologic
processes involve the eyes and periocular structures, from
something as simple as colonization of the skin and lashes alone
without invasive disease to necrotizing bacterial keratitis. The site
of infection may be the periocular skin or lid or an anaerobic
environment such as the canalicular system or the capsular bag.
The source of bacteria may be local (i.e., from the lids and
lashes), or it may be from a remote site (as in metastatic
endophthalmitis) or from the nasopharynx or sinuses. In recent
years, significant advances in our understanding of the
mechanisms of bacterial diseases have been made. Bacterial
antibiotic resistance has been on the increase, and newer
antibiotics that are more specific in their coverage have become
available. We are constantly understanding more and more
about the host–bacterial interaction, its effect on bacterial
virulence and pathogenicity, and the resultant therapeutic
implications. The methods of identifying bacteria are gradually
shifting away from traditional staining and culture techniques to
newer automated or rapid-identification techniques. More
recently, atypical bacteria have been found to be associated with
infectious keratitis following refractive surgery. The role of ‘slime’
is increasingly recognized to play an important role in the
pathogenesis of ocular infections, particularly with regard to
contact lens and intraocular lens related infections. The ability to
diagnose and treat infections correctly is critical. One might
ask, ‘What should I know that will help me in the management of
my patient with a bacterial infection’? In this chapter, we
attempt to give the reader the basis for understanding this
ever-changing field.
ANATOMY, PHYSIOLOGY, AND LIFE CYCLE
Bacteria belong to the kingdom Protista, which encompasses
fungi, protozoa, and algae as well. The more complex euka-
ryotic organisms are the fungi, protozoa, and algae; the simpler
prokaryotic organisms are the bacteria. The taxonomy of the
bacteria is extensive, having undergone frequent revisions in the
past but now requiring the approval of an official international
body.1 With newer techniques such as deoxyribonucleic acid
(DNA) typing and sequencing, the heterogeneity of bacteria
within their various groups becomes more apparent. The
determination of DNA composition by identifying the G + C
(the amino acids guanine, G, and cytosine, C) content of DNA
has shown that the whole phylum of vertebrates ranges only
from 36% to 44% G + C, whereas bacteria range from 25% to
75%. For example, in the genera Staphylococcus and Micro-
coccus, which are in the family Micrococcaceae, the former has
30–40% G + C, whereas the latter has 65–75% G + C.2 Such a
variation in DNA sequencing among bacteria is now being used
clinically to develop rapid diagnostic systems.
The most practical method of classifying bacteria still
depends on their Gram-staining properties and their cell mor-
phology. Also important, however, are their fermentation
products, their ability to metabolize various substrates, their
sensitivity to different antibiotics, and their colonial morpho-
logy. Bacteria lack any nuclear or mitotic apparatus; their
DNA is organized into a single, naked, circular chromosome
that is ~1 mm in length. Some bacteria, such as Borrelia
burgdorferi, which causes Lyme disease, have a linear
chromosome. Smaller molecules of DNA known as plasmids
are significant, because they may carry information for drug
resistance or they may code for toxins that can affect human
cellular functions. The structure of bacterial cells is termed
prokaryotic; whereas those with a membrane-bounded nucleus
are called eukaryotic. Owing to their small size, there is a limit
to the number of molecules that can be present in the cell at
any given time. Prokaryotic cells have come to regulate their
synthesis by induction, regression, or end product inhibition
to produce only what is required for metabolism or growth in a
particular environment.3
Phospholipids and proteins make up the bacterial cell mem-
brane, and in contrast to eukaryotic cells, bacterial cell walls
(except for those of mycoplasmas) do not contain sterols.
Because prokaryotic cells lack both mitochondria and an endo-
plasmic reticulum, electron transport systems are located in
the cell wall itself.
The cell wall or cell envelope plays an important role in
many bacterial cell functions. Besides containing the electron
transport systems, the envelope also serves as an osmotic
101
 MICROBIOLOGY
barrier and regulates the transport of solutes. Thus, the cell wall
protects the cell against rupture from the high internal osmotic
pressure. In hypertonic environments, bacteria may survive as
spheroplasts, or L forms, without their rigid cell wall, but as
a result they may lose their pathogenicity. A macromolecule
unique to the cell wall of many bacteria is the peptidoglycan
(PG). This component of the cell wall is responsible for shape
definition and maintenance.3
The cell wall is the site of many antigenic determinants of
the various bacteria. Moreover, when endotoxin is present, it
is located in the cell wall. The cell envelope of Gram-positive
bacteria has only a thick (15–80 nm) PG layer surrounded by a
polysaccharide capsule. PG is a cross-linked heteropolymer
of amino acids and amino sugars that constitute ~50% of
the cell wall by weight.4 Teichoic acid (TA) is a negatively
charged ribitol-phosphate polymer that attaches to PG by
covalent bonds, accounting for 40% of the cell wall.5 The cell
envelope of Gram-negative bacteria is more complex than that
SECTION 3
of Gram-positive bacteria. Although the PG layer is thinner
(only 1–2 nm), there is a phospholipid outer membrane that
forms a protective barrier, making Gram-negative bacteria more
resistant to hydrolytic enzymes and toxic substances. Mem-
brane proteins that are present in the outer membrane serve to
regulate transport through transmembrane prefixing, or porins,
allowing the passive diffusion of low-molecular-weight com-
pounds such as sugars and amino acids. Antibiotics are much
larger molecules and therefore have difficulty penetrating the
outer membrane and in part are responsible for Gram-negative
bacterial antibiotic resistance. For example, Pseudomonas
aeruginosa are highly resistant to antibiotics due to the outer
membrane. The number and diameter of the porin channels
vary among different Gram-negative species, which helps explain
some of their intrinsic differences in antibiotic susceptibility.6
Gram-negative bacteria possess a periplasma between the inner
and outer walls of the cell membrane. The periplasma contains
at least 50 different properties. Important among these may be
b-lactamase and aminoglycoside phosphorylase that function
to inactivate certain antibiotics.3 Also found in the outer
membrane of Gram-negative bacteria is endotoxin, composed of
lipopolysaccharide (LPS). It is endotoxin that confers virulence
and species specificity. Variability of this surface polysaccharide
allows serologic differentiation of bacterial isolates. The lipid A
portion is mainly responsible for toxicity.7 Mycoplasmas lack a
rigid cell wall, and agents such as Treponema, Borrelia, and
Leptospira have flexible thin walls.
The outer capsule that encloses many bacteria can be well
organized, as in Streptococcus pneumoniae, or it can consist
of a diffuse layer known as glycocalyx, or ‘slime layer’, as in
Staphylococcus epidermidis. This outer capsule can prevent
phagocytosis and the glycocalyx aids in the adherence of
bacteria to tissues and to artificial devices such as prostheses,
catheters,8 and intraocular lenses. The capsules of
N. meningitidis group B and the capsule of Escherichia coli
are the two best known examples. Biofilm is an accumulation
of bacteria encased in an exopolysaccharide matrix, allowing
the bacteria to adhere to each other or to a solid surface. This
biofilm is potentially important in ophthalmology, because it
prevents skin antisepsis.9 Biofilm may also play a role in
staphylococcal adherence to plastic polymers such as intra-
ocular and contact lenses.10 Streptococci appear to use biofilms
to strengthen their adherence to mucosal surfaces.11 First the
bacteria attach to the surface and initiate cellular division to
colonize the surface. Once a threshold is reached, specific genes
are turned on to secrete an extracellular polysaccharide. The
bacteria within the extracellular polysaccharide matrix are
protected from the host immune system as well as antibiotics.
102 This may explain the high resistance of bacteria to antibiotic
treatment in diseases such as endocarditis or infection of
prosthetic devices.
Bacterial flagella allow bacteria to swim through liquid and
move over solid surfaces (aprocytophaga exhibits gliding motility
that may contribute to its potential to produce infections in
immunocompromised patients). Flagella are complex machinery
allowing bacteria to migrate toward specific nutrients, or away
from toxins, a process called chemotaxis. The bacteria are able
to detect a difference in the concentration of specific molecules
over a period of time. Fimbriae also aid in bacterial adherence
to tissues.12 Shorter and more hairlike than the longer flagella
that provide bacteria mobility, the fimbriae function as adhesins,
mediating adhesion to specific surfaces. This is important in
pathogenesis, especially for gonococcus and E. coli. In Neisseria
gonorrhoeae, at least two surface components have been identi-
fied aiding in attachment to genitourinary cells. These com-
ponents are protein II and type-specific pili. Piliated strains
attach much better than nonpiliated strains. E. coli type 1
fimbriae potentiate the uptake of nutrients from and the
delivery of toxins to eukaryotic cells.13 Bacteria can shift rapidly
between a form that possesses fimbriae and one that does not.
Although the fimbriae help bacteria initially to establish colon-
ization in a host, they also increase the bacterial susceptibility
to phagocytosis. Loss of the fimbriae after adherence may
therefore aid in tissue invasion. Different types of fimbriae vary
in specificity for the host glycoprotein receptor to which they
attach. S. pyogenes also possess a nonfimbrial adhesin, protein
F, which mediates attachment of the bacteria to fibronectin.
Most adhesins are lectins and have a high affinity for binding to
specific carbohydrates.
Bacteria reproduce by an asexual process called binary fission.
Cell division begins with an ingrowth of the cytoplasmic mem-
brane, called septal mesosomes, which eventually produces a
complete cross-wall. Bacteria lack mitotic spindle. The chromo-
somes are replicated and attached to the cell membrane during
cellular division. Differences in cross-wall formation and
cleavage account for the bacterial shape and arrangement.
Incomplete cleavage results in bacterial chains. Streptococci
form long chains by producing parallel cross-walls, whereas
staphylococci form clumps by beginning each new septum
perpendicular to the preceding one.14
Although much remains to be discovered about the growth of
the individual bacterial organisms, we do know that bacterial
growth depends on DNA synthesis controlled by RNA and that
it depends on messenger RNA. Under unbalanced or adverse
conditions which are frequently present in the body, DNA
synthesis can occur in the absence of RNA once the growth
cycle has already begun. Typically, at least in the laboratory, the
bacterial growth cycle has four phases: the lag phase, the
logarithmic growth phase, the stationary growth phase, and a
decline phase. Bacteria vary in their temperature requirements
for growth and can be divided into three categories according to
the temperature at which their growth or generation time is
optimal. Psychrophiles grow best at a temperature of 0–20.5°C;
mesophiles thrive from 20–40°C; and thermophiles multiply
best at higher temperatures of 40–90°C. Most bacteria are
mesophiles; some important mesophiles can grow at tempe-
ratures below their normal range. Staphylococci grow slowly at
5°C and may contaminate donor corneas in preservative media
or nonpreserved drops stored in the refrigerator. Because anti-
biotics may not inhibit their growth at these low temperatures,
it is recommended that corneal tissue and its storage media be
allowed to come to room temperature before transplantation.
Streptococci and Proteus vulgaris also possess the ability for
psychrophilic growth.15
Iron is an essential nutrient for bacteria. In the human body,
transferrin in the blood and lactoferrin in external secretions
 Ocular Bacteriology

bind most of the iron.16 Lactoferrin is able to bind iron even

TABLE 11.1. Bacteria Commonly Associated with Ocular
under the more acidic conditions that are present at sites of
Infections
infection.17 Organisms unable to obtain iron in vivo will not
proliferate, but it is clear that pathogens can circumvent this • Gram-Positive
problem. For example, the Neisseria species possess a major • Cocci
iron-regulated protein (MIRP) to help the pathogen in iron • Micrococcaceae
• Staphylococci
acquisition and subsequent pathogenicity.18 Other organisms • Coagulase-positive (e.g., Staphylococcus aureus)
such as Branhamella catarrhalis possess iron-acquisition • Coagulase-negative (e.g., Staphylococcus
proteins that aid in virulence.19 Iron availability may influence epidermidis)
the nature of the disease and whether it stays in one place or • Streptococcaceae
disseminates; it may also determine whether the disease is • Streptococci (e.g., Streptococcus pneumonia,
extracellular or intracellular and the site of pathogenicity. Streptococcus viridans)
• Bacilli
Owing to its avascularity, the eye is iron deficient, and this may • Bacillus cereus
aid in its resistance to bacteria.20 Bacteria undergo phenotypic • Propionibacterium acnes
changes in metabolism and outer membrane proteins that • Listeria
enable them to acquire iron. N. meningitidis becomes more • Actinomyces
virulent after growth in iron-restricted conditions at low pH.21 • Nocardia
Under conditions of iron-restricted growth, pathogenic bacteria • Gram-Negative

CHAPTER 11
• Cocci
appear to produce exotoxins.22 These exotoxins include toxin A,
• Neisseriaceae
elastase, alkaline phosphatase, protease, and hemagglutinin • Neisseria
from P. aeruginosa, a-toxin from Clostridium perfringens, and • Branhamella
b-toxin from Serratia marcescens.23 Bacteria can break down • Moraxella
almost any organic compound into usable components. For • Kingella
example, some Pseudomonas species can grow on camphor and • Acinetobacter
• Bacilli
naphthalene, and this may explain the propensity of Pseudo- • Enterobacteriaceae
monas for growing in make-up.24 • Escherichia
• Shigella
CLASSIFICATION OF COMMON OCULAR • Salmonella
• Klebsiella
BACTERIA • Enterobacter
• Serratia
Identification of bacteria is a time-consuming and laborious
• Proteus
task and not without controversy and debate. After a pure • Yersinia
bacterial culture has been isolated and undergone a Gram • Vibrionaceae
stain, the bacterium is further identified as to genus and species • Pseudomonadaceae
by the results of various physiologic and biochemical tests • Pseudomonas
(Table 11.1). Commercially available kits are being used • Pastereurellaceae
• Haemophilus
frequently, especially in nonreference laboratories for the rapid • Actinobacillus
identification of bacteria; there are however, some who question • Pasteurella
the accuracy and cost of such methods. Bergey’s Manual is the
definite taxonomy source. Recent developments have seen a
shift from conventional phenotypic identification methods to
modern molecular techniques.25
Conventional dehydration methods utilize morphology, is more complex. McCulley and Dougherty have shown that
cultured appearances, requirements for growth, metabolism and blepharitis can be divided into several distinct clinical forms
biochemical activities, and susceptibility to physical and and that CNS, as well as S. aureus, are important in the pro-
chemical agents. duction of staphylococcal blepharitis and seborrheic blepharitis
with a staphylococcal component.26,28–31 Meibomian gland
secretions from patients with meibomian gland involvement
GRAM-POSITIVE COCCI have an abnormality in the free fatty acid component that may
Staphylococci be mediated by the normal ocular flora. Assays of the most
Staphylococci belong to the family Micrococcaceae, which common bacterial lid flora in normal subjects and patients with
encompasses two genera: Staphylococcus and Micrococcus. chronic blepharitis have shown that strains of CNS isolated
The species in the genus Staphylococcus are divided into those from patients with a meibomian gland abnormality more fre-
that are coagulase-positive and those that are coagulase- quently produced both a fatty wax esterase and a cholesterol
negative. Coagulase-positive staphylococci include S. aureus, esterase.32,33 Tetracycline and minocycline have been shown to
S. intermedius, and S. hyicus. At least 17 species of coagulase- decrease or eliminate bacterial flora, resulting in an improve-
negative staphylococci (CNS) have been identified. The best- ment of blepharitis.34–36 These findings point out the important
known member of this family and the most common bacterium relationship among indigenous flora, environmental factors
cultured from the eyelids and conjunctiva is S. epidermidis.26 (e.g., temperature and pH), bacterial virulence factors, and
The absence of coagulase should not be equated with lack of exoenzyme production.
virulence, because members of this group (e.g., S. haemolyticus)
can have pathogenic potential.27 Streptococci
Both coagulase-positive and -negative staphylococci are The genus belongs to the family Streptococcaceae. Species are
responsible for various ocular diseases. That staphylococci are classified according to the presence of certain surface antigenic
the organisms responsible for infection in some conditions such and physiologic characteristics.37 Important ophthalmic
as dacryocystitis, keratitis, and endophthalmitis is obvious, but pathogens in this group include S. pneumoniae (formerly
their role in blepharitis, marginal keratitis, and phlyctenulosis diplococcus), which is part of the respiratory flora, b-hemolytic 103
 MICROBIOLOGY
streptococci, and group D enterococci, which are part of the
enteric flora. Streptococci can be classified based on the type
of hemolysis produced on blood agar. S. pneumoniae is an
a-hemolytic streptococcus. Viridans streptococci is optochin-
resistant and insoluble in bile. Differentiation of the species and
the sensitivity to various antibiotics have become crucial as a-
streptococci have been found to be resistant to aminoglycoside
and polymyxin B and they are becoming increasingly so to
penicillin and fluoroquinolones.38 A type of nutritionally
deficient streptococci has recently been described. They require
pyridoxine for growth and as a result will not grow on blood agar
or in broth without the addition of pyridoxine. Nutritionally
deficient streptococci are a known cause of endocarditis and
can invade the eye as well, producing infectious crystalline
keratitis.39 Crystalline keratitis is most commonly caused by
streptococci but also occurs with other bacteria such as
nontuberculosis mycobacterium.40
SECTION 3
GRAM-NEGATIVE COCCI
Neisseriaceae
The family Neisseriaceae includes the genera Neisseria,
Branhamella, Moraxella, Kingella, and Acinetobacter, all of
which are potential ocular pathogens. The organisms are either
diplococci or short bacilli. Their laboratory diagnosis is based
on sugar fermentation reactions or serologic techniques.41 All
members of the Neisseriaceae are oxidase- and catalase-positive
(except for Acinetobacter, which is oxidase-negative). Neisseria
species and ~50% of Acinetobacter species ferment glucose.
The differentiation of Neisseria from Branhamella can be
difficult. Branhamella will typically grow on blood agar but not
on Thayer–Martin medium, and it does not ferment glucose,
dextrose, maltose, or lactose. N. gonorrhoeae are commonly
resistant to penicillin, as well as tetracycline, with increasing
resistance to fluoroquinolones.42 Gonococcal ophthalmia neo-
natorum is prevented by the application of topical erythromycin
ophthalmic ointment immediately after birth in newborns.
Acinetobacter species are commensal organisms of the upper
respiratory tract, skin, and genitourinary tract that can be con-
fused with Neisseria. They are Gram-negative aerobic bacteria
that appear coccobacillary or coccal in shape. However, a negative
oxidase test result will readily differentiate Acinetobacter from
Neisseria.
Moraxella species are either bacillary or coccobacilli, forming
either pairs or short chains of pairs in smears. Presumptive
identification in smears can usually be made owing to the large
size and end-to-end configuration of Moraxella organisms,
although they may appear to be Gram-positive on thick smears.
Moraxella species grow on MacConkey agar and do not ferment
carbohydrates. Most species are susceptible to penicillin.
Moraxella are part of the normal flora of the upper respiratory
tract, but can cause conjunctivitis, keratitis, and endoph-
thalmitis. Kingella species were formerly classified as Moraxella
and, like Moraxella, are nonmotile Gram-negative rod, cocco-
bacillary, or diplococcal in shape and oxidase-positive. Kingella
can rarely cause endophthalmitis.43
GRAM-NEGATIVE BACILLI
Enterobacteriaceae
The family Enterobacteriaceae comprises at least 27 genera
and seven enteric groups, with more than 110 species.44 Mem-
bers of this family are either motile with peritrichous flagella or
nonmotile, and they do not form spores. All members grow
both aerobically and facultatively anaerobically. The Entero-
bacteriaceae ferment glucose, reduce nitrates to nitrites, and are
104
catalase-positive and oxidase-negative. They also lack cyto-
chrome oxidase activity. Important genera include Escherichia,
Shigella, Salmonella, Klebsiella, Enterobacter, Serratia, tribe
Proteae (Proteus, Morganella, and Providencia), and Yersinia.
Escherichia coli has rarely caused endogenous endoph-
thalmitis following septicemia.45 However, E. coli can acquire
and transmit multiple antibiotic-resistant plasmids. Serratia
was once considered to include a nonpathogen and was used to
study air currents by being released from air balloons and blown
through hospital ventilation systems.15 Today, we know that
Serratia causes infectious keratitis and endophthalmitis.
Members of the tribe Proteae, especially Proteus mirabilis,
can produce ocular disease and are typically resistant to poly-
myxins and tetracycline.46 On blood agar, P. mirabilis produces
gray, swarming colonies that are oxidase- and indole-negative.
Yersinia pestis causes bubonic plague, which had a devastating
effect on Western civilization in the fourteenth century.
Although now it is not commonly associated with ocular
disease, Yersinia species have been cultured from patients with
Parinaud’s oculoglandular syndrome.47
Vibrionaceae
Members of the family Vibrionaceae are non-spore-forming
Gram-negative bacilli that are oxidase-positive. They move by
means of a polar flagellum and are capable of aerobic or anae-
robic growth. Although they are rarely found to be the cause of
ocular disease, three genera, Vibrio, Aeromonas, and Plesio-
monas, do sometimes cause keratitis and endophthalmitis.48–50
Pseudomonadaceae
The genus Pseudomonas comprises ubiquitous Gram-negative
bacilli. The presence of cytochrome oxidase distinguishes them
from the Enterobacteriaceae. A polar flagella may be present.
The growth requirements of Pseudomonas are simple: They can
use a variety of compounds for nutrition, and some strains can
even grow in distilled water. This may explain the incidence of
Pseudomonas infections associated with homemade saline
solution and soft contact lenses and inadequately sterilized
intraocular lenses. The most common organism causing cornea
ulcers associated with contact lens wear are Pseudomonas and
Serratia. Pseudomonas can cause rapid destruction of the
cornea resulting in poor visual outcome.51
Pasteurellaceae
The bacteria of the family Pasteurellaceae are small non-spore-
forming, Gram-negative bacilli. They are nonmotile and either
aerobic or facultative anaerobic. Most are fastidious, requiring
enriched media in the laboratory. The family has three genera:
Haemophilus, Actinobacillus, and Pasteurella. Haemophilus
species are the most common pathogens. They require hemin
(X factor) and nicotinamide-adenine dinucleotide (NAD). The
cell wall of Haemophilus is typical for a Gram-negative bac-
terium showing endotoxic activity. Many H. influenzae possess
a polysaccharide capsule and can be divided into serotypes
based on the capsular reaction.
Many other species of the Pasteurellaceae can produce
ocular disease, and they can be differentiated on the basis of
their individual requirements for hemin and NAD. A variety
of tests including indole production, urease activity, ornithine
decarboxylase reactivity, and carbohydrate fermentation of
glucose, sucrose, and lactose can also be used.52 Many Haemo-
philus influenzae produce b-lactamase. Effective treatment
includes new generations of cephalosporins, aminoglycosides,
and fluoroquinolones.
Actinobacillus species require carbon dioxide for growth.
The only known pathogen of the genus is A. actinomycetem-

comitans, which can cause endophthalmitis.53 Pasteurella
infections, which are usually transmitted through contact with
animals that are carrying the bacilli, can cause conjunctivitis,
corneal ulceration, and endophthalmitis.54
MISCELLANEOUS GRAM-NEGATIVE
BACTERIA
E. corrodens is a normal inhabitant of the human mouth and
upper respiratory tract. It can cause infection following a
human bite, and it can be the culprit in an opportunistic dis-
ease. Eikenella species are non-spore-forming, facultatively
anaerobic, moderately sized, Gram-negative bacilli. These
bacteria grow slowly on common media with CO2, and about
half of the isolates form distinctive pits on the agar. Certain
strains are mobile on moist surfaces and produce an endo-
toxin. E. corrodens is susceptible to ampicillin, newer peni-
cillins and cephalosporins but resistant to aminoglycosides and
clindamycin. E. corrodens have been reported to cause keratitis
and endophthalmitis.55 Another common member of the oral
flora, Capnocytophaga, has been documented as the cause of
keratitis and endophthalmitis.56–58
Although Debre first recognized cat-scratch disease in 1931,
his findings were not reported until 1950. Ocular involvement
typically takes the form of Parinaud’s oculoglandular syndrome
with a conjunctival granuloma at the inoculation site.59 Cat-
scratch bacilli have been identified in conjunctival granulomas.
The differential diagnosis of Parinaud’s oculoglandular syn-
drome is quite long, including a number of bacterial and viral
infections. Bartonella henselae has been found to be the prin-
cipal cause of cat-scratch disease.60 It is a small, pleomorphic,
Gram-negative rod.61 Treatment of cat-scratch disease is
usually supportive with spontaneous resolution over
2–4 months. Oral ciprofloxacin may speed resolution of the
disease.62
ANAEROBIC GRAM-NEGATIVE BACILLI
Anaerobic Gram-negative bacilli are a group of non-spore-
forming bacteria that comprises part of the normal anaerobic
oral and intestinal flora. Bacteroides fragilis is the most com-
monly isolated organism. Unlike most anaerobes, B. fragilis is
resistant to many antibiotics, including penicillin. Cuchural
reviewed the antibiotic sensitivities of a number of strains of
B. fragilis.63 Resistance rates to imipenem and ticarcillin-
clavulanic acid were 0.2% and 1.7%, respectively. No isolates
were resistant to either metronidazole or chloramphenicol.
The rate of resistance to clindamycin was 5% and to cefoxitin
11%. B. fragilis rarely cause ocular infection, with one case of
endophthalmitis reported.64
GRAM-POSITIVE BACILLI
Gram-positive bacilli are comparatively large spore-forming
bacilli that grow on nonselective media producing nonhemolytic
rapidly growing colonies. They are ubiquitous and have been
known to cause a severe endophthalmitis after trauma has
occurred.65 Bacillus cereus is the most common pathogen.
Vancomycin, clindamycin, and the aminoglycosides are usually
the drugs of choice.65
The most important of the non-spore-forming Gram-positive
bacilli are the genera Corynebacterium and Propionibacterium.
The organisms are small, nonmotile, and catalase-positive, and
they ferment carbohydrates producing lactic acid (Corynebac-
terium) or propionic acid (Propionibacterium). Propioni-
bacterium species are anaerobic and are a common isolate from
Ocular Bacteriology
the eyelid and the conjunctiva.29 P. acnes is an important cause
of chronic endophthalmitis.66
Anaerobic, Gram-positive bacilli that are spore-forming
belong to the genus Clostridium. They can cause several serious
diseases, including botulism and tetanus. In addition,
C. difficile causes pseudomembranous colitis.
Listeria species are short, Gram-positive, facultatively
anaerobic (but not strictly) bacilli and they exhibit charac-
teristic tumbling motility in suspension or in a hanging drop.
L. monocytogenes, the most common species, is catalase-
positive and Voges-Proskauer-positive; it hydrolyzes esculin
but does not produce hydrogen sulfide or reduce nitrite. Listeria
species are known ocular pathogens. Zaidman and co-workers
developed a rabbit model of L. monocytogenes infection and
concluded that the best treatment is a combination of penicillin
and gentamycin.67 Listeria can also cause endogenous
endophthalmitis.68
CHAPTER 11
Actinomyces and Nocardia
Actinomyces species are facultatively anaerobic or strictly
anaerobic Gram-positive bacilli that are usually arranged in
hyphae but can fragment into short bacilli. A. israelii, the most
common opportunistic species, grows on blood agar enriched
with vitamin K. The organisms can cause a chronic canali-
culitis.69 Penicillin remains the most effective treatment.
Similar in appearance to Actinomyces and almost indis-
tinguishable on Gram’s stains is the genus Nocardia. Nocardia
species are strict aerobic bacilli that are Gram-positive, yet
they may appear to be Gram-negative with intracellular
Gram-positive beads. They have a cell wall similar to that of
mycobacteria and are acid-fast with weak acids, which helps to
distinguish them from Actinomyces species. Members of the
Nocardia are catalase-positive and grow on nonselective media.
Norcardia is a known cause of kerititis and the treatment of
choice is amikacin.70 Endophthalmitis caused by Norcardia has
poor prognosis.71
MYCOBACTERIA
Mycobacterium tuberculosis and M. leprae remain two of the
most prevalent and serious causes of infections worldwide.
They are acid-fast, although M. leprae is more sensitive to
decoloration. The growth of these nonmotile slender rods is
slow, with some species taking 2–6 weeks, although growth of
fast-growing species can occur in 3–5 days. Runyon classified
mycobacteria into four groups based on their rate of growth and
chromogenicity. In ophthalmology, it is probably more practical
to divide mycobacteria into two groups: M. tuberculosis and
atypical mycobacteria. Atypical mycobacteria (especially
M. fortuitum and M. chelonei) are emerging as a frequent cause
of keratitis following refractive surgery. These bacteria are
sometimes difficult to diagnose and treat, with potentially poor
visual outcome.72,73 Topical amikacin has been effective in the
treatment of corneal ulcers. Newer generations of fluoroquino-
lones, such as gatifloxacin, have been shown to be effective
against M. chelonae in a rabbit model.74
MOLLICUTES
Mollicutes are a class of microorganism bounded only by a
membrane. The two most important genera are Mycoplasma
and Ureaplasma. Three pathogen strains have been identified:
M. pneumoniae, M. hominis, and Ureaplasma urealyticum.
They can be differentiated by their ability to metabolize glucose
(M. pneumoniae), arginine (M. hominis), or urea (U. urealyticum).
M. pneumoniae causes pneumonia. M. hominis causes post-
105
 MICROBIOLOGY
partum fever.75 U. urealyticum is associated with urethritis in
men and lung diseases in premature infants.76 Mycoplasmas
resemble chlamydiae, rickettsiae, and viruses in passing through
450-nm filters but, like bacteria, they are Gram-negative,
able to grow on artificial media, and capable of dividing by
binary fission. Erythromycin and tetracycline are usually
effective, although some M. hominis are resistant to erythro-
mycin and some ureaplasmas are resistant to tetracycline.75,77
Mollicute-like organisms (MLO) are found in chronic uveitis,
especially gastrointestinal tract-associated disease.78
INFECTION OF THE HOST
Bacteria produce a variety of ocular diseases. Bacterial conjunc-
tivitis and bacterial keratitis are commonly seen. Endo-
phthalmitis presents a challenging clinical problem. Blepharitis
in its various forms may constitute an imbalance in the
normal relationship between bacteria and the skin of the eyelid.
SECTION 3
The exact roles of CNS and their toxin production, and of
Propionibacterium acnes in meibomian gland dysfunction
continue to be studied and defined. Infections of the periocular
tissue include canaliculitis, dacryocystitis, and preseptal
and orbital cellulitis. Bacteria also can have remote effects
such as syphilitic interstitial keratitis and mycobacterial
phlyctenulosis.
The virulence of a pathogenic organism depends on its
potential to produce disease. One important factor is its ability
to adhere to mucosal surfaces and to enter epithelial cells.
Invasive properties are carried in various ways in plasmids,
bacterial phage, and DNA segments in the bacterial chromo-
some. These properties can be exchanged between bacteria,
rendering noninvasive bacteria invasive. Characteristics of
bacteria important in ocular infections include: virulence of
the organism, the invasiveness of the organism, the number
of organisms entering the host, and their site of entry. Certain
extracellular enzymes may be important in the establishment of
infection and in its spread through tissues. These include
collagenase (C. perfringens), coagulase (staphylococci), hyalu-
ronidases (staphylococci, streptococci, clostridia, pneumococci),
streptokinase or fibrinolysis (hemolytic streptococci), hemo-
lysins and leukocidins (streptococci, staphylococci, clostridia,
Gram-negative rods), and proteases (neisseriae, streptococci)
that can hydrolyze immunoglobulins, such as secretory IgA.75
In blepharitis, staphylococci and P. acnes produce lipases and
esterases.
The host determines the effect of many virulence factors.
That is, certain characteristics of the host can influence the
development of disease. For example, the host’s age, use of
drugs, and sexual habits can all determine the effect of virulence
factors. The use of contact lens or surgical trauma increase
the risk of ophthalmic disease. Blepharitis, dry eye states, cana-
liculitis, chronic nasolacrimal duct obstruction, and previous
ocular disease also increase the risk. Damaged epithelium in
the cornea is particularly susceptible to bacterial adherence;
bacteria adhere to the epithelial edge rather than the bare
stroma.79 Tissue injury results from: the direct action of the
bacteria, from microbial toxins, from indirect injury, from
inflammation, or from immunopathologic processes. In response
to an injury, polymorphonuclear cells, as well as macrophages
and lymphocytes, enter the site. Tissue fluids provide plasma
proteins, including immunoglobulins such as IgG, comple-
ment, and properdin. The primary mediators of inflammation
include histamine, tumor necrosis factor, cytokines, leuko-
trienes and prostaglandins. The phagocytic cells play a key role
in the interaction with the microorganism, ingesting and killing
bacteria. The inflammatory process releases chemokines which
106 attract additional inflammatory cells.
ADHERENCE, COLONIZATION, AND INVASION
Cellular microbiology is a rapidly developing field that deals
with the interaction of bacteria and their host cells. Epithelial
cells with their tight cellular junctions act as a barrier to
bacterial adherence, penetration, and the entry of soluble
toxins. Epithelial cells may respond to bacterial adherence by
secreting cytokines, causing a major cytoskeletal rearrangement
and playing an important role in the mucosal immune
response. However, the relationship between the host and the
potential pathogen is complex and still incompletely under-
stood regarding why some bacteria are invasive and others
colonize the cell surface. Some produce exotoxins that destroy
host cell functions, whereas others utilize the host cell to
advance their pathogenic potential.80
Microbial adhesion to host tissue is a primary event in
colonization and an important stage in microbial pathogenesis.
Adhesive ligands in bacteria range from rod-like structures (pili
or fimbriae) to outer membrane proteins and polysaccharides.
Individual bacteria may possess multiple adhesins that target
distinct host cell molecules and deliver diverse signals resulting
in extracellular location or internalization. Both the nature
and the density of the target receptor on the host cell may be
determining factors in the outcome of the bacteria–host
interaction.80
The invasion of mucosal surfaces and ocular tissues by
bacteria occurs in several steps. First, bacteria must establish
themselves in close proximity to the ocular surfaces, such as
the lids and lashes. This, by the way, is why the cleansing and
isolation of these surfaces is so critical in ocular surgery.
Second, the bacteria must avoid being swept away, which is one
of several reasons why patients with severely dry eyes are at
increased risk of infection. Next, bacteria must acquire essen-
tial nutrients for growth, especially iron, and be able to replicate
at a rate sufficient to maintain or expand their population.
Finally, the bacteria must resist local host defenses.
Association, that is localization of bacteria on a surface, must
take place before adherence can occur. Most bacteria and host
tissue carry negative charges. In order to overcome the repelling
forces, many mechanisms are utilized by the bacteria to adhere
to the host surface. This may be as simple as possessing
hydrophobic forces which help adhere to host tissue. Motility of
bacteria may enhance association. Bacteria may associate with
mucus or exudates, forming noncovalent bonds. Chemotaxis
may help bacteria to penetrate the mucous barrier, thus
enhancing contact with receptors on the epithelial surface.80
Bacterial attachment is essential in order for colonization to
occur in environments with a surface exposed to a fluid flow.
Adhesion of bacteria to the epithelial surface depends upon
adhesins, the complex polymers on the bacterial surface. The
presence of pili, hair-like appendages that extend from the
surface of the cell, aid in the adhesion of bacteria to host cells.81
For example, E. coli have pili that allow the bacteria to adhere
to the epithelial cells in the intestinal wall.82 The presence of
fimbriae assist in bacterial adhesiveness.83 These are frequently
present on Gram-negative organisms. A variety of bacteria
produce adhesins that tend to be outer membrane proteins.
Outer membrane proteins, as well as fimbriae, aid in adhesion
of N. gonorrhoeae to epithelial cells. Staphylococci and
streptococci can adhere to epithelial cells and thus colonize
skin and mucous membranes.110 The important components
of fimbriae consist of lipoteichoic acid (LTA), protein F, and
M protein.83 Lipteichoic acid and protein F adhesion to
epithelial cells are mediated by fibronectin. The M protein
prevents phagocytosis.81 S. aureus produces a surface protein
with specific affinity for fibronectin.84 A variety of streptococci
and staphylococci species can bind fibronectin, probably
through affinity with their cell wall LTA. The presence of

fibronectin on the cell surface appears to enhance bacterial
adhesion as well.85 LTA can interfere with the killing or
phagocytosis by polymorphonuclear leukocytes.86 Some isolates
of S. epidermidis can inhibit the bacterial phagocytic activity of
neutrophils, independent of adherence. This inhibition of
neutrophils may represent another virulence factor.87
Adherence of P. aeruginosa to the corneal epithelium may be
the first step in the pathogenesis of infection.88–90 Pseudomonas
adheres to the basal epithelial cells through the interaction of
a specific adhesion-receptor. In order for bacterial adherence to
occur, several steps must take place. First, van der Waals forces
produced by surface molecules overcome the normal repulsive
forces of two similarly charged cells.91 Then, once the cells
become close enough, hydrophobic binding holds the bacteria to
the surface, and strong bonds form between the exopoly-
saccharides of the bacteria and the substrate glycoprotein of
the target cell. The significant differential adherence between
basal and nonbasal corneal epithelial cells is probably the rea-
son why superficial trauma or epithelial cell damage allows
Pseudomonas infections to develop.92 This may play a sig-
nificant role in contact lens-associated Pseudomonas keratitis.
Using a rabbit model, Koch and associates showed that a
bacterial suspension of P. aeruginosa alone caused no inflam-
mation but that corneal infection developed in 11 of 14 eyes
wearing new or worn contaminated soft contact lenses.93
Trancassini and associates demonstrated that strains of
P. aeruginosa that produce alkaline protease and elastase adhere
better.94 Bacterial adherence may also depend on nonbacterial
factors. Deighton and Balkau investigated the adherence of
strains of S. epidermidis to glass and plastic material.95 They
found that the degree of adherence depended mainly on the
growth media; adherence was enhanced by the addition of
glucose or oleic acid and it was inhibited by serum. After
attachment takes place, penetration of the epithelial cells must
occur. LPS core with an exposed terminal glucose residue
expressed in P. aeruginosa has been shown to highly correlate
with the level of adhesion to epithelial cells.96 In the case of
E. coli, this is a process similar to phagocytosis.97
When they are present, bacterial cell wall capsules are
important virulence factors.98 While cell wall capsules are more
commonly seen in Gram-negative bacteria, encapsulated
staphylococci may be seen in vivo.99 The primary virulence
factor of H. influenzae surface antigen, the type b capsular
polysaccharide, is polyribosylribitol phosphate (PRP).100 Some
bacteria, such as Bacteroides species, become encapsulated
during an inflammatory process, further increasing their
pathogenicity as a result.101 The capsule thus formed inhibits
phagocytosis by covering and thus making the recognition sites
of opsonins (C3b and IgG) inaccessible to phagocytic cells.102
M-protein inhibits opsonization and impairs complement
activation and binding of C3b to the bacterial cell wall.103,104
Surface sialylation of the bacterial capsule also helps micro-
organisms to resist host defenses.105 In a mouse model of
Campylobacter infections, Pei and Blaser demonstrated that
virulence was enhanced when S-protein was present on the
bacterial cell surface as a capsule.106
Bacterial glycocalyx also may aid in colonization and infec-
tivity by protecting the bacteria from antibiotics and from the
host’s immune system and phagocytic cells.107 Glycocalyx
production is important in the adhesion of certain P. aeruginosa
strains to respiratory tissues.108 For staphylococcal strains, pro-
tein A and clumping factor may be important mediators of
adherence.109 Protein A interferes with opsonic activity of anti-
bodies, because it binds to the Fc portion of IgG (except IgG3),
and to a lesser extent, IgM and IgA2.110 Streptococci also carry
an Fc binding protein on the cell wall and therefore evade the
natural host defense mechanisms.111
Ocular Bacteriology
The ability of specific bacteria to adhere to the sites at which
they produce clinical disease has been shown in various situ-
ations, including S. pneumoniae to human pharyngeal
epithelial cells, S. pyogenes to pharyngeal epithelial cells, and
E. coli to bladder epithelium. S. aureus, P. aeruginosa,
H. influenzae, and S. pneumoniae adhere to mucus in the
respiratory tract. S. aureus, S. pneumoniae, and P. aeruginosa,
three of the most common causes of corneal ulceration, exhibit
markedly greater adherence to human corneal epithelial cells
than do other bacterial species.112 S. aureus produces a number
of cell surface proteins that bind to host protein. These include
fibronectin, fibrinogen, vitronectin, bone sialoprotein, throm-
bospondin, collagen, IgA, elastin, prothrombin, plasminogen,
laminin, and mucin.113 Protein A binds IgG in such a way that
F1-receptors on phagocytic cells cannot bind to the F1 protein
of the immunoglobulin. After establishing adhesion, some
bacterial pathogens enter epithelial cells by endocytosis.
Intracellular invasion provides a new source of nutrients and
CHAPTER 11
affords protection from some host defenses; however, the
bacteria must survive inside an endocytic vacuole, and, while
exposed to products such as lysozyme, they must multiply and
spread to other cells.114 Many pathogenic microbes may invade
the host by inducing their own endocytosis. This phenomenon
has been designated as parasite-directed endocytosis. Although
still poorly understood for most pathogens, it is thought that in
the case of most bacteria, this represents biologic mimicry, with
the bacteria producing a molecule that resembles a natural host
ligand for which there is a host cell receptor.115 Organisms such
as Mycobacterium, Actinomyces, Corynebacterium, Listeria,
and Francisella species contain large quantities of structural
lipid that protects them from digestion by the lysosomes of
phagocytes, probably because of their ability to scavenge oxygen
radicals.116
The virulence of bacteria also depends on their ability to
produce enzymes that are directed at host defenses. Coagulase
produced by staphylococci forms a fibrin clot from fibrinogen,
thus protecting the bacteria from phagocytosis. Streptococci
can produce a streptokinase that dissolves fibrin clots and
allows further spread of the bacteria. Streptokinase activation of
plasminogen produces fibrinogen degradation products.117
Whitnack and co-workers showed that the binding of fibrinogen
and fibrinogen degradation products to M-protein enhances its
antiopsonic property.118 S. pneumoniae pneumolysin inhibits
polymorphonuclear leukocyte chemotaxis and the ability to kill
opsonized pneumococcus.119,120 Neuraminidase may also be an
important virulence factor of S. pneumoniae. Neuraminidase
might alter glycoproteins on the ocular surface, thus enhancing
bacterial attachment. Pneumococci can adhere to corneal
epithelial cells in vitro.112 Hyaluronidase digests hyaluronic
acid, which is an important ‘tissue cement’ and aids in the
spread of some streptococci and staphylococci. Leukocidin,
produced by some staphylococci and streptococci and some
bacilli, disintegrates neutrophils and tissue macrophages.
Catalase destroys the hydrogen peroxide present in lysosomes.
N. gonorrhoeae, N. meningitides, H influenzae, and
S. pneumoniae produce an IgA protease that destroys
immunoglobulin IgA1.81 Other bacteria produce cytolysins,
such as hemolysins that kill red blood cells or leukocidins,
that lyse leukocytes.81 Streptococci group A produce
streptolysin O and S, which lyse red blood cells and are lethal
for mice.121 Endotoxin activity is an important aspect of Gram-
negative virulence. P. aeruginosa produces an elastase, alkaline
protease, exotoxin A, and LPS endotoxin. The P. aeruginosa
exotoxin A has a cytopathic effect, and alkaline protease is
active against collagen.122–128 Gram-positive bacteria, although
they do not contain LPS, do have PG that can lead to vascular
dilation and hypotension similar to LPS but not as severe. 107
 MICROBIOLOGY
Burns and associates have shown that a metalloproteinase
inhibitor (HSCH2) inhibits P. aeruginosa elastase and that, in a
rabbit model, delayed the onset of corneal melting and
perforation.129
HOST DEFENSES
Several defense systems are important in the prevention of
microbial infection. The first barrier consists of the skin and its
indigenous flora that help to create a milieu inhospitable to
most pathogens. Lactic acid and fatty acids in sweat and
sebaceous glands serve to lower the pH to a point at which
most pathogenic bacteria will not survive. The mechanical
flushing action of the lids and tears, in addition to antibody,
lactoferrin, b-lysin, and lysozyme present in tears, serve as the
next major barrier to infection. The conjunctiva and mucous
membranes are important in preventing bacterial adherence
and in allowing ‘natural antibodies’ such as IgM, humoral
SECTION 3
immunity, and cell-mediated immunity (CMI) access to the
ocular surface.
NONSPECIFIC DEFENSES
The normal conjunctiva contains all immunologic components
and high levels of inflammatory cells (~300 000 per mm2).130
Although immunoglobulins and complement system are the
most important factors in the host’s defense against bacteria,
other factors include fibronectin, C-reactive protein, lysozyme,
and transferrin play a significant role. Immunoglobulins G
and M (IgG and IgM) have the greatest bactericidal activity,
whereas IgA is very effective in restricting bacterial adhesion
on mucosal surfaces.131,132 These components contribute to
specific as well as nonspecific defense mechanisms. Tears
usually contain IgA, IgE, IgG, and complements. Secretory IgA,
usually in conjunction with complement activated by the alter-
nate pathway, can be bacteriolytic.133,134 IgA has an important
role in preventing infections as evidenced by an increased
incidence of staphylococcal infections observed in atopic dis-
ease with its associated defects in IgA and CMI.135
The complement system is also very important in defending
against bacterial infections. The main outcomes of complement
activations are: (1) lysis of bacteria, (2) production of inflamma-
tory mediators, (3) opsonization of organisms for phagocytosis,
and (4) facilitate antibody-mediated immune responses.81
Complement assists phagocytic cells by depositing an opsonic
protein (C3b) on the bacterial surface that then interacts with
specific receptors on the phagocytic cell surface. It is clear that
phagocytic killing by leukocytes is an important defense
mechanism against bacterial infection, because patients with
abnormalities of polymorphonuclear leukocyte function are
susceptible to recurrent or persistent infections.136 Pneumolysin
can activate the classic complement pathway, whereas the
alternate pathway may be activated by the PG of group A
streptococci or the TA of S. pneumoniae.4,137,138 In Gram-
negative infections, complement can be directly bactericidal
through the assembly of a membrane attack complex (C5b-9)
that can lyse susceptible Gram-negative bacteria. Complements
are also chemotactic, drawing leukocytes into the cornea. Typi-
cally, an antigen–antibody complex activates the complement
reaction, but interaction of bacteria directly with C1q can also
activate complement.139,140 Bacterial cell wall components such
as LPS can activate the alternate complement pathway.141
Through its interaction with specific antibody, LPS can activate
complement via both the classic and alternate pathways; LPS
alone activates the alternate pathway.142 Deposition of
LPS–antibody complexes may cause ring infiltrates in Gram-
108 negative corneal infections.143
Neutrophils are the primary cells found at the site of bac-
terial corneal infections.144 During phagocytosis they release
prostaglandins, which increase vascular permeability and
induce degranulation of mast cells and basophils. Mast cells
in turn release histamine, eosinophil chemotactic factor,
prostaglandins, and SRS-A. Neutrophil lysosomal products
include cationic proteins, acid proteases, and neutral proteases.
The cationic proteins increase vascular permeability and are
chemotactic for mononuclear phagocytes. The acid proteases
degrade basement membrane, and neutral proteases degrade
fibrin, elastin, and collagen. Neutrophils also contain lysozyme,
hydrolytic enzymes, collagenase, lactoferrin, and toxic nitrogen
oxides.145 Antimicrobial neutrophil peptides (defensins) have been
isolated in the tear film.146 Cullor and associates have demon-
strated that neutrophil defensins possess both bacteriostatic
and bactericidal activity against various ocular pathogens.147
Lysozyme is an enzyme that can lyse certain bacteria by
acting as a muramidase to cleave the glycosidic bond of the
N-acetylmuramic acid residues in the bacterial cell wall.148
Lysozyme makes up 40% of the tear protein, with levels in
normal adults ranging from 1.3 to 1.4 ± 0.6 mg/mL.149,150 The
lysozyme content in tears decreases with age and decreases in
several eye diseases, including keratoconjunctivitis sicca, chro-
nic conjunctivitis, and nutritional deficiency with xerosis.151–153
Lysozyme is primarily effective against saprophytic Gram-
positive bacteria such as micrococci. Some coagulase-positive
staphylococcal strains can produce lysozyme, which may help
them overcome any inhibitory effect of the indigenous flora.154
Lysozyme may also interact with a recently described substance
called lysostaphin. Certain staphylococcal strains produce
lysostaphin. In contrast to lysozyme, lysostaphin inhibits many
strains of staphylococci including S. aureus, but it does not
inhibit micrococci.155 Lysozyme appears to increase the
antistaphylococcal activity of lysostaphin from 16- to 200-
fold.156 In Gram-negative bacteria, lysozyme aids the action of
complement on the cell’s cytoplasmic membrane.157
HUMORAL IMMUNITY
Normal tears contain antibodies against bacteria. Local anti-
body synthesis takes place in the lacrimal gland, but some
antibodies originate from lymphocyte sensitization in the
mucosal immune system.158 In P. aeruginosa infections, Berk
and associates showed that mice develop IgM and IgG anti-
bodies corresponding to their ability to recover from corneal
infection.159 Antibodies attach to the outer membrane proteins
(porin protein F) and protect the cornea.160 IgA at the ocular
surface can prevent bacterial attachment to epithelial cells.150
However, not all antibody responses are beneficial to the host.
Griffiss and associates have reported that serum IgA directed
against N. meningitidis blocks the lytic activity of IgG and IgM
for this organism.161
Complement and opsins, discussed earlier, are necessary for
the adherence of bacteria to polymorphonuclear leukocytes.
Complement can destroy bacteria directly or by causing
chemotaxis of neutrophils. Antibody-coated bacteria may be
unable to adhere to corneal epithelium. Antibodies can also
neutralize the exotoxins released by some bacteria.
CELL-MEDIATED IMMUNITY
CMI contributes to the defense against microorganisms. When
a T lymphocyte becomes sensitized to a bacterial antigen, it
releases a soluble factor (lymphokine) that can help to activate
the macrophage and localize it at the site of an infection.
The sensitized lymphocyte can also release chemotactic factors
for macrophages, neutrophils, basophils, and eosinophils.

Cytokines are released by inflammatory cells and have multiple
effects, such as activation and differentiation of other inflam-
matory cells, chemotaxis, and cytotoxic in bacteria. Upon entry
of the invading bacteria, the antigen is engulfed by macro-
phages. The antigen is processed and presented on the cell
surface to the T lymphocytes. Once recognized by the
T lymphocytes, the lymphocytes are activated and start to
proliferate. PG, TA, and other cell wall components may be
polyclonal activators of both B and T cells. Polyclonal activation
of human lymphocytes may be useful to the host as a mech-
anism of resistance to infectious diseases; however, the process
could also have adverse effects by triggering or perpetuating
chronic inflammatory disease. Studies in animals indicate
that immunization with the capsular polysaccharide provides
a T-cell-dependent immunity to abscess development when
challenged with Bacteroides fragilis. Also, it appears that the
killing of B. fragilis is T-cell dependent.162 Group A strep-
tococcal cell membranes appear to enhance certain T-cell
functions.163
DIAGNOSTIC TESTS
The diversity of infectious processes that involve the eye makes
it necessary for the ophthalmologist to be aware of a variety of
basic microbiologic techniques. Jones and associates have
written what still remains the most comprehensive approach to
ocular laboratory diagnosis.164 Both the ophthalmologist and
laboratory must be knowledgeable in determining which
bacteria are considered pathogens in ocular disease versus
contaminants or normal flora. Frequently, the material obtained
from cultures is small and must be inoculated onto media
immediately. The specific technique to be used and the cultures
taken will depend on the clinical diagnosis and setting; it is
useful to have protocols written out beforehand in order to
avoid needless errors. It is also helpful to maintain a culture
tray that is readily available. Routine culture media can be
stored in a refrigerator, but only fresh plates of media should
be used. Media that appear dry or that have pulled back from
the edges of the Petri dish should be replaced. Plates should be
brought to room temperature before inoculating them with
clinical material.
The method used to collect a specimen depends upon the site
and etiology of the infection. Cultures of the cornea, conjunc-
tiva, and eyelids can be done either with the Kimura platinum
spatula or with swabs. For eyelid cultures, our procedure is to
use a moistened calcium alginate swab. The use of a moistened
swab helps to prevent drying of the material and to create a
capillary attraction may enhance bacterial pickup. Furthermore,
the moistened swab allows release of the material over several
plates and avoids cutting into the media surface, which can
make recognition and isolation of colonies more difficult. If the
blepharitis is ulcerative, the platinum spatula may be used to
remove the fibrin scale, and this material may be cultured as
well. In cases of conjunctivitis, we will again use the swab
moistened in sterile saline or nutrient broth, reserving the
spatula to obtain specimens for cytology.
In cases of suspected microbial keratitis, a four-step approach
to the culture is taken. First, a moistened swab is used to
culture the ulcer base. Next the ulcer is scraped, usually with
a platinum spatula, but in some cases a Bard-Parker No. 15
blade or a Beaver blade may be required to obtain sufficient
material. The material obtained should then be immediately
inoculated onto culture media transferred to a moistened swab
and streaked onto appropriate media. The spatula is used to
obtain material for smears and slides, and finally a moistened
swab is again applied to the ulcer in order to pick up any bac-
teria brought to the ulcer surface. It should be emphasized that
Ocular Bacteriology
this is the minimum number of samples that should be taken.
Whenever there is a large, fulminating ulcer or sufficient
material is available, separate scrapings of the ulcer should be
done for each plate. In our laboratory, we have had more success
using separate plates for each site cultured. Although it requires
more plates and labeling, this technique facilitates the isolation
and identification of individual pathogens, particularly in
polymicrobial infections.
In cases of endophthalmitis, both aqueous and vitreous
should be cultured.165 Compared to aqueous fluid or fluid from
the vitrectomy cassettes, undiluted vitreous provide the high-
est yield of positive cultures. If there is sufficient material,
smears should also be performed for Gram-stain for bacteria or
KOH stain for fungus. Although smears may not always be con-
sistent with culture results, they may nevertheless be invaluable
in confirming a bacterial process in cases of culture-negative
endophthalmitis. A positive Gram-stain is useful information;
whereas a negative Gram-stain result had little correlation with
CHAPTER 11
culture results.165
MEDIA
Media can be divided into two broad types: broad-spectrum and
selective. All of the media used in ophthalmology are enriched
and nonselective, because selective media contain chemical
substances or antibiotics to inhibit the growth of all but the
desired organism. The basic media used for culture and
identification of most ocular bacterial pathogens are listed in
Table 11.2.
BLOOD AGAR
Blood agar consists of a Brucella agar base with a peptic digest
of animal tissue, dextrose, and yeast extract. Most aerobic bac-
teria (and fungi) will grow on it except for the more fastidious
pathogens, especially Neisseria, Haemophilus, Moraxella, and
atypical mycobacteria. When incubated under anaerobic
conditions, most anaerobic organisms will grow on blood agar
as well but it must be supplemented with hemin, vitamin K,
and sometimes cysteine. It also has the advantage of revealing
the hemolytic reaction of the organism. This is the best single
general purpose culture medium for the diagnosis of ocular
pathogens.
CHOCOLATE AGAR
Chocolate agar is prepared by using GC agar base and bovine
hemoglobin. Growth factors, hemin (X factor), and nico-
tinamide adenine dinucleotide (V factor) are added to the
TABLE 11.2. Bacterial Culture Media
Routine
Blood agar
Chocolate agar
Enriched thioglycolate broth
Sabouraud dextrose agar (for fungi)
Optional (Depends on Availability and the Clinical Situation)
Brain heart infusion broth
Lowenstein Jensen medium
Middlebrook agar
109
 MICROBIOLOGY
agar.166 These nutrients are essential for the growth of
Haemophilus, N. gonorrhoeae, N. meningitidis, and Moraxella.
When one suspects N. gonorrhoeae, Thayer–Martin medium
should also be used. Thayer–Martin medium contains 3 mg of
vancomycin, 7.5 mg of colistin, and 12.5 U of nystatin per
milliliter of agar to inhibit other bacteria or yeasts that could
inhibit the growth of gonococcus. However, Thayer–Martin
medium is only a supplement to and not a replacement for
chocolate agar, because potentially nongonococcal strains of
Neisseria may be inhibited by the added antibiotics. Incubation
of Thayer–Martin plates should be done in an atmosphere
containing 3–10% CO2.
BRAIN–HEART INFUSION BROTH
A highly nutritious and buffered liquid is a useful adjunct to
solid media for several reasons. Material picked up by the swab
but not released onto the solid agar thus has an opportunity to
SECTION 3
grow. Any antibiotics or other inhibitors of bacterial growth
will be diluted and, therefore, have less effect. Inoculation of
broth also allows the use of antimicrobial removal devices, such
as those developed by Osato. However, they do not permit one
to confirm that growth is occurring along the inoculum streak
nor do they allow one to quantify the amount of growth.
Other useful selective media include eosin methylene blue
(EMB) agar and MacConkey agar. These media are primarily
useful for the isolation of Gram-negative bacteria. Methylene
blue agar inhibits Gram-positive bacteria and has carbohydrates
that can be fermented by Escherichia coli and other Gram-
negative bacteria. MacConkey agar contains the carbohydrate
lactose, a fermentable carbohydride, as well as bile salts, which
inhibit the growth of Gram-positive bacteria.
Anaerobic cultures are routinely done in thioglycollate broth
without indicator. The broth is supplemented with hemin and
vitamin K. At times, aerobes also grow in thioglycollate, usually
near the surface; anaerobes, on the other hand, grow below the
surface. A disadvantage is that an anaerobic pathogen can be
overgrown by other anaerobic bacteria or by aerobic bacteria.167
In cases in which anaerobic cultures are especially important,
such as a possible P. acnes endophthalmitis or chronic cana-
liculitis, other anaerobic media should be used. Prereduced
anaerobically sterilized media (PRAS), anaerobic blood agar, or
chocolate agar can be used.168 In cases in which one obtains a
fluid sample, such as in endophthalmitis, the sample can
be injected through the rubber stopper into a chopped meat
glucose medium. Aerobic and anaerobic blood culture bottles
can also be used.
Lowenstein–Jensen medium is used for the isolation of
mycobacteria. It contains ribonucleic acid adequate for micro-
bacterial growth, along with penicillin and nalidixic acid, which
inhibit contaminating organisms. Nocardia species will also
grow on this medium.169 Middlebrook agar are used for the
detection of mycobacteria, and may be more sensitive than
Lowenstein–Jensen medium.170 These two media are especially
important in patients diagnosed with an infectious keratitis
following refractive surgery given that nontuberculous myco-
bacteria are common causes of the infection.73 Many of the
Mycobacterium chelonae–Mycobacterium abscessus complex
will also grow on blood agar media.
Proper conditions during incubation are essential. Aerobic
and anaerobic cultures should be kept at 35°C. Blood and
chocolate agar should be incubated under higher carbon dioxide
tension (3–10%). Routine cultures should be kept for 1 week,
but anaerobic cultures should be incubated for 2 weeks. Fungal,
actinomycete, and mycobacterial cultures should be held for
8 weeks. Mycobacteria grow best under a carbon dioxide tension
110 of 5–10%.
STAINS
While the results of smears may not always be consistent
with the final cultured organisms, smears are an important
component of bacterial diagnosis. Although one could base
initial therapy on Gram stain findings, given the incongruity
between smear and culture results, it would seem most pru-
dent to use the smear results to add to therapy rather than
delete from the standard initial treatment. Smears are also use-
ful in identifying polymicrobial processes in which one type of
bacteria may inhibit or delay the identification of other bac-
terial pathogens. Furthermore, smears may identify the pre-
sence of organisms that do not appear on culture for days or
even weeks. Smears are invaluable whenever cultures prove to
be negative, especially in patients who have previously received
antibiotics. In the laboratory, stains are essential in order to
identify cultured bacteria.
The proper preparation and examination of smears requires
both experience and patience. Smears are prepared by spreading
a thin film of the specimen over a defined area of the slide.
Smears that are too thick can obscure many important
details. Smears spread out over an entire slide increase the
length of time required to completely examine the slide and
increase the possibility of overlooking pathogens. The slide
should be free of lint and fingerprints, air-dried, and gently
heat-fixed. One must look at a large number of slides in order
to be able to distinguish between the occasional bacteria of
the ‘normal’ flora and an actual pathogen. In repertory results,
microbiologists should report only cell morphology and a Gram
reaction, not whether they think they see ‘pathogens’ or
‘normal flora’.
One of the oldest and most commonly used stains is the
Gram stain. As we have discussed earlier, this is a differential
stain in that bacteria are either Gram-positive (blue-purple) or
Gram-negative (orange-red). There are several theories to explain
why bacteria respond differently to a Gram stain. One theory
suggests that crystal violet and iodine form a chemical complex
in the bacterial cytoplasm. Alcohol in the staining process may
dissolve lipid, allowing the crystal violet–iodine complex to leak
out of the cytoplasm. Gram-negative bacteria with their high
lipid content in the cell wall would therefore lose more stain
than would Gram-positive bacteria. The cell walls of Gram-
positive bacteria are less permeable to small molecules than are
those of Gram-negative organisms. PG in the cell wall of Gram-
positive bacteria may trap the crystal violet–iodine complex.
Because Gram-negative bacteria have less PG, they would trap
considerably less stain.171 In any case, knowing whether an
organism is Gram-positive or Gram-negative continues to have
important diagnostic and therapeutic implications. Variable
Gram staining may occur with excessive decolorizing, with
smears that are too thick, or with older cultures. Gram-positive
organisms may appear Gram-negative if there has been previous
antibiotic treatment, leukocytic destruction, or excessive
heating of the slide.169 The safranin counterstain can replace
crystal violet, thus the slide should not be counterstained for
a prolonged time. Giemsa staining is not as important in
bacterial infections, because it has no differential value, but its
ability to delineate cellular types and detect inclusion bodies or
multinucleated giant cells make it an important investigative
tool in ocular diagnosis. Bacteria generally stain blue. The
Brown–Hopps stain is a Gram stain modified for tissues.
Aniline can be added to the Gram stain to improve iden-
tification of actinomycetes.
Acridine orange (AO) stains all DNA and RNA regardless
of organism. AO has recently received renewed interest owing
to its ability to stain Acanthamoeba species. The AO stain is
very good for bacteria too and is more sensitive than a Gram

stain, requiring fewer organisms to yield a positive result.172
Bacteria can stain red, orange, or green depending on relative
amounts of DNA versus RNA, whereas nonbacterial cells such
as squamous cells and polymorphonuclear leukocytes stain
green-yellow.173 If bacteria are detected, then a Gram stain
can be performed on the same slide without decolorization.
The major disadvantage is that the AO stain requires a
fluorescent microscope. Acid-fast staining is useful to detect
Mycobacterium species. The brilliant green counterstain allows
for improved contrast between acid-fast organisms and the
background. These include the Carbol-fuschsin or Ziehl–
Neelsen stains for acid-fast organisms. If Nocardia is suspected,
then an aqueous solution of 1% sulfuric acid rather than 3%
hydrochloric acid in 95% ethanol must be used as the
decolorizing agent. Fluorescein-conjugated lectins have been
used to identify microorganisms, primarily fungi, but do not
offer any advantages over existing stains in bacteriologic
diagnosis.174
HIGH-TECHNOLOGY DIAGNOSTIC
METHODS
Newer diagnostic methods may be used increasingly in
bacteriologic diagnosis. Antigen detection tests have been devel-
oped utilizing a variety of techniques, including counter-
immunoelectrophoresis (CIE), coagglutination (CoA), latex
agglutination (LA), enzyme immunoassay (EIA), enzyme-linked
immunosorbent assays (ELISA), radioimmunoassay (RIA),
solid-phase immunofluorescence and fluorescence polarization
immunoassay (FPIA), and immunoblotting (‘Western blot’).
These tests have tremendous potential and to date have been
useful in detecting cerebral spinal fluid pathogens, especially if
there has been pretreatment with antibiotics.81 Las and Western
blot have been used for the detection of Lyme disease and
Chlamydia trachomatis, respectively. In ophthalmology, these
tests are used most commonly for the detection of Chlamydia,
viruses, fungi, and ocular protozoal disease.
DNA probes are particularly useful when looking for a par-
ticular organism such as a mycobacterium. These probes are
also helpful for the detection of organisms that are present
in small numbers or are fastidious and difficult to cultivate.
Radiolabeled DNA probes are more sensitive and more specific,
but results take several days. Nonradioactive probes are gen-
erally less sensitive but faster. Various kits based upon the use
of specific nucleic acid probes are now available commercially
for identifying specific bacteria in a sample. They combine
high specificity with speed.175 These procedures do not dis-
tinguish between viable and nonviable bacteria, which may be
an advantage, especially when prior antibiotic treatment has
been used. The problem of sample size can be overcome by
nucleic acid amplification. The most widely accepted method is
the polymerase chain reaction (PCR). These methods rely on
the hybridization of a specific nuclei acid probe to a specific
DNA sequence of the organism. Despite the need for specific
primers, the main problem with the use of PCR is its exquisite
sensitivity, making contamination a real possibility. The 16S
rRNA is a highly conserved portion of bacteria RNA with many
copies present in each organism. This allows for rapid and
specific identification of the microorganisms. These tests are
available for many bacteria such as mycobacterium species,
C. trachomatis and N. gonorrhoeae. Commercially available
systems of ligase chain reaction (LCR) are available for
C. trachomatis and N. gonorrhoeae. PCR can also be performed
for the detection of RNA targets called reverse transcriptase
PCR. Other systems of RNA amplification include transcrip-
tion-mediated amplification (TMA) and the nucleic acid
sequence-based amplification (NASBA).81
Ocular Bacteriology
Gas-liquid chromatography (GLC) and high-pressure liquid
chromatography (HPLC) have been useful in the clinical micro-
biology laboratory, especially in the identification of quinones
and in carbohydrate analysis for taxonomic classification.176
Also, analysis of cell wall phospholipid fatty acid has shown
that each genus has a unique lipid fingerprint. Several auto-
mated bacteria identification systems are currently marketed.
ANTIBIOTIC SUSCEPTIBILITY AND
SENSITIVITY
Susceptibility tests help to determine the most effective
therapeutic agent available. These tests are somewhat arti-
ficial, because they do not consider the host’s defenses and
immune status, the number and accessibility of the organisms,
and whether the bacteria are intra- or extracellular, all of which
may influence antibiotic selection. In serious ocular infections,
bactericidal rather than bacteriostatic antibiotics should be
CHAPTER 11
utilized whenever possible. In bacterial keratitis, sensitivity
testing does not take into account the antibiotic levels
obtainable through the use of fortified drops. Antibiotic drug
levels can be much higher on the ocular surface than in serum,
where the cut-off susceptibility is determined. Therefore, even
if the bacteria are reported to be resistant to a specific anti-
biotic, the organisms may still be killed by topical antibiotic due
to the high drug level achieved with frequent topical appli-
cations. Clinical response is the most important parameter in
evaluating patients with infectious keratitis. Just as it is
important for the clinical microbiology laboratory to report
and identify all bacteria present in ocular cultures, it is vital to
make sure that the clinical laboratory performing the sensitivity
testing is aware of the specific agents available for ophthalmic
use so that these antibiotics can be routinely tested. Antibiotics
such as polymyxin B, bacitracin, and neomycin are no longer
included in most clinical laboratories’ sensitivity panel, but they
remain important ocular therapeutic agents.
Susceptibility testing using either disk diffusion or dilutional
tests should be performed on all potential pathogens. In order
to accelerate the selection of appropriate antibiotics, direct
susceptibility testing has been advocated.177 A pure culture is
required for the test to be reliable and several factors, including
the density of the inoculum and the presence of other micro-
organisms, can make the results misleading. It is probably
better to prescribe broad-spectrum antibiotics and then, once
the microorganism has been identified, modify therapy, if
necessary, based on clinical response and antibiotic sensitivities
of the organism. Disk diffusion tests are the most commonly
used technique.178 Antimicrobial-containing disks are placed
on the agar surface inoculated with a pure culture of the
organism. A zone of inhibition occurs around the disk. The
extent of this inhibition determines whether the bacteria are
sensitive to the particular antibiotic. The significant zone of
inhibition is different for each antibiotic owing to differences
in diffusion rates between antibiotics. Disk diffusion techniques
do have some limitations. They depend upon rapidly growing
organisms. The disk does not measure bactericidal activity,
and combinations of agents cannot be assayed. The disks only
reflect the usually obtainable serum concentrations and not
the higher levels obtainable within the tear film or cornea or
intraocularly. Therefore, organisms reported as resistant may be
susceptible in the ophthalmic setting. The most common
clinical setting in which this occurs is in the patient in the ICU
or burn unit who is infected with multiple aminoglycoside-
resistant Pseudomonas organisms and may respond to fortified
aminoglycosides, especially when they are combined with car-
benicillin or ticarcillin.179,180 A recently introduced BIOGRAM
(Giles Scientific, New York, NY) translates disk diffusion zone 111
 MICROBIOLOGY
sizes into minimal inhibitory concentrations (MICs), using
regression line analysis. A printed report is produced that
includes calculated MICs, Kirby–Bauer interpretations, and
inhibitory quotients that are based on achievable serum, urine,
bile, and cerebrospinal fluid concentrations.181 Potential
advantages include the ability to select from 34 antibiotics, the
ability to read results for many organisms in just 5–6 h, and
90–95% correlation with reference laboratory results.182
Another approach for determining antibiotic susceptibility
is an elution method. The antimicrobial elutes from paper disks
into broth or agar, thus providing a desired concentration of the
antimicrobial agent in the medium. This approach is used in
some automated systems for susceptibility testing of aerobic
and facultatively anaerobic bacteria as well as in susceptibility
testing of anaerobic bacteria and mycobacteria.183 Paper
diffusion methods are superior for the detection of methicillin-
resistant strains, provided that either a medium with a high
sodium chloride content is used or plates are incubated at 30°C
SECTION 3
for at least 24 h.184
Dilutional tests have several advantages over disk diffusion
testing. Besides determining the MIC, the minimal lethal con-
centration (MLC), or minimal bactericidal concentration (MBC)
can also be determined. Microdilution methods that place the
antimicrobial agents in microtiter tray wells are more practical
and lend themselves more to automation, because the trays can
then be read photometrically. The small sample size may make
detection of resistant subpopulations less likely, especially as
incubation times are reduced. Clinically, this is important in
detecting third-generation cephalosporin resistance because of
depressed b-lactamase production in Enterobacter, Serratia, and
P. aeruginosa.185 In order to consider the organism susceptible,
the peak obtainable concentration should be two to four times
higher than the MIC. The MBC level assumes greater
importance in clinical situations in which the cure of an
infection depends entirely on the antibiotic and bactericidal
activity. This is important for immune-deficient patients and
for those with CNS infections, but it also may be an important
consideration in endophthalmitis. Serum bactericidal activity
can be measured by the Schlichter test. Although not entirely
standardized, this test considers other factors that influence
antibiotic activity (especially serum protein binding) and has
been used primarily in the treatment of endocarditis and
osteomyelitis.186 Interpretation of MIC data is confusing to
many clinicians; one should encourage the laboratory to include
interpretative data with the report. Other pharmacodynamic
factors in bacterial infections of importance are the rate and
extent of bactericidal action, postantibiotic effect, minimal
antibiotic concentration, and postantibiotic leukocytic effect.187
Bacteria have shown great ability to develop resistance to
antibodies usually by the transfer of DNA between bacteria of
the same or different species. Much of the antibiotic resistance
encoded by genes is carried on plasmids. The production of
b-lactamase by H. influenzae, N. gonorrhoeae, and staphyl-
ococci correlates well with resistance to penicillin. Tests such as
the nitrocefin test can provide results in a matter of minutes
rather than overnight.188 This is increasingly important as anti-
biotic resistance is seen more frequently in clinical situations,
for example, in coagulase-negative staphylococcal endophthal-
mitis.189 Pericellular resistance has now been found in
S. pneumoniae not due to b-lactamase production but due to
changes to the genes encoding the target enzymes.190 There has
also been an increasing number of bacteria resistant to fluoro-
quinolones, a commonly prescribed ophthalmic antibiotic.191
112
ANTISEPTICS AND DISINFECTION
Sterilization and disinfection are important concepts that are
taken for granted every day. Sterilization implies destruction of
all forms of life, including spores, and generally requires a
physical agent such as pressurized steam or ethylene oxide.
Disinfection refers to the destruction of pathogens and fre-
quently involves the use of a chemical agent. Antimicrobial
agents are used daily in ophthalmic practice to preserve
medicines, sterilize instruments, and prepare the operative field
for surgery. There are numerous factors to be considered in the
selection of an appropriate antiseptic. The chemical must be
bactericidal and nontoxic to the host. The length of exposure,
pH, and temperature are also taken into account. Some
methicillin-resistant strains of S. aureus (MRSA) containing
plasmids encoding gentamicin resistance (MGRSA) also have
increased MIC values toward biocides such as GACs, chlor-
hexidine, acridines, and propamidine isethionate.107,192 Gram-
negative bacteria such as Pseudomonas are usually less sensitive
to chemical biocides (antiseptics, disinfectants, preservatives,
and sterilants) than are Gram-positive cocci. The main reason
is due to the great complexity of the outer cell membrane.193
Recent reports suggest that there is an increase in the resistance
of organisms to biocides, with increasing pressure for selecting
out antibiotic-resistant organisms.194
Key Features
• Most common ocular surface bacteria flora are Gram-positive
cocci, mainly CNS.
• The most common causes of ocular infections such as
infectious keratitis and endophthalmitis, are due to Gram-
positive cocci, such as staphylococci and streptococci.
• Pseudomonas are frequent causes of infectious keratitis in
contact lens associated infections.
• Minimizing the risk of postoperative infections is achieved by
eliminating bacteria from the ocular surface with the use of
antiseptic and antibiotics in the perioperative period.
Skin asepsis is important in ophthalmic surgery, because, as
noted earlier, most cases of endophthalmitis arise from the
patient’s own flora.195,196 Hendley and Ashe evaluated the effec-
tiveness of various antimicrobial agents in eradicating CNS
from the surface and stratum corneum of the skin.197 They
evaluated five antiseptic solutions and four antimicrobial
ointments. The skin surface was effectively sterilized by eight
of the nine agents tested. A soap-and-water wash was ineffec-
tive, but solutions of povidone-iodine, chlorhexidine-ethanol,
and 2% tincture of iodine eliminated surface bacteria. However,
sterilization of the stratum corneum was much more difficult to
accomplish. The rates of eradication of CNS from the stratum
corneum after surface treatment with chlorhexidine-ethanol
and povidone-iodine were not different from the control sites.
Only triple antibiotic ointment (neomycin, polymyxin B sulfate,
and bacitracin) was effective initially and inhibited overnight
repopulation from occurring. Only povidone-iodine has been
demonstrated to decrease the risk of endophthalmitis following
intraocular surgery.198,199 However, multiple studies have
demonstrated the effectiveness of povidone-iodine and
antibiotics in eliminating bacteria from the ocular surface at the
time of ocular surgery.200–202

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 CHAPTER

12 Chlamydial Disease
Irmgard Behlau

ANATOMY, PHYSIOLOGY, AND LIFE CYCLE MICROBIAL CHARACTERISTICS

OF THE MICROORGANISM
TAXONOMY
Historically based on different phenotypic properties,1 all
chlamydiae were classified into the order Chlamydiales, one
family Chlamydiaceae, and one genus Chlamydia, which
was composed of four species, C. trachomatis, C. psittaci,
C. pneumonia, and C. pecorum.1 Only the first three species are
associated with human disease. Humans are the natural hosts
of C. trachomatis and C. pneumoniae. These species have no
animal reservoirs, and transmission is from human to human.
Birds and some mammals are the natural hosts of C. psittaci
(Table 12.1).1–6 Based on recent analysis of 16S and 23S ribonu-
cleic acid (rRNA) analysis, a new taxonomic classfication7,8
proposes that the order Chlamydiales be divided into four
families, with the family Chlamydiaceae divided into two
genera, Chlamydia and Chlamydophila. The genus Chlamydia
is composed of three species: C. trachomatis, C. muridarum,
and C. suis; the genus Chlamydophila is composed of six
species: C. pneumoniae, C. psittaci, C. pecorum, C. abortus,
C. caviae, and C. felis.7,8
For many years Chlamydia organisms were considered viruses,
due to their obligate intracellular replication and small size
(diameter 200–1500 nm). They contain both DNA and RNA,
replicate by binary fission, possess a cell wall, and are inhibited
by antimicrobial agents. These bacteria are nonmotile and have
morphologic similarities to Gram-negative organisms with a
trilaminar outer membrane, but lack classic peptidoglycan.9,10
At this time, seven chlamydial genomes have been sequenced;
the molecular mass of the chlamydial genome is 660 µ 106
Da which is smaller than any other prokaryote except for
Mycoplasma sp.11 Certain metabolic pathways are missing
including amino acid and purine–pyrimidine biosynthesis,
anaerobic fermentation, and transformation competence
proteins.12 Chlamydiae possess the metabolic pathways to
synthesize adenosine triphosphate (ATP), but are unable to
synthesize ATP or to produce metabolic energy;13 therefore,
these use the ATP produced by the host cell for their own energy
requirements. Although chlamydiae’s dependence on the host
cell may appear restrictive, chlamydiae are proving to be highly
evolved pathogens. They are capable of infecting warm- and
cold-blooded animals and a variety of cell types from soil

TABLE 12.1. Characteristics of Human Chlamydial Species

C. trachomatis C. pneumoniae C. psittaci

Genus Chlamydia Chlamydophila Chlamydophila

Natural hosts
Serovars
Mode of transmission
Human diseases and
associated serovar
Elementary body
Morphology of Inclusion body
Synthesize folate
Sulfa sensitivity
Iodine-staining glycogen in
inclusions
Humans Humans Animals and birds
18 1 ≥4
Person to person, mother to infant Airborne person to person Airborne bird excreta to humans
Trachoma: A, B, Ba, C Upper and lower respiratory Pneumonia (psittacosis), fever of
Genital infections: D, Da, E, F, G, tract disease; coronary artery unexplained origin
H, I, Ia, J, K disease; ?age-related macular
LGV: L1, L2, L2a, L3 degeneration
Coccoid Pear shaped Coccoid
Single, round-vacuolar Multiple, uniform-dense Multiple, variable-sized dense
inclusions
+ — —
+ — —
+ — —
117
 MICROBIOLOGY
protists to brain microglial cells.1 Their high prevalence rate
of infection in humans and birds suggests that adaptation of
Chlamydia to obligate intracellular parasitism offers some
evolutionary advantage. Most recently, they appear to be able to
enter an alternative nonreplicative and persistent life-cycle,14,15
allowing them an optimal survival mechanism, thereby
allowing recurrent, relapsing, and persistent infections.
MORPHOLOGY AND LIFE CYCLE
The evolutionarily distinct, intracellular biphasic life cycle
shared by all Chlamydiae has been well characterized under
favorable environmental conditions; it consists of inactive
infectious elementary bodies (EBs) and metabolically active but
noninfectious reticulate bodies (RBs). All EBs are of similar size
(300 nm); C. trachomatis and C. psittaci are spherical particles,
while the EB of C. pneumoniae is pear-shaped (Fig. 12.1a). The
chlamydial life cycle (Fig 12.2) begins when infectious,
SECTION 3
metabolically inert EBs attach to cells of a susceptible host
epithelial cell via uncertain mechanisms.16 EBs stimulate
uptake and entry into the cell by receptor-mediated endocytosis
via postulated clarithin-coated pits16–18 but pinocytosis via
noncoated pits and use of heparin-like bridging molecules are
also speculated. Ingestion by the host cell results with the
internalized EB within a host-derived vacuole termed inclusion.
Through an unknown process requiring bacterial protein syn-
thesis, inclusions are stable, not maturing into late endosomes
or fusing with lysosomes. Phagolysosomal fusion does not occur
and the organism is protected from digestion by lysozymes.19
The chlamydial phagosome, or inclusion body, is transported to
a juxtanuclear position that corresponds to the peri-Golgi
region. The inclusion body then intercepts cellular metabolites
being transported from the Golgi apparatus to the cell mem-
brane via the trans-Golgi exocytic pathway.20,21 Approximately
8 h after entering the cell, the EB reorganizes into a reticulate
body (RB), so-called because of the dispersed fibrillar pattern of
its nucleic acids (Fig. 12.1b).22,24 The RB is the replicative phase
in the life cycle of chlamydiae. Transition of EB to RB is
associated with: loss of infectivity, an increase in diameter to
800–1000 nm, and an increase in ratio of DNA to RNA from
1:1 in the EB to 3:1 in the RB,23 an increase in the rate of
metabolic activity compared to a metabolically inert EB, and a
change in the cell wall from rigid and impermeable in the EB to
flexible and permeable in the RB. These cell wall changes are
thought to result from reduction of cross-linked disulfide bonds
in the outer membrane proteins by the intraphagosomal reduc-
ing conditions to which the EB is exposed after endocytosis.24–28
The increased permeability of the RB cell wall permits uptake
a b
118
of ATP and nutrients from the host cell. RBs typically line the
inner margin of the inclusion body membrane which contrasts
with the EBs that are distributed randomly throughout the
inclusion.4,29 The RBs initiate RNA and DNA synthesis and
multiply by binary fission until the original phagosome
becomes distended by its content of several hundred to more
than 1000 chlamydial cells. After 8–12 rounds of multiplica-
tion, the RB asynchronously differentiate to EBs.29 As RB repli-
cation proceeds, the reducing power of the microenvironment
probably decreases, and free sulfhydryl groups are oxidized,
forming disulfides. This restores the rigidity and imper-
meability of the cell wall and produces a decrease in the rate
of metabolism, coincident with reorganization of RB into EB.24
At 48–84 h postinfection (depending on the infecting species),
the host cell and its intracytoplasmic inclusions rupture, and
the newly formed EB progeny are released into the extracellular
milieu, infecting other cells or a new host to begin a new
cycle.29a,29b,29c
The recognition that chlamydiae may cause persistent
infections in their hosts dates back to 1933 with latent
psittacosis in birds.30 There is increasing evidence in vitro and
in vivo that chlamydiae persist in an altered form during
chronic disease.31 Under adverse conditions, such as glucose or
amino acid limitation, elevated temperatures, or sublethal
antibiotic concentrations, chlamydiae are capable of conversion
to a noncultivable growth stage with nonreplicating persistent
bodies (PBs) which appear aberrant and display altered gene
expression.14 The different in vitro persistence systems share
altered growth and ultrastructural characteristics with enlarged,
pleomorphic RBs that are inhibited in binary fission, but accu-
mulate chromosomes and do not differentiate to EBs. These
changes are reversible27,38,39 once either the factor that inhibits
growth is removed (antibiotics,28,32,33 cytokine-induction,
particularly interferon gamma [IFN-g],34–36 or infection with
phage),37 or replacement of a missing nutrient.27,29,39 In contrast
to other persistence model systems, chlamydiae become
spontaneously persistent following infection of monocytes40,41
and when maintained under continuous culture conditions.42,43
Supportive in vivo observations for chlamydial persistence
include epidemiologic reports of recurrences which are most
likely due to reactivation of persistent infections rather than
reinfections15 (active trachoma decades after the initial
infection,44 altered morphological forms in vivo (recent electron
microscopic visualization of C. pneumoniae aberrant RBs
(resembling those seen in vitro) within macrophages in patients
with degenerative aortic valve stenosis),45 detection of
chlamydial macromolecules in diseased hosts in the absence
of cultivability (Chlamydia pneumoniae in human choroidal
FIGURE 12.1. Electron micrographs of
Chlamydia trachomatis showing (a) an EB with
cell wall and electron-dense core of nucleic
acids and (b) an RB with DNA and ribosomes
distributed in a fibrillar pattern. RB is
characteristically larger than EB (bars =
0.1 mm).
From Clark RB, Nachamkin I, Schatzki PF, et al:
Localization of distinct surface antigens on Chlamydia
trachomatis HAR-13 by immune electron microscopy
with monoclonal antibodies. Infect Immun 1982;
38:1273.

Proposed
Persistence
B C
Phase
A
D including a new family of polymorphic outer membrane proteins
J
Life Cycle
of
Chlamydia E
I
K
H therefore isolates from this species are serologically identical.
F
G
Elementary Body (EB)
Reticulate Body (RB)
Aberrant RB
Nucleus
Golgi Apparatus
FIGURE 12.2. Life cycle of Chlamydia organisms.
neovascular membranes due to age-related macular degener-
ation),46 and clinical antibiotic resistance.15 Further evidence to
discount that the in vivo evidence may represent enhancement
of an inapparent low-grade infections, are the demonstrated
similarities in chlamydial gene or protein expression between
persistent cell culture systems and tissue samples from sites of
chronic disease.47–53 The mechanism by which chlamydiae
enter and exit the persistent phase is yet to be defined, but the
important survival advantage of a persistent phase warrants
the addition of this phase to the well-accepted biphasic life cycle
of chlamydiae (Fig 12.2).
ANTIGENS
Chlamydiae contain both common antigens and species-
specific antigens that play a role in pathogenesis and diagnosis
of infection. All chlamydiae share the genus-specific common
antigen which is a glycoprotein that is similar to the
lipopolysaccharide (LPS) found in the outer membranes of
Gram-negative bacteria.54 It contains a ketodeoxyoctanoic acid-
reactive moiety55 and is present in the outer membranes of both
EBs and RBs. Type-specific antigens have been characterized in
C. trachomatis and C. psittaci. The microimmunofluorescence
(MIF) test has identified 15 serovars of C. trachomatis:56,57
serovars A, B, Ba, and C are usually isolated in areas of endemic
trachoma,58 serovars D through K are the most prevalent
sexually transmitted59–61 and ocular infection with these
serovars results in inclusion conjunctivitis, and serovars L1, L2,
and L3 are the agents of lymphogranuloma venereum58 (Table
12.1). Three additional serovars (Ba, Da, Ia, and L2a) of
C. trachomatis have more recently been identified.62 Species-
specific and type-specific antigens of C. trachomatis are located
in the major outer membrane protein (MOMP),63–65 encoded by
the ompA gene of C. trachomatis constitutes ~60% of its outer
membrane, has a molecular mass of 38–42 kDa,66–68 and has
four surface-exposed variable domains which confer serotype-
specific epitopes, and are immunodominant.63,65,69 Part of
the reason that C. trachomatis evades the host’s immunologic
defenses is MOMP antigenic variation resulting from allelic
polymophism at the omp1 locus70 Molecular evaluation of the
major outer membrane protein (MOMP) gene (omp1) offers a
more precise method of characterizing C. trachomatis than does
immunotyping by MIF.70 Determination of omp1 genotypes will
be useful in epidemiologic studies to identify reservoirs and
transmission patterns of C. trachomatis and to select candidate
Chlamydial Disease
strains for vaccine development.33 There are other antigens
associated with species and serotype specificity which are
incorporated into the cell wall of C. trachomatis. In addition,
soluble antigens that are released into the supernatant fluids of
cell cultures infected by C. trachomatis have been described,71,72
(POMPs). IncA is the prototype of exported proteins which
localize in the cytoplasmic surface of the inclusion membrane,73
inject the host cell by a type III secretion mechanism,74–76 and
may provoke immunopathogenic responses in the host. Type-
specific antigens have not been characterized for C. pneumoniae,
SYSTEMIC INFECTION OF THE HOST
NATURAL HISTORY OF CHLAMYDIAL
INFECTIONS
CHAPTER 12
Spectrum of Chlamydia trachomatis Infections
Since C. trachomatis can infect columnar or transitional
epithelium at any anatomic site, multiple-organ involvement
is possible. The most frequently infected sites are those most
accessible to infected mucosal secretions such as the external
genital tract, conjunctivae, and upper respiratory tract. From
these external sites, infection can spread within an organ
system and result in infection of structures (e.g., salpingitis,
epididymitis, pneumonitis, perihepatitis) that are protected
against primary contact.77–80 Infection can also spread from one
infected external site to another (e.g., urethra, cervix, rectum,
conjunctivae) by natural drainage of infected secretions or poor
personal hygiene. The oculogenital serovars of C. trachomatis
(A through K) can infect any squamocolumnar epithelial
mucosa. LGV serovars are more invasive and can infect lymph
nodes and associated structures.
INFECTION AND INFLAMMATORY RESPONSE
Natural infection with C. trachomatis appears to confer little
protection against reinfection. Multiple or persistent infections
are essential characteristics in the pathogenesis of ocular
trachoma. Chlamydial infections elicit an inflammatory
response that is characterized by PMN predominance with a
shift to lymphocyte predominance and the formation of
lymphoid follicles on infected mucosal surfaces as the infection
progresses. PMNs have been shown to phagocytose chlamydial
EBs81–83 during initial exposure of the host, and impede spread
of infection by EBs released into the extracellular milieu during
subsequent chlamydial growth cycles. The role of lymphocytes
is incompletely understood, but intact lymphocyte function is
apparently important, because duration of infection and
infection-related mortality rates from the mouse pneumonitis
strain of C. trachomatis were greater in athymic nude mice than
in immunocompetent animals.84–88 Similarly, guinea pigs
treated with antithymocyte serum to suppress cell-mediated
immune function were unable to eliminate genital infection by
the guinea pig inclusion conjunctivitis strain of C. psittaci.89
Lymphoid follicle formation is characteristic of human ocular
and genital chlamydial infections.90–96 There is thinning or loss
of epithelium overlying the follicles and they may become
necrotic as the disease progresses with resultant fibrosis and
scarring.
ANTIBODY RESPONSE
Our understanding of the role of antibody in natural infection
is incomplete. C. trachomatis infections cause immunoglobulin
M (IgM) and IgG antibodies to appear in the serum and IgG 119
 MICROBIOLOGY
and IgA antibodies to appear in mucosal secretions.97–99 These
antibodies are directed against several chlamydial antigens,
including MOMP, as well as 60-kDa and 75-kDa proteins.100–102
In vitro, EBs that have been exposed to antibodies fail to
replicate in cell culture, although they attach to the cells and
induce endocytosis.103–105 In the mouse, high levels of serum
antibodies protect against the mouse pneumonitis strain of
C. trachomatis.87 In contrast, preexisting serum antibodies in
humans do not appear to protect against infection, but may
be important for containment and resolution of chlamydial
infections. Most persons in groups at high risk for sexually
transmitted infections have serum antibodies but are subject to
repeated infections from both previously unencountered
chlamydial serovar or genotype and reinfection with preexisting
serovar-specific antibody.100 Consistent with these findings is
the observation that infants become infected with maternal
serovars of C. trachomatis even if they acquired maternal IgG
antibody transplacentally.106 In guinea pig inclusion conjunc-
SECTION 3
tivitis (GPIC), produced by a strain of C. psittaci, disease was
more prolonged, severe, and invasive when the humoral
antibody response was suppressed.107,108 In a study of women
with cervical C. trachomatis infection who underwent elective
abortion without prior antichlamydial treatment, ascending
infection and salpingitis occurred less frequently in patients
who had higher titers of serum antibodies.101 Although infec-
tion occurs at birth in infants with congenital C. trachomatis
infection, the incidence of pneumonia is highest during the
second and third months of life, a period that coincides with the
decline in titer of transplacentally acquired antibodies.109
CELL-MEDIATED IMMUNE RESPONSE
Cell-mediated immune responses (CMIs) to chlamydial infec-
tions, as detected by antigen-directed lymphocyte proliferation
assays, have been demonstrated in both humans and
animals.109,110 CMIs in animals have also been demonstrated
by induction of footpad swelling in response to local antigen
injection in the mouse pneumonitis model of chlamydial
infection.111 CMI appears to contribute to control and resolution
of infection. For example, transfer of T cells from mice with
normal immune function confers protection against the
prolonged infection and high mortality otherwise observed in
athymic mice infected with the mouse pneumonitis agent.86
The same serovar of C. trachomatis also produces nonresolving
genital infections in athymic mice but not in mice with an
intact CMI.88 Induction of cytotoxic T lymphocytes is another
CMI mechanism that may be important in the resolution of
chlamydial infections.112–115 Although cytotoxicity was directed
principally against Chlamydia-infected cells mediated by the
cytokine IFN-g,116 nonspecific cytotoxicity against uninfected
cells was also noted (mediated by tumor necrosis factor alpha
(TNF-a)).117 Further studies are needed to delineate the role of
CMI in chlamydial infections.
SUSCEPTIBILITY TO ANTIMICROBIAL
DRUGS
The macrolide (erythromycin, azithromycin, and clarithromycin)
and the tetracycline (tetracycline, doxycycline, and minocycline)
antibiotics are structurally unrelated, but block chlamydial
protein synthesis by inhibition of the 50S and 30S ribosomal
subunits, respectively.118 Although their action is bacteriostatic,
they are the most effective therapeutic agents in the treatment
of chlamydial infections.119 Azithromycin given as a single dose
has become the treatment of choice for uncomplicated lower
genital infections with C. trachomatis120 and trachoma.121
120 Community wide treatment with azithromycin is part of efforts
to control trachoma.121,122 Fluoroquinolones may also be effec-
tive but are second- or third-line agents. Due to rapid develop-
ment of resistance, rifampin cannot be recommended despite
good in vitro activity. Since chlamydial cell walls do not contain
peptidoglycan, it is not surprising that b-lactam antibiotics
remain ineffective against chlamydial infections.119 Aminogly-
cosides and cephalosporins are also not active against Chlamydia.
Key Features: Recommended Treatment for
Lymphogranuloma Venereum225
Recommended Regimen
• Doxycycline 100 mg orally twice a day for 21 days
Alternative Regimen
• Erythromycin base 500 mg orally four times a day for 21 days
• Azithromycin 1.0 g orally once weekly for 3 weeks is probably
effective, although clinical data are lacking
Key Features: Recommended Treatment of Chlamydial
Urethritis/Cervicitis in Adults and Adolescents225
Recommended Regimens
Azithromycin 1 g orally in a single dose OR
Doxycycline 100 mg orally twice a day for 7 days
Alternative Regimens
Erythromycin base 500 mg orally four times a day for 7 days OR
Erythromycin ethylsuccinate 800 mg orally four times a day for
7 days OR
Ofloxacin 300 mg orally twice a day for 7 days OR
Levofloxacin 500 mg orally once daily for 7 days
Key Features: Recommended Treatment Regimens for
Chlamydial Infections in Pregnancy225
Recommended Regimens
Azithromycin 1 g orally in a single dose OR
Amoxicillin 500 mg orally three times a day for 7 days
Alternative Regimens
Erythromycin base 500 mg orally four times a day for 7 days OR
Erythromycin base 250 mg orally four times a day for 14 days OR
Erythromycin ethylsuccinate 800 mg orally four times a day for
7 days OR
Erythromycin ethylsuccinate 400 mg orally four times a day for
14 days
Erythromycin estolate is contraindicated during pregnancy
because of drug-related hepatotoxicity. The lower dose 14-day
erythromycin regimens may be considered if gastrointestinal
tolerance is a concern
Key Features: Recommended Treatment Regimens for
Chlamydial Infections in Children225
Recommended Regimens for Children Who Weigh < 45 kg
Erythromycin base or ethylsuccinate 50 mg kg–1 day–1 orally
divided into 4 doses daily for 14 days
Recommended Regimen for Children Who Weigh >45 kg but
Who Are Aged <8 Years
Azithromycin 1 g orally in a single dose
Recommended Regimens for Children Aged >8 years
Azithromycin 1 g orally in a single dose OR
Doxycycline 100 mg orally twice a day for 7 days
Sexual assault or sexual abuse of children must be considered.
Follow-up cultures are necessary to ensure that treatment has
been effective

Key Features: Recommended Management of
Chlamydial Infections in Infants225
Ophthalmia Neonatorum Caused by C. trachomatis
Considered for all infants aged <30 days who have conjunctivitis,
especially if the mother has a history of untreated Chlamydia
infection.
Diagnostic Considerations
Sensitive and specific methods used to diagnose chlamydial
ophthalmia in the neonate include both tissue culture and
nonculture tests (e.g., DFA tests, EIA, and NAAT).
The majority of nonculture tests are not FDA-cleared for the
detection of Chlamydia from conjunctival swabs, and clinical
laboratories must verify the procedure according to CLIA
regulations. Specimens must contain conjunctival cells, not
exudate alone. Specimens for culture isolation and nonculture
tests should be obtained from the everted eyelid using a Dacron
tipped swab or the swab specified by the manufacturer’s test kit.
A specific diagnosis of C. trachomatis infection confirms the need
for treatment not only for the neonate but also for the mother and
her sex partner(s). Ocular exudate from infants being evaluated for
chlamydial conjunctivitis also should be tested for N. gonorrhoeae.
Recommended Regimen
Erythromycin base or ethylsuccinate 50 mg kg–1 day–1 orally
divided into 4 doses daily for 14 days.
Topical antibiotic therapy alone is inadequate for treatment of
chlamydial infection and is unnecessary when systemic treatment
is administered.
The efficacy of erythromycin treatment is ~80%; a second
course of therapy might be required and follow-up is necessary.
Infant Pneumonia Caused by C. trachomatis
Diagnostic Considerations
Specimens for chlamydial testing should be collected from the
nasopharynx. Tissue culture is the definitive standard for
chlamydial pneumonia. Nonculture tests (e.g., EIA, DFA, and NAAT)
can be used, although nonculture tests of nasopharyngeal
specimens have a lower sensitivity and specificity than nonculture
tests of ocular specimens. DFA is the only FDA cleared test for the
detection of C. trachomatis from nasopharyngeal specimens.
Tracheal aspirates and lung biopsy specimens, if collected, should
be tested for C. trachomatis. Because of the delay in obtaining test
results for Chlamydia, the decision to provide treatment for
C. trachomatis pneumonia must frequently be based on clinical
and radiologic findings.
The results of tests for chlamydial infection assist in the
management of an infant’s illness and determine the need for
treating the mother and her sex partner(s).
Recommended Regimen
Erythromycin base or ethylsuccinate 50 mg kg–1 day–1 orally
divided into 4 doses daily for 14 days.
The effectiveness of erythromycin in treating pneumonia caused
by C. trachomatis is ~80%; a second course of therapy might be
required. Follow-up of infants is recommended.
HOST–MICROBE INTERACTION IN
THE EYE
NATURAL HISTORY OF TRACHOMA
Blinding trachoma, the end-stage of a chronic process caused by
repeated infections with C. trachomatis, occurs in impoverished
populations living under conditions of poor hygiene.123–125 The
disease is particularly prevalent in the Middle East and parts
of southeast Asia. In hyperendemic areas, infection is acquired
during infancy, and most children are infected by 2 years of
age.126 Primary infection induces purulent follicular conjunc-
tivitis (except during the neonatal period). The follicles consist
of lymphoid germinal centers.127 Because lymphoid tissue is
Chlamydial Disease
absent from the conjunctivae of neonates, lymphoid follicles
do not form. Infection at this age produces acute purulent
conjunctivitis, but the tissue reaction is one of papillary hyper-
trophy.128 Primary infection resolves spontaneously and induces
transient protective immunity; in endemic areas, however,
reinfection is inevitable. The same serovar of C. trachomatis is
often transmitted reciprocally among members of a household.129
With repeated infections, healing is associated with central
degeneration and necrosis of lymphoid follicles, thinning of
the overlying conjunctival epithelium, and proliferation of
fibroblasts, resulting in fibroses and scarring.130 Uninterrupted
progression of this process eventually converts the normally
smooth and lubricating conjunctival epithelium into one that
is xerotic and cicatrized. Extensive fibrosis produces entropion
and trichiasis. End-stage blindness is the result of corneal
drying, ulceration, and scarring.
CHAPTER 12
PATHOGENESIS OF TRACHOMA
Studies in Humans
The observation that repeated chlamydial infections are
characteristic of the course of blinding trachoma has led to the
concept that the disease constitutes an immunopathologic
response of the host to C. trachomatis infections.129,131 Initial
infection presumably induces immune sensitization of the host
but only transient or incomplete protective immunity.
Reinfections or relapses result in intensified inflammatory
reactions, fibrosis, scarring, and pannus formation. In vaccine
studies using inactivated EB as antigen, recipients immunized
with an antigen dose that proved to be inadequate to induce
immunity against infection developed more severe disease
with subsequent infections than did unvaccinated controls.132
Reinfection also frequently results in exacerbation of
trachoma.129–131 Consistent with this observation is a report
that trachoma did not progress further in persons who moved
from an endemic to a nonendemic area where they were no
longer exposed to the pathogen.133 Immunopathogenesis is
further evidenced by the finding that in trachoma-endemic
areas, proliferative responses of peripheral blood lymphocytes to
stimulation by chlamydial antigens, a marker of CMI, are more
common in patients with trachoma than in controls without
disease.134
The apparent genetic susceptibility to trachoma further
supports this concept. In a study in Gambia, the frequencies of
the human leukocyte antigen (HLA) complex class I antigen,
HLA-A28, and the A„6806 allele were significantly greater in
patients with trachoma than in age-, sex-, and location-matched
controls.135 Immunopathology may be associated with HLA-
A„6802-restricted T-lymphocyte responses. In Chlamydia-
associated involuntary tubal infertility, another disease of
suspected immunopathogenic origin, antibodies to the 60-kDa
C. trachomatis heat shock protein, a putative immunopathogenic
antigen, are more common in affected individuals than in
controls.136–139 Heat shock or stress proteins are produced by
all prokaryotic and eukaryotic cells in response to damaging
stimuli such as elevated environmental temperature.125 They
are major antigens of many pathogens and appear to be important
to the immune response, including immune surveillance and
autoimmunity.140,141 In mice, the immune response to the
60-kDa heat shock protein of C. trachomatis is genetically
controlled.142 This observation adds support to the concept that
the outcome of chlamydial infections in humans may also have
a genetic component.
Studies in Animals
Animal experiments support the hypothesis that trachoma is
an immunopathologic process induced by repeated ocular 121
 MICROBIOLOGY
infections with C. trachomatis.143 In primate studies, progressive
conjunctival and limbal scarring and pannus formation
occurred only in animals that had received more than one
chlamydial inoculation or that had previously been immunized
with an experimental trachoma vaccine.143–149 Similar results
were seen regardless of the serovar involved (serovar A or
serovar E),150 which suggests that repeated ocular infection
induces trachoma. The inflammatory reaction decreased in
severity with repeated inoculations of both serovars, and
Chlamydia could not be reisolated from the eyes after six to
eight weekly inoculations, despite continuation of the inocu-
lations. This is consistent with the fact that C. trachomatis
can seldom be isolated from the eyes of humans with advanced
trachoma. This progression of disease in the absence of
detectable Chlamydia organisms suggests that the immune
response is partially protective, but continued antigenic
stimulation elicits a pathologic immune response. Repeated
inoculation with live organisms was essential to development
SECTION 3
of chronic disease.151,152
Taylor and co-workers, by infecting cynomolgus monkeys
with C. trachomatis,153,154 determined that internal antigens
(isolated by a soluble triton extract) rather than surface antigens
(MOMP, LPS) are the stimuli involved in the pathogenesis of
trachoma, not surface antigens.152,155 Ocular delayed hyper-
sensitivity was similarly demonstrated in guinea pigs156 and the
ability of a triton extract of GPIC EBs to produce an inflam-
matory response in the eyes of monkeys previously infected
with C. trachomatis, suggests that the sensitizing antigen is
genus-specific rather than species-specific.153 Lymphocytes
in the inflammatory response were antigen-specific for
Chlamydia.149 In guinea pigs, infection of the conjunctivae,
vagina, or intestine, but not intramuscular injection of live
GPIC EBs, resulted in ocular sensitization and a delayed hyper-
sensitivity reaction on subsequent conjunctival challenge with
triton-extracted antigen.157 This suggests that ocular delayed
hypersensitivity can be induced by prior infection of mucosal
surfaces, not only of the eye but other anatomic sites.158,159
Cytokines elaborated by the host in response to chlamydial
infections may also be important to the progression of trachoma.
In animal studies, chlamydial infections induce host production
of both IFN-g and TNF-a.160,161 TNF-a stimulates collagenase,
prostaglandin E2, and hyaluronic acid production by human
fibroblasts.162,163 IFN-g also stimulates hyaluronic acid
production.163,164
INCLUSION CONJUNCTIVITIS
Neonatal Inclusion Conjunctivitis
C. trachomatis is the most frequent cause of neonatal
conjunctivitis.165,166 When a pregnant woman has culture-
positive cervical infection with C. trachomatis at the time of
labor and delivery, the infant born per vaginal birth has an
18–50% chance of becoming clinically infected.167 The serocon-
version rate of infection may be as high as 70%.106 The con-
junctivae of the infant delivered via an infected birth canal
appear to be the usual site of initial infection; subsequently,
infection spreads to the nasopharynx.168 If untreated, the
infection may involve the lower respiratory tract and cause
pneumonia.167,169 The rectum and vagina may also become
colonized.169,171 Almost all infants with conjunctival infection
develop conjunctivitis within the first 3 weeks of life, which,
even if it is not treated, is usually self-limited.168 In industri-
alized nations, infants seldom become reinfected, and progres-
sion to trachoma does not occur. In cases of persistent or
untreated infection, however, corneal micropannus and palpebral
conjunctival scarring occur occasionally.172–174
122
Inclusion Conjunctivitis in Adults
Studies in western Europe and in the United States identified
C. trachomatis via culture as the pathogen in as many as 9% of
cases of acute conjunctivitis and 19% of cases of chronic
conjunctivitis.175–181 In one study that limited patients to 20–25
years of age, the isolation rate was as high as 23%.177 Adults
with chlamydial conjunctivitis frequently have a concurrent
genital infection. Presumably, poor personal hygiene results in
contamination of the conjunctivae by infected genital secretions.
Because repeated ocular infections are rare, corneal scarring,
although reported, appears to be unusual.
LABORATORY DIAGNOSIS
Key Features: Diagnostic Tests for Chlamydia Infections
Cell Culture
• Clinical specimen cultured on cell monolayer (McCoy or HeLa)
• Sensitivity is 75–80% by expert laboratories; specificity is
~100%
• Advantages are highly specific and all Chlamydia species can
be cultivated
• Disadvantages are expense, high level of technical expertise,
stringent cold-chain transportation, and time until results
(3–7 days) have limited its use
Direct Fluorescent Assay
• An antigen in the membrane of Chlamydia trachomatis (usually
MOMP) is detected directly by an antibody labeled with a
fluorochrome, examined under ultraviolet light
• Sensitivity is 80–90%; specificity is 95% compared to culture
• Advantages are direct assessment of specimen adequacy,
cost-effective, rapid results (30 min), and no special
transportation
• Disadvantages are highly trained personnel, performance
variability due to fixation technique, number of EBs present,
serotype and antibody used
Nucleic Acid Amplification Test
• Important advance in diagnosis of Chlamydia infection; uses
species-specific primers to amplify Chlamydia DNA
• Highest sensitivity 90%; highest specificity for nonculture test
99–100%
• Advantages are not dependent on the viability of the organism
and able to detect to as low as 10 copies of Chlamydia DNA
• Disadvantages are the inhibition by substances (problem
overcome by Amplicor, Roche); stringent lab conditions to
avoid ‘carry-over’ lab contamination
Detection by Cell Culture
Cell cultures have been considered the gold standard for
detection of C. trachomatis, but the definition of gold standard
has been now defined by a combination of tests (culture, DFA,
PCR). The principal disadvantages of cell culture are that (1) it
may give false-negative results if the organism is inactivated by
improper collection, transport, or storage; (2) it requires special
laboratory facilities and experienced personnel; (3) it takes
several days to perform the test and obtain results; and (4) it is
expensive. Chlamydiae are relatively labile organisms and
viability is enhanced by keeping specimens cold and minimizing
transport time to the laboratory. Because Chlamydia organisms
are present in infected epithelial cells and not in the exudate
produced by infection, the specimen should contain as many
epithelial cells as possible. To collect conjunctival specimens,
one should cleanse the eye of exudate and swab the conjunctival
surface with pressure sufficient to exfoliate cells. Swabs with
 Chlamydial Disease

metal or plastic shafts rather than wood shafts are preferred,

because toxic products from wood may be leached into the
collection medium and have toxic effects on the cell culture into
which it is inoculated. Sucrose phosphate buffer is frequently
used as a collection medium.182 Antibiotics (usually aminogly-
cosides) and fungicides to which C. trachomatis organisms are
resistant are usually incorporated into the collection medium to
inactivate contaminating bacteria and yeast that otherwise
would grow in and destroy inoculated cell cultures. After
collection, specimens may be stored at 4°C if they are to be
cultured within 24–48 h. Specimens that cannot be cultured
within that time frame should be stored at –70°C to retard
inactivation. Isolation rates are highest when specimens are
cultured promptly after collection.
Since C. trachomatis is an obligate intracellular parasite, it a
replicates only in living cells. Although the organism was first
successfully cultivated in 1957 in the yolk sacs of embryonated
eggs, this method is labor-intensive and less sensitive than the

cell culture technique that was developed later.183,184 The yolk
sac method is only used to prepare antigens for the MIF test
discussed below. Most laboratories use cell culture for isolation
and demonstration of intracytoplasmic inclusion by various
staining procedures. The cell types most frequently used for
cultivation and detection of C. trachomatis are McCoy cells
and HeLa 229 cells185,186 A nutrient-rich cell culture medium
is employed, and the cultures are treated with metabolic
inhibitors such as cycloheximide or cytochalasin B to prevent
the cells from competing with the parasite for nutrients.187,188
Despite this favorable microenvironment, C. trachomatis,
except for serovars L1, L2, and L3, does not readily infect cell
cultures. Infection requires enhancement by centrifugation of
inoculated cultures at 2500–3000 µ g for 60 min.189–194 After
inoculation, cultures are usually incubated for 72 h at 35°C
and then stained and examined for chlamydial cytoplasmic
inclusion bodies. Giemsa’s or iodine stains can be used to stain
the inclusions; however, the sensitivity of the method is
increased by staining with fluorescein-conjugated monoclonal
antibody prepared against C. trachomatis.195,196 Chlamydial
inclusions fluoresce with a bright apple-green color. Figure
12.3a shows an example of inclusions in an infected McCoy cell
culture stained with fluorescein-conjugated monoclonal
antibody.
C. psittaci can be isolated from respiratory tract secretions,
blood, and tissue biopsy specimens (spleen, liver) from patients
with ornithosis (psittacosis). The organism can be isolated by
inoculation of the yolk sac of embryonated eggs or of cell
cultures of L cells or McCoy cells. C. psittaci inclusion bodies
are detected by Giemsa staining of infected cell culture mono-
layers or impression smears of infected yolk sac membranes.
For isolation of C. pneumoniae, throat swabs or specimens
of respiratory tract secretions are obtained and placed in the
same transport medium that is used for C. trachomatis.
C. pneumoniae was originally isolated in HeLa 229 cells, but
HL, HEp-2, and H292 cell cultures have been reported to be
more sensitive.197–202 Inclusions in infected cells can be
specifically identified by staining with fluorescein-conjugated
monoclonal antibodies.
C. trachomatis is a biosafety level 2 (BL2) agent and is not
considered a dangerous pathogen in the laboratory. Occasional
reports of laboratory associated follicular conjunctivitis have
been reported. The LGV biovar is more invasive and after
aerosolization by sonication or centrifugation, pneumonia and
lymphadenitis has been reported. C. psittaci is a biosafety level
3 organism and needs to be handled in laboratories with BL 3
containment. C. pneumonia infections in the laboratory have
occurred, but these are mild.2
CHAPTER 12
b
FIGURE 12.3. Diagnosis of Chlamydia trachomatis infections by
immunofluorescence test with monoclonal antibodies. (a) Fluorescein-
conjugated antibody was reacted with McCoy cell culture 48 h after
infection with C. trachomatis. Fluorescing structures are
intracytoplasmic chlamydial inclusions (µ400). (b) A direct cervical
specimen from a patient with culture-confirmed chlamydial infection.
Fluorescing material consists of single or clumped chlamydial EBs or
RBs from infected and disrupted cervical mucosal cells (µ630).
From Tam MR, Stamm WE, Handsfield HH, et al: Culture-independent diagnosis
of Chlamydia trachomatis using monoclonal antibodies. N Engl J Med 1984;
310:1146.
Direct Cytological Examination
C. trachomatis was discovered in 1907 by cytologic exami-
nation of conjunctival cells from patients with trachoma.203 In
patients with ocular trachoma or acute chlamydial inclusion
conjunctivitis, the juxtanuclear cytoplasmic inclusions of
C. trachomatis can often be detected in Giemsa-stained smears
of conjunctival cell scrapings.204 In inclusion conjunctivitis,
stained scrapings are positive in up to 90% of infants, but only
in 50% of adults.205–207 In mild active ocular trachoma, it is
relatively insensitive with inclusion-bearing cells found in only
10–30% of scrapings. In a study of genital infections, the
Giemsa method detected only 15% of infections of the male
urethra and 41% of cervical infections.208 Papanicolaou-stained
cervical smears are also insensitive and nonspecific for
detection of cervical infections.209,210
Antigen Detection
Direct staining of specimens by fluorescein-
conjugated monoclonal antibody (DFA)
In this test, smears of cells obtained by swabbing infected
mucous membranes are stained with fluorescein-conjugated
123
 MICROBIOLOGY
monoclonal antibodies prepared against C. trachomatis. When
examined under a fluorescent microscope, intact inclusion
bodies or scattered EBs from ruptured cells fluoresce a bright
apple-green. The technique was first used to detect urethral and
cervical infections, but it is equally useful for detection of
conjunctival infections.211–216 Figure 12.3b shows a positive
cervical smear. The test can also be used for rectal specimens,
but the typically high concentrations of other bacteria in such
specimens sometimes produce false-positive results from cross-
reactive staining.217 Compared with cell culture, the sensitivity
of DFA testing in various reports has ranged from 70% to 100%,
and specificity appears to be greater than 95%.218 A study of
neonatal conjunctivitis reported sensitivity of 100% and
specificity of 94%.165 DFA testing has the following advantages:
(1) Unlike cell culture, DFA detects both viable and nonviable
Chlamydia organisms, therefore, the rigorous transport and
storage conditions that are essential for prevention of
inactivation are not as necessary; (2) The test is more rapid and
SECTION 3
results are available in hours; (3) The cost of a DFA test is
approximately a fourth that of culture; (4) The adequacy of the
specimen can be assessed during the procedure by noting the
presence or absence of columnar or cuboidal epithelial cells.
Absence or paucity of these cells indicates an inadequate
specimen. The technique also has certain disadvantages: (1) It
requires a fluorescent microscope and an experienced micro-
scopist who can distinguish between fluorescing chlamydial
particles and nonspecific fluorescence. (2) Cross-reactive
staining sometimes occurs in specimens that contain large
numbers of other bacteria. This is most common with rectal
specimens and is seldom a problem in conjunctival specimens.
Several DFA assays are commercially available. The anti-
MOMP monoclonal antibodies (Syva Microtak; Trinity Biotech)
are species-specific for C. trachomatis, and will not stain
C. psittaci or C. pneumoniae. Since MOMP is distributed
evenly on the surface of chlamydiae, the quality of fluorescence
is good and it takes only 30 min to perform. Monoclonal
antibodies to LPS (Pathfinder; Kallestad) will stain all
chlamydiae and are distributed unevenly.
Enzyme immunoassay
In enzyme immunoassay (EIA), C. trachomatis antigen is
detected by a colorimetric signal generated by antigen–antibody
reactions. A number of EIAs are commercially available and
they use either monoclonal or polyclonal antibodies to detect
chlamydial LPS, which is more soluble than MOMP. Like DFA,
EIA is quicker and less expensive than culture, and the viability
of C. trachomatis organisms in the specimen is irrelevant to the
validity of the test. Most EIAs take several hours to perform and
are suitable for batch processing.219 The test has an objective
end-point (photometric measurement of color intensity), in
contrast with the subjective interpretation required by
microscopic examination in DFA. However, the adequacy of the
specimen (presence of epithelial cells) cannot be assessed by
EIA.220 Like DFA, EIA is less sensitive and specific than
isolation of the organism in cell culture by an experienced
laboratory.221 When large numbers of specimens are processed,
however, EIA requires less technologist time per specimen than
DFA does because the objective (photometric) end-point of EIA
makes the test much less labor-intensive than the microscopic
examination required by DFA. The performance of commercial
EIAs for C. trachomatis varies considerably, but increases in
sensitivity have been achieved by using cycling enzymes to
amplify the signal component in the IDEA PCE test (DAKO
Ltd, Ely, UK).222–224 These tests have a specificity of only 97%
which makes them not amenable to screen low prevalence
124 populations due to a low predictive value. With the use of
confirmatory tests, the specificity approaches 99.5%. Two types
of confirmatory tests are used. In one assay, all positive results
are repeated in the presence of a monoclonal antibody directed
against the type-specific epitope on the LPS.225 Another
approach is to use a second test by a different method such as a
DFA test based on MOMP detection to confirm an LPS-based
EIA.226
Nucleic acid tests
Nucleic acid hybridization (NAH) tests for C. trachomatis are
used in parts of the world as extensively as EIAs. One utilizes
DNA–RNA hybridization (PACE 2, Gen-Probe, San Diego, CA)
to enhance sensitivity to detect chlamydial RNA. It is about
as sensitive as the better antigen detection and cell culture
methods and is relatively specific.227–228 Another NAH test uses
signal amplification to increase the sensitivity up to 90% of
the nucleic acid amplification (NAA) tests. Five NAA methods
are currently licensed for detection of C. trachomatis. They
are based on detection of chlamydial DNA or RNA using
amplification procedures such as polymerase chain reaction
(PCR), ligase chain reaction (LCR), chlamydial ribosomal RNA
using transcription-mediated amplification or strand displace-
ment amplification. The PCR, LCR, and strand displacement
amplification assays amplify nucleotide sequences of the cryptic
plasmid present in each C. trachomatis EB. The transcription-
mediated amplification is directed against rRNA. Both the
cryptic plasmid of EB and rRNA are present in multiple copies,
so theoretically they should be able to detect less than one EB.
Sampling and specimen variability cause the actual sensitivity
to be lower.229 All assays are highly specific if cross-
contamination is kept minimal. The NAA tests are more
sensitive than culture and other nonculture techniques. The
NAA methods are becoming the tests of choice in routine
clinical laboratories, especially for urogenital chlamydial
infections. However when organisms are needed for further
study, isolation in cell culture will continue to be used.
Serologic diagnosis
Chlamydial antibodies can be detected by complement fixation
(CF), MIF testing, and enzyme-linked immunosorbent assay
(EIA),218 using group or species-specific antigens or a com-
bination of these to measure immunoglobulin G (IgG), IgA,
IgM, or total classes of antibodies to individual or multiple
chlamydial serovars. The CF test is rarely performed today, is
based on the group-specific chlamydial LPS, which is relatively
insensitive, and was used for LGV. The genus-specific CF test
can be used for serologic diagnosis of psittacosis (C. psittacosis).
MIF, in contrast, is a sensitive and specific test that detects both
IgG- and IgM-class antibodies in serum, tears, and genital
secretions.230 The MIF test is most useful in epidemiologic
studies; it has limited diagnostic application in C. trachomatis
infections due to many high-risk patients having already expe-
rienced a primary infection and it often requires retrospective
pairing of sera.230 Two exceptions are: (1) chlamydial pneumonia
with detection of IgM-class antibodies (primary infection)
especially in infants and up to 70% sensitivity in adults231,232
and (2) C. trachomatis ocular infections where the presence of
IgG or IgA chlamydial antibodies in tears appears to correlate
with disease activity.230,233–235 Several recombinant EIA tests are
commercially available for detection of chlamydial antigens by
either monoclonal or polyclonal antibodies to detect a
chlamydiae-specific recombinant fragment of LPS, 3-deoxy-D-
manno-2-octulopyranosonic acid. This reduces cross-reactivity
from other Gram-negative bacteria containing LPS. Com-
parisons of these recombinant immunoassays with traditional
CF or the gold standard MIF test has shown a slightly lower
sensitivity and specificity for these serum antibodies to peptides
 Chlamydial Disease

or recombinant antigens than obtained using whole EBs as the
antigen.236–238 It has been postulated that these results reflect
individual variability in humoral response in a population to
single chlamydial antigens.
Key Features: Antimicrobial Susceptibility
• Beta-lactam antibiotics are ineffective
• Mechanism of action is inhibition of 50S and 30S ribosomal
subunits
SUMMARY
Despite the long recognition of chlamydial infections, our
knowledge of its pathogenesis and immunology, detection,
treatment, and most importantly prevention, continues to lag.
C. trachomatis and C. pneumoniae are pathogens of humans
infection when transmitted to humans. C. trachomatis is the
most prevalent sexually transmitted pathogen in Western
societies and an important cause of acute and chronic
conjunctivitis, including trachoma. Protective immunity is
incomplete; repeated infections often cause fibrosis and scarring
of affected tissues, believed as a result of an immunopathologic
process. The most recent advances described here are a new
taxonomic classification, an additional pathway of persistence
and latency to its previously described biphasic life-cycle, and
newer molecular diagnostics for detection.239 The treatment
choices (tetracyclines, macrolides) remain essentially un-
changed,240 and, an effective vaccine continues to be elusive by
our incomplete understanding of the immunology and
pathogenesis of chlamydial infections.
ACKNOWLEDGEMENT
The author of this chapter acknowledges Joseph M. Thomas, Alfred D.

CHAPTER 12
and have no animal reservoirs. C. psittaci is principally a Heggie, and Jonathan H. Lass for their contributions from Albert &
Jakobiec’s Principles and Practice of Ophthalmology, Second edition.
pathogen of birds that causes pneumonia and systemic

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207. Sandstrom KI, Bell TA, Chandler JW, et al:
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217. Rompalo AM, Suchland RJ, Price CB, et al:
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CHAPTER 12
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SECTION 3
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 CHAPTER
13 The Spirochetes
Marlene L. Durand
INTRODUCTION
Spirochetes are mobile, corkscrew-shaped bacteria that represent
a phylogenetically ancient bacterial group.1 They are 10 times
longer and thinner than most pathogenic bacteria (Table 13.1),
and nearly all are invisible on Gram stain. With rare exception,
they cannot be cultured in clinical microbiology laboratories. As
a consequence, the diagnosis of most spirochetal diseases relies
on serologic tests or microscopy using special techniques (e.g.,
silver stain, dark-field microscopy).
Some spirochetes, such as nonpathogenic treponemes, are
members of the normal human oral or gastrointestinal flora,
while others are pathogenic. Pathogenic spirochetes include
Treponema, Borrelia, Leptospira, and Spirillum (Table 13.2).
Those that can cause disease of the central nervous system
(CNS) can also cause ocular disease, as would be expected. Sys-
temic spirochetal infections that may involve the eye include
syphilis, Lyme disease, relapsing fever, and leptospirosis.
TREPONEMES
NONPATHOGENIC TREPONEMES
The treponemes include both nonpathogenic and pathogenic
species. Nonpathogenic oral treponemes, such as Treponema
denticola, Treponema vincentii, and Treponema medium, are
normal colonizers of the mouth. They play important roles in
subgingival plaque and periodontal disease.2 Oral treponemes
differ from the pathogenic treponemes in many ways. Most oral
treponemes may be readily cultured anaerobically if selected
media are used,3 while pathogenic treponemes such as the
syphilis treponeme, Treponema pallidum, cannot be propogated.
The genome of T. denticola was recently sequenced and was found
to be much larger than that of T. pallidum, with little DNA
sequence homology.4 The pathogenic treponemes include the
nonsexually transmitted endemic treponemes as well as the
agent of syphilis.
SYPHILIS
History
Syphilis is a disease of great historical significance. It was first
reported in Europe in the late fifteenth century and coincided with
the return of Columbus’s ships from the New World. Syphilis
quickly reached epidemic proportions in Europe, and spread across
the world with the explorations of the sixteenth century. By the
turn of the twentieth century, syphilis was the leading cause of
neurologic and cardiovascular disease among middle-aged people.5
With the advent of the Wasserman test in 1906, the prevalence
of the disease was appreciated; between 8% and 14% of the
urban population were infected, and 25% of patients progressed
to a chronic illness.5
Epidemiology
Syphilis is found worldwide, and there are more than 12 million
cases. In the US, the incidence has declined dramatically since the
advent of penicillin in the 1940s. It is primarily a sexually trans-
mitted disease, although it can be acquired transplacentally
(congenital syphilis), by kissing or other close contact with an
active skin or mucous membrane lesion, and by blood transfu-
sion. Transfusion-related transmission is now very rare both
because blood donors with positive serologic tests are excluded,
and because the organism cannot survive more than 24–48 h
under conditions of blood bank storage.
Microbiology and Pathogenesis
Syphilis is caused by T. pallidum subspecies pallidum. The spiro-
chete has outer and cytoplasmic membranes, a thin peptidoglycan
layer, and flagella that lie in the periplasmic space. It contains a
circular chromosome of ~1000 kbp, making it one of the smallest
bacterial genomes. The mechanism of T. pallidum pathogenesis is
poorly understood, and no known virulence factors have been
identified. The outer membrane is mostly lipid with few surface
proteins. This has led to the hypothesis that this is a ‘stealth’
organism that minimizes the number of surface membrane-
bound targets in order to evade the host immune system.6
The number of organisms required to establish infection varies
between patients, but an inoculum of only four bacteria can
establish infection in rabbits. The dividing time is 30 h, and cli-
nical lesions appear when there are 107 organisms per milligram
of tissue.7 A larger inoculum will therefore lead to a clinically
apparent lesion sooner than a small inoculum.
Organisms gain entrance to the body through mucous mem-
branes or abraded skin, and a lesion appears at the site on ino-
culation an average of 3 weeks later. However, T. pallidum has
already spread throughout the body by this time, since there is
a spirochetemia within hours to days of the initial inoculation.
Any organ may be infected, although the CNS is especially
targeted. Evidence of organ infection may not become clinically
apparent until decades later, however.
Stages
Syphilis has long been divided into stages (Table 13.3), and
clinical manifestations, serologic results, and treatment depend
on the stage of disease. Although untreated syphilis is a life-long
infection, it is only contagious during the early stages (up to
4 years after initial infection).
‘Primary syphilis’ includes the development of a chancre at
the inoculation site, usually the external genitalia. A chancre is
a painless, ulcerated lesion with a smooth base. There is no 131
 MICROBIOLOGY

TABLE 13.1. Characteristics of Spirochetes in Comparison with Common Pathogenic Bacteria.

Organism Size ( µm) Cultivable? Usual Diagnostic Method

Spirochetes:
Treponemes (pathogenic) 0.15 µ 5–15 No Serology, microscopy
Borrelia 0.2 µ 20–30 Difficult Serology
Lyme relapsing fever 0.2 µ 8–30 Difficult Microscopy
Leptospira 0.1 µ 6–20 Yes Culture, serology
Spirillum 0.2 µ 3–5 No Microscopy
Common Bacteria:
Staphylococcus 1 (sphere) Yes Culture
Pseudomonas 0.5 µ 2 Yes Culture
SECTION 3

TABLE 13.2. Overview of Spirochetes

Organism Disease Transmission Locale Eye Disease

Treponemes
T. pallidum* Syphilis Sexual contact, congenital,
transfusion Worldwide Yes
T. pertenue* Yaws Direct contact† Tropical, worldwide No
T. endemicum* Bejel Direct contact, fomite Arid, North Africa,
Arabian peninsula No
T. carateum Pinta Direct contact Amazon No
Borrelia
B. burgdorferi Lyme Tick Europe, North America Yes
Borrelia species Relapsing fever Tick louse Worldwide No
Central/East Africa,
Andes
Leptospira
L. interrogans Leptospirosis Zoonosis Worldwide India, Yes
Hawaii
Spirillum minus Rat-bite fever Rat bite Asia No
* Syphilis, yaws, and bejel are all caused by the same genus and species, T. pallidum, but by different subspecies. Therefore the correct names for these spirochetes are
T. pallidum subsp pallidum, T. pallidum subsp pertenue, T. pallidum subsp endemicum. Treponema carateum is a separate species, rather than a subspecies of
T. pallidum.
† Yaws, bejel, and pinta are endemic treponematoses that are transmitted by direct contact with skin lesions, rather than by sexual contact. In bejel, transmission may
also be by mucous membrane contact or fomites (sharing drinking cups).

exudate, and the chancre does not bleed when scraped. In some
cases no chancre develops, and in others only a small papule
occurs. Multiple chancres may occur, especially in HIV-infected
patients. The chancre heals spontaneously in 3–6 weeks. Serologic
tests may be negative, since these tests cannot detect antibodies
until 1–3 weeks after the development of the chancre.8 Diagnosis
is usually made by finding the treponemes in chancre scrapings
using either dark-field microscopy or immunostaining with
fluorescent antibodies (DFA-TP).
‘Secondary syphilis’ begins 2–8 weeks after the chancre appears
and is the phase most associated with constitutional symptoms.
A rash develops in the majority of patients and usually involves
the palms and soles. Painless moist plaques called condoloma
lata may develop in intertriginous areas; these are highly con-
tagious. Constitutional symptoms such as fever, sore throat,
arthralgias, and malaise develop in 70% of patients. The CNS is
132 involved in 40% of patients, although fewer are symptomatic.
This is called acute neurosyphilis to distinguish it from tertiary
neurosyphilis. An aseptic meningitis is seen in 1–2% of
patients. Ocular involvement, usually uveitis, may occur. The
RPR is reactive, usually at high titer, in virtually all patients
with secondary syphilis. The symptoms of secondary syphilis
may resolve and then relapse; relapses are usually milder.
‘Latent syphilis’ is, by definition, that stage when the patient is
asymptomatic and there are no signs of the disease (other than
positive serology). This stage is divided into early latent and late
latent. Early latent usually comprises the first 4 years of infec-
tion, during which a relapse may occur and the patient may still
be contagious.7 However, a recent publication by the Centers for
Disease Control and Prevention (CDC) considers early latent
syphilis as infection acquired within the preceding 1 year.9 If the
date of onset of syphilis cannot be determined, as is usually the
case, patients are treated as late latent syphilis. Late latent
syphilis may last decades. Although the specific treponemal

TABLE 13.3. The Stages of Syphilis and Their Treatment
Stage Symptoms/Signs*
Primary Painless chancre
Secondary Rash, flu-like symptoms;
may have aseptic meningitis
Early latent None
Late latent None
Tertiary Cardiovascular, neurologic,
ocular, otosyphilis
* Symptoms and signs listed are those typical for the stage; exceptions except.
†
RPR or VDRL = nontreponemal tests.
‡
FTA-abs or TPPA = specific treponemal tests.
§
For details, including treatment in special hosts (e.g., pregnant patients, penicillin-allergic patients, children, etc.) see Workowski KA, Berman SM for the Centers for
Disease Control and Prevention. Sexually transmitted treatment guidelines, 2006. Morbid Mortal Weekly Report 2006;55 (RR 11):1–94.
IM = intramuscular; IV = intravenous; PCN = penicillin; MU = million units.
#
HIV-infected patients who have late latent syphilis, or latent syphilis of unknown duration, should have a lumbar puncture to determine if asymptomatic neurosyphilis is
also present. If the cerebrospinal fluid is abnormal, they should be treated for neurosyphilis with IV penicillin.
tests (e.g., FTA-abs, TPPA) are positive during this stage, the
nonspecific tests (e.g., RPR, VDRL) may wane with time, so that
many patients with late latent syphilis have a nonreactive RPR.
‘Tertiary syphilis’, also called ‘late syphilis’, is primarily mani-
fested by cardiovascular or CNS symptoms. In the preantibiotic
era, up to 25% of patients progressed to tertiary syphilis. Tertiary
syphilis is seen even in the antibiotic era, and often represents
unrecognized infection acquired decades earlier. It also may
represent failure of benzathine penicillin therapy given for the
early stages of syphilis. Benzathine penicillin, the standard
treatment for primary, secondary, and latent syphilis, does not
cross the blood–brain barrier. As a consequence, a patient may
develop late neurosyphilis despite having been treated for
syphilis years earlier. Such failures are known to occur in one
patient per 333–1000 treated patients.7
Cardiovascular syphilis will occur in 10% of untreated patients
with syphilis. It is mainly an aortitis, and the classic finding is
a fusiform aortic aneurysm of the ascending aorta. Concurrent
late neurosyphilis is common.
Late neurosyphilis, as distinguished from the acute neuro-
syphilis that may be seen during secondary syphilis, is a chronic
meningitis involving all parts of the CNS. Asymptomatic neuro-
syphilis is the most common form of late neurosyphilis and is
diagnosed by an abnormal cerebrospinal fluid (CSF). The CSF
VDRL is positive in only half of the cases of neurosyphilis, so
other abnormalities (e.g., pleocytosis, elevated CSF protein) are
significant. Symptomatic late neurosyphilis includes findings of
meningovascular or parenchymatous involvement. There may
be personality changes, memory loss, slurred speech, and psy-
chiatric manifestations such as megalomania. The patient may
be misdiagnosed with Alzheimer’s disease. There may be
demyelination of the posterior columns of the spinal cord, leading
to an ataxic gait, loss of bladder or bowel function, ‘shooting’
pains, and peripheral neuropathy.
Ocular syphilis or otosyphilis may occur as part of tertiary
syphilis and are often considered subsets of neurosyphilis. This
may lead to confusion, since ocular or otosyphilis may occur
without involvement of the brain or meninges. A normal CSF
formula does not exclude ocular or otosyphilis.
Nonspecific tests for syphilis (RPR or VDRL) may be negative
in up to 50% of patients with tertiary syphilis, because these
reactions wane with time. Specific treponemal tests (FTA-abs,
TPPA) usually remain positive for life, however.
The Spirochetes
RPR† FTA-abs‡ Treatment§
+ or — + or — IM benzathine PCN||
2.4 MU µ 1 dose
+ + IM benzathine PCN µ 1;
IV PCN if neurosyphilis
or ocular syphilis
+ + IM benzathine PCN µ 1
+ or — + IM benzathine PCN
weekly µ 3 weeks#
IV PCN µ 10–14 days
+ or — + (usual dose 4 MU q4h)
CHAPTER 13
Ocular Syphilis
Ocular syphilis may occur either during secondary or tertiary
syphilis. The findings of ocular syphilis are protean, and are
discussed in detail in other chapters (see Chapters 345 and 351).
General recommendations for serologic diagnosis and treatment
in ocular syphilis are listed in Table 13.4. The details of treatment
in various groups (HIV, penicillin-allergic, children, pregnant
patients, etc.) are given by the CDC in their 2006 guideline.9
All patients with ocular syphilis should be screened for asymp-
tomatic neurosyphilis. If CSF abnormalities exist, treatment
with IV penicillin is the same, but a follow-up lumbar puncture
is required at 6 months to determine adequacy of therapy. If the
CSF is still abnormal at that point, the patient should be
retreated. All patients with ocular syphilis should be screened
for HIV, as there is a higher incidence of ocular syphilis in HIV-
infected patients than in the non-HIV-infected patients.10 A
recent study of 320 HIV-positive patients receiving highly active
antiretroviral therapy at a Washington, DC, infectious disease
clinic and screened for syphilis found that 7.5% had syphilis,
TABLE 13.4. Ocular Syphilis: Recommendations for Serologic
Diagnosis and Treatment
1. Screen with both RPR and FTA-abs. A nonreactive RPR does
not exclude ocular syphilis.
2. Confirm a reactive FTA-abs with a TPPA (to exclude false-
positive FTA-abs).
3. A patient who has eye findings consistent with ocular syphilis as
well as a reactive TPPA should be treated for presumed ocular
syphilis. A history of prior treatment for syphilis with IM benzathine
penicillin does not exclude this diagnosis.
4. Test for HIV, as there is a higher incidence of ocular syphilis in HIV.
5. Perform a lumbar puncture (LP) to exclude concomitant
neurosyphilis. A normal CSF does not exclude ocular syphilis, but
an abnormal CSF will require a follow-up LP 6 months after
treatment to ensure adequacy of therapy for neurosyphilis.
6. Treat ocular syphilis the same as for neurosyphilis, with IV
penicillin 4 million units every 4 hours for 10–14 days in adults with
normal renal function. Patients with penicillin allergy may require
desensitization with the help of an allergist. At the end of IV
therapy, some experts also prescribe IM benzathine penicillin
2.4 million units once weekly for 3 weeks.
133
 MICROBIOLOGY
and 13% of these patients with syphilis had ocular syphilis.11
All patients in this study with ocular syphilis also had an
abnormal CSF, consistent with coexisting neurosyphilis.
Syphilis in HIV-Infected Patients
In general, syphilis in patients with HIV is more severe and
protracted. These patients are especially likely to develop neuro-
syphilis and ocular syphilis, and relapses with these manifesta-
tions despite standard benzathine penicillin are well described.
Therefore, a more vigorous or protracted treatment regimen is
recommended for HIV-coinfected patients with syphilis. The
CDC recommends that HIV-positive patients with late latent
syphilis or syphilis of unknown duration have a lumbar puncture.9
Patients with abnormal CSF should be treated for neurosyphilis.
Syphilis Serology
Syphilis is diagnosed primarily by serologic tests. Nonspecific tests
for syphilis include rapid plasma regain (RPR) and Venereal
SECTION 3
Disease Research Laboratory (VDRL). These tests vary with the
stage of disease and response to treatment. A VDRL or RPR
should become nonreactive within 1 year of treatment for pri-
mary syphilis and 2 years for secondary syphilis. The RPR or
VDRL may be negative in primary syphilis, but ~100% of
patients have a reactive test in secondary syphilis, usually at
high titer. The highest titers occur during untreated secondary
and early latent syphilis and decline thereafter, usually to less
than 1:4. Between 25% and 50% of patients with late latent or
neurosyphilis have negative RPR or VDRL test results. All posi-
tive RPR or VDRL results should be confirmed by a specific trepo-
nemal test, as false-positive results occur. Specific treponemal
tests measure antibodies against specific treponemal antigens.
The most commonly used tests are FTA-abs (fluorescent trepo-
nemal antibody absorbed) and TPPA (T. pallidum particle agglu-
tination). The FTA-abs is an older test but has occasional
false-positive results, so the TPPA is preferred but may not be
as readily available. The specific treponemal tests usually become
positive during early syphilis and usually remain positive for
life, even after successful treatment.
False-positive tests for RPR or VDRL are more common than for
FTA-abs, but occur in both. Other spirochetal diseases can cause
false-positive results. The endemic treponematoses cause identical
serologic results as syphilis. Lyme disease is a well-known cause
of a false-positive FTA-abs, although the RPR is usually negative.
Rheumatologic conditions frequently cause false-positive RPR or
VDRL reactions, and may also produce false-positive FTA-abs.
A second specific test, such as the TPPA or syphilis Western
blotting method, should be used to confirm a positive FTA-abs,
especially in patients with rheumatologic diseases. A study
using the Western blot as the gold standard in 107 patients with
rheumatologic disease found that the FTA-abs had a specificity
for syphilis of only 68%, with 32 false-positive results.12
ENDEMIC TREPONEMATOSES
Endemic treponematoses include yaws, bejel, and pinta. These
are non-sexually transmitted infections with skin lesions as their
early manifestation. Yaws and bejel are caused by treponemes
that are very closely related to syphilis: T. pallidum subspecies
pertenue and endemicum, respectively. Pinta is caused by a
separate species, Treponema carateum. The spirochetes of
endemic treponematoses are indistinguishable morphologically
and serologically from syphilis.
Prior to mass treatment programs of 30–50 years ago, endemic
treponematoses were prevalent especially in impoverished and
rural communities. Yaws was a worldwide disease of the tropics
and subtropics, including the Caribbean islands such as Haiti.
134 Bejel was seen in arid regions in North Africa, the Middle East, and
the Arabian peninsula. Pinta was found only in the Caribbean
and South America. In 1950, there were an estimated 50 million
cases of yaws worldwide, and from 1952 through 1969, procaine
penicillin G was administered in mass treatment campaigns
conducted by the World Health Organization (WHO) and the
United Nations Children’s Fund. These campaigns resulted in
a marked decrease in this disease and other endemic trepone-
matoses, although 2.5 million people are still affected. Today,
yaws-endemic foci persist in west and central Africa, Southeast
Asia, on some Pacific Islands such as Papua New Guinea, and
Central America. Foci of bejel exist in the Middle East and the
Sahel region of Africa. Pinta is found only in some Indian tribes
in the Amazon region.13
Yaws and bejel are seen mainly in children under age 15, while
pinta may affect young adults. Transmission in all three is by
direct contact with infected skin or mucous membrane lesions.
In yaws, skin lesions begin as a papule, usually on the legs, and
slowly enlarge into a raspberry-like mass. Lesions spontaneously
regress, followed by the appearance of secondary skin lesions.
Secondary lesions also usually resolve, but in 10% of patients,
late disease occurs characterized by destructive bony or cartila-
ginous lesions. Bejel has similar manifestations, although ini-
tial lesions are most often painless patches on oral mucosa. Late
disease also involves chronic destructive lesions involving carti-
lage or bone. Pinta only involves the skin and does not have late
destructive lesions.
Treatment of the endemic treponematoses is with penicillin.
In mass campaigns, IM penicillin was used, but the need for refri-
geration of the medication makes this difficult in many areas.
Recently, a trial using oral penicillin in Guyana was found to be
effective.14
False-Positive Syphilis Tests
Endemic treponematoses are not sexually transmitted yet pro-
duce serologic results (RPR, FTA-abs, TPPA) identical to those
of syphilis. For patients who grew up in a yaws-endemic area such
as Haiti prior to the mass treatment programs of the 1950s and
1960s, for example, a positive syphilis serology may reflect this
early exposure to yaws rather than infection with syphilis. How-
ever, the patient should always be treated for the possibility of
syphilis given the serious sequelae of untreated disease. Unlike
syphilis, the endemic treponematoses do not involve the CNS at
any stage of disease.13 As a consequence, it seems unlikely that
late yaws, bejel, or pinta would cause ocular disease. Patients in
yaws- or bejel-endemic areas with findings consistent with ocular
syphilis and positive syphilis serologies most likely have ocular
syphilis. However, some reports have attributed these eye findings
to late yaws or bejel even though syphilis cannot be excluded.15
BORRELIA
Summary: Treponemes
• Nonpathogenic treponemes are part of the normal oral flora
and play a role in dental plaque and periodontal disease.
• Syphilis, caused by T. pallidum, silently invades the CNS in
many patients soon after infection.
• Clinical signs of early syphilis may be missed by the patient,
so late syphilis may occur decades later in a patient with no
known history of syphilis.
• Patients with clinical findings consistent with late syphilis (e.g.,
ocular syphilis, neurosyphilis, cardiovascular) should be tested
by both TPPA and RPR, as the RPR titer may be negative.
• Yaws, bejel, and pinta are primarily childhood skin diseases
that are not sexually transmitted but lead to identical serologic
test results (RPR, FTA-abs) as syphilis.

LYME DISEASE
Lyme disease is caused by Borrelia burgdorferi, an organism that
is the longest and thinnest of the spirochetes. The disease is trans-
mitted to humans by ticks, and it is now the most common vector-
borne disease in the US and Europe. The disease was recognized
as a new entity in the US in 1976, when a cluster of children in
Lyme, Connecticut appeared to have juvenile rheumatoid arthritis.
Diseases with similar characteristics had been previously recog-
nized in Europe as Bannworth’s syndrome, erythema chronicum
migrans, and acrodermatitis chronica atrophicans. The recovery
of the organism from ticks and infected humans established the
link between these diseases.
Epidemiology
The spirochete is transmitted by the Ixodes tick, which has a larval,
nymphal, and adult stage. These ticks require a blood meal for
each stage. Nymphs are the size of the head of a pin and are
responsible for most disease transmission to humans. The tick
bite is painless and the tick may go unrecognized. The peak
months of human disease mirror the peak months of nymphal
feeding, May through July. The main foci of US disease are in
the Northeast from Massachusetts to Maryland, Wisconsin and
Minnesota, and northern California. Deer and white-footed
mice are the major mammalian hosts for the tick.
Microbiology
There are three different groups of B. burgdorferi. The strain
found in North America is B. burgdorferi (sensu strictu).
Although all three groups have been found in Europe, Borrelia
garinii and Borrelia afzelii cause most disease there, and these
are the only two groups found in Asia. Clinical manifestations
of Lyme disease vary somewhat in these different regions of the
world and may be due to this strain variability.
The complete genome for B. burdorferi has been sequenced.16
It contains a linear chromosome of 950 kbp plus nine circular
and 12 linear plasmids. The organism uses plasmid-encoded outer
surface proteins (Osp) A through F to adapt to different environ-
ments.17 The spirochete expresses OspA in the tick midgut but
OspC when in the mammalian host. Another surface lipoprotein
(VIsE) undergoes significant antigenic variation during dissemi-
nation in the host. The spirochete depends on the host for most
of its nutritional requirements.
The organism may be cultured in special Barbour–Stoenner–Kelly
media, though such cultures are not available in most clinical
labs. Organisms are usually cultivable only from patients with
early disease, usually from the initial rash of erythema migrans,
and occasionally from plasma or CSF.
Clinical Manifestations
Lyme disease resembles syphilis in that it has three stages.
Stage 1 occurs 3 days to 1 month after the tick bite, and is char-
acterized by a local erythema migrans skin lesion at the site of
the bite. Over half of the patients are unaware of the bite. The
skin lesion is initially homogeneously red, then the center may
become intensely red, indurated vesicular, or necrotic. Often the
circular lesion expands leaving a lighter center, giving a bulls-
eye appearance.
Stage 2 occurs days to weeks after stage 1. Multiple secondary
annular lesions may develop, and they are usually smaller than
the initial lesion. The patient may have flu-like symptoms with
fatigue, headache, fever, myalgias, and lymphadenopathy. After
several weeks, 15% of untreated patients in the US develop
neurologic signs and 5% develop cardiac abnormalities. The
neurologic manifestations most often include an aseptic
meningitis with lymphocytic pleocytosis (~100 cell/mm3) and
an associated facial palsy. The facial palsy may be bilateral.
The Spirochetes
Other manifestations include encephalitis, motor or sensory
radiculoneuritis, mononeuritis multiplex, cerebellar ataxia, and
myelitis. Untreated, these symptoms may last weeks to months.
The most common cardiac manifestation is heart block. This
may be first degree, Wenckebach, or complete heart block, and
usually resolves in a few days so a permanent pacemaker is not
indicated. Ocular disease other than conjunctivitis is rare, may
occur during stage 2, and may include interstitial keratitis,
iritis, or choroiditis.
Stage 3 represents the chronic stage of disease, and occurs
months after infection. It is characterized by either arthritis or
chronic neurologic abnormalities. Approximately 60% of
untreated patients will develop arthritis, usually involving the knee
or other large joints. Recurrent attacks, separated by periods of
remission, are typical, but eventually these resolve in most
patients. Joint fluid shows a neutrophil-predominant, inflam-
matory infiltrate. Arthritis resolves with antibiotic treatment in
90% of patients, but in 10% a chronic arthritis develops. This
CHAPTER 13
may be a postinfectious immune response, as testing of joint
fluid or synovial tissue for Borrelia DNA is often negative.
Chronic neuroborreliosis may occur years after the primary infec-
tion, often following an asymptomatic latency period. In the
US, the characteristic symptom is a subtle cognitive disturbance,
often with a mild memory loss. There are usually no abnormalities
in the CSF, although intrathecal antibody tests may be positive.
Diagnosis
The diagnosis is made primarily by serology. Serologic testing is
performed in two stages, with a screening ELISA (enzyme-linked
immunosorbant assay) followed by a Western blot confirmation
of any positive ELISA results. The screening test has many
false positives, so only those confirmed by Western blot are con-
sidered true positives. Serologic tests are often negative during
the first 1–2 weeks of primary infection, and IgM antibodies
appear subsequently. IgM antibodies may persist for years, and
are not recommended for diagnosis of chronic infections. Most
patients develop IgG antibodies within 1 month of infection,
and these also may remain positive for years despite treatment.
Treatment
The treatment of early Lyme disease is with oral doxycycline or
oral amoxicillin for 2–3 weeks. Doxycycline is preferred because
it will also treat other tick-borne diseases (e.g., babesiosis,
ehrlichiosis) that may have been simultaneously introduced by
the tick bite. Patients with arthritis should be treated with these
agents for 1–2 months, or with IV ceftriaxone 2 g once daily for
2–4 weeks. Neurologic disease, either during early or late stages
of Lyme disease, is treated with IV ceftriaxone 2 g once daily for
2–4 weeks; most experts treat late neuroborreliosis for 4 weeks.
RELAPSING FEVER
Relapsing fever is an infection characterized by recurrent fevers
and flu-like symptoms interspersed with periods of apparent
health. It is caused by Borrelia species, and there are two types
of disease, tick-borne and louse-borne.
Louse-Borne Relapsing Fever
Louse-borne relapsing fever (LBRF) is caused by B. recurrentis
and usually occurs in epidemics during wartime, famine, or
other upheavals. The last large epidemic occurred during World
War II when 50 000 people died of this disease. The disease still
occurs in northeastern and central Africa, especially Ethiopia,
Sudan, and Somalia. The disease is transmitted by the human
body louse, which ingests the organism during a blood meal from
an infected person, then releases Borrelia to another person when
the louse is crushed. The Borrelia can then penetrate intact skin 135
 MICROBIOLOGY

and cause disease after an incubation period of ~1 week. Onset LEPTOSPIRA

of symptoms is usually abrupt, and symptoms include high
fever, myalgias, headache, hepatomegaly, splenomegaly, and cough.
Hemoptysis, hematemesis, or hematuria may also present.
Neurologic involvement occurs in 30% cases. After an average
of 5 days, the patient becomes asymptomatic for ~9 days and
then suffers a relapse. Diagnosis is by clinical suspicion and
demonstration of the spirochetes on peripheral blood smears of
febrile patients. Serodiagnosis by detecting antibodies against a
surface protein of the spirochete has recently been proposed.18
Treatment is with tetracycline or penicillin, but this often induces
a dangerous Jarisch–Herxheimer reaction.19 The latter may be
prevented by pretreatment with antibodies against tumor necrosis
factor alpha.20 Untreated, up to 40% of patients may die.
Tick-Borne Relapsing Fever
Tick-borne relapsing fever (TBRF) is seen sporadically and in occa-
sional outbreaks. It has been reported worldwide. In the US,
SECTION 3
LEPTOSPIROSIS
Leptospirosis is a worldwide zoonosis most common in tropical
regions. In the US, it is most common in Hawaii. It is caused by
various Leptospira species, most often Leptospira interrogans.
Leptospires are motile, tightly coiled spirochetes with pointed
ends. They are best seen by dark-field microscopy and can be
cultured on polysorbate–albumin media. The leptospires are main-
tained in nature by chronic renal infection of carrier animals,
such as rodents and cattle, and human infection usually occurs
after exposure to contaminated water or damp soil. Clinical
disease is manifested either as a self-limited flu-like illness or as
a severe illness characterized by renal and liver failure as well as
a hemorrhagic pneumonia (Weil’s disease). Leptospirosis is a
biphasic illness in 50% of cases, with an asymptomatic period
between the two acutely symptomatic phases. Uveitis may occur

most cases have occurred after patients have stayed in mountain
cabins in the Western US. The illness is caused by at least 15
Borrelia species, with B. hermsii most commonly reported. All
species are transmitted by soft ticks of the genus Ornithodoros.
These ticks require blood meals but can survive without a meal
for up to 15 years. Animal reservoirs for the ticks include mice,
rats, squirrels, rabbits, owls, and lizards. The tick cannot travel
more than 50 yards except on an animal host, so most cases of
infection occur near a particular locale. The same location may
be a source of subsequent cases. An outbreak occurred in 62 cam-
pers staying in log cabins on the north rim of the Grand Canyon
in 1973,21 and another cluster of 15 cases occurred in the same
location in 1990.22 The tick feeds at night and its painless bite
transmits the Borrelia to humans. Symptoms of disease are
similar to those of LBRF, although more relapses usually occur
in TBRF and case fatality rates are lower (2–5%).
Uveitis
Iritis and iridocyclitis may occur during the acute illness of LBRF.
Uveitis may also occur in TBRF. A case of anterior and inter-
mediate uveitis recently occurred in a 12-year-old boy in Oregon
who had developed TBRF several weeks earlier.23
and may be anterior, posterior, or panuveitis.24 Retinal vasculitis
is seen in 5–50% of cases.
SPIRILLUM MINUS (RAT-BITE FEVER)
Spirillum minus is one of two causes of a relapsing, febrile illness
that follows a rat bite (the other being due to a Gram-negative rod,
Streptobacillus moniliformis). Spirillum minus is a short thick
spirochete (Table 13.1), and is carried by 25% of rats. Rat-bite
fever is rare. Most cases in the US are caused by Streptobacillus
moniliformis, while cases in Asia are caused by Spirillum minus.
In Japan, the illness is called sodoku (‘so’ = rat, ‘doku’ = poison).
The illness occurs 1–4 weeks following a rat bite. The site of the
bite becomes swollen and purple, and subsequently ulcerates
and develops an eschar. There is regional lymphadenopathy and
a flu-like febrile illness, often accompanied by a maculopapular
rash. Fevers follow a relapsing course, with febrile episodes lasting
3–4 days and interspersed with afebrile periods lasting 3–9 days.
The organism cannot be cultured, and diagnosis is made by
microscopic visualization of the organism in blood, exudate, or
lymph node samples. Treatment is with penicillin. Eye disease
has not been reported.

Summary: Borrelia Summary: Leptospirosis and Rat-Bite Fever

• Lyme disease, caused by B. burgdorferi, is transmitted by a
tick and is endemic in Massachusetts to Maryland, Wisconsin
and Minnesota, and northern California.
• There are three different groups of B. burgdorferi; the two
found in Europe and Asia cause a slightly different
manifestation of disease than the group (sensu strictu) found in
the US.
• ELISA screening tests for Lyme have many false-positive
results and must be confirmed by a Western blot.
• Relapsing fever is caused by Borrelia species and may be
either louse-borne or tick-borne. Uveitis has been described
in both forms. The tick-borne form is seen in the US, primarily
in patients who have camped in mountain cabins in the West.
• Leptospirosis is a zoonosis seen most often in tropical or
subtropical regions.
• In the US, leptospirosis has been most often seen in Hawaii.
• Leptospirosis is biphasic in half of patients, with an initial flu-
like illness, recovery, then a late immune phase.
• Uveitis may occur during the immune phase, weeks to months
following the initial illness.
• Rat-bite fever is rare, and in Asia it is caused mainly by a
spirochete, Spirillum minus. There are no reports of eye
disease.

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137
 CHAPTER
14 Parasitic and Rickettsial Ocular Infections
Michael S. Gilmore and Juan-Carlos Abad
PARASITIC INFECTIONS
INTRODUCTION TO PARASITOLOGY
Terminology
Parasitology is the study of different species from the animal
kingdom that live together or in close association (on or in the
body of another).1 A parasite living on the surface of its host is
an ectoparasite; an internal parasite is an endoparasite. Infes-
tation is associated with ectoparasitism and infection with
endoparasitism. Parasites are either obligate (they exist only as
parasites) or facultative (they may also exist in a free-living
state). Parasites can be permanent (complete life cycle within
the host) or temporary.
Parasite Classification
Morphology, life cycle, genetics, reproduction, and aspects of
parasite growth and development are used to classify and cate-
gorize parasitic species. Serology, biochemistry, electron micro-
scopy, isoenzyme electrophoresis, DNA, RNA, and protein
analysis techniques may be required to differentiate members of
a species that are otherwise indistinguishable.
Key Features: Parasitic Infection
Protozoa
• Acanthamoeba, Trypanosoma, Leishmania, Giardia,
Toxoplasma, and Plasmodium
Metazoa
• Platyhelminthes
• Taenia and Schistosoma
• Nematoda
• Trichinella, Ascaris, Toxocara, and Onchocerca
• Arthropoda
• Sarcoptes and Demodex
The single-celled Protozoa, long considered to be one phylum,
have recently been divided into a number of groups assigned
phylum rank.2 These phyla are: Sarcomastigophora, Labyrin-
thomorphorpha, Apicomplexa, Microspora, Acestospora,
Myxozoa, and Ciliophora. Examples of human parasitic proto-
zoans are Acanthamoeba, Trypanosoma, Leishmania, Giardia,
Toxoplasma, and Plasmodium.
The phylum Platyhelminthes are worms characterized by
bilateral symmetry with rudimentary development of sensory
and motor nerve elements. Platyhelminthes are divided into
four classes: Turbellaria, Monogenea, Cestoidea, and Trematoda.
Adult cestodes, commonly called tapeworms, have a head
(scolex) and a segmented body (strobila) and live within the
digestive tract of their host. Examples of Cestoidea are Taenia,
Echinococcus, and Spirometra. Adult trematodes in the sub-
class Digenea are commonly called flukes, and their develop-
ment occurs in at least two hosts. Examples of Trematoda are
Schistosoma and Paragonimus.
The phylum Nematoda comprises a large number of organ-
isms commonly known as roundworms. Nematodes are divided
into two classes, Phasmidia and Aphasmidia, based on the pre-
sence or absence of cuticle-lined organs (phasmids). Examples
of nematodes are Trichinella, Ascaris, Toxocara, Dracunculus,
Loa, and Onchocerca.3
The phylum Arthropoda includes organisms from the classes
Arachnida, Insecta, and Crustacea; all have a hard cuticle
exoskeleton. Examples of Arthropoda are Sarcoptes, Demodex,
Phthirus, Oestrus, Dermatobia, and Hypoderma.
Table 14.1 is a summary of parasites that cause major ocular
diseases.
HOST–PARASITE INTERACTIONS
Interactions between the host and the parasite are crucial for
maintenance and continued transmission of parasitic infec-
tions. Parasitic adaptations that limit the host response include:
(1) life-cycle stages (eggs, larvae, adult organisms, cysts) that
evoke different host immune responses; (2) parasite surface
composition variation;4 and (3) tissue location (i.e., intracellular
versus extracellular). Host factors that render humans
particularly susceptible to infection include: (1) nutritional
status/malnourishment, (2) genetic susceptibility (a relative
resistance to Plasmodium vivax occurs in African-Americans,
and it has been attributed to the Duffy-negative phenotype
present in this population5), and (3) endogenous or exogenous
immunosuppression.
PROTOZOA
ACANTHAMOEBA
Several genera of free-living amebae cause disease in humans.
Acanthamoeba infections are the most important among ocular
pathogens. They cause keratitis in healthy persons. In immuno-
suppressed patients, Acanthamoeba infections may result in
granulomatous amebic encephalitis (GAE) and disseminated
infection. Vahlkampfia and Hartmannella have also been impli-
cated as a cause of infectious keratitis.6
Distribution
Acanthamoeba species are widespread in nature. They are
found in fresh, sea, tap, bottled, and brackish water,7 as well as
in dust, sewage, sludge, swimming pools (especially in warmer 139
 MICROBIOLOGY

TABLE 14.1. Ocular Parasitic Diseases in Humans

Geographic
Parasite Ocular Lesions Distribution Laboratory Tests Therapy
Protozoa
Acanthamoeba Indolent, painful corneal Worldwide Calcoflour white stain, Polyhexamethylene
ulcer and infiltrates, culture on Escherichia coli biguanide or
iridocyclitis chlorhexidine;
propamidine or
hexamidine;
itraconazole
American trypanosomiasis Bipalpebral edema, Central and South Blood smears Nifurtimox
(Tripanosoma cruzi) unilateral conjunctivitis, America
Romaña’s sign
Giardia lamblia Retinal vasculitis Worldwide Cysts and trophozoites in stool Metronidazole
Leishmania tropica, Lid ulcer Middle East, Asia Scrapings of skin lesions Antimony sodium
braziliensis (Oriental Minor, Central gluconate,
SECTION 3

sore, espundia) and South allopurinol, or

America ketoconazole
Malaria (Plasmodium Retinal hemorrhages, papille- Equatorial region Blood smear Chloroquine,
species) derma, retinal edema primaquine
Microsporidiosis Superficial epithelial Worldwide Corneal scrapings Debridement, topical
(Encephalitozoon keratopathy fumagillin,
species in immuno- itraconazole
suppressed patients)
(Nosema species in Stromal keratitis Worldwide Corneal scrapings and biopsy Trimethoprim-
immunocompetent sulfamethoxazole
patients)
Pneumocystis carinii Choroidal granulomas Worldwide Bronchial washings, sputum Pentamidine
cultures, tissue biopsy isothionate,
trimethroprim-
sulfamethoxazole
Toxiplasma gondii Retinochoroiditis, papillitis, Worldwide Serum ELISA, aqueous or Pyrimethamine,
retinal vasculitis, uveitis, vitreous PCR trisulfapyrimidine
secondary glaucoma or sulfadiazine,
clindamycin;
steroids, laser,
cryotherapy
Intestinal Nematodes
Ascaris lumbricoides Rare intraocular worm, Worldwide Eggs in stool, complement Mebendazole,
vitamin A deficiency fixation larva in ocular piperazine
granuloma or histopathology
Extraintestinal Nematodes
Baylisascaris procyonis Diffuse unilateral Southeastern Direct observation Laser
subacute retinitis United States photocoagulation;
and Caribbean thiabendazole or
ivermectin
Dracunculus medinensis Eyelid and orbital mass Africa and India Examination of the worm Surgical excision
Filariasis
1. Dirofilaria species Periobital or intraocular Worldwide ELISA Surgical excision
worm
2. Lymphatic filariasis Elephantiasis, anterior Tropical areas, Peripheral blood Diethylcarbamazine
(Wuchereria chamber or subretinal Far East
bancrofti, Brugia microfilaria (rare)
malayi, Brugia
timor)
3. Loa loa Subcutaneous nodule, Central Africa Blood smear, tissue biopsy Diethylcarbamazine
subconjunctival worm,
periobital swelling and
pain
4. Onchocerca Skin and eye nodules, Africa, Central and Skin snip, nodule biopsy Ivermectin
volvulus keratitis, uveitis, South America
chorioretinitis, optic
atrophy

140 Continued
 Parasitic and Rickettsial Ocular Infections

TABLE 14.1. Ocular Parasitic Diseases in Humans—Cont’d

Geographic
Parasite Ocular Lesions Distribution Laboratory Tests Therapy

Thelazia callineda or Conjunctivitis, extraocular Central America Biopsy lesion for worm Surgical excision
californiensis muscle paresis, orbital
granuloma
Toxocara canis, cati Posterior and peripheral Worldwide ELISA on serum, aqueous Thiabendazole,
retinal granuloma, or vitreous; CT mebendazole
panuveitis
Trichinella spiralis Lid and periorbital edema, Worldwide Serology, skin biopsy Thiabendazole and
extraocular muscle steroids
paresis and pain
Trematodes (Flukes)
Paragonimus westermani Periocular cyst Far East, India, Eggs in feces or sputum, Praziquantel
Africa, Central serum ELISA
and South

CHAPTER 14
America
Schistosoma haematobium Dacryoadenitis, conjunctival Africa, Middle Eggs in urine, lesion biopsy, Praziquantel,
and japonicum (bilharzia, and orbital granulomas East, Far East CT niridazole
schistosomiasis)
Tapeworms
Coenuriasis (Multiceps Lids and intraocular cyst Sheep-raising areas Casoni’s intradermal test Surgical excision
multiceps, Taenia (New Zealand),
brauneri) Argentina,
California
Echinococcus granulosus Orbital cyst (common), Sheep-raising Skin test, indirect Praziquantel
intraocular cyst (rare) areas (Africa) hemagglutination or
immunofluorescent serology,
radiography, CT
Sparganum proliferum Orbit or anterior chamber Far East DIrect observation Surgical excision
cyst
Cysticercus cellulosae Intraocular granuloma Worldwide Skin test, radiograph for Praziquantel,
calcified cysts niridazole
Arthropods
Demodex folliculorum Chronic blepharitis Worldwide Direct observation Lid hygiene
Myasis
1. Ophthalmomyasis Lid furuncule and cellulitis, Central and South Direct observation Mechanical removal
externa orbital invasion America,
(Dematobia Old World
hominis,
Chrysomia
bezziana)
2. Ophthalmomyasis Subretinal tracks, Tropical areas Direct observation, Laser
interna intravitreal invasion parasite recovery photocoagulation,
(Hypoderma removal of the
lineaturm) parasite
Ophthalmia nodosa Conjunctival nodule Worldwide Histopathology Surgical excision
(caterpillar hairs)
Phthirus pubis Chronic blepharitis Worldwide Direct observation Lid hygiene, antibiotic
or eserine ointment
CT, computed tomography; ELISA, enzyme-linked immunosorbent assay; PCR, polymerase chain reaction.

months), hot tubs, air conditioning ducts, dialysis units, human
and animal feces, human oral cavities, and contact lenses
and associated paraphernalia. Acanthamoeba cysts are stable
and still infective after being stored in water at 4°C for
24 years.8
Acanthamoeba keratitis has been associated with corneal
trauma, exposure to contaminated water and dust,9 and contact
lens wear. The use of homemade saline solutions, improper
contact lens care, and eye exposure to contaminated water
while wearing lenses are responsible for the association of
Acanthamoeba with contact lens use.10 Males and females are
affected equally. Since the first documented case of Acantha-
moeba keratitis was reported in 1973,11,12 the number of cases
has increased steadily.3,13 A recent series using a confocal micro-
scope as a diagnostic aid suggests that Acanthamoeba keratitis
may be more common than previously thought.14
GAE remains infrequent.15 Several cases of disseminated
Acanthamoeba infection in patients with acquired immuno-
deficiency syndrome (AIDS) with mainly cutaneous manifesta-
tions have been reported.16 141
 MICROBIOLOGY
Morphology, Biology, and Life Cycle
Acanthamoeba exists in two stages: trophozoite and cyst.
Trophozoites are the proliferative, active forms; and size
depends on species (20–40 mm).7 They have irregular shape and
pseudopodia with characteristic spine-like processes (Fig. 14.1).
The cytoplasm contains a single nucleus with a large, dense,
central nucleolus surrounded by a clear zone called the zona
pellucida. Cytoplasmic organelles are evident, as is a character-
istic large contractile vacuole. Trophozoites move by gliding in
straight lines and feed on Escherichia coli and other enteric
Gram-negative bacilli. The trophozoite, when exposed to
unfavorable conditions (desiccation, lack of food, contact
with toxic substances or solutions), undergoes immediate
encystment. Acanthamoeba proliferate by binary fission.
Acanthamoeba cysts are the resistant, dormant stage of this
parasite. Cysts are characterized by a double-walled envelope.
The outer wall, the exocyst, is wrinkled, and the inner wall,
the endocyst, is smooth. There is a space between the two
SECTION 3
walls except at the ostiole, where the exocyst is joined to the
endocyst. Cyst morphology and size are species-specific
(12.5–19.2 mm), and encystment states can be differentiated by
shape (e.g., spherical, polygonal).7 The cytoplasm of the cyst
contains a single nucleus located centrally, several lipid
droplets, mitochondria, and other cytoplasmic organelles but
a b
c
d e
FIGURE 14.1. Acanthamoeba trophozoites; unstained culture, fresh
wet preparation, phase contrast (µ400). Acanthamoeba species (a),
A. polyphaga (b), A. culbertsonii (c), A. astronyxis (d), and
142 A. castellani (e).
lacks a functioning contractile vacuole. Excystment occurs
when favorable environmental conditions return.
Infection of the Host
The mechanism for development of Acanthamoeba keratitis
may be related to epithelial trauma, strain virulence, the number
of organisms present, and favorable ameba–cornea contact
conditions.17 The proliferation and binding of Acanthamoeba to
contact lenses is enhanced by co-contamination of the contact
lens care system with Gram-negative bacteria.18 Acanthamoeba
infection causes destruction of the corneal epithelium and
stroma, with subsequent infiltration of inflammatory cells,
descemetocele formation, and corneal perforation.19 The
cellular reaction around necrotic organisms may be more
intense.20 Acanthamoeba castellani has been shown to produce
a plasminogen activator21 and nonspecific collagenases,22 which
might be related to its pathogenicity.
Diagnosis
In cases of Acanthamoeba keratitis, smears and culture isola-
tion are the initial diagnostic steps. Generally, deep corneal
scrapes are necessary to detect Acanthamoeba. The confocal
microscope has been used for in vivo diagnosis of Acanthamoeba
keratitis.23,24 If these diagnostic measures are unrewarding and
clinical suspicion is high, corneal biopsy is recommended.25
Corneal Smears
In Giemsa-stained or Gram-stained samples, Acanthamoeba
may resemble leukocytes, macrophages, and other mononuclear
cells (Fig. 14.2). Gomori-methenamine silver (stains the cyst
wall black) as well as periodic acid-Schiff (stains the cyst wall
red) may help in identifying the organisms. Calcofluor white, a
chemofluorescent dye, has proved useful in detecting Acantha-
moeba cysts.26 Smear preparations can be fixed in methyl alcohol
and processed using an aqueous solution of 0.1% calcofluor
white with Evans blue counterstain. The slides are examined by
fluorescent microscopy. The cyst wall appears bright apple-green;
trophozoites and other cells appear red-brown. Fluorescent
antibody staining of corneal scrapes can also provide a rapid
diagnosis of Acanthamoeba keratitis with the added advantage
of species differentiation.27 Slides can be fixed in 10% buffered
formaldehyde, incubated with diluted rabbit anti-Acanthamoeba
serum, followed by second-labeled antirabbit serum. Cysts and
trophozoites fluoresce brightly. More recently, isoenzyme
profiles28 and restriction fragment length polymorphisms of
mitochondrial DNA29 have been used in differentiating
Acanthamoeba.
FIGURE 14.2. Corneal scraping from a patient with Acanthamoeba
keratitis shows double-walled polygonal cysts. Giemsa stain µ400.

Acanthamoeba Culture
Acanthamoeba grows at 25–35°C. For corneal culture recovery,
nonnutrient agar overlaid with E. coli is a common culture
medium. The scraped specimen is placed on the agar surface
without streaking or cutting the agar. The plates are sealed with
adhesive tape to prevent dehydration and observed for a mini-
mum of 2 weeks. If culture plates are not available, transport
solutions can be used. Page’s saline solution (a low-osmolarity
solution) allows trophozoites to survive transportation at
ambient temperature for up to 48 h.30
Corneal Biopsy
If corneal smears and cultures from the corneal scrapings are
negative, corneal biopsy is the next viable diagnostic approach.
A 3–4-mm dermatologic punch is used to make a half-thickness
corneal trephination straddling the lesion and normal cornea.
The specimen is split in half. One part is fixed in glutaraldehyde
for light and electron microscopy studies. The other half is
hand-carried to the microbiology laboratory for bacteria, fungi,
and Acanthamoeba culture. The same diagnostic stains and
culture media used in the scrapings are used in addition to
fluorescent antibody stains. Electron microscopy may be used
as well.31
Prevention
Acanthamoeba keratitis, because of its association with contact
lenses, may be prevented by meticulous lens care and
sterilization precautions. Thermal disinfection solutions are
effective against Acanthamoeba.32 For lenses requiring chemical
disinfection, solutions containing chlorhexidine killed Acan-
thamoeba in 30 min, benzalkonium chloride systems required
at least 1 h, and hydrogen peroxide systems required up to 2 h.33
Solutions containing sorbate, polyaminopropylbiguanide, or
polyquaternium-1 may not be effective in killing Acanthamoeba
organisms.33 Contact lenses should not be worn during activities
that may increase exposure to potentially contaminated water.
Treatment
Cationic antiseptics such as polyhexamethylene biguanide
(Baquacil)34 and chlorhexidine35 kill Acanthamoeba cysts and
trophozoites by disrupting the parasite’s plasmalemma.
Aromatic diamidines, such as propamidine isethionate (Brolene)
and hexamidine (Desomedine),36 inhibit the parasite’s DNA
synthesis and can be used in combination. Aminoglycosides
(neomycin, paromomycin) and the antifungal imidazoles (mico-
nazole, clotrimazole37) have some efficacy as topical agents.
Oral itraconazole has been used by some authors,38 and higher
doses of antimicrobials may provide additional value in
treatment.39 Early animal work suggested that corticosteroids
block the conversion of trophozoites to cysts, hence enhancing
the effect of the amebicidal medications, although this remains
controversial.40 Steroids suppress the host’s immune response
and decrease inflammatory signs, making the patient more
comfortable,41 but they may be associated with a poor out-
come.42 A subconjunctival vaccine composed of Acanthamoeba
antigens was successfully evaluated in a pig model.43
AMERICAN TRYPANOSOMIASIS
American trypanosomiasis (Chagas’ disease) is caused by the
protozoan Trypanosoma cruzi. South and Central America are
endemic areas of Chagas’ disease.
Morphology, Biology, and Life Cycle
In Chagas’ disease, triatomid insects are infected with the
parasite during a blood meal from a contaminated human.
They are also called besadores (‘kissing bugs’) because of their
Parasitic and Rickettsial Ocular Infections
tropism to bite in the head region. During the next blood meal,
the insect defecates near the bite wound; the host experiences a
mild itching sensation and rubs the feces contaminated with
trypomastigotes into the insect bite. If the insect bites near the
eye or mouth, the parasites can penetrate directly into the host
via mucosal membranes. Trypomastigotes enter a wide variety
of cells (cardiac, striated muscle fibers, and cells of the reticulo-
endothelial system), where they transform into amastigotes
(1.5–5 mm in length; aflagellated). Intracellularly, the amasti-
gotic forms replicate by binary fission and destroy the cell.
Amastigotic forms released in the peripheral blood rapidly
transform into trypomastigotes and infect other cells or are
ingested by triatomid insects. American trypanosomiasis can be
transmitted congenitally and in blood transfusion.
Infection of the Host
In Chagas’ disease, acute-phase reactions depend on the route
of entry of the parasite. When the trypanosomes enter via the
CHAPTER 14
conjunctiva, Romaña’s sign (unilateral bipalpebral edema with
conjunctivitis and lymphadenopathy) may be observed.44 If
trypanosomes enter through the skin, a hypersensitivity
reaction, called chagoma (furuncle-like lesion with swelling of
the regional lymph nodes), may be present. There is a mild
febrile illness that usually goes unnoticed. In the chronic phase,
cardiomyopathy and motility alterations of the digestive tract
(megaesophagus and megacolon) are common complications.
Diagnosis
During the acute stage of Chagas’ disease, direct examination of
peripheral blood smears can confirm the diagnosis of trypano-
somiasis. Fresh anticoagulated blood may demonstrate motile
trypomastigotes, or the parasite may be identified on Giemsa-
stained blood smears. During chronic disease, the parasite is
rarely found in the peripheral blood. Xenodiagnosis (the feeding
of uninfected triatomids on an infected patient and subsequent
demonstration of parasites in the insect), hemoculture, or
animal inoculation are limited by the time lag until they
become positive.45 Serologic examinations are affected by cross-
reactivity with antileishmaniasis antibodies.46 Clinical findings
of cardiac arrhythmias, right bundle branch block, and heart
failure in conjunction with megaesophagus and megacolon in a
patient from an endemic area suggest trypanosomiasis.
Prevention
For Chagas’ disease, elimination of triatomid insects in endemic
areas is useful. Chemoprophylaxis is controversial. The use of
insect repellents and appropriate clothing decreases the chances
of acquiring the infection.
Treatment
Nifurtimox and benznidazole can be used in the treatment of
acute trypanosomiasis.47 They have no proven effect on the
chronic manifestations of the disease.
LEISHMANIASIS
Leishmaniasis is a cutaneous, mucocutaneous, or visceral
infection caused by protozoa of the genus Leishmania (family
Trypanosomatidae).
Distribution
Four major clinical syndromes are caused by several species of
leishmania: cutaneous leishmaniasis of the Old (L. tropica) and
New (L. mexicana and L. braziliensis) Worlds; mucocutaneous
leishmaniasis or espundia (L. braziliensis braziliensis); diffuse
cutaneous leishmaniasis in patients with decreased immunity;
and visceral leishmaniasis, or kala-azar (L. donovani). 143
 MICROBIOLOGY
Morphology, Biology, and Life Cycle
Leishmania organisms are found in two stages: promastigote
(flagellated) and amastigote (nonflagellated). The life cycle alter-
nates between the vector sandfly Phlebotomus (Old World) or
Lutzomyia (New World) and a mammal host. The female fly
acquires the parasite during a blood meal from an infected host.
The promastigotic form (infectious stage for humans) proli-
ferates extracellularly in the intestine of the sandfly and is
introduced into the mammalian host by the fly bite. Pro-
mastigotes in the host enter macrophages and transform into
obligate intracellular amastigotes ((2–5.5) µ (1–2 mm)). Disease
spread occurs through infection of new macrophages, following
lysis of parasite-infected cells.
Infection of the Host
The human cutaneous infection, in the early form of the
disease, is a single nodule at the site of the bite. The nodule can
progress centrifugally, ulcerate, and scar. Mucocutaneous leish-
SECTION 3
maniasis is characterized by lesions involving the lower
extremities, followed by lesions of mucous membranes and
cartilage of the oral cavity, nasal septum, and larynx. Ocular
infection may result in eyelid edema, ulceration, and scarring.
Conjunctival granuloma and interstitial keratitis have been
reported.46,48
Diagnosis
Definitive diagnosis of leishmaniasis is by direct identification
of the parasite. Stained smears (Wright’s or Giemsa stain) or
biopsy (H&E or Wilder’s reticulin stain) may demonstrate
amastigotic or intracellular forms. Needle aspiration culture
from the lesion edge or inoculation of a tissue biopsy specimen
in appropriate culture media may demonstrate the promasti-
gotic form. Serologic tests provide only indirect evidence of
Leishmania infection. The leishmanin skin test (Montenegro
test) is a delayed hypersensitivity reaction to dead promasti-
gotes injected intradermally. Negative hypersensitivity results
occur in cases of diffuse cutaneous leishmaniasis, and strongly
positive results occur in leishmaniasis recidivans. In visceral
leishmaniasis, the leishmanin skin test result is negative during
active disease and positive in most patients several months to
1 year after recovery.
Prevention
Insect repellents, appropriate clothing, and fly netting may
provide protection.
Treatment
The drugs of choice for all forms of the disease are pentavalent
antimonials: sodium stibogluconate or meglumine antimoniate
(Glucantime). Alternatives for cutaneous leishmaniasis include
allopurinol49,50 or ketoconazole.51 Amphotericin B and penta-
midine can be used in severe cases.47
MALARIA
Malaria is an infection caused by the protozoan Plasmodium.
Four species have been identified as human pathogens:
P. falciparum, P. vivax, P. ovale, and P. malariae. P. vivax, the
species most commonly infecting humans, causes benign tertian
malaria. P. falciparum is the most dangerous species, causing
malignant tertian malaria.
Distribution
Malaria is endemic in hot and humid (tropical or subtropical)
regions of Africa, Asia, and Central and South America,
affecting an estimated 200 million people and causing over
144 1 million deaths every year, especially among children.52
Morphology, Biology, and Life Cycle
The parasites are transmitted through the bite of the infected
female anopheline mosquito, the definitive host for all
Plasmodium species. The mosquito becomes infected when it
ingests the macrogametocytic and microgametocytic forms of
the parasite in the peripheral blood of an infected human, the
intermediate host. After fusion of the gametocytes (sexual
cycle), a zygote develops into an ookinete, forms an oocyst, and
then differentiates into sporozoites. The sporozoites (2–3 mm),
the infectious form of the parasite, remain in the mosquito’s
salivary glands and are inoculated into humans along with the
salivary secretions during blood feeding. The sporozoites, once
in the human circulatory system, rapidly enter the hepatic
parenchymal cells, differentiate into merozoites (1.5 mm),
replicate, rupture the cells, and are released back into the
circulatory system. Alternatively, in infections by P. vivax and
P. ovale, hepatic merozoites can differentiate into hypnozoites,
a dormant form that can cause disease relapse many years later.
Merozoites released into the circulatory system cannot enter
new parenchymal cells but enter red blood cells instead,
initiating the erythrocytic cycle. In red blood cells, merozoites
transform into trophozoites, which enlarge and then give rise to
multiple merozoites (schizogony) that rupture the red blood
cells and are released into the circulatory system to enter new
red blood cells. Trophozoites can also differentiate into macro-
gametocytes (female presexual stage, 10 mm) or microgameto-
cytes (male presexual stage). The macrogametocytes and
microgametocytes are ingested by the anopheles mosquito
during the blood feeding and reinitiate the sexual life cycle.
Infection of the Host
Sudden attacks of headaches, spiking fever, perspiration, and
shaking chills, interspersed with asymptomatic normal periods,
are clinical symptoms of acute-phase malaria. Subacute,
chronic, and recurrent forms of the disease also can occur.
Ocular manifestations of malaria include blotchy preretinal and
retinal hemorrhages believed to be caused by cytoaggregation of
the parasitized erythrocytes.53,54 In children with cerebral
malaria, papilledema or retinal edema beyond the arcades are
markers of a poor prognosis.55
Diagnosis
Malaria is diagnosed by detection of the trophozoite or gameto-
cyte in blood smears. Several smears should be collected at
hourly intervals and stained with Giemsa or Gram’s stain.
Two smears should be prepared at each time interval, one
thick, for parasite detection, and another thin, for morphologic
analysis. Diagnostic serologic techniques are not routinely
available.56
Prevention
Prevention of malaria is achieved by personal protection from
mosquito exposure and by the use of insecticides. Chemo-
prophylaxis can also be used in endemic areas. Blood banks
should follow the American Association of Blood Banks regula-
tions in screening donors for preexisting malarial infection.56 A
malaria vaccine against the merozoite has shown variable
results.57,58
Treatment
Chloroquine is the drug of choice for the erythrocytic phase of
the infection. In cases of chloroquine-resistant P. falciparum,
quinine or the antiarrhythmic quinidine could be used. Alter-
natives include mefloquine and pyrimethamine/sulfadoxine
(Fansidar). Primaquine is used to eradicate the hypnozoites in
cases of infections by P. vivax or P. ovale. Caution should be
taken in patients with glucose-6-phosphate deficiency. A

number of antibiotics, including the tetracyclines, rifampin,
clindamycin, trimethoprim, sulfonamides, and doxycycline,
have some effect.47
MICROSPORIDIOSIS
‘Microsporidia’ is the nontaxonomic term given to a group of
eukaryotic, obligate intracellular protozoan parasites. They
infect a wide variety of life forms, ranging from protozoa to
humans. Only two genera of Microsporidia, Encephalitozoon
and Nosema, cause infection that affects the ocular tissues.
Morphology, Biology, and Life Cycle
Microsporidia are endemic in the tropics,59 but it seems that
not all healthy people are susceptible to this disease. Recog-
nition of this disease has increased because of the AIDS
pandemic. Horizontal transmission is believed to take place in
animals and possibly in humans. Infection with Microsporidia
is believed to occur after ingestion or inhalation of spores from
fecal or urine contamination. The spores that infect humans
usually measure 1–2 mm by 2–4 mm.60 Organisms usually infect
the epithelial cells in the intestinal or respiratory tracts, and
from there they could disseminate to other organ systems.61
The most common presentation of Microsporidia in humans is
chronic diarrhea in AIDS patients.61 Two forms of keratitis are
recognized. The first type is caused by Nosema, which affects
immunocompetent people and produces stromal keratitis.62,63
Only four cases have been reported. The second type is caused
by Encephalitozoon, and it affects the corneal epithelium in the
form of punctate epithelial keratitis in AIDS and immuno-
suppressed patients.64
Diagnosis
In corneal scrapes, the acid-fast and Gomori-methenamine silver
stains demonstrate the organism well.63 Electron microscopy
might be required for the diagnosis. Histopathologic features of
keratoplasty specimens in patients with corneal nosematosis
demonstrate invasion of the stroma by multiple organisms,
areas of necrosis, and multinucleated giant cells. In cases of
AIDS, the parasites seem to be confined to the corneal epithe-
lium with absent inflammation.65
Treatment
In cases of Encephalitozoon keratitis, local debridement65 could
be combined with topical fumagillin.66 Oral itraconazole or
albendazole67 has been used as an adjuvant.
TOXOPLASMOSIS
Toxoplasmosis is an infection caused by the protozoan Toxo-
plasma gondii. Cats are the only known definitive host of the
parasite, but intermediate hosts, including humans, are at risk
of infection.
Distribution
Both animals and humans demonstrate serologic evidence of
Toxoplasma infection worldwide. Toxoplasmosis can be
congenital or acquired. In the United States, 30–60% of adults
have positive serology results for Toxoplasma.68 In developing
countries, acquired toxoplasmosis occurs at a younger age with
a higher prevalence in the adult population.68 In congenital
toxoplasmosis, 45% of untreated women that develop primary
toxoplasmosis during gestation give birth to infected infants;
8% of these infants are severely affected.69 Estimates of fetal
infection in the United States range from 4200 to 16 800 cases
per year.70 T. gondii is one of the most frequent causes of
retinochoroiditis and posterior uveitis,71 occurring mainly in
Parasitic and Rickettsial Ocular Infections
the second and third decades of life.72 In contrast with intra-
cranial disease, toxoplasmic retinochoroiditis appears to be
uncommon in patients with AIDS.73
Morphology, Biology, and Life Cycle
T. gondii exists in three forms. Trophozoites (tachyzoites) are
the propagative form of the parasite. Tissue cysts (bradyzoites)
occur in the chronic stage of the disease. Oocysts are shed in the
cat’s feces after sexual reproduction of the parasite (Fig. 14.3).
Intestinal phase
When cats are infected by ingestion of bradyzoite cysts from an
infected intermediate host, such as rodents and birds, brady-
zoites rapidly transform into tachyzoites, penetrate the cat’s
intestinal mucosa, and undergo an enteroepithelial cycle of
sexual proliferation, resulting in the development of oocysts.
Oocysts detach from the intestinal epithelium and are shed in
the feces. Each oocyst ((11–14) µ (9–11) mm) contains four
CHAPTER 14
sporozoites. In the external environment, the oocyst undergoes
sporulation within 1–3 days and then becomes infectious. Cats
can shed 3–100 million oocysts after primary infection.
Tissue phase
Intermediate hosts (as well as cats) can be infected by: (1)
ingesting bradyzoites or tachyzoites from uncooked meat,
unpasteurized milk, or contaminated water from an inter-
mediate host; (2) ingesting or inhaling oocysts shed in the cat’s
feces; and (3) congenital transmission of tachyzoites (see
Fig. 14.3). After exposure, the host immune defenses are
initiated, and the proliferative stage of the infection is curtailed.
Organisms encyst and remain viable in the cell tissues, where
they can reactivate at a later date.
Infection of the Host
Toxic products from Toxoplasma and hypersensitivity reactions
are responsible for the tissue damage. Inflammatory reactions
are not usually observed around the bradyzoite cysts, owing
possibly to incorporation of host elements into the cyst walls,
masking the parasite antigens.74 The infection recurs when a
cyst ruptures, releasing parasites that proliferate and invade
neighboring cells. Bradyzoite cysts can be located in many
FIGURE 14.3. Toxoplasmosis. Life cycle of Toxoplasma gondii. The
human as an intermediate host could get infected by ingesting
oocysts shed in the cat’s feces, by eating meat contaminated with
tissue cysts, or by transplacental (congenital) infection. 145
 MICROBIOLOGY
tissues and are most numerous in the brain, skeletal muscle,
myocardium, and retina.68
Infection in immunocompetent patients
The acute infection in healthy persons leads to a mononucleosis-
like clinical picture with fever, malaise, headache, arthralgia,
hepatosplenomegaly, and lymphadenopathy. It is transient and
usually of no consequence, except in cases of placental
transmission or delayed retinochoroiditis.
Infection in immunocompromised patients
Toxoplasmosis in the immunocompromised host is most
probably reactivation of a previous latent infection,75 although
in certain circumstances (leukemia and organ transplantation),
infection can be acquired from blood transfusions and conta-
minated donor tissue. The cell-mediated immune response is
an important mechanism for resistance to T. gondii infection.
Chronic immunosuppression can reactivate latent infection.
SECTION 3
Retinochoroiditis
The most common form of retinal involvement is necrotizing
retinochoroiditis, although cases of neuroretinitis75 and pro-
gressive panophthalmitis76 have been reported. Elderly patients
seem to be prone to a particularly severe form of Toxoplasma
retinochoroiditis.77 Ocular disease in healthy persons is mainly
the result of reactivation of encysted organisms after congenital
infection,72 although several cases of acquired retinochoroiditis
have been reported from endemic areas.78 Ruptured retinal cells
sensitize lymphocytes and initiate the production of auto-
antibodies that may contribute to the retinitis.79
Congenital infection
Congenital transmission of toxoplasmosis occurs when a
Toxoplasma infection is acquired during pregnancy or 6 months
earlier. The neonate of a woman with previous antibodies to
Toxoplasma will not have congenital toxoplasmosis.72 The
disease is usually more severe in the fetus than in the mother.
Transplacental transmission of Toxoplasma increases when the
infection is acquired in the second and third trimesters of
pregnancy. Severe fetal disease, however, is more prevalent when
the infection is acquired in the first trimester of pregnancy.69
Diagnosis
Laboratory diagnosis of T. gondii infection includes serologic
analysis and its histologic identification.
Serologic tests
The high prevalence and persistence of Toxoplasma antibodies
in the general population makes interpretation of serologic test
results difficult. Diagnosis of acquired infection requires
demonstration of seroconversion and a rise in antibody titer in
samples taken 4–6 weeks apart. The presence of Toxoplasma-
specific IgM indicates a recently acquired infection. Because
IgM does not cross the placenta, an increase in IgM titers in the
neonatal period is an indicator of congenital toxoplasmosis.
Recurrent Toxoplasma chorioretinitis may not increase IgG
levels, and IgM antibody is not detected. When ocular lesions
suggest toxoplasmosis, serum antibodies are considered to be
significant at any level of detection, although a positive serologic
test result is not conclusive proof of toxoplasmosis. A negative
serologic test result in an undiluted sample should exclude the
diagnosis of toxoplasmosis, although exceptions have occurred,
especially in patients with AIDS.76 No association between
serologic Toxoplasma antibody titers and eye disease severity
has been reported. ELISA is used to identify and quantify IgM
and IgG antibodies individually. Toxoplasma antibodies can be
146 detected in ocular fluids, and the ELISA can demonstrate local
ocular production of antibodies, thus aiding in the diagnosis of
difficult ocular toxoplasmosis cases.80,81 Other serologic tests,
such as complement fixation, hemagglutination, latex agglu-
tination, and immunofluorescent antibody, have been largely
replaced by the ELISA test. The PCR may be useful in detecting
Toxoplasma parasite DNA when cysts cannot be visualized.82 In
cases of retinochoroiditis, the diagnostic yield of PCR is higher
in the vitreous than in the aqueous.83
Histologic identification
The parasite is identified by routine microscopic examination of
H&E-stained or Giemsa-stained tissue sections. Identification
of tachyzoites indicates an active infection; detection of cysts
indicates a chronic stage of the disease (except for identification
of cysts in placental or fetal tissues). Fluorescent antibodies84
and peroxidase–antiperoxidase techniques85 are reliable methods
for Toxoplasma detection.
Prevention
Oocyst contamination
Toxoplasma oocysts can be destroyed by exposure to heat in
excess of 60°C; chemical disinfectants are usually ineffective.
Hand washing is indicated after contact with soil contaminated
by cat feces and when changing cat litter boxes.
Bradyzoite contamination
Bradyzoite cysts in tissues may remain viable in meat for several
days at room and refrigerator temperatures. All bradyzoites are
destroyed by cooking meat to 70°C. Hands should be washed
after handling raw meat. Soap, alcohol, and chemical disinfec-
tants inactivate bradyzoites on the skin.
Congenital toxoplasmosis
Pregnant women should be cautioned about exposure to Toxo-
plasma. Seronegative pregnant women in high-incidence areas
may be tested repeatedly; if seroconversion is detected, prompt
therapy should be initiated with nonmutagenic drugs. To facili-
tate early diagnosis and treatment, pregnant women in high-
incidence areas should be familiarized with the clinical
symptoms of acquired toxoplasmosis.
Treatment
Although Toxoplasma eye disease is self-limiting, some cases
may require treatment. The combination of sulfadiazine and
pyrimethamine86 (given concomitantly with folinic acid) is
usually the first line of treatment in cases of toxoplasmic retino-
choroiditis. Clindamycin,87 spiramycin, and trimethoprim–
sulfamethoxazole are alternative drugs. Steroids can be added to
the antimicrobial therapy if the ocular lesions threaten the
macula or the optic nerve. Cryotherapy and laser photocoagu-
lation may be indicated in special cases.
METAZOA
INTESTINAL NEMATODES
Ascariasis
Ascariasis is a nematode infection caused by Ascaris
lumbricoides.
Distribution
Ascariasis occurs worldwide, more frequently where hygiene
and sanitary conditions are inadequate.
Morphology, biology, and life cycle
Ascaris infection occurs when fertilized eggs (45–70 mm µ
35–50 mm) are ingested from contaminated soil or vegetables.

Ingested eggs hatch in the host intestine after the outer coating
is dissolved by gastric acid. The larvae penetrate the intestinal
mucosa and are disseminated via the lymphatic and circulatory
systems. The larvae become trapped in the lung’s circulation,
penetrate the alveolar wall, migrate to the trachea and
esophagus, and are swallowed. In the small intestine, the larvae
mature and mate. Adult A. lumbricoides are large parasites
(female, 20–40 cm µ 3–6 mm; male, 15–30 cm µ 2–4 mm). The
female passes an average of 200 000 eggs a day.
Infection of the host
The adult parasite inhabits the small intestine, where it can
cause symptoms that range from vague abdominal pain to com-
plete intestinal obstruction. Single worms can migrate to the
biliary tree, pancreatic duct, or appendix, causing obstruction.
In cases of massive ascaris infection, vitamin A absorption may
be decreased, which in turn causes xerophthalmia.88 Systemic
manifestations can occur during the larval migration stage,
including fever, pneumonitis, and even invasion of the intra-
ocular or periocular tissues.
Diagnosis
The diagnosis of ascariasis is made by identification of eggs in
feces or, more rarely, larvae in sputum. Occasionally, adult
worms are expelled from the mouth or rectum. Abdominal
radiographs may demonstrate parasites as worm outlines; chest
radiographs may show fleeting infiltrates (Löffler’s pneumonia)
owing to migrating larvae. The ELISA test can also be used.89
Prevention
Adequate hygienic and sanitary conditions contribute to pre-
vention of ascariasis. Water should be boiled and uncooked
vegetables avoided in endemic areas.
Treatment
Mebendazole and albendazole inhibit glucose uptake by the
parasite.90,91 Mebendazole is slowly and only slightly absorbed
from the gastrointestinal tract.92 Mebendazole is teratogenic in
rats and should not be given to pregnant women.92 In cases of
massive parasite load, these drugs should be used with caution
because they might promote parasite migration (i.e., biliary
duct or appendix obstruction). Pyrantel pamoate is effective
against Ascaris. It produces spastic paralysis and could lead to
intestinal obstruction in cases of massive infection. In these
cases, piperazine citrate, which produces flaccid paralysis of the
parasite, should be used. Most of the anthelmintics kill the
adult parasite, not the larvae, so a second course of treatment
is often given 2 weeks after the first to allow time for the larvae
to complete the pulmonary cycle and mature into adult
parasites.89
EXTRAINTESTINAL NEMATODES
Diffuse Unilateral Subacute Neuroretinitis
Diffuse unilateral subacute neuroretinitis93 is a syndrome caused
by the subretinal migration of the larval or adult form of a
parasite of the class Nematoda. Most reported cases have been
from the southeastern United Stated and the Caribbean. Several
nematodes have been implicated, including Toxocara species
and Ancylostoma caninum. Recent reports have implicated the
raccoon and skunk roundworm Baylisascaris procyonis.94–96 The
migration of the parasite causes unilateral damage to the retina,
pigment epithelium, and optic nerve along with vitreal inflam-
mation. There is usually severe loss of visual acuity. If the parasite
is seen, photocoagulation is an effective means of treatment.96,97
If no parasite is seen and clinical suspicion is high, thiabendazole98
or ivermectin9 can be used but their value is controversial.99
Parasitic and Rickettsial Ocular Infections
Filariasis
Human filarial parasites infect an estimated 200 million people
and cause a range of disease manifestations. Adult filarial
worms are threadlike, live in the subcutaneous tissues and
lymphatics, and reproduce sexually to produce microfilariae, the
first larval stage. Microfilariae are ingested by hematophagous
arthropods, in which they develop into infective larvae that
molt in the vertebrate host and mature into male or female
worms.
Dirofilariasis
Dirofilaria immitis is the heartworm of dogs; D. repens is found
in cats and dogs in Asia, Europe, and South America; and
D. tenuis infects raccoons in North America. They are acciden-
tally transmitted to humans by the same vectors that infect the
animal hosts, Aedes and Culex mosquitos. The parasite is
unable to produce microfilariae in the human host. Subcuta-
neous nodules and cardiopulmonary ‘coin’ lesions have been
CHAPTER 14
reported. Ophthalmic dirofilarial infections are more common
in the eyelids and periorbital tissues,100 conjunctiva,101 orbit,102
vitreous,103 and anterior chamber, in that order. The most
common clinical presentation is a well-encapsulated nonviable
parasite, although an occasional viable parasite has been detected.
Diagnosis is serologic using a highly specific ELISA test.104
Surgical removal is the mainstay of therapy.
Lymphatic Filariasis
Wuchereria bancrofti, Brugia malayi, and Brugia timori are
filarial nematodes with a propensity for lymphatic invasion.
W. bancrofti is distributed throughout Africa, Asia, the
Caribbean, Latin America, and Western and South Pacific
Islands. B. malayi and B. timori are found in the Far East.
Infection of the mosquito vector occurs when the insect takes a
blood meal of an infected host. Ocular filariasis by these
organisms is rare. Adult B. malayi worms have been found in
the conjunctiva and probably result from direct inoculation to
the eye rather than migration. Elephantiasis of the eyelid has
been reported. One case of a subretinal worm,105 and a second
of an immature W. bancrofti in the iris,106 represent rare intra-
ocular cases. The finding of living adult worms in lymphatic
vessels is suggestive. A single dose of 100 mg of diethyl-
carbamazine (DEC) provokes the emergence of microfilariae
into the peripheral circulation–blood should be drawn 1 h after
the administration of DEC. Treatment consists of a 21-day
regimen of DEC, although infection may recur. Topical 1%
atropine solution has been described as an agent capable of
killing microfilariae in the anterior chamber.106
Loiasis
Loiasis is a nematode infection caused by the filaria Loa loa.
Distribution
Endemic areas of loiasis are the rain forests of West and Central
Africa.
Morphology, biology, and life cycle
The vectors, female flies of the genus Chrysops (family
Tabanidae), are infected by ingesting human blood contami-
nated with the parasitic microfilariae. The larvae become
infectious in the arthropod and penetrate the host skin during
the next blood meal. Larvae develop into adult roundworms
(male, 4–7 cm in length; female, 2–3 cm) in the subcutaneous
tissues of the host. After mating, gravid females release micro-
filariae, which enter the circulatory system and, after trans-
mission to another fly, initiate a new life cycle. The microfilariae
exhibit diurnal activity, appearing in the peripheral blood only
from dawn to dusk. 147
 MICROBIOLOGY
Infection of the host
The disease is often asymptomatic, although transient pruritic
or painful subcutaneous swellings (known as Calabar swellings)
are a classic manifestation of the disease. Adult worms can
sometimes be observed beneath the skin or conjunctiva
(Fig. 14.4).107
Diagnosis
Definitive diagnosis is made by identification of either micro-
filariae in the blood or adult worms in subcutaneous tissues or
conjunctiva. Blood should be drawn during daylight because of
the diurnal periodicity of microfilaremia. Serologic testing for
specific IgG immunoglobulin may be useful in the diagnosis of
L. loa in amicrofilaremic cases.108
Prevention
Loiasis is prevented by protection against fly bites (appropriate
clothing, insect repellents).
SECTION 3
Treatment
Diethylcarbamazine citrate is the drug of choice in the treat-
ment of loiasis. Adult worms should be surgically removed from
the subconjunctiva.109
Onchocerciasis
Onchocerciasis, or river blindness, is a chronic filarial disease
caused by the nematode Onchocerca volvulus. It is one of the
major causes of infectious blindness worldwide.
Distribution
Onchocerciasis is an endemic disease with over 18 million
infected persons worldwide, of whom ~2 million have some
form of visual impairment and ~400 000 suffer from blind-
ness.110 Endemic areas include Equatorial Africa and several
foci in Central America, South America, and the Arabian
peninsula. All age groups are affected. The intensity of infection
increases with host age and reaches a plateau during the second
decade of life. In hyperendemic areas in West Africa, approxi-
mately one-third of people over the age of 15 years have micro-
filariae in the anterior chamber of the eye, and half of those
over the age of 40 become blind from the disease.111 Men are
more commonly affected than women because of occupational
exposure.110
FIGURE 14.4. Loa loa. Note the adult worm in the subconjunctival
space.
Courtesy of Roberto Pineda II, MD, and Susannah Rowe, MD.
Photo by Kit Johnson.
148
Morphology, biology, and life cycle
Black flies, members of the family Simuliidae (order Diptera),
are the only known vectors for O. volvulus. The flies are found
mostly near fast-flowing rivers in tropical and subtropical
regions. Female black flies are blood feeders, and it is during
the blood meal that the fly can transmit or receive the infection
from humans. When a black fly (1–5 mm long; black, gray, or
tan) bites an infected person, microfilariae in the circulatory
system are ingested along with the blood meal. In the insect
vector, microfilariae (300–360 µ 5–9 mm, unsheathed) develop
into infectious larvae and are retransferred to human skin
during the next blood meal. They enter humans via the fly
bite wound and develop into adult nematodes within 2–3
months. Adult worms (females, 25–50 cm µ 0.25–0.50 mm;
males, 1.9–4.2 µ 0.13–0.15 mm) are white or cream-colored,
threadlike roundworms, living in the subcutaneous tissues,
deep fasciae, or joints, commonly in clusters; they may be
encapsulated (onchocercoma) by a host immune response.
The worms reproduce sexually, and new microfilariae appear
within a year after primary infection. The adult female can
produce millions of microfilariae during her lifetime (15 years).
O. volvulus can be transmitted congenitally from severely infected
mothers, but this is rare. Parasitic nodules are usually concen-
trated in the area of the original black fly bites. African black
flies more frequently bite on the hips and legs; Central and
South American black flies usually bite the head area.
Infection of the host
Living Onchocerca microfilariae cause little adverse reaction in
humans and appear to be undetected by the host immune
system. Damage caused by onchocerciasis is due to dead or
dying microfilariae. The pathogenicity varies with the species of
Onchocerca.112 If a large number of microfilariae die at the same
time (e.g., after DEC treatment in heavily infected persons), an
inflammatory/immune response called the Mazzotti reaction
may result.113 The reaction causes a localized or generalized
skin pruritic rash, fever, lymph node inflammation, headache,
nausea, joint and muscle pain, tachycardia, respiratory distress,
and hypotension. Deaths caused by the Mazzotti reaction have
been reported.
In an important new advance, it has been shown that much
of the inflammation that occurs upon death of microfilariae is
attributable to the release of lipopolysaccharide from the cell
wall of an endosymbiont of the bacterial genus Wolbachia.114
Wolbachia belong to the order of Rickettsiales and are essential
for reproduction of the fliaria. Antibiotics that kill the endo-
symbionts stop embryogenesis in female worms. Tetracyclines,
rifampicin, and chloramphenicol are active against Wolbachia.
Doxycycline (100 mg/day) for 6 weeks blocked embryogenesis
of worms over a period of 18 months, and higher doses
(200 mg/day) were effective for 24 months without severe side
effects. Combined with ivermectin treatment, more than 90%
of the patients were free of microfilaridermia for 18 months.114
In cases where Wolbachia elimination is not attempted, the
anterior segment manifestations of ocular onchocerciasis, such
as sclerosing keratitis and iritis, as well as the presence of optic
neuritis and atrophy, are sometimes reversible after ivermectin
therapy.115
Diagnosis
Clinical diagnosis Detection of typical subcutaneous nodules
suggests the diagnosis of onchocerciasis, which must be con-
firmed by histologic examination.116 Detection of intraocular
O. volvulus microfilariae is diagnostic for onchocerciasis.111
Serologic tests are nonspecific; blood analysis usually reveals
moderate eosinophilia.

Skin biopsy Skin biopsy is used not only for diagnosis, but also
to assess the intensity of infection (number of microfilariae per
milligram of skin).89 Usually, 1 mg of healthy skin is sliced to a
depth of 0.5 mm from several sites (shoulders, buttocks). The
skin snips are placed immediately into 0.5 mL of saline
solution, where they are held for 3 h to allow the microfilariae
sufficient time to migrate from the tissue. Detection of a single
microfilaria is a definitive diagnosis; a moderately infected
patient has 20–100 microfilariae per milligram of skin.
Prevention
Areas of black fly infestation should be avoided because no
prophylactic drug is effective against the infectious larvae.
Personal protection, such as appropriate clothing and insect
repellents, should be used. The Onchocerciasis Control
Program established by the World Health Organization has
been effective in reducing transmission of onchocerciasis in a
700 000-km2 area involving seven countries in Central and
West Africa.110
Treatment
Additional clinical trials to determine optimum antibiotic
activity for eliminating Wolbachia from the worms and
rendering them sterile, are currently underway. Previously,
ivermectin has been the drug of choice.115,117 It causes a spastic
paralysis of microfilariae, thus reducing the side effects of
treatment related to migration of the parasites. It does not affect
adult worms.111 The drugs formerly used in the treatment of
onchocerciasis, suramin and DEC, can cause severe reactions
related directly to the patient load of microfilariae and are
not currently recommended. Nodulectomy may be useful to
decrease the adult worm load.
Thelaziasis
Nematode members of the family Spiruroidea, genus Thelazia,
are parasites of birds and mammals and are usually located in
the conjunctiva and lacrimal gland ducts. Adult worms are
cream-colored and measure 0.75 µ 17 mm. Some species
(T. callipaeda, Asia, China, and Korea; T. californiensis, North
America) have been reported in humans. Flies of the genera
Musca and Fannia are the intermediate hosts for this parasite.
Definitive hosts include dogs, cats, horses, sheep, bears, and
deer. In humans, the worms invade the conjunctiva, causing
pain and watery conjunctivitis.118 They can be seen as creamy
white, threadworm masses coiled in the conjunctival sac or
migrating over the cornea. Eyelids and extraocular muscles
can also be compromised. Intraocular penetration does not
occur. Therapy for ocular thelaziasis is surgical removal of
the parasite.
Toxocariasis
Dogs and cats are the definite hosts for Toxocara canis and
Toxocara cati, which are members of the nematode family
Ascarididae. Toxocariasis in humans (an intermediate host)
is caused predominantly by T. canis, and it is manifested clini-
cally as either visceral larva migrans (VLM) or ocular larva
migrans (OLM).
Distribution
T. canis has a worldwide distribution in dogs and is uniformly
prevalent in North America.119 Pregnant and lactating dogs are
the most important factors in Toxocara infection. In puppies,
intestinal infection rates can reach 100%; in adult dogs, the rate
falls to less than 20%.120 T. cati infection also appears to occur
worldwide in cats, with a prevalence in North America varying
between 24% and 67%.119 Toxocara infection in humans is
Parasitic and Rickettsial Ocular Infections
sporadic but widespread. Demographic factors, such as socio-
economic status, hygiene practices, and association with dogs,
influence infection rates.121 Seroprevalence rates of toxocariasis
in children (1–11 years) in different geographic regions of the
United States range between 4.6% and 7.3% and are higher in
warmer climates.121 The frequency of seropositive titers declines
markedly with increasing age; peak infection occurs at 1–5 years.
Children with geophagic behavior and who are exposed to dogs
are most likely to develop OLM.122
Morphology, biology, and life cycle
Dogs and other canines (definitive hosts) are infected by several
routes: ingestion of infectious eggs, ingestion of late-stage larvae
or immature adult worms (during maternal grooming of the
litter), ingestion of larvae in tissues of paratenic hosts (e.g.,
mice), and transplacental or transmammary transmission.
Infection in cats is similar to that in dogs, although there is no
evidence of transplacental infection. The life cycle in puppies
CHAPTER 14
initiates with ingestion of Toxocara eggs (75–85 mm, spherical
with a thick shell) that hatch in the stomach or small intestine
of the definitive host and release infectious larvae (20 µ
400 mm).123 The larvae burrow into the intestinal mucosa, enter
the lymphatic and circulatory systems, and migrate to the lung
capillary bed within 3–5 days. In the lungs, the larvae enter the
bronchioles, trachea, and pharynx and are swallowed to develop
into adult worms (T. canis, 4–18 cm; T. cati, 3–12 cm) in the
intestine. Adult worms produce eggs (200 000/day)124 that are
shed in the feces 4–5 weeks after infection. Eggs are non-
infective when shed and require appropriate soil conditions for
development of the infectious larvae.
Transmission to humans may occur by ingestion of eggs from
the soil, contaminated hands, and fomites, or less frequently by
ingestion of the larval stage from undercooked meat. If the host
is large enough (adult dogs and humans), larvae pass through
the pulmonary capillaries and are distributed to somatic tissues
instead of being trapped in the alveoli. Humans are paratenic
hosts, with larvae migrating aimlessly in the tissues for varying
time periods. The larvae reach the eye via the choroidal blood
vessels, where they migrate into the subretinal space or vitreous
cavity.125
Infection of the host
The tissue damage observed in toxocariasis results from
larva migration (mechanical) and immune reaction. Clinical
manifestation of the disease depends on the organ and the
number of invading larvae. Several larvae in the liver may
cause no disease, whereas a single larva in the eye can cause
blindness.
Diagnosis
Serology ELISA is used for serodiagnosis. Titers may be equal126
but are usually lower127 in patients with ocular infections com-
pared with patients with systemic disease. ELISA titers of 1:32
are indicative of VLM (78% sensitivity, 92% specificity),128 and
titers of 1:8 are indicative of OLM (90% sensitivity, 91% speci-
ficity).129 ELISA can also be used on intraocular fluids.130 High
titers can be detected in the aqueous humor and the vitreous
when concomitant serum titers are low or absent, suggesting
localized antibody production.131,132 Aqueous humor (especially
when cells are observed at the clinical examination) and vitreous
cytology can demonstrate eosinophils, suggesting a parasitic
infection.133
Blood analysis Patients with VLM may have leukocytosis,
hypereosinophilia, and hypergammaglobulinemia (IgG, IgM, or
IgE); blood findings are usually normal in patients with OLM.
149
 MICROBIOLOGY
Ocular imaging studies Detection of intraocular calcifications
by computed tomography may provide a differential diagnosis
with retinoblastoma, although small retinoblastomas can
remain uncalcified, and cases of toxocariasis with calcium
deposits have been reported.134 Echographic findings such as a
solid, highly reflective peripheral mass; a vitreous band or
membranes extending between the posterior pole and the mass;
and a traction retinal detachment or fold from the posterior pole
to the mass suggest ocular toxocariasis.135
Histopathology In tissue sections, circumscribed granulo-
matous reactions with neutrophil and eosinophil infiltrates
are seen, occasionally with the larvae located in the center of
the reaction (Fig. 14.5). Fibrinoid necrosis may occur in the
central area of recent lesions, whereas older lesions may reveal
fibrous encapsulation. Giant cells, epithelioid cells, macro-
phages, and lymphocytes are usually present around degener-
ating larvae.136
SECTION 3
Prevention
Newborn litters and lactating dogs and cats should be
dewormed at regular intervals. Because deworming medication
does not eradicate all somatic larvae, pregnant dogs require
repetitive prophylaxis and deworming with each new litter.
Treatment
Thiabendazole is controversial because the death of the parasite
entices an intense inflammatory response.137 Steroids are fre-
quently used to decrease it. Photocoagulation, cryopexy, and
vitrectomy have been employed.
Trichinosis
Trichinosis is a nematode infection caused by the roundworm
Trichinella spiralis. Humans are infected by eating raw or
improperly cooked meat, especially pork.
Distribution
Trichinosis is endemic where pork is consumed, especially in
the Western hemisphere and Western Europe. Several cases
have been attributed to the consumption of wild carnivores,
such as bear and wild boar. Between 1982 and 1986, the annual
average number of cases in the United States was 57.138
FIGURE 14.5. Intraocular toxocariasis. Fibrotic mass with many
eosinophils. Toxocara canis larvae within the fibrotic proliferation.
Masson’s trichrome stain µ250.
Courtesy of Miguel Burnier Jr, MD.
150
Morphology, biology, and life cycle
There are no intermediate hosts, and both the adult and larval
stages develop in the same animal. After ingestion of contami-
nated meat, encysted Trichinella larvae (0.4 µ 0.26 mm) are
freed by gastric digestion of the cyst wall. The larvae develop
into adult worms (females, 2–3.6 mm µ 75–90 mm, are approxi-
mately twice the length of males) in the small intestinal
mucosa. Following copulation, the male dies, and within a week
the viviparous female releases larvae (100–160 mm µ 6–7 mm),
which enter the mucosal vascular channels and are distributed
throughout the body. Larviposition continues for ~4–6 weeks.
Only larvae that encyst in skeletal muscles mature and become
infectious. The muscles of the diaphragm, tongue, and eye are
mostly affected. Calcification of cysts begins in 6–18 months.
The cycle is repeated when the host is eaten by another
carnivore.
Infection of the host
Disease severity is directly related to the numbers of larvae
ingested, varying from completely asymptomatic to severe with
neurologic, pulmonary, and cardiovascular complications. In
the intestine, the adult worms cause inflammation and mucus
production. Muscle invasion by the larvae can cause myalgia
and weakness. Encysted larvae, localized in extraocular muscles,
cause periorbital inflammation with conjunctivitis, hemorrhage,
edema, pain, and photophobia.3 Eosinophilia is frequent.
Diagnosis
Definitive diagnosis is made by direct observation of encysted,
coiled larvae in tissue biopsy specimens. Serologic test results
are positive after the third week of infection.
Prevention
Trichinosis is prevented by proper cooking of pork.
Treatment
Mebendazole and thiabendazole are available for the treatment
of trichinosis. Thiabendazole therapy has been associated more
frequently with side effects, such as dizziness, mental changes,
rash, nausea, and Stevens–Johnson syndrome in children.139
The administration of corticosteroids is indicated for the treat-
ment of the allergic reaction to dead parasites.
Schistosomiasis
Schistosomiasis is an infection caused by three species of
Schistosoma: S. mansoni, S. japonicum, and S. haematobium.
Distribution
S. mansoni is prevalent in Africa, the Middle East, and South
and Central America; S. japonicum in the Far East; and
S. haematobium in the Middle East and Africa.
Morphology, biology, and life cycle
The intermediate host of Schistosoma is the snail (Biompha-
laria species). Humans are the only definite host and only
significant disease reservoir. Schistosoma eggs in fresh water
release miracidium larvae that enter the snail and differentiate
into cercariae (final larval stage). Cercariae pass from the snail
to the water and penetrate the human skin. After penetration,
the cercariae migrate to the lungs and then to the liver as
worms, where they mature and mate. Females of S. mansoni
and S. japonicum lay their eggs in the smallest venules of the
intestinal wall, and the eggs are shed with the feces. Females of
S. haematobium lay their eggs in the smallest vessels of the
vesical plexuses, and the eggs are shed in the urine. The eggs
reach fresh water, and the cycle is repeated again.

Infection of the host
The prepatent period in humans (from cercaria penetration
until appearance of eggs in the feces or urine) is ~50 days.140
Local dermatitis after contact with infested water is common
(‘swimmer’s itch’). In cases of S. mansoni or S. japonicum infec-
tion, the acute phase may include abdominal pain, chills, fever,
cough, diarrhea, and eosinophilia; during chronic phases,
hepatosplenomegaly, ascites, and esophageal varices, with
recurrent episodes of hematemesis, can occur. In cases of
S. haematobium infection, dysuria, hematuria, and suprapubic
pain, as well as obstructive uropathy, may occur. Infection of the
eye includes granulomatous choroiditis,141,142 dacryoadenitis,143
and conjunctivitis,144 and lid masses145 in endemic areas.
Diagnosis
Definitive diagnosis is made by detecting the eggs in feces or
urine. Biopsy of the rectal or urinary bladder mucosa is rarely
indicated.
Prevention
Prevention can be accomplished by improving sanitation and
reducing egg contamination in fresh water. Snail control with
molluscicides may be useful in endemic areas.
Treatment
Praziquantel, oxamniquine, metrifonate, and niridazole are
available for the specific treatment of schistosomiasis.47
Tapeworms
Taeniasis and cysticercosis
Tapeworms of the genus Taenia can cause two different human
diseases: taeniasis and cysticercosis. Taeniasis is an intestinal
infection caused by the adult T. solium and T. saginata.
Cysticercosis is a tissue infection caused by the larval form of
T. solium (Cysticercus cellulosae).
Distribution Taeniasis and cysticercosis occur where sanitary
conditions are poor and where raw or undercooked conta-
minated pork and beef are routinely consumed. Endemic foci of
the disease are South and Central America and Africa.
Morphology, biology, and life cycle Taeniasis is acquired by
ingestion of raw or poorly cooked meat contaminated with the
larval form of the parasite (cysticerci). Taenia larvae attach to
the host intestinal mucosa and develop into adult worms
(3–9 m) in the intestinal lumen. Terminal gravid segments
of the worm, called proglottids (T. saginata, 20 µ 5–7 mm;
T. solium, 12 µ 5 mm), are shed in feces and contain
50 000–100 000 viable eggs. Eggs (30–40 mm) in proglottids are
infectious immediately after shedding. Ingestion of eggs by
intermediate hosts (pigs, cattle, or humans) results in hatching
of the eggs into larvae (5 µ 10 mm, with a scolex) and
penetration through the intestinal wall. The larvae are
transmitted through the lymphatic and circulatory systems,
where they invade various organs and develop into cysticerci
(infectious form). Humans develop cysticercosis via ingestion of
T. solium eggs, either from exogenous sources or from their own
stools. Only larvae of T. solium penetrate the human intestine;
T. saginata does not cause human cysticercosis because the
larvae cannot penetrate the intestinal wall.
Infection of the host Patients with taeniasis are usually
asymptomatic. Patients with cysticercosis may also be asympto-
matic, although clinical manifestations of neurocysticercosis
(epilepsy, intracranial hypertension, and mental disturbances),
ophthalmocysticercosis (loss of vision, periorbital pain, scotoma,
Parasitic and Rickettsial Ocular Infections
and photopsia),146 and subcutaneous and muscular cysticercosis
(subcutaneous nodules) may be noted. In the eye, the cysti-
cercus cyst may be localized in the orbit,147 the subconjunctival
space, or intraocularly in the anterior or posterior chamber.
Larvae can be identified in the subretinal space, where they
cause hemorrhage and edema.148
Diagnosis Taeniasis is diagnosed by isolation and identifica-
tion of the proglottids in feces. If T. solium proglottids are
identified, additional evaluation for potential cysticercosis is
warranted. Clinical findings, such as brain calcifications, cystic
lesions in the CNS, and demonstration of larvae with scoleces
within the eye, are diagnostic of cysticercosis. Ocular ultra-
sonography may be an alternative to computed tomography and
magnetic resonance imaging in the evaluation of patients of
suspected intraocular or orbital cysticercosis.149 Indirect hemag-
glutination and ELISA may be helpful, although false-positive
results can occur.150
CHAPTER 14
Prevention Appropriate sanitation and personal hygiene are
important in the control of fecal contamination of water and
food. Raw or improperly cooked pork should be avoided,
especially in endemic areas.
Treatment Anthelminthic drugs used in the treatment of
taeniasis and cysticercosis include praziquantel (drug of choice),
niclosamide, and paromomycin. Mebendazole and albendazole
are effective against Taenia but not against Cysticercus. In cases
of ocular cysticercosis, surgical removal of cysts is often
necessary.151
ARTHROPODS
DEMODICOSIS
Demodex folliculorum and D. brevis are two species of follicle
mites causing demodicosis in humans. D. folliculorum lives on
hair follicles in the facial region, and D. brevis inhabits seba-
ceous glands. The disease is extremely common, with infesta-
tion rates reaching 97% in endemic areas.152 Demodicosis is
usually a benign infestation, although follicle mites have been
associated with blepharitis.152
MYIASIS
Ophthalmomyiasis refers to the involvement of the ocular
tissues by larvae from flies of the order Diptera.
Distribution
Myiasis is a worldwide disease, occurring more frequently in
warm climates. The prevalence of the different species of
flies varies according to the locale. Dermatobia hominis is
endemic in transequatorial coffee-growing areas of South
America. Chrysomyia bezziana is primarily a cattle parasite in
the Old World. Calliphora vomitoria organisms are present in
decaying animal or vegetable matter worldwide. Ophthalmo-
myiasis is the infestation that occurs in the ocular or periocular
tissues.
Morphology, Biology, and Life Cycle
Larvae from several fly species can cause ophthalmomyiasis.
These larvae are usually obligatory parasites, requiring host
tissue for completion of their larval stages. Eggs or larvae may
be transported to the eye by the adult fly, by a secondary vector
such as a tick or mosquito, or by the patient’s hands. D. hominis,
C. vomitoria, and Chrysomyia bezziana infection occurs via
151
 MICROBIOLOGY
oviposition on periocular tissue. Hypoderma lineatum larvae, a
cattle parasite, penetrate the skin and migrate aimlessly,
causing painful abscesses.
Infection of the Host
Ocular disease may be external or internal. In external ophthal-
momyiasis, lid edema,153 furuncular lesions,154 orbital involve-
ment,155 and even loss of the eye156 can occur (Fig. 14.6).
Internal ophthalmomyiasis is caused predominantly by larvae
of H. lineatum. Subretinal tracks (trails of depigmentation in
the retinal pigment epithelium) are the result of maggot migra-
tion in the subretinal spaces and are pathognomonic of internal
ophthalmomyiasis.157 The larvae could migrate into the vitreal
cavity. Visual compromise varies from nonexistent158 to severe
visual loss.159
Diagnosis and Treatment
Myiasis is diagnosed on the basis of recovery or visualization of
SECTION 3
the larvae. In cases of ophthalmomyiasis externa, covering of
the skin lesion with bland medicinal oil or petroleum jelly
forces the larvae to the skin surface, facilitating removal with a
forceps. In cases of ophthalmomyiasis interna, laser photo-
coagulation of the subretinal larvae160 or extraction by vitrectomy
of the intravitreal larvae has been attempted.
OPHTHALMIA NODOSA
Ophthalmia nodosa is a condition caused by an immune reaction
to caterpillar hairs or other insect matter. Caterpillar hairs are
acquired by direct contact or via airborne transmission. The
hairs induce a granulomatous inflammatory response with pain
and foreign body sensation. The most commonly affected tissue
is the conjunctiva, where nodules have been occasionally
reported.161 The caterpillar hairs may penetrate into the deeper
ocular tissues, causing keratitis, iridocyclitis, and even endo-
phthalmitis.162 Ophthalmia nodosa is treated by surgically
removing the caterpillar hair and by topical steroids.
PHTHIRIASIS
Phthiriasis is a lice infestation caused by the arthropod Phthirus
pubis.
a b
FIGURE 14.6. Ophthalmomyasis externa. A 94-year-old woman from Cundinamarca (Colombia) with altered mental status found with massive
orbital infestation by Dermatobia hominis. Note the marked lid edema and distorted anterior segment (a). The larvae had destroyed all the
intraocular contents (b).
152 Courtesy of Pedro I Navarro, MD.
Distribution
Lice infestation is cosmopolitan; transmission occurs by direct
physical contact with infected persons. The 15–40-year-old age
group is more commonly affected. In children, infestation with
P. pubis results from contamination from an adult.163
Morphology, Biology, and Life Cycle
Phthiriasis is considered a venereal disease. The source of lice
is generally the hair in the pubic area of an affected person. The
lashes become infected by either direct contact or by contact
with contaminated bedding and clothes. Other species of lice,
such as P. humanus capitis (head louse) and P. humanus
humanus (body louse), do not affect the eyelashes. The reason
the lashes are affected by P. pubis seems to be related to the
parasite’s arm span. There is itching and erythema of the lid
margin. Chronic follicular conjunctivitis is common. The oval
and transparent parasite’s eggs or nits are glued to the
eyelashes. The adult louse is frequently overlooked because of
its transparency.
Diagnosis
The diagnosis of lice infestation is based on the demonstration
of nits and adult lice in the lashes. Wood-light illumination can
be used to demonstrate the fluorescence of the nits.164
Treatment
Physostigmine (Eserine) ointment can be used to suffocate the
parasite.165 Lindane should be used in the pubic area.
RICKETTSIAL INFECTIONS
Rickettsial infection is an acute disease caused by the bacteria-
like microorganisms of the family Rickettsiaceae. In addition to
Wolbachia mentioned above, three genera are involved:
Rickettsia, Rochalimaea, and Coxiella, with human infections
caused primarily by Rickettsia prowazekii, Rickettsia typhi,
Rickettsia rickettsii, Rickettsia tsutsugamushi, Coxiella burnetii,
and Rochalimaea quintana. Rickettsia can infect a wide number
of hosts, from invertebrates to vertebrates. Rickettsial diseases
in humans can be divided clinically into the typhus group
(epidemic typhus, murine typhus), the spotted fever group (Rocky
Mountain spotted fever, boutonneuse fever, rickettsialpox), and

other rickettsial diseases (scrub typhus or chigger-borne typhus,
Q fever, trench fever).
DISTRIBUTION
Key Features: Rickettsial Infection
• Small, Gram-negative coccobacillary bacteria
• Replicate intracellularly
• Use host ATP
• Athropod vectors
Rickettsial infections occur worldwide. Improved treatment and
prevention methods have decreased the incidence of rickett-
sioses, but they have not been completely eliminated.
MORPHOLOGY, BIOLOGY, AND LIFE CYCLE
Rickettsia are pleomorphic, Gram-negative organisms (0.2–0.5 mm
µ 0.8–2 mm) that resemble bacteria in their structural and
chemical characteristics but are distinct organisms, because
several species have an obligate intracellular nature. They
multiply by binary fission in the cytoplasm of infected cells or,
as with the spotted fever group organisms, replication can also
occur in the cell nucleus. R. prowazekii replicates until the cell
lyses, whereas R. rickettsii does not cause cell lysis and leaves
the host cell early in the course of infection to infect other cells.
Disease transmission is via arthropods.166
Lice (Pediculus humanus) are the vectors of the epidemic
typhus caused by R. prowazekii. The organisms invade the
louse’s intestinal epithelial cells and multiply, causing cell lysis.
The louse does not survive more than 10 days after the primary
infection, and during this period it sheds rickettsiae in its feces.
Contaminated louse feces are deposited on the skin during
insect blood meals, and the rickettsia gains entrance into the
body via wounded or scratched skin. Humans are an important
reservoir host for epidemic typhus.
Ticks (several Dermacentor species) are the vectors of the
Rocky Mountain spotted fever caused by R. rickettsii. The
vector is contaminated by feeding on infected animals (e.g.,
rodents), with rickettsiae remaining in the arthropod salivary
glands. Humans are only accidentally infected. R. rickettsii are
not pathogenic for the ticks; infection is maintained among
ticks by transovarial transmission.
Several species of Leptotrombidium (mites) are the vectors of
the scrub, or chigger-borne, typhus, caused by R. tsutsugamushi.
Adult mites and larvae (chiggers) are infected by feeding on
contaminated vertebrates (e.g., mice). Rickettsiae are located in
the arthropod salivary glands and are inoculated into the host
during the blood meal. R. tsutsugamushi is not harmful to the
mites; infection is maintained among mites by transovarial
passage. The mites function as both reservoirs and vectors of
the disease. Because R. tsutsugamushi has strain variations,
some patients may experience a second attack of scrub typhus.
Lice (P. humanus) are the vectors of the trench fever caused
by R. quintana. The body louse acquires and passes the
infection by feeding on a rickettsemic human. Organisms grow
extracellularly in the louse intestinal lumen; humans are
contaminated through louse feces deposited on the skin.
Humans are reservoirs for trench fever. Transovarial trans-
mission of R. quintana among lice has not been observed.
Fleas (Xenopsylla cheopis) are the vectors of the murine
typhus caused by R. typhi. Humans are accidentally infected.
Organisms proliferate in the flea intestinal cells, and the disease
is transmitted by contaminated flea feces deposited on the skin.
Fleas do not transmit R. typhi to offspring transovarially.167
Parasitic and Rickettsial Ocular Infections
Mites (Allodermanyssus sanguineus) are the vectors of
the rickettsialpox caused by R. akari. Humans are only acci-
dentally infected. The mite also transmits the infection
transovarially.
Q fever is caused by C. burnetii. Ticks transmit the infection
to domestic animals that shed the rickettsia in milk, urine, feces,
and placental products. C. burnetii is highly resistant to extremes
of temperature and desiccation. Humans and other animals are
infected by inhalation or mucosal contact with dust containing
the rickettsiae. In ticks, infection with one species may prevent
subsequent infection with other rickettsial species.168
INFECTION OF THE HOST
In humans, rickettsiae multiply in endothelial cells of small
blood vessels, causing endothelial proliferation and perivascular
infiltration, subsequent extravasation of fluid with edema, and
hypotension. If untreated, the disease can progress to gangrene
CHAPTER 14
and disseminated intravascular coagulation. Formation of a
typhus nodule or glial nodule (a perivascular aggregation of
mononuclear cells such as lymphocytes and macrophages) in
the CNS is characteristic of the disease.166,169 Skin and several
other organ tissues (kidney, heart, lung) can be involved,
causing skin rash, encephalitis, and renal and liver failure, and
may lead ultimately to death of the host. Rickettsial infection
may induce resistance to reinfection or, in contrast, persistent
lymphoid tissue disease as in Q fever and recrudescent epidemic
typhus. Table 14.2 summarizes the epidemiology and clinical
findings of some human rickettsial diseases.
CLINICAL FINDINGS
The clinical spectrum of rickettsial disease varies widely
according to the organism involved and the host response.
Fever, rash, and history of arthropod exposure suggest the
disease, although these signs are not always present.170 Other
signs, including prostration, nausea, vomiting, abdominal and
back pain, myalgia, arthralgia, cough, photophobia, and
conjunctivitis, may be present. A primary cutaneous lesion
(eschar) may be observed at the site of the insect bite or
attachment. In epidemic typhus, a recrudescent mild form of
the disease, called Brill–Zinsser disease, can occur. Classic Q
fever presents as atypical pneumonia or with influenza-like
symptoms. Ocular findings in all rickettsial diseases may
include sore, red eyes with conjunctival papillae, chemosis,
and petechiae; iritis, retinitis (edema, hemorrhage, exudate);
venous engorgement; arteriole occlusion; and optic nerve
edema.171
DIAGNOSIS
Demonstration of rising antibody titers to rickettsial antigens
using paired acute and convalescent sera is the most widely
used method of clinical diagnosis of rickettsial infection. A
fourfold or higher rise in titer suggests acute disease. Serologic
methods include indirect immunofluorescent antibody, com-
plement fixation, indirect hemagglutination, and ELISA. The
Weil-Felix reaction is an agglutination test using Proteus
mirabilis strains OX19, OX2, or OXk with antigens similar to
those of Rickettsia. The Weil-Felix reaction is not completely
reliable, and rickettsialpox and Q fever are not associated with
Weil-Felix antibody rises.
Rickettsiae stain poorly with Gram’s stain but can be
visualized using Giemsa or Macchiavellos stain. Culture using
enriched blood-agar media can be used for recovery of
R. quintana. All other rickettsiae require living cells (embryo-
nated eggs or other tissue culture systems) for culture. 153
 MICROBIOLOGY

TABLE 14.2. Epidemiology and Clinical Characteristics of Rickettsial Diseases

Mammalian Geographic Incubation

Organism Transmission Reservoir Distribution Disease (Days) Clinical Signs*

Rickettsia Louse feces Humans North and South Epidemic typhus 5–23 Generalized
prowazekii America, Africa, maculopapular
Asia rash; central
nervous system
involvement,
myocarditis, renal
insufficiency; no
eschar; may be
recrudescent
Rickettsia Flea feces Rodents Worldwide Murine typhus 4–15 Generalized
typhi maculopapular
rash; no eschar
Rickettsia Tick bite, dogs Rodents Western hemisphere Rocky Mountain 2–14 Maculopapular
rickettsii spotted fever (petechial) rash
SECTION 3

on extremities
and later on
trunk; eschar
Rickettsia Mite bite Rodents Asia Scrub typhus 8–12 Maculopapular rash
tsutsuga- on trunk
mushi spreading to
palms and
soles; eschar
Coxiella burnetii Inhalation, goats Cattle, sheep Worldwide Q fever 8–39 Interstitial
pneumonia; no
eschar; rare rash;
chronic form:
hepatitis and
endocarditis
Rickettsia akari Mite bite Mice USA, former USSR, Rickettsialpox 10–24 Mild condition;
Korea vesicular lesions
on initial papular
rash; eschar
Rochalimaea Louse feces Humans Europe and Africa Trench fever 8–30 Splenomegaly;
quintana macular rash
*All patients usually present with high fever and headache that may be accompanied by prostration, myalgia, arthralgia, and conjunctivitis.

PREVENTION
Personal protection against vector contact (protective clothing)
and use of insect repellents in endemic areas are preventive
measures. Lice infestation can be avoided by frequent changes
of clothing or by application of insecticides. Forceps and hand
protection while removing ticks are recommended because both
tissues and fluids from crushed ticks are contaminated. Vector
and reservoir control may be indicated in endemic areas.
Milkborne transmission, observed in Q fever, can be prevented
by pasteurization. Chemoprophylaxis is not recommended.170
Effective vaccines for the major rickettsial infections (e.g., Rocky
Mountain spotted fever) have been developed but are not used
frequently168 because rickettsial diseases, if promptly recognized
and treated, are no longer lethal.169
TREATMENT
Tetracyclines are preferred drugs in the treatment of rickett-
siosis. Chloramphenicol is also effective.171

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CHAPTER 14
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157
 CHAPTER
15 Fungal Infections of the Eye
Wiley A. Schell, Gary N. Foulks, and John R. Perfect
The first reported case of fungal infection of the cornea dates
back to 1879, involving a farmer who was struck in the eye by
oat chaff with resultant keratomycosis caused by Aspergillus
glaucus.1 Physicians and microbiologists subsequently have
realized the unique relationship between fungi and human
ocular disease. The frequent association of fungal ocular
infection with occupational trauma and exposure to vegetable
material is well documented.2–10 Increasing recognition of
fungal ocular infection in the 1950s and 1960s concurrent with
the increased use of topical antibiotics and corticosteroids on
the eye led to more than 148 case reports by 1962 and firmly
established the association of fungal infection with impaired
host defenses or physical trauma.11 Subsequent work has
confirmed the importance of impaired host defenses or broken
anatomic barriers; and, has examined fungal growth charac-
teristics as they relate to expression of clinical disease, providing
insight into improved therapy against these infrequent but
extremely tenacious invaders.
Many fungal species have been identified in human ocular
diseases.12–14 Chorioretinal or orbital diseases are most often a
result of systemic mycoses contracted through respiratory tract
exposure (Histoplasma, Cryptococcus, Blastomyces, Coccidioides)
or dissemination from the gastrointestinal tract or an intra-
vascular catheter (Candida).15–24 In contrast, the fungal species
associated with lacrimal, corneal, or traumatic intraocular
infections are found in soil and vegetable matter and can be
cultured from 2.5% to 52% of normal eyes, depending on
climate and occupation. Fungi are not part of the normal flora
of the lids or conjunctiva of normal eyes but are only transient
colonizers. When specimens are taken from the conjunctiva or
lids, the same fungus is rarely isolated sequentially in an
individual, and most cultures grow only one or two fungal
colonies, suggesting a very low burden of organisms.25,26 Almost
half of the reported cases of ocular surface infections are
attributed to environmentally common species of the genera
Aspergillus, Penicillium, Fusarium, and to Candida albicans, a
commensal of humans. This finding correlates with epidemio-
logic studies in which these fungi have been transiently isolated
from normal eyes.
Typically, environmental fungi cause keratitis after pene-
trating into the cornea through trauma. Also, topical therapy
with antibiotics and corticosteroids generally increases fungal
colonization of the eyelids and conjunctiva and is thus a major
predisposing factor for oculomycosis through superinfection.27,28
Isolation of fungal species in eyes with known underlying
abnormalities such as dacryocystitis has increased. An asso-
ciation of seborrheic blepharitis with Malassezia furfur colo-
nization or infection has been suggested. Finally, an increase in
colonization of eye structures may result from exogenous
factors, including the use of mascara contaminated by fungi
such as Candida parapsilosis.
HOST–FUNGI INTERACTIONS IN THE EYE
Key Features: Fungal Infections of the Eye
• Exogenous
• Keratomycosis
• Chorioretinitis
• Endophthalmitis
• Endogenous
• Chorioretinitis
• Endophthalmitis
• Orbital
Ocular defenses to fungal infection are numerous, and
oculomycosis is common only when anatomic structures are
breached. Normal flora of the eyelids, the conjunctival sac with
normal lacrimation, and the mechanical movements of the
eyelids create an unfavorable environment for the growth of
most opportunistic fungi, such as Aspergillus and Candida
species. Alteration of the normal flora with systemic or topical
antibacterial agents or corticosteroids, however, can decrease
this barrier and allow colonization and growth of fungi. Because
many fungi do not grow at elevated temperatures, normal body
temperature is high enough to prevent many environmental
fungi from becoming pathogenic. The lower temperature of the
cornea relative to the rest of the body and eye, and its exposure
to potential trauma, may partially explain why keratomycosis is
the most common ocular fungal infection. The intact corneal
epithelium is generally resistant to fungal penetration and
infection; this affords great protection. Breach of the epithelial
barrier is often a prerequisite for keratomycosis, which explains
its association with trauma through occupational, recreational,
or surgical exposures. First, direct inoculation by trauma may
occur when the fungus is carried on a projectile. Second,
colonizing fungi may invade the wound after trauma; such
invasion is particularly enhanced by the use of antibacterials,
corticosteroids, or both. Third, surgical procedures such as
keratoplasty, corneal transplantation, or radial keratotomy are
occasionally associated with introducing fungi into the eye via
transplant or contaminated irrigating solutions.29–35 Several
well-described outbreaks of ocular fungal infections with
C. parapsilosis and Paecilomyces lilacinus have been associated
with lens implants and contaminated irrigation solutions.36–39
Finally, soft contact lenses can act as a nidus for fungal invasion
159
 MICROBIOLOGY
into the cornea if they are not properly cleaned and disinfected
(Fig. 15.1).8,40–41 Corneal infection allows extension to the
sclera or intraocular space because there are few subsequent
tissue barriers. The role of local antibodies and complement
in protection against fungal infections of the eye is uncertain.
The polysaccharide nature of the fungal cell wall can activate
complement, and secretory immunoglobulin A (IgA) can protect
against mucosal infection with Candida species, but the
importance of such local immunity protection in the eye is not
well understood. On the other hand, clinical experience demon-
strates that topical and systemic corticosteroids enhance the
risk of ocular fungal infections and clearly suggests that local
immunity factors are important in protecting the eye from
fungal invasion.
The second avenue for fungal invasion is through the blood
stream (endogenous rather than exogenous). This oculomycosis
generally occurs when there is some systemic host immune
depression. The most common example is white blood cell
SECTION 3
defects, particularly chemotherapy-induced neutropenia.
During neutropenia, invasion of the eye is particularly difficult
to diagnose because general hallmarks of infection, such as an
inflammatory response in the chorioretina or vitreous body, are
not always visible.42 Candida and Aspergillus species, however,
can reach the retina in the presence of a normal granulocyte
count if the systemic inoculum is high, as occurs in certain
human infections and in experimental animal models. For
example, fungal ocular infections have occurred during hyper-
alimentation, post partum, during prolonged antibiotic therapy,
in the neonatal period, and with intravenous drug use.43–61 The
cell-mediated immune system is a well-characterized protective
system against fungal infection and obviously is important in
preventing and fighting established ocular fungal infections.
Debilitating diseases or generalized impairment of the immune
system are predilecting factors for fungal infection, both
systemically and ophthalmically. Rhino orbital zygomycosis in
the diabetic or cancer patient represents invasion of blood
vessels within the orbit secondary to an underlying immune
depression. C. neoformans invasion of the orbit or chorioretinal
area has become more common in severely immunocom-
promised hosts with cell-mediated defects, such as patients
with acquired immunodeficiency syndrome (AIDS) and those
on high-dose corticosteroids.
Although ocular involvement with C. neoformans has
increased during the AIDS epidemic, infection with this fungus
was frequently reported in prior years. One study found ocular
160 FIGURE 15.1. Fusarium solani growing from soft contact lens.
signs and symptoms in 45% of all patients with meningitis.17
Manifestations range from ocular palsies to involvement of the
choroid–retina.17,62 In one-fourth of cases, eye involvement is
diagnosed before meningitis.63 Simultaneous infections with
C. neoformans and other pathogens such as human immuno-
deficiency virus and cytomegalovirus can occur in severely
immunosuppressed patients.64,65 Although most cases of ocular
cryptococcosis arise from bloodstream dissemination, the eye
has been the direct portal of entry in such cases as donor
transmission through a corneal transplant30 and cryptococcal
keratitis after keratoplasty procedures.32,33 Thus, some cases of
disseminated cryptococcosis might originate in the eye rather
than the lung.
Ocular cryptococcosis can lead to visual loss. In fact, most
cases of cryptococcal endophthalmitis lead to severe visual loss;
successful management is rare.66,67 The AIDS epidemic has
given rise to reports of catastrophic loss of vision in patients
with cryptococcosis without evidence of endophthalmitis.68,69
The funduscopic examination yields either normal results or
evidence of papilledema. The clinical manifestations suggest
two pathogenic processes. First, some patients experience rapid
visual loss within 12 h to a few days. This clinical syndrome
suggests optic neuritis in which the optic nerve and its vessels
are infiltrated by large numbers of yeast cells. No successful
therapeutic strategies are known for this form of visual loss.
Other patients can present with slow visual loss that generally
begins later during antifungal therapy and gradually progresses
over weeks to months. Symptoms may be related to increased
intracranial pressure in these patients, and treatment with
central nervous system shunts or optic nerve fenestrations may
halt the progression of visual loss.69
In contrast with infections, in which ocular defenses clearly
fail to prevent fungal inoculum from replicating, there is a
syndrome called ‘presumed ocular histoplasmosis’, which is
characterized by chorioretinal scars, hemorrhages, and neovas-
cularization. It has been suggested that these host reactions
are due to the presence of the yeast cells or antigens of
H. capsulatum, but only rarely have viable organisms been
documented for this syndrome.70 The thrust of treatment has
been corticosteroids or laser therapy to stop the lesion’s
advancement;71 antifungal therapy rarely has been helpful.70
When oculomycosis occurs, the fungus tends to invade
directly into tissue planes. This is particularly apparent in
keratomycosis, as demonstrated in Figure 15.2, a case of
C. parapsilosis infection in a keratoplasty patient on long-term
topical steroid therapy. The host response to the organism can
be acute suppurative inflammation, chronic inflammation, or
granulomatous inflammatory reaction, depending on the fungal
species and tissue location (Fig. 15.3). The organism can
actively damage host tissue by stimulating the host to elaborate
inflammatory mediators such as oxidative products. The fungus
may also secrete products that injure the eye. For example, a
potential virulence factor for C. albicans is its production of
extracellular acid proteases and phospholipases, which may
further aid in tissue destruction.72–74 Aspergillus species can
produce elastase, which most likely facilitate hyphal invasion
into blood vessels and may further contribute to damage of eye
tissue.75 Certain fungi produce mycotoxins under specific
conditions, but to date no such products have been detected in
or shown to contribute to destruction of ocular tissue.
Fungi possess poorly understood factors that allow a certain
tropism for eye structures during bloodstream invasion. For
instance, during fungemia with C. albicans in the rabbit model
of candidiasis, yeast cells consistently localize in the eyes and
kidneys while other tissues are spared. In humans, the propen-
sity for ocular invasion during candidemia is high.46,49–60 This
may be related to the unique vascular arrangements of the eye,

a b
FIGURE 15.2. (a) Clinical picture of stromal keratitis in corneal graft (Candida parapsilosis). (b) Histologic section demonstrating deep lamellar
infiltration of yeast (C. parapsilosis) with acute and chronic inflammatory cellular infiltrate. Methenamine silver stain, µ33.
a1 a2
b1 b2
but specific fungal factors for this localization also are likely.
Findings suggest that early pseudohyphal formation plays a role
in establishing an endogenous ocular infection. This propensity
for C. albicans ocular infections has been corroborated in
human infections, of which the vast majority are associated
with this Candida species. However, other Candida species
occasionally cause endogenous eye infection, particularly when
the inoculum is as large as can occur with C. parapsilosis
infection during hyperalimentation. Spores from Aspergillus
species, which are found on fomites such as drug paraphernalia,
can reach ocular structures and establish infection when
inoculated intravenously.53,76–78
DIAGNOSTIC TESTING
The diagnosis of fungal etiology in ocular infection can be
difficult. Certain clinical characteristics may be helpful to
ophthalmologists, including duration and features of the ocular
lesions. These are reviewed elsewhere in this book. However, it
must be emphasized that there remains no substitute for the
proper collection of specimens for histologic and cultural iden-
tification (Fig. 15.4). Infections are diagnosed in the laboratory
by culture or microscopy performed on clinical specimens.
Recently, it also has been shown that polymerase chain reaction
Fungal Infections of the Eye
CHAPTER 15
FIGURE 15.3. Histopathology of mycotic
ocular infections. (a1) Stromal keratitis due to
Candida parapsilosis with acute and chronic
inflammatory infiltrate. (a2) Methenamine silver
stain, µ132. Keratitis with infiltration by
Cryptococcus neoformans showing
granulomatous reaction. Papanicolaou stain,
µ600. (b1) Endophthalmitis due to a
zygomycetous fungus. H&E, µ132.
(b2) Chorioretinitis due to Aspergillus species.
PAS, µ132.
(b1 and b2) Reprinted from Perry HD, Donnenfeld ED:
Cryptococcal keratitis after keratoplasty. Am J
Ophthalmol 1990; 110:320.
(PCR) amplification of fungal DNA can be used to detect and
identify the infecting fungus.79–84
Microscopy of clinical specimens can be performed by
various methods. Calcofluor white/KOH is one extremely
sensitive technique. It is rapid and easy to perform but is not a
permanent preparation. Giemsa stain, periodic acid-Schiff, and
methenamine silver stain are sensitive and permanent
preparations. Gram’s stain detects yeasts such as Candida
species but is not reliable for other fungi such as molds and
should not be relied on for detecting mycotic infection. Gram-
stained slides can be decolorized and reused with one of the
preferred reagents. Microscopy may reveal yeast or hyphae of
the infecting organism, but specific identification of the species
of fungus requires culture. Fluorescein-conjugated lectins or
fluorescent antibody conjugates have been used to allow
differentiation among species such as Candida, Aspergillus, and
Fusarium, but these stains are not commercially available.85
Certain molds, particularly Paecilomyces lilacinus, sometimes
form spores within the infected tissue, and this can be a useful
differential characteristic.86 These spore forms can be mistaken
for Candida species. Superficial infections can be identified by
scraping surface lesions, with organisms identified by culture
and often corroborated by microscopy of stained smears prepared
from the scrapings. Definitive diagnosis of deep keratitis or 161
 MICROBIOLOGY
a b
c d
SECTION 3
e f
FIGURE 15.4. Colony and microscopic appearance of pathogenic
fungi. (a) Creamy, round colony growth of Candida albicans.
(b) Gram’s stain of yeast cells, Candida species. (c) Filamentous
colony of Aspergillus fumigatus, with characteristic blue-green color
due to sporulation. (d) Cotton blue stain of hyphae and sporulating
structures of A. fumigatus. (e) Filamentous white colonies of Fusarium
solani. (f) Microscopic view of spores of F. solani.
intraocular infection often requires culture of an aspirate,
because direct smears do not always reliably correlate with
culture-proved infection.9,10,86–88 Biopsy of deep corneal lesions
may be required to demonstrate the organism by special
histologic stains. In deep infections of the cornea, superficial
scraping may not yield enough organisms to identify or culture.
Corneal biopsy with histopathologic examination may be
required. In such situations, the use of periodic acid-Schiff,
methenamine silver, or calcofluor white stains is helpful in
demonstrating the organism; detection with fluorescein-
conjugated lectins or fluorescent antibody conjugates also is
possible.89
Although microscopy can be the most rapid laboratory test
for establishing a diagnosis, culture is required in order to
perform in vitro antifungal susceptibility testing. Also, because
of a high error rate for named sequences in public databases,
it is strongly recommended that PCR results be correlated with
results obtained by traditional culture methods. Because ocular
infections are often caused by common saprobes in the
environment and access to tissue or other diagnostic specimens
is limited, special techniques for specimen evaluation must be
used to diagnose fungal infections of the eye. The clinicians and
laboratory personnel must communicate effectively, to agree on:
protocols for using media that do not inhibit fungal growth,
inoculation techniques that help differentiate infective
organisms from contaminants, and prolonged incubation times
and optimal temperature to allow for the slow growth of some
fungal species.
Media for culture should not include cycloheximide, which
inhibits fungal growth, but inclusion of gentamicin or chloram-
phenicol may be needed to suppress bacterial overgrowth. A
streak inoculation technique on media with specimens obtained
162 from the examining room or operative suite should allow
laboratory personnel to determine whether growth is on or off
the inoculation streaks and thus differentiate possible patho-
gens from airborne contaminants. Specimens should be
collected from external ocular surface infections or lacrimal
infections with a moist applicator and inoculated by streaking
directly onto culture media.88 Scrapings from corneal ulcers in
cases of keratomycosis or aspirates from the anterior chamber
and the vitreous cavity in cases of endophthalmitis should be
directly inoculated onto both Sabouraud’s (or similar) agar
media and brain-heart infusion broth medium as well as blood
agar plates.86,88 Incubation should be at 24–30°C (30°C is
preferred) and should be prolonged. Although species of some
genera, such as Candida, Fusarium, Paecilomyces, Curvularia,
and Alternaria, normally are visible within 3 days, as many as
25% of fungal isolates may require up to 2 weeks of incubation.88
When Histoplasma or Blastomyces is suspected, cultures should
be incubated for at least 4 weeks.
THERAPEUTIC CONCEPTS
Key Features: Categories of Primary Therapeutic
Agents
• Polyenes (amphotericin B, pimaricin)
• Pyrimidines (5-fluorocytosine)
• Imidazoles/triazoles (fluconazole, itraconazole, ketoconazole,
posaconazole, voriconazole)
• Echinocandins (anidulafungi, caspofungin, micafungin)
• Biguanides (polyhexamethyl biguanide)
Therapy of fungal infections can be difficult and prolonged. The
difficulty in treatment is due to a combination of the growth
characteristics of fungi, the limited availability of effective
antifungal agents, and the poor tissue penetration of these
agents. Until 1950, safe and reliable treatment for deep fungal
infections did not exist, and treatment of superficial infections
depended on empirical topical preparations. Nystatin was first
introduced in the mid-1950s, and amphotericin B came to
dominate treatment of deep mycoses in the 1960s. In the 1970s,
5-fluorocytosine was introduced as treatment for candidiasis
and cryptococcosis, but drug resistance became a problem.
Since the mid-1980s, several N-substituted imidazole or
triazole compounds have been introduced and modified with
significant improvement in activity and pharmacokinetics.
The most useful antifungal agents are of two groups: those
affecting cell walls or membranes, and those interrupting nucleic
acid or protein synthesis. The polyene macrolide antibiotics
interact with the sterols in the fungal cell membranes to impair
their barrier function and thus produce leaking of cellular
substances with subsequent metabolic disturbance and result-
ing cell death. The toxicity of amphotericin B, however, is
related to similar interactions with sterols in host cells.
Resistance of fungi to amphotericin B is rare and probably
occurs by alterations in the sterol composition of the cell
membrane.90 The azole antifungals share an imidazole or
triazole ring with N-carbon substitution that allows interaction
with primary target sites within the fungal cell. At low concen-
trations, these compounds inhibit cytochrome P-450 enzymes,
which leads to the accumulation of 14-g-methylsterols and
reduced biosynthesis of ergosterol. At higher concentrations,
some azoles can cause direct cell membrane damage. The
fluorinated pyrimidine, 5-fluorocytosine, is deaminated once
inside the susceptible yeast cell: A cytosine deaminase converts
it to 5-fluorouracil for incorporation into fungal RNA and thus
disruption of protein synthesis. The echinocandin class of

antifungals is the most recent of antifungal systemic agents and
it targets a cell wall synthesis enzyme, 1,3-b-glucan synthetase.
This enzyme inhibition results in fungicidal activity against
many Candida species and inhibition of hyphal tip growth in
Aspergillus species.
In vitro testing of antifungal susceptibility and its correlation
with in vivo response historically have been difficult because
minimum inhibitory concentrations vary greatly under different
test conditions. However, progress has been made and stan-
dardized protocols now are in effect for susceptibility testing of
yeasts and molds.91–93 Standardization of in vitro antifungal
testing involves comparing direct antifungal activity, pharma-
cokinetics of the agents, and prior clinical experience on
treatment of certain fungal infections. Efforts are ongoing to
establish standardized testing for new antifungal drugs effective
against a wider range of fungi. Despite the concerns about
clinical validation, our opinion is that yeasts and possibly molds
from serious oculomycoses should be evaluated comparatively
by in vitro susceptibility testing with available antifungal
agents. This can allow detection of possibly drug-resistant fungi
and can provide the grounds for clinical judgment of the best
antifungal regimen.
Response to therapy depends on several factors. Host factors
include the integrity of the immune defense mechanisms
(especially cell-mediated functions) and the location and extent
of infection. Pharmacokinetic factors include penetration and
tissue distribution of the antifungal agent as well as predilection
for tissue binding. Antimicrobial factors include the observable
effect on the fungal organisms and the response in growth
characteristics of the fungus in the presence of the antifungal
agent. A further clinical problem in treatment is that when the
organism encounters adverse conditions (elevated temperature,
anaerobiosis, chemotherapeutic agents), it may revert to a
dormant or slow growth state that is more difficult to eradicate
with cell wall- or cell membrane-active antifungals and thus
requires longer treatments. Finally, clinical experience – both
that of the attending clinician and that gleaned from references
in the literature – can be a helpful guiding factor. The following
discussion summarizes specific therapeutic concepts in
management of oculomycoses.
The single most important factor in the success of treatment
for oculomycosis is early diagnosis and treatment. Fungal infec-
tions can have an indolent course, and the longer these infec-
tions remain untreated, the more difficult they are to eradicate.
For this discussion, infections are divided into three categories:
(1) keratomycosis, (2) endophthalmitis, and (3) orbital infection.
KERATOMYCOSIS
Fungal keratitis is usually caused by environmentally wide-
spread molds such as Aspergillus species, Fusarium species,
Paecilomyces species, and Curvularia species, but other fungi,
such as Candida species and Cryptococcus neoformans, also
can cause keratitis in susceptible hosts. Identification of the
fungus and comparative in vitro susceptibility testing to
available antifungal drugs usually are important. For fungal
corneal ulcers, pimaricin remains the most reliable topical
antifungal agent in a 5% suspension or as a 1% ointment for
treatment of superficial ocular injuries or prophylaxis with
high-risk injuries for oculomycosis. It also is not as irritating
to the eye as the other polyenes, such as amphotericin B.
Unfortunately, pimaricin therapy has two drawbacks. First,
although it has broad-spectrum antifungal activity across many
species, isolates may be relatively resistant to its antifungal
activity, with only half of studied strains being inhibited by
3 mg/mL or less.94 Second, it has limited ability to penetrate the
cornea. Third, the film formed on the cornea by pimaricin is
Fungal Infections of the Eye
not conducive to simultaneous use of multiple antifungal
eyedrops. Nystatin is less active in vitro than the other polyenes
but is reasonably well tolerated in a 3% ointment. Amphotericin
B can be irritating to the eye, and in high concentrations (5%)
can lead to punctate epithelial erosions. Even so, it is frequently
used in a topical solution for serious infections. Topical anti-
fungals are likely to be most successful early in the infection,
before it has extended into deeper layers of the cornea. Novel
topical disinfectants, notably polyhexamethylene biguanide
(Bacquacil) have shown efficacy in experimental testing and in
limited clinical use.95,96 Although evidence-based studies are
lacking, use of multiple antifungal eyedrops to achieve potential
synergistic or additive activity might be tried in particularly
refractive cases. It is emphasized that proper cultures for
isolation and identification of the fungus should be taken before
beginning therapy.
The second approach to therapy of keratomycosis is the use
of systemic antifungal agents. For superficial fungal ulcers, this
CHAPTER 15
second line of therapy may not be necessary, but deeper corneal
infections may require it. The azole compounds have become
attractive candidates for systemic administration. They are safe
and relatively broad-spectrum. The ocular pharmacology of
these azole compounds (miconazole, ketoconazole, fluconazole,
and itraconazole) has been examined in both humans and
animals.97,98 The rank of penetration into eye structures such as
vitreous body and aqueous humor, from highest to lowest, is
fluconazole, ketoconazole, miconazole, and itraconazole. The
azoles’ penetration into the eye appears to be improved by
inflammation, as is the case with other drugs. Azoles have been
shown to penetrate into corneal tissue of rabbits and can be
found in corneal tissue even when the eyes are not inflamed.98,99
Therefore, it is reasonable to anticipate that future reports will
show the success of these agents in the management of fungal
keratitis and scleritis. Flucytosine is another agent with excellent
penetration into eye structures and has shown some success in
Candida keratitis. Its major limitation is its narrow spectrum
of activity. It inhibits only a portion of Candida species,
C. neoformans, and some dematiaceous molds. For corneal
infections, systemic amphotericin B therapy has not been
widely used. It has been used, however, as a topical preparation
and in fungal scleral infections as a subconjunctival injection.
The success of subconjunctival injection of amphotericin B
remains unclear, and it can be extremely painful and sometimes
produces tissue necrosis and nodules.100,101 Its limited eye
penetration and toxicity have reduced enthusiasm for its use in
infection at this site except for the most difficult cases.
Voriconazole has been successfully used as a topical and
systemic drug to treat Fusarium keratitis that was refractory to
amphotericin B and itraconazole.102 Voriconazole concentration
in the anterior chamber of the eye in this case was 160% of that
measured in the plasma. Another study showed voriconazole
concentrations in the vitreous and aqueous that were 38–53%
that of the plasma in patients who received two 400 mg doses
12 h apart.103 Other reports of clinical efficacy for voriconazole
in keratomycosis include three cases caused by Scedosporium
apiospermum.104–106
Econazole (2%), in a randomized trial of 112 patients, was
shown to be as effective as 5% natamycin in the treatment of
keratomycosis caused by molds, mainly A. flavus and Fusarium
(species not specified).107
Caspofungin experience in keratomycosis is scant, but com-
parison of 0.5% caspofungin with 0.15% amphotericin B in a
rabbit model of C. albicans keratomycosis showed equal
efficacy.108
In addition to antifungal therapy, some eyes require excisional
keratoplasty, particularly in cases of impending perforation.
Even in these cases, however, aggressive antifungal chemotherapy 163
 MICROBIOLOGY
before and after surgery may improve the final level of visual
acuity.
There is no strong evidence that topical or systemic steroids
help in the management of fungal eye infections. In fact, they
are often the major risk factor for these infections and their
progression. Prevention of inflammation and resultant tissue
destruction and the preservation of visual acuity are vital
objectives, but there are no guidelines to balance the positive
effects of steroids on inflammation versus the negative effects of
stimulating fungal growth. Therefore, adjunctive corticosteroid
therapy should not routinely be used in fungal eye infections.
ENDOPHTHALMITIS
There are two types of fungal endophthalmitis. Exogenous
endophthalmitis is associated with trauma or surgery in which
the organism is introduced directly into the ocular structures.
Endogenous endophthalmitis is generally produced by Candida
SECTION 3
species or Aspergillus species from a chorioretinal lesion, and
extension into the vitreous body accompanies systemic dissemi-
nation of the fungus. It may also occur with the endemic
mycoses, such as blastomycosis, after the initial pulmonary
infection. The need to manage these infections has significantly
intensified over the last decade because of expanding immuno-
compromised populations, complex surgical procedures, and
increasing use of antibiotics and intravenous catheters. The
most important therapeutic principle in endophthalmitis is
early diagnosis and correct identification of the fungus.109 For
instance, in patients with candidemia who are not neutropenic,
a prospective evaluation of the eye may identify an early ocular
infection in a third of the patients.45 Early treatment is more
likely to yield a better visual outcome. Animal models of endo-
genous C. albicans endophthalmitis suggest that early treat-
ment with either azoles or amphotericin B is more successful
than delaying treatment for a week despite similar numbers of
yeasts at each time period.98,110 Correct identification of the
organism by blood or ocular fluid cultures and determination of
in vitro susceptibility to various antifungal agents helps identify
the most promising antifungal agents for successful treatment.
Candida remains the most common invasive ocular pathogen
for endophthalmitis. Because there are no comparative studies
on therapeutic regimens, it remains reasonable to select the
antifungal agent with the most successful experience, ampho-
tericin B. Systemic amphotericin B in doses of 0.5–1 mg kg–1 day–1
has been used to control Candida endophthalmitis but at the
higher doses toxicity is substantial. Amphotericin B has very
low levels as measured in the vitreous body and aqueous humor,
but these measurements do not account for drug that is bound
to tissue.111,112 Because the penetration of amphotericin B is
poor, however, intraocular therapy combined with vitrectomy
frequently has been used. In a primate model, up to 3 mg of
intravitreal amphotericin B was tolerated without permanent
retinal toxicity, and a human took 50 mg of amphotericin B over
a 6-month period without serious retinal toxicity.113 A slowly
given 1–5 mg intravitreal injection is probably not toxic to the
retina. Now that liposomal amphotericin B at 3–5 mg kg–1 day–1
is available, it may be possible to deliver even more drug to
this site of infection safely.114,115 The value of intravitreal
amphotericin B is not proved and toxicity questions do remain,
but it may be of particular benefit when the vitreous body is
significantly involved, as in cases requiring vitrectomy and in
Aspergillus infections extending into the vitreous body.115
Flucytosine remains a possible agent for ocular Candida
infections with its high penetration into the vitreous body and
aqueous humor.116 There has been little experience with its use
alone, and concern over primary resistance in a portion of
164 Candida isolates remains.117 An attractive regimen for Candida
endophthalmitis would be combination chemotherapy with
amphotericin B and flucytosine.118 This combination regimen
has been successful in prospective studies in the treatment
of cryptococcal meningitis, and its in vitro synergy against
Candida by virtue of different mechanisms of antifungal action
theoretically could eradicate the fungus more rapidly and
improve visual outcome. However, no prospective studies have
proved this hypothesis.
With the advent of the azole compounds, clinicians have
another treatment avenue. The early azoles (ketoconazole and
miconazole) had some successes and failures. The newer azole
(fluconazole) has excellent ocular pharmacokinetics and may be
helpful in managing ocular fungal infections. The only com-
parative data regarding the efficacy of these compounds are
from animals.98,110 These models suggest that amphotericin B
may still be more potent in eradicating Candida from the eye
than the azole compounds are. There have also been case reports
of Candida and Coccidioides infections in which miconazole
was not effective but patients improved after receiving
amphotericin B therapy.119 Such results, however, should not
necessarily dissuade clinicians from carefully using these newer
azole compounds in fungal endophthalmitis, because more
clinical experience with these compounds in ocular infections is
needed. For example, one report on ocular candidiasis in drug
addicts cited an excellent response to ketoconazole treatment.47
Voriconazole reports thus far suggest this new azole can
be effective in treating some cases of endophthalimitis caused
by Aspergillus species,120,121 Scedosporium apiospermum,
Paecilomyces lilacinus,122 Scytalidium dimidiatum,123 Fusarium
verticilloides (as F. moniliforme),124 and Candida sp.125 In addi-
tion to high intraocular levels being measured in cases of
systemic administration, an animal model indicates that
intraocular injections of voriconazole are well tolerated.126
Although amphotericin B and flucytosine remain the most
attractive combination regimen for Candida, a polyene–azole
combination might be useful in certain eye infections,
particularly if both antifungals have in vitro activity against the
fungus. The concern about polyene–azole antagonism in vitro
has not been proved in vivo. Another combination regimen that
may be considered is fluconazole plus flucytosine. These two
oral agents reach high drug levels within ocular tissue. Finally,
the regimen of amphotericin B plus rifampin has been used
successfully both in animals and in humans.127,127a,128 The
point of this discussion is that combination antifungal chemo-
therapy can be considered rational treatment if proper
identification and comparative in vitro susceptibility testing on
the fungus are performed.
The newest class of antifungal compounds targets the
synthesis of 1,3-b glucan within the fungal cell wall. One of
these, caspofungin, is now available but clinical experience is
very limited at this time. One prospective study reported
success in all seven Candida endophthalmitis patients using
caspofungin.129 Another reported successful use of caspofungin
in treatment of C. glabrata endophthalimitis130 A. fumigatus
was successfully treated by adding caspofungin to a regimen
of voriconazole.124 A retrospective review of endophthalmitis
suggested that combination voriconazole–caspofungin can be
effective in the treatment of Candida endophthalmitis as
well.131 In contrast, however, treatment failure of C. albicans
endophthalmitis accompanied by poor ocular penetration has
been reported.132 Two additional echinocandins, mycafungin
and anidulafungin, have been approved for use with esophageal
and invasive candidiasis but experience with thse drugs has not
yet been reported for fungal eye infections. Therapeutic
vitrectomy may be helpful in certain patients and likely clears
the eye of inflammatory debris.48,49,58,61,78,133–143 For this treat-
ment, our current understanding makes it reasonable to select
 Fungal Infections of the Eye

patients with extensive vitreous involvement and likely visual
impairment from scarring, with progressive inflammation
despite antifungal agents, and patients with extensive vitreal
involvement but an unclear underlying pathogen.
ORBITAL INFECTION
Fungal infections in the orbit that do not initially invade ocular
structures are generally caused by a member of Zygomycetes
such as Rhizopus species or by Aspergillus species.16,21,144 The
rhinocerebral form of zygomycosis is a characteristic acute
progression of infection into the orbit, causing orbital swelling
and eventual paralysis of orbital structures.21,145 Generally
caused by R. arrhizus, this infection primarily affects diabetics,
particularly if acidosis has occurred; cancer patients; or patients
receiving chelation or steroid therapy. The infection starts in
the nasal or sinus cavities and invades the regional arterial
vessels by direct extension, causing thrombosis and leading to
Early debridement of infarcted tissue is essential to a suc-
cessful outcome and may obviate the need for subsequent orbital
exenteration. The goal of treatment remains the prevention
of extension into the brain. The immediate control of the
underlying disease, such as acidosis, is also important; finally,
amphotericin B at 0.7–1 mg kg–1 day–1 or a lipid formulation of
amphotericin B at 5 mg kg–1 day–1 is usually given. The lipid
formulation of amphotericin B offers reduced toxicity compared
to the non-lipid formulation. Posaconazole, a new triazole, is
gaining positive experience in treatment of zygomycosis and
may become part of the management strategy.146 The length of
therapy should be tailored to the patient’s response and extent
of infection.
CONCLUSION
Fungal infection in the eye is most often of exogenous origin in
an immunocompetent host whose local tissue defenses have

ischemic necrosis. Extension through the orbital apex into the
brain occurs as infection progresses. A black eschar in the nasal
area or drainage of ‘black pus’ from the eye suggests this
diagnosis.21 Identifying the patient at risk and performing an
early examination of the nasal and sinus areas for signs of
disease often leads to diagnosis before the orbit becomes involved.
Aspergillus infections of the sinus have eroded through bone or
invaded local vessels and entered the orbit, producing proptosis.
Therefore, evaluation of recent proptosis of ocular structures
should include a careful examination of the sinuses.
CHAPTER 15
been damaged. The growth characteristics of the fungus can
result in superficial infection or invasion into deep tissues,
where it may alter its growth pattern in response to the local
milieu. Effective therapy of such infections must be selected
from the small number of antifungal agents and requires
recognition of the limitations of susceptibility testing, the
importance of tissue penetration and absorption, and the need
for protracted treatment. Because of these limitations, success
of therapy primarily depends on early diagnosis of the fungal
infection and correct identification of the particular fungus.

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child with normal immune function. Eye
(London) 2000; 14:670–671.
141. Tsai CC, Chen SJ, Chung YM, et al:
Postpartum endogenous Candida
endophthalmitis. J Formos Med Assoc
2002; 101:432–436.
142. Sato Y, Miyasaka S, Shimada H: Prognosis
of endogenous fungal endophthalmitis and
utility of Ishibashi’s classification. Jpn J
Ophthalmol 2001; 45:181–186.
143. Weissgold DJ, Orlin SE, Sulewski ME, et al:
Delayed-onset fungal keratitis after
endophthalmitis. Ophthalmology 1998;
105:258–262.
144. Sacho H, Stead KJ, Klugman KP, Lawrence
A: Infection of the human orbit by
Aspergillus stromatoides. Mycopathologia
1987; 97:97–99.
145. Gass JDM: Ocular manifestations of acute
mucormycosis. Arch Ophthalmol 1961;
65:226.
146. Van Burik JA, Hare RS, Holomon HF, et al:
Posaconazole is effective as salvage
therapy in zygomycosis: a retrospective
summary of 91 cases. Clin Infect Dis 2006;
42:61.
 CHAPTER

16 Ocular Virology
James Chodosh

INTRODUCTION TABLE 16.1. Classification of Virus Families by Nucleic Acid

Type and Strandedness, and Presence of an Envelope
Viruses are obligate intracellular pathogens without the capacity
to replicate outside the host. They may cause clinically evident Examples
infection, establish latent infections with or without significant RNA Viruses
disease, or less commonly, induce encephalitis and other life-
threatening illnesses. This chapter focuses on the general Single-stranded, positive sense, Astroviridae, Caliciviridae,
description of viruses and elucidates common mechanisms nonenveloped Picornaviridae
relevant to ocular viral pathogenesis. Single-stranded, positive sense, Coronaviridae, Flaviviridae,
enveloped Retroviridae, Togaviridae
DESCRIPTION AND CLASSIFICATION Single-stranded, negative sense, Arenaviridae, Bunyaviridae,
enveloped Filoviridae, Orthomyxoviridae,
Viruses are small (10–400 nm in diameter) infectious units Paramyxoviridae, Rhabdoviridae
each consisting of a nucleic acid genome and a protein capsid Double-stranded, positive sense, Birnaviridae, Reoviridae
shell. Some virus families also express an external lipid enve- nonenveloped
lope. Viruses lack any independent means of energy metabo-
lism, molecular biosynthesis, or replication. Viral genes are DNA Viruses
transcribed and viral progeny produced only inside a permissive Single-stranded, nonenveloped Circoviridae, Parvoviridae
host cell. The existence of viruses as distinct infectious
Double-stranded, nonenveloped Adenoviridae, Papovaviridae
organisms was first suggested by early experiments in which
specific infections were transmitted experimentally by a filtrate Double-stranded, enveloped Herpesviridae, Iridoviridae,
of secretions from an infected animal using filter pore sizes Poxviridae
small enough to exclude bacteria.1 In the absence of detailed Single/double-stranded, Hepadnaviridae
knowledge of viruses beyond their associated clinical syn- enveloped
dromes, initial schemes of viral categorization grouped human (Reproduced with permission from reference 38, with permission from Lippincott,
viruses by the affected organ or other clinical criteria. Thus, all Williams & Wilkins.) Reproduced from Chodosh J, Stroop WG: Introduction to
viruses associated with hepatitis were considered together. We viruses in ocular disease. In: Tasman W, Jaeger EA, eds. Duane’s foundations of
clinical ophthalmology. Philadelphia: Lippincott Williams & Wilkins; 1998:1–10.
now know that the hepatitis viruses are diverse.
In 1966, the International Committee on Nomenclature of
Viruses (ICNV), later to become the International Committee
on Taxonomy of Viruses (ICTV), began to classify the myriad by -virus. Family members share a characteristic morphology,
of different viruses into groups. In generating a taxonomy of replicate in a similar fashion, and have relatively conserved
viruses, the ICTV considers virus morphology, physical prop- nucleic acid sequences. The recently published VIII report of
erties, nucleic acid type and strandedness, physical state of the the ICTV classified a total of three orders, 73 families, nine
genome, proteins expressed, antigenic properties, and serologic subfamilies, 287 genera and more than 6000 viruses.4 Genbank
cross-reactivity, as well as biologic effects of infection.2 Viruses contains the genomes of an additional 3142 viruses which have
are then classified broadly by the type of nucleic acid, its strand- not yet been classified.5 As more viruses are sequenced, more
edness, and if single-stranded, whether positive- or negative- viruses will be classified, others possibly reclassified, and phylo-
sense, and by the presence or absence of an external lipid bilayer genetic relationships clarified. In the near future, rapid
envelope (Table 16.1). In most cases, classification by ultra- identification of viruses by viral microarray analysis,6,7 followed
structural appearance correlates well. For example, the eight by sequence analysis may reduce or render unnecessary less
human herpes viruses so far identified all have an identical direct and more time-consuming methods of identification and
electron microscopic appearance and a high degree of genomic classification.
homology.3 Viral culture with negative staining transmission
electron microscopy to directly examine virus morphology and VIRAL COMPONENTS
size, and thin section electron microscopy of infected tissues to
directly observe viruses during viral infection remain time- A virion is a single viral infectious unit including nucleic acid,
honored means of identifying previously unknown viruses. capsid, and if present, an external envelope. Viral nucleic acids
By agreement, virus orders are designated by the suffix consist of either RNA or DNA. Viral RNA genome may be
-virales, families by -viridae, subfamilies by -virinae, and genera either single- or double-stranded, and in the case of single- 169
 MICROBIOLOGY
stranded viruses, either positive-sense, with the same polarity
as the viral messenger RNA (mRNA), or negative-sense, with
opposite polarity to the viral mRNA. Furthermore, RNA viral
genomes are either segmented, with discrete nucleic acid mole-
cules, or nonsegmented, with all of the genetic information on
a single nucleic acid molecule. Finally, DNA and RNA genomes
exist in either linear or circular (episomal) form. These
characteristics of nucleic acid structure determine much of the
specific mechanics of viral replication.
The viral capsid is a protein shell that surrounds the viral
nucleic acid. The capsid interacts internally with the genome to
stabilize it, protects the genome from the external environment,
and in the case of nonenveloped viruses, expresses on its surface
the necessary ligand(s) for virus–host cell binding.8 The viral
capsid proteins also assist in delivery of the viral genome to the
intracellular site of viral replication. Thus, viral capsid structure
is integrally related to key viral functions, in particular, trans-
mission, attachment, and entry into host target cells, but also
SECTION 3
virion assembly and egress. The capsid and nucleic acid
together are referred to as the nucleocapsid. Occasionally, as
with herpesviruses, the nucleocapsid is surrounded by an
additional protein layer, the tegument.
Capsid structure is specified by the viral genome, and the
economy of genome size frequently dictates a capsid of repeat-
ing protein subunits. Simplicity further dictates that subunits
interact in symmetrical forms with conserved subunit inter-
actions.9 Common capsid structural motifs include the icosa-
hedron with its 20 plane surfaces and the helix.10 Electron
microscopy and X-ray diffraction crystallography, in conjunc-
tion with nucleic acid and protein sequencing, can delineate the
components of capsid structure at the molecular level.
A viral envelope surrounds the capsid of some virus families.
The envelope consists of viral genome-encoded glycoproteins
and a few host cell proteins embedded in a host cell-derived
lipid bilayer.11 Viral glycoproteins act as ligands for receptors on
host targets, as well as antigens for neutralizing antibodies
directed against the virus. In the initial stages of infection,
envelope glycoproteins mediate attachment of the virus to its
receptor on the host cell surface and fusion of the viral envelope
with the host cell membrane. During viral replication, viral-
encoded glycoproteins are targeted on a molecular level to
specific membranes in the host cell in order to serve as sites of
interaction between the viral nucleocapsid and the host cell
membrane prior to budding. Cell membranes used by enveloped
viruses include the nuclear envelope, endoplasmic reticulum,
Golgi apparatus, and plasma membrane. Polarized epithelial
cells, such as those found at mucosal surfaces, maintain tight
intercellular junctions, and possess biochemically and
morphologically distinct apical and basolateral cell membranes.
Due to differential targeting of viral glycoproteins to apical
versus basolateral membranes, polarized cells typically release
enveloped viruses from either the apical or basolateral cell
surface. Virus shed apically into mucosal secretions such as the
tear film creates the potential for transmission. Virus shed
basolaterally may infect deeper tissues and/or disseminate.12
Because of the lipid component of their viral envelopes, viruses
such as herpes simplex virus or human immunodeficiency virus
are intrinsically vulnerable to damage by ultraviolet light,
detergents, alcohols, and general-use antiseptics. Nonenveloped
viruses such as adenoviruses may be quite resistant to
degradation even under relatively harsh conditions.13
VIRAL RECEPTORS AND VIRAL TROPISM
Viral tropisms for specific cell types and tissues require a ligand
on the viral capsid surface (nonenveloped viruses) or envelope
170 (enveloped viruses) that can bind to a receptor on the target cell.
TABLE 16.2. Selected Ocular Viruses and Their Possible
Receptors
Virus Host Cell Receptor
Adenovirus type 37 CD4625
Epstein–Barr virus CD2115,16
Herpes simplex virus Heparan sulfate26,27
Human cytomegalovirus Heparan sulfate28
Human papillomavirus Integrin a622
Influenza virus Sialic acid29,30
Rhinovirus ICAM-117–19
Vaccinia virus EGF receptor31
Reproduced from Chodosh J, Stroop WG: Introduction to viruses in ocular
disease. In: Tasman W, Jaeger EA, eds. Duane’s foundations of clinical
ophthalmology. Philadelphia: Lippincott Williams & Wilkins; 1998:1–10.
Viral ligands are typically glycoproteins. Host cell virus
receptors are diverse and may be protein, glycoprotein, lipid, or
carbohydrate.14 Although viral ligand–host cell receptor inter-
action is essential for adsorption of the virus to the cell surface,
the ligand receptor complex also often mediates subsequent
internalization of the virus and uncoating of the capsid. The
polarized location of the virus receptor on epithelial tissues with
distinct apical and basolateral cell surfaces, and the changes in
receptor expression during cell differentiation largely determine
tissue susceptibility to infection in vivo. For example, virus
receptor expression only on the basolateral surfaces of less
differentiated epithelial cells would permit infection by virus
presented across an underlying basement membrane, but not by
virus present in mucosal fluids or on undamaged skin.12
Viruses presumably evolved the capacity to bind constitutive
host cell membrane components with essential primary cellular
functions (Table 16.2). Therefore, binding of virus to a cell
surface component subverts the natural function of that cellular
molecule. For example, the B lymphocyte receptor for
Epstein–Barr virus is the C3d complement receptor, CD21.15,16
Rhinoviruses bind to intercellular adhesion molecule-1 (ICAM-
1),17–19 present on nasopharyngeal20 and conjunctival21
epithelial cells. Human papillomavirus (HPV) appears to bind
the a6 component of the a6b4 integrin complex.22 Adenovirus
type 2 utilizes the CAR protein for attachment23 and integrins
for internalization,24 while the corneal pathogen adenovirus
type 37 binds the C3b complement binding protein CD46.25
In classic lytic viral infections, virus replication diverts
cellular protein production machinery for the synthesis of viral
proteins. However, before shutdown of host macromolecular
synthesis, the cell may respond to viral infection by upregu-
lation of specific genes. For instance, binding of cytomegalovirus
to cells in vitro stimulates production of protooncogenes.32
Adenovirus binding stimulates the rapid induction of host cell-
derived proinflammatory cytokines by an intracellular signal
transduction pathway.33–35 The cellular function of each host
cell virus receptor likely influences initial molecular responses
to infection.
VIRAL INFECTION AND REPLICATION
Viruses may infect the human host via the placenta and birth
canal, ingestion of breast milk, inhalation of airborne secre-
tions, contaminated food, by insect bite, inadvertent intra-
vascular injections, or intimate and/or sexual contact. Ocular
infection by viruses most often follows direct contact with virus
 Ocular Virology

externally, either from infected secretions in the birth canal
(herpes simplex virus, HPV), on fomites (adenovirus), or
airborne particles (rhinovirus), or is acquired during viremia
(human cytomegalovirus, measles virus). Other mechanisms
of ocular viral infection include extension from contiguous
adnexal disease (herpes simplex virus), neuronal spread down
trigeminal sensory nerve fibers (herpes simplex virus),36 spread
from the upper respiratory tract via the nasolacrimal duct
(rhinovirus), and transplacental passage of infectious virus
(rubella virus). Rarely, ocular infection may disseminate
elsewhere (enterovirus 70).37 A summary and classification of
viruses infecting the human eye and adnexa is presented in
Table 16.3.
The ultimate objective of infection for a virus, whether latent
or not, is the generation of viral progeny. The synthesis of viral-
encoded proteins is essential to the ability of the virus to
replicate and be transmitted, and largely determines the specific
effects of viral infection on the cell. Although differences exist
between enveloped and nonenveloped viruses in the mechanics
of infection, the replicative cycle of all viruses can be divided
into six stages: (1) attachment, (2) penetration, (3) uncoating,
(4) replication, (5) assembly, and (6) release (Figure. 16.1).

TABLE 16.3. Classification Table of Viruses Affecting the Human Eye

Virus Family Subfamily/Genus Nucleic Acid Envelope Capsid Ocular Target

Herpes simplex Herpesviridae Alphaherpesvirinae/ dsDNA + Icosahedral Eyelid

virus, type 1 Simplexvirus Conjunctiva

CHAPTER 16
(HHV1) Cornea
Trabecular meshwork
Uvea
Retina
Herpes simplex Herpesviridae Alphaherpesvirinae/ dsDNA + Icosahedral Eyelid
virus, type 2 Simplexvirus Conjunctiva
(HHV2) Cornea
Trabecular meshwork
Uvea
Retina
Varicella zoster Herpesviridae Alphaherpesvirinae/ dsDNA + Icosahedral Eyelid
virus (HHV3) Varicellovirus Conjunctiva
Cornea
Trabecular meshwork
Uvea
Retina
Optic nerve
Epstein–Barr Herpesviridae Gammaherpesvirinae/ dsDNA + Icosahedral Lacrimal gland
virus (HHV-4) Lymphocryptovirus Conjunctiva
Cornea
Uvea
Retina
Optic nerve
Human Herpesviridae Betaherpesvirinae/ dsDNA + Icosahedral Retina
cytomegalovirus Cytomegalovirus Optic nerve
(HHV5)
Human herpes Herpesviridae Betaherpesvirinae/ dsDNA + Icosahedral Retina
virus 6 (HHV6) Roseolovirus
Human herpes Herpesviridae Betaherpesvirinae/ dsDNA + Icosahedral ?
virus 7 (HHV7) Roseolovirus
Human herpes Herpesviridae Gammaherpesvirinae dsDNA + Icosahedral Conjunctiva
virus 8 (HHV8) (Kaposi’s sarcoma)
Adenovirus Adenoviridae Mastadenovirus dsDNA – Icosahedral Conjunctiva
Cornea
HPV Papovaviridae Papillomavirus dsDNA – Icosahedral Eyelid
Conjunctiva
Cornea
Smallpox (variola) Poxviridae Orthopoxvirus dsDNA + Complex Eyelid
virus Conjunctiva
Cornea
Uvea
Optic nerve
Vaccinia virus Poxviridae Orthopoxvirus dsDNA + Complex Eyelid
Conjunctiva
Cornea
Molluscum Poxviridae Molluscipoxvirus dsDNA + Complex Eyelid
contagiosum virus Conjunctiva
Cornea

Continued 171
 MICROBIOLOGY

TABLE 16.3. Classification Table of Viruses Affecting the Human Eye—Continued

Virus Family Subfamily/Genus Nucleic Acid Envelope Capsid Ocular Target

Orf virus Poxviridae Parapoxvirus dsDNA + Complex Eyelid

Enterovirus(es) Picornaviridae Enterovirus ssRNA – Icosahedral Conjunctiva
(includes Poliovirus, Cornea
Coxsackievirus,
Echovirus,
Enterovirus)
Rhinovirus Picornaviridae Rhinovirus ssRNA – Icosahedral Conjunctiva
(+)
Rubella virus Togaviridae Rubrivirus ssRNA + Icosahedral Cornea
(+) Uvea
Lens
Trabecular meshwork
Retina
Globe
Alphavirus/Flavivirus Togaviridae Rubrivirus ssRNA + Icosahedral Conjunctiva
SECTION 3

(encephalitis, enceph- (+)

alomyelitis, yellow
fever, dengue viruses)
Influenza virus Orthomyxo- Influenzavirus ssRNA + Helical Lacrimal gland
viridae (A, B, C) (–) Conjunctiva
Episclera
Cornea
Uvea
Retina
Optic nerve
Cranial nerves
Human coronavirus Coronaviridae Coronavirus ssRNA + Helical Conjunctiva
(+)
Newcastle disease Paramyxoviridae Paramyxovirus ssRNA + Helical Conjunctiva
virus (–) Cornea
Parainfluenzavirus(es) Paramyxoviridae Paramyxovirus ssRNA + Helical Conjunctiva
(–)
Respiratory syncitial Paramyxoviridae Pneumovirus ssRNA + Helical Conjunctiva
virus (–)
Mumps virus Paramyxoviridae Paramyxovirus ssRNA + Helical Lacrimal gland
(–) Conjunctiva
Sclera
Cornea
Uvea
Optic nerve
Cranial nerves
Measles (rubeola) Paramyxoviridae Morbillivirus ssRNA + Helical Conjunctiva
virus (–) Cornea
Uvea
Retina
Optic nerve
Cranial nerves
Rift Valley fever Bunyaviridae Bunyavirus ssRNA + Coiled Retina
virus (–)
Colorado tick fever Reoviridae Coltivirus dsRNA – Icosahedral (?: reported to cause
virus (+/–) photophobia,
retroocular pain)
Rabies virus Rhabdoviridae Lyssavirus ssRNA + Helical (Transmission via
(–) corneal button)
Human immuno- Retroviridae Lentivirus ssRNA + Coiled Lacrimal gland
deficiency virus (+) Retina
+, enveloped; –, Nonenveloped; (+), Ppositive sense RNA genome; (–), Nnegative sense RNA genome.
Reproduced from Chodosh J, Stroop WG: Introduction to viruses in ocular disease. In: Tasman W, Jaeger EA, eds. Duane’s foundations of clinical ophthalmology.
Philadelphia: Lippincott Williams & Wilkins; 1998:1–10.

172
 Ocular Virology

FIGURE 16.1 Representations of the stages of

RNA and DNA virus replication. Attachment of
virus to susceptible cells is followed by
DNA viruses RNA viruses penetration and uncoating. Most DNA viruses
undergo transcription, replication, and
assembly in the nucleus. The DNA virus shown
is released from the cell by lysis. Most RNA
Attachment Attachment viruses replicate in the cytoplasm. The dashed
line illustrates the transcription pathway of the
Penetration Penetration negative-sense viruses, and the solid line
indicates the pathway taken by the positive-
sense RNA viruses. The RNA virus shown is
released from the cell by budding through the
plasma membrane.
Adapted from Chodosh J, Stroop WG: Introduction to
viruses in ocular disease. In: Tasman W, Jaeger EA,
Uncoating Uncoating eds. Duane’s foundations of clinical ophthalmology.
Philadelphia: Williams & Wilkins; 1998:1–10.
(–) Sense (+) Sense

CHAPTER 16
Early Transcription Translation
transcription

Translation Replication Translation Proteins

Late
transcription Proteins

Translation Replication

Capsid Translation
proteins
Capsid
Assembly Assembly
proteins

Release Release

Following adsorption to the host cell receptor, penetration
occurs by endocytosis or translocation, or in the case of
enveloped viruses, fusion of the envelope with the host plasma
membrane. Virus capsid components play an active role in
transport of the virus into the cell. Uncoating, or shedding of
capsid components, typically occurs in the cell cytoplasm.
Replication takes place in the nucleus for most DNA viruses
and in the cytoplasm for most RNA viruses. Mechanisms of
viral replication are summarized in Figure 16.2. Assembly of
the virus, the process by which capsid is added to newly
replicated genome, typically occurs in the cytoplasm. Release of
virus from the cell occurs by budding or cell lysis.
Transcription of viral nucleic acid to produce the enzymes
and structural proteins necessary for replication varies with
the type of viral genome. With the exception of the positive-
sense single-stranded picornaviruses, alphaviruses, and flavi-
viruses, it is necessary to first transcribe an mRNA. DNA
viruses that replicate in the cell nucleus utilize cell-derived
polymerases. Otherwise, generation of a viral-encoded RNA
polymerase is required. Lastly, because eukaryotic host cells
do not recognize internal initiation sites within mRNA
molecules, posttranslational modifications of viral proteins by
cellular or viral enzymes are often used to produce the indi-
vidual proteins necessary for replication and maturation of
the virion.
Assembly of infectious virus and subsequent release of virus
from the cell are tightly linked and largely determine the out-
come of infection. The assembly of nonenveloped viruses in the
cell nucleus or cytoplasm typically exposes the cell to capsid
components that may inhibit cell function and cause cell death.
To acquire envelopes, viruses encode proteins for insertion into
host cell membranes that then act as binding targets for
immature virions. Egress of the virus via budding may itself
lead to cell lysis, as with herpesviruses. 173
 MICROBIOLOGY

dsDNA Positive-sense RNA Negative-sense RNA Retrovirus

Early transcription Early transcription Early transcription

+ Strand RNA – Strand RNA tRNA primer

+ Strand RNA

Early mRNAs Translated by

Reverse
host ribosomes
transcription

Early proteins Proteins needed + Strand RNA

( ) initiate for replication
genome synthesis and encapsidation
SECTION 3

Translated by RNA: DNA

Replication Transcription
host ribosomes duplex
+ Strand RNA

Proteins needed
for replication
and encapsidation
Replication

– Strand RNA Replication

+ Strand RNA
dsDNA
Late transcription
provirus

Integration into host DNA

Replication

mRNAs Progeny + strand

genome

Capsid proteins

FIGURE 16.2 Representations of viral transcription and replication strategies. dsDNA viruses: dsDNA virus early mRNAs are transcribed from
separate promoters (two such transcripts are shown). The mRNA is translated in the cytoplasm and the proteins are returned to the nucleus.
Replication involves binding of early-produced transcriptases to the genome; new DNA strands are synthesized by semi-conservative strand
displacement (as illustrated) or discontinuous mechanisms. Late transcription follows DNA replication and involves transcription of mRNAs
encoding structural proteins. Positive-sense RNA viruses: the RNA genome is directly translated by host ribosomes, producing the proteins
needed for replication. Transcription of the nascent positive-sense RNA by genome-encoded RNA-dependent RNA transcriptase produces a
negative-sense RNA, which serves as a template for synthesis of new genomes. Negative-sense RNA viruses: Negative-sense RNA viruses carry
RNA-dependent RNA polymerase in the virus particle, which transcribes the negative-sense genome into positive-sense molecules. These are
translated into the proteins needed for replication and encapsidation. The positive-sense molecules also serve as templates for generation of
new negative-stranded genomes. Retroviruses: Retroviruses carry reverse transcriptase, which converts the single-stranded RNA genome into a
circular, double-stranded DNA proviral molecule. Transcription of the first strand of DNA is initiated at the tRNA primer; circularization of the RNA
allows transcription to proceed along the length of the RNA strand. The genomic RNA is degraded by the RNAse property of reverse
transcriptase, and the second DNA strand is synthesized using the first DNA strand as a template. The fully dsDNA circular molecule integrates
into host chromosomal DNA; host DNA flanking sequences are indicated by the broken lines. Replication involves transcription of mRNAs
encoding viral proteins and transcription of full-length, positive-sense RNA from the integrated provirus.
Adapted from Chodosh J, Stroop WG: Introduction to viruses in ocular disease. In: Tasman W, Jaeger EA, eds. Duane’s foundations of clinical ophthalmology.
Philadelphia: Williams & Wilkins; 1998:1–10.
174

Clinical illness is inadequate as a criterion to assess viral
infection, because viral infection may be subclinical or essen-
tially asymptomatic. Viruses cause disease by a variety of mech-
anisms, including altered cellular metabolism due to viral gene
products, altered host gene expression mediated by interactions
between viral proteins and the host genome, and host immune
response to viral infection of the cell. The end results of viral
infection range from frank destruction of host tissues, disrupted
function on cellular, tissue, organ, and/or systemic levels,
recurrent disease due to intermittent viral expression over time
from latently infected cells, neoplastic transformation, and
immunologically mediated disease.
VIRAL DIAGNOSTICS
Ocular viral infections are often diagnosed on clinical criteria.
However, when atypical, particularly severe, or when a correct
diagnosis will alter subsequent treatment, laboratory investi-
gation may be indicated. Multiple approaches are available
to achieve laboratory confirmation of a specific viral entity, but
confirmation of ocular viral infections depends on the clinician
to obtain specimens at appropriate times during the course
of infection and on the proper specimen handling after
collection.38–40 Commonly used techniques to identify viral
pathogens include viral culture, microscopy, antigen detection,
nucleic acid detection, and serology. Communication between
the physician and laboratory staff regarding the differential
diagnosis generally improve the likelihood of identifying a viral
pathogen.
Viral culture followed by direct or indirect immuno-
flourescent antigen detection remains the gold standard of
virus detection against which all other methods are compared,
although in cases of latent viral infection with intermittent
virus shedding, isolation of a virus may be misleading with
regards to causality of disease.41–43 Skin, conjunctival, or
corneal scrapings, or intraocular fluids (in exceptional cases) are
obtained during the acute phase of infection. In the laboratory,
the inoculum is transferred onto the appropriate cell line for
growth of the virus. The choice of cell type depends on the virus
one wishes to cultivate. Any given cell line is generally capable
of supporting the growth of only a limited range of viruses.
Clinical laboratories typically grow viruses in primary, diploid,
or heteroploid continuous cell lines derived from human
cancers or animals. When the virus is inoculated onto a
susceptible cell line, it produces a characteristic change in the
host cell, termed cytopathic effect (CPE). The specific appear-
ance of CPE varies between virus families and may allow a
presumptive identification of the virus. Although rapidly
growing viruses such as herpes simplex virus can produce a
detectable CPE within a day or two, others, such as CMV,
rubella, and some adenoviruses, can take 1–4 weeks. Once CPE
is evident, the virus is usually identified with direct or indirect
immunofluorescence techniques. When the identity of the virus
is unknown or the CPE is uncharacteristic, morphologic
examination with electron microscopy may be helpful. A
relatively recent innovation in viral cell culture, the shell vial
technique, allows the rapid identification of viruses. Cultures
are centrifuged at low speed for 1–3 days and stained by direct
immunofluorescence for viral antigen, prior to development
of CPE.
Other methods of virus identification include microscopic
examination of scrapings or tissue samples with Giemsa stain,
electron microscopy, or with antigen detection systems using
immunofluorescence or immunoperoxidase. Cytology may in
some circumstances allow the initial, early recognition of viral
infection. Scrapings from clinical lesions (skin or ocular tissues)
Ocular Virology
streaked onto a glass microscope slide, fixed, and subsequently
stained with hematoxylin and eosin, Tzanck, Giemsa, or
Papanicolaou stain and examined by light microscopy, may
show distinctive inclusions that represent abnormal accumu-
lations of host cellular material caused by the virus-induced
disruption of host cell metabolic activity. Cytology may be
helpful in herpes simplex virus, varicella-zoster virus, CMV,
measles, and rabies infections. Multinucleated giant cells and
ballooning cytoplasm may be observed in herpes simplex,
varicella-zoster, and cytomegaloviral infections and are
characteristic of these human herpes virus infections.
Antigen detection requires the technician to know what virus
is suspected. The immunoperoxidase technique is useful in
laboratories without access to a fluorescent microscope.44
Agglutination tests to detect viral antigens are based on visible
agglutination of particles, such as latex, red blood cells, or
polystyrene, to which virus-specific antibody has been adsorbed.
Agglutination methods are easily performed, but few com-
CHAPTER 16
mercial kits have been shown to detect ocular viral pathogens.
Electron microscopy is limited by the need for large quantities
of virus in the specimen, and is relatively insensitive for clinical
specimens. Solid phase immunoassays are rapid, available,
quantifiable, and relatively inexpensive, but have not been
widely adopted for ocular infections. Nucleic acid detection
by hybridization or polymerase chain reaction (PCR) are
promising, in particular those PCR techniques that are able to
detect and differentiate several different viruses in one
experimental run.45 The high sensitivity of PCR is also a
detriment as the technique does not differentiate bystander
viruses, for example herpes simplex shed into the tear film,
from viral pathogens. In situ hybridization on tissue sections for
viral gene expression within pathologic tissue cells then
becomes the gold standard to prove that an abnormal cell is
actually infected with the virus. The same conclusion may be
obtained by immunohistochemistry for viral antigen.
Serologic tests for virus-specific IgG antibody require patient
sera during both the acute and convalescent periods – 2–4
weeks after the onset of clinical disease – at which time the
information may no longer be clinically useful. Increases in
virus-specific IgG may be sufficiently robust to assist in
diagnosis of primary infection and in re-infection, but not
in viral reactivation. On the other hand, serology for virus-
specific IgM is very useful in primary infections. Therefore,
serology for herpes simplex virus might be useful in children
with recent onset of suspicious but not classic skin and/or
corneal lesions, but herpes simplex virus serology is rarely
informative in older adults in whom the prevalence of herpes
simplex virus-specific IgG antibody is high. Unlike IgG, IgM
does not cross the placental barrier. Therefore, in a neonate, the
finding of IgM antibodies indicates infection of the child.46
Intraocular antibody titers can be useful in intraocular
infections when the serology is suggestive of past infection. The
Goldmann–Witmer coefficient compares the ratios of pathogen-
specific antibody to albumin between intraocular fluid and
serum. A ratio of antibody (eye)/albumin (eye) to antibody
(serum)/albumin (serum) of greater than three indicates
intraocular infection with the specific pathogen.47
VIRAL IMMUNOPATHOGENESIS
Virus infections may be suppressed by neutrophils, natural
killer cells, B lymphocyte-derived antibodies, and effector T
lymphocytes.48 Unlike B and T lymphocytes, natural killer cells
can act without antigen specificity or immunologic memory.49
Interferon-stimulated natural killer cells limit the extent of viral
infection early on, before the machinery of acquired antigen-
175
 MICROBIOLOGY
specific immunity has fully engaged. Additionally, activated
natural killer cells attack cells with reduced MHC class 1
expression to counter viral evasion of MHC class 1 presentation
(see further ahead). In the eye, chemokine expression by virus-
infected host cells induces rapid migration of leukocytes into
virus-infected tissues, but may serve to increase local tissue
damage and lead to reduced vision.50
In patients with secondary immunity, virus-specific anti-
bodies can neutralize free virus in blood or mucosal secretions.
They also mediate cell death of infected cells through
complement-mediated killing and by antibody-dependent cell-
mediated cytotoxicity. Viral neutralization by antibody depends
on recognition of viral epitopes present on virus surfaces such
as envelope glycoproteins, or, in the case of nonenveloped virus,
surface capsid proteins. High quantities of virus–antibody
immune complexes in the blood can induce immune complex-
mediated immunopathology at distant tissue sites.
CD8+ cytotoxic T lymphocytes (CTLs) typically recognize
SECTION 3
viral epitopes in the context of MHC class 1 molecules
expressed on the surface of virus-infected cells, and are critical
to the elimination of virus-infected cells. Nucleated cells
express class 1 molecules, and so any virus-infected cell may be
a target for CTLs. Killing occurs through a directional release
of perforin and granzymes. However, stimulation of T cell
immunity may be accompanied by production of tumor
necrosis factor and other cytokines that have deleterious effects
at local and systemic levels.51
Some viruses possess the means to evade the host immune
system. A herpes simplex virus-encoded protein, ICP47,
successfully competes with antigenic viral peptides for transport
into the endoplasmic reticulum where peptides are loaded
onto the MHC complex.52 Thus, herpes simplex virus-infected
cells can be resistant to CTL lysis. Similarly human
cytomegalovirus-encoded US11 dislocates MHC molecules into
the cytosol where they are degraded by cell proteases.53 These
and other examples of the means by which ocular viruses may
evade the immune system are presented in Table 16.4.
Certain viruses produce homologs of human proteins that
can influence host immunity. Epstein–Barr virus encodes a
homolog of human IL-10.61 Expression of viral IL-10 by infected
cells inhibits interferon-g production and T cell immunity, and
results in enhanced survival of virus-infected cells. Human
herpesvirus 8 encodes a structural homolog of IL-6, suggested
to influence the pathogenesis of Kaposi’s sarcoma.62 Human
cytomegalovirus encodes a homolog for a human chemokine
receptor, providing the capacity to divert host cell-derived
chemokines and thereby prevent inflammation and viral
clearance.63,64
Pathogenic roles for viruses in a variety of autoimmune
diseases have been suggested.65–69 Some viruses stimulate
polyclonal B cell activation and lead to excessive deposition of
immune complexes in sensitive tissues. The altered cytokine
milieu associated with viral infection can stimulate autoreactive
T cells, resulting in inadvertent damage to normal tissues.
Inflammation of immunologically sequestered tissues such as
those present in the central nervous system or the eye in the
mature adult could expose previously hidden epitopes and lead
to local hyperimmunologic responsiveness with devastating
functional consequences.70 Finally, shared antigenic determi-
nants between virus and host can lead in genetically susceptible
individuals to immunologic recognition of self epitopes
(molecular mimicry),71 with immune-mediated damage at
distant and ostensibly normal sites.
Viruses that infect the eye or its adnexa tend to produce
stereotypic pathologic changes in target tissues. Infection of the
eyelid skin by viruses typically induces the formation of vesicles
176 and ulceration. Infection of the conjunctiva results in increased
TABLE 16.4. Ocular Viruses and Molecular Means by Which
They Evade Host Immunity
Examples of Immune Escape
Virus Mechanisms
Herpes simplex virus Virus-encoded ICP47 blocks peptide
translocation to MHC class 152
Virus-encoded proteins bind and
neutralize complement components54
Latency in sensory neurons
Human cytomegalovirus Virus-encoded US11 causes cytosolic
degradation of MHC class 1 heavy
chains53
Viral MHC class I homolog inhibits
NK cell attack55
Latency in glandular tissue
Epstein–Barr virus Exclusive EBNA-1 expression in type 1
latency reduces recognition by
cytotoxic T lymphocytes56
Adenovirus type 2 Prevents MHC class I transport to cell
surface57
Protection from TNF-mediated
cytolysis58
Influenzavirus Inhibits cytolysis by interferon59
Antigenic shift and drift
Vaccinia virus Blocks antiviral effect of interferon60,61
Reproduced from Chodosh J, Stroop WG: Introduction to viruses in ocular
disease. In: Tasman W, Jaeger EA, eds. Duane’s foundations of clinical
ophthalmology. Philadelphia: Lippincott Williams & Wilkins; 1998:1–10.
numbers and size of conjunctival lymphoid follicles along with
the enlargement of corresponding draining lymph nodes. Viral
infection of the corneal epithelium invariably causes punctate
epithelial cytopathic effect evident biomicroscopically as
isolated swollen epithelial cells (punctate epithelial keratitis)
and loss of individual epithelial cells (punctate epithelial
erosions). When extensive, the punctate erosions may coalesce
to form confluent epithelial ulcers (dendritic, dendritiform, and
geographic ulcers).72 Corneal stromal infection results in white
blood cell recruitment to the site of infection,73 with resultant
stromal infiltration. Retinal infection leads to retinal necrosis.
Viral encephalitis, encephalomyelitis, and meningitis may lead
to cranial nerve inflammation and dysfunction of vision and/or
extraocular motility.
LATENCY, CARCINOGENESIS, LOSS OF
FUNCTION
Although some virus species may cause self-limited infections
with complete clearance of the virus, others can persist
indefinitely in the host.74,75 For example, adenoviruses persist
within nasopharyngeal lymphoid tissue, Epstein–Barr virus in
nasopharyngeal epithelial cells and B lymphocytes, herpes
simplex and varicella zoster viruses in sensory ganglia, and
HPV in skin and mucosal epithelia. An infection is said to be
latent when persistent but not currently productive of infectious
virus. In latent infections, only limited viral gene expression
occurs, and the immune system’s response to the few gene
products of the latent virus is absent or altered.76 Interestingly,
latent infection frequently occurs in cell types poorly permissive
for lytic infection by the virus, such as with herpes simplex
virus and sensory neurons, Epstein–Barr virus and B lym-
phocytes, and HPV and basal skin epithelial cells. Latent
infections also tend to occur in slow-cycling cells. Persistent
infections may consistently or intermittently produce infectious
 Ocular Virology

virus in tears or at skin and mucosal surfaces. When virus is
produced in what had been a latent infection, the infection is
said to be reactivated.
Persistent viral infection of susceptible cells can lead to
malignant transformation. Viral proteins, whether directly
through interaction with the host genome or by interaction
with cellular proteins, can induce transformation of the cell and
loss of senescence. HPV-induced squamous cell carcinoma is an
elegant example of tumor induction by viruses.77–79 HPV
tropisms for skin and mucosa derive in part from tissue-specific
gene expression.80 HPV types 6 and 11 are maintained in a
latent state within basal epithelial cells as circular episomes
with very limited viral gene transcription and low copy number.
Early viral gene products stimulate cell growth and lead to a
skin wart or a conjunctival papilloma. As HPV-containing basal
epithelial cells mature and differentiate into superficial
epithelial cells, they become permissive for complete viral gene
expression and produce infectious virus. Carcinomatous
from its complex with cellular E2F transcription factor. E2F
then activates transcription of genes that initiate the cell cycle.
Hence, increased cellular levels of E6 and E7 proteins contribute
to the malignant phenotype of HPV-16- and HPV-18-infected
squamous epithelium.
The pathologic consequences of viral infection depend on a
complicated array of factors. The presence of viral receptors
on host cells at a surface exposed to infectious virus, the
permissiveness of the cell to viral gene expression, the capacity
of the host to eliminate the virus as balanced by the damage to
host tissue due to the immune response, and finally the fine
function of the host cell and its tissue, all determine the
functional and anatomic derangements associated with viral
infection. For a virus like herpes simplex, tropic for almost all
ocular tissues, the morbidity of ocular infection varies with the
tissue infected. Herpes simplex virus infection of the
conjunctiva is self-limited and leaves no visual deficit, while
infection of the corneal stroma may result in varying degrees of

transformation due to HPV-6 or HPV-11 is very rare. In
contrast, HPV-16 and HPV-18 stereotypically integrate their
viral genome into host chromosomal DNA, and this in turn has
been strongly associated with malignant transformation and
squamous cell carcinoma. In the episomal state, transcription
of HPV protooncogenes E6 and E7 is effectively repressed by the
HPV E2 gene product. When HPV genome integrates into host
cell chromosomal DNA, the circular (episomal) viral DNA
molecule breaks at a recombination site within the E2 open
reading frame, resulting in a truncated E2 protein, and
disinhibition of E6 and E7 transcription. The E6 protein binds
to and initiates the degradation of the cellular p53 tumor
suppressor gene product. The E7 protein displaces cellular pRB
CHAPTER 16
vision loss, and infection of the retina may result in complete
loss of useful vision. In contrast, HPV ocular tropism is limited
to the conjunctiva, limbus, and eyelid skin. Blinding sequelae of
HPV infection occur with malignant transformation of infected
tissues. As classification of viruses proceeds on a molecular
genetic level, the mechanisms by which viruses infect ocular
cells, destroy critical ocular structures, evade the immune
system, and induce cancer may be better understood.
ACKNOWLEDGMENT
The author wishes to acknowledge Thomas J Liesegang, MD for his work
as author of this chapter in the previous (second) edition of this book.

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 SECTION 4 PHARMACOLOGY AND TOXICOLOGY
Edited by Mark B. Abelson

CHAPTER

17 Ocular Pharmacokinetics
Denise K. Chun, Aron Shapiro, and Mark B. Abelson

INTRODUCTION DOSING FREQUENCY

Getting a particular drug to a receptor often requires that it be The frequency with which drugs are administered is typically
administered at a site that is remote from the target, such as by governed by how rapidly the drugs are removed by metabolism
injection into the blood stream or via oral administration. Oph- or clearance of the unmetabolized drug. The loss of a drug is
thalmic delivery for diseases of the frontal eye area is unique in described by the half-life of the drug, i.e., the time for the tissue
that medication in the form of an eye drop can be delivered concentration to fall to one-half of its value. Dosing frequency
directly to the diseased tissue. However, diseases of the pos- is commonly every half-life so that a series of peaks and valleys
terior areas of the eye remain challenging therapeutic targets. is established (Fig. 17.1). One factor that affects a drug’s half-
The administered drug must diffuse across several tissues life, and thus the dosing frequency, is the affinity the drug has
(absorption), distribute into a variety of tissues and fluids (dis- to its receptor. Drug receptor affinity can be taken as the inverse
tribution), be subject to a wide array of metabolizing enzymes of the dissociation rate constant, and the half-life of a drug can
(metabolism), and then be eliminated from the area (elimination). be easily calculated from the dissociation rate constant.
The study of these processes makes up the field of pharmaco- Timoptic (timolol maleate ophthalmic solution), used in the
kinetics, which is essential for choosing the appropriate design, treatment of ocular hypertension or open-angle glaucoma, is an
delivery system, and dosing regimen for any therapeutic agent. example of a topically administered drug in which dosing fre-
Pharmacokinetics describes the quantitative relationship between quency can be decreased after the start of treatment from twice
the administered dose and dosing regimen and the observed a day (bid) to once a day (qd), if intraocular pressure (IOP) is shown
plasma and/or tissue concentration of the drug as a function of to be maintained at satisfactory levels. Similarly, azithromycin,
time. Pharmacodynamics can be defined as the quantitative a topical antibiotic in development for the treatment of bacterial
relationship between the observed plasma and/or tissue con- conjunctivitis, is under evaluation for dosing regimen of bid on
centration, of the active form of the drug and the pharmacologic days 1–2 and qd days 3–5.
effect or biologic response.1,2 These terms are more loosely
described as what the body does to the drug (pharmacokinetics)
and what the drug does to the body (pharmacodynamics).3 TIME TO REACH STEADY STATE
There are unique limitations to classic pharmacokinetic Figure 17.1 shows that the tissue drug level rises because each
approaches when designing and evaluating ocular drug therapies. subsequent dose adds to the quantity of drug left from the earlier
Numerous factors that affect the bioavailability (i.e., how much dose. Steady state is achieved at a specific dosing rate when the
drug was instilled or injected versus how much actually got in) of tissue concentration equals the rate of elimination. The half-life
ocular drugs are based on constraints imposed by the anatomy of
the eye, as well as by formulations of the drug itself. Nevertheless,
there has been significant progress in novel topical and vitreo-
retinal ophthalmic pharmaceuticals during the last couple of
decades.4–10 Many products have been or are being developed in
this area, including suspensions, ointments, gels, newly formulated
solutions, intravitreal, subconjunctival, and intravenous inject-
ables, liposomes, micro- and nanoparticles, iontophoretic systems,
mucoadhesives, and erodible and nonerodible inserts. One of the
most important tools for developing and assessing these products
is an accurate pharmacokinetic model. The primary objective of a
pharmacokinetic model must be to enhance the accuracy of esti-
mates of the dynamic state of drug behavior in an actual clinical
situation.11 Many pharmacokinetic models have been reported
in the literature and represent varied levels of sophistication.
Several excellent reviews on this subject are available.12–17

CLINICAL UTILITY
Following is a brief explanation of the applications of pharma- FIGURE 17.1. Hypothetical tissue drug concentration versus time for
cokinetics to clinical practice. multiple doses of the same drug at set time intervals. 179
 PHARMACOLOGY AND TOXICOLOGY
FIGURE 17.2. Plot of hypothetical tissue drug concentration versus
time for multiple doses of the same drug given at time intervals either
before or after the half-life of the drug. - - - - before the half-life;
— after the half-life.
of a drug determines the time to reach steady state. In most
SECTION 4
cases, the time it takes to reach steady state is about four to five
times the half-life of a drug.
STEADY-STATE MAXIMUMS AND STEADY-
STATE MINIMUMS
Suppose the dosing interval is much earlier, or later, than the
half-life of the drug. Figure 17.2 gives a few hypothetical
examples of the impact of dosing interval. It is easy to see that
dosing too soon can push the drug into the toxic range, while
dosing too late can give periods of time when the levels are
subtherapeutic. The importance of the dosing interval cannot
be underestimated in achieving therapeutic effectiveness and
minimal toxicity.
METHODS OF DRUG APPLICATION
The eye is an extraordinarily protected organ that excludes foreign
chemicals, such as drugs, through a variety of mechanisms.
Understanding the various loss pathways of a topically applied
drug can ensure that therapy is maximized and both local and
systemic toxicity minimized. These loss pathways and potential
remedies are discussed in detail later in this chapter.
PHARMACOKINETIC PARAMETERS
A profile of drug concentration in ocular tissue can be dissected
to provide important information. Figure 17.3 shows a typical
profile.
Cmax The maximal concentration of drug in the tissue is
Cmax. The level that is reached dictates therapeutic and
toxic responses and is directly related to the applied
drug concentration and the absorption and elimination
rate constants.
Tmax The time to reach a maximal level of drug in the
tissue is Tmax. This parameter is a function of only the
absorption and elimination rate constants and is
independent of the applied concentration.
180
FIGURE 17.3. Typical profile of drug concentration versus time in an
ocular tissue. Cmax, maximal level of drug in tissue; Tmax, time to reach
maximal level of drug in tissue.
Area under the curve (AUC) The AUC yields the total
amount of drug absorbed from an applied dose. The
bioavailability of a drug is computed from the AUC,
which is most important in determining therapy for
chronic medication.
PHARMACOKINETIC METHODS
Key Features
• In the eye, it is difficult to clearly define classic
pharmacokinetics parameters such as clearance and volume
of distribution, as well as rate and extent of absorption
• Human pharmacokinetics studies are limited to noninvasive
means of data collection
• Pharmacodynamic, instead of pharmacokinetic measurements,
can be taken with the caveat of patient variability with such
responses
• The use of animal models, especially rabbits, is integral for
testing ophthalmic drugs
HUMANS
Unlike systemic pharmacokinetic studies where the absorption,
distribution, metabolism, and elimination rate constants can
easily be calculated from measurements of drug concentration
in blood samples, human ocular pharmacokinetic studies are
limited to noninvasive observation of fluorescence or gamma-
scintigraphic probes, as well as determination of drug concen-
trations from the aqueous humor during cataract surgery, or
from explanted tissues, conjunctival biopsies, or enucleation.
Attempts are being made to find new methods for evaluating
ocular pharmacokinetics with the hopes of eventual application
to humans. For example, magnetic resonance imaging (MRI)
has been used to study the real-time release of a drug surrogate
(Gd-DTPA) from a polymer-based intravitreal implant in
rabbits.18 Also, cell culture models of ocular tissues are being
developed in order to test drug transport into the tissues, and
for potential toxicological screening of compounds.19
As an alternative to direct pharmacokinetic measurements,
ophthalmic pharmacodynamic responses such as miosis and
mydriasis,20,21 light reflex inhibition,22–27 and IOP have been
 Ocular Pharmacokinetics

used as parameters for investigating the effectiveness of ocular
drug administration. One caveat on using pharmacodynamic
measurements in designing ophthalmic drugs is that the same
dose often produces a different magnitude of effect in various
individuals. Some of the factors that can contribute to this varia-
tion include eye pigmentation, whether or not the individual
wears contact lenses, patient compliance, the clinical state of
the eye (i.e., age of the individual and disease status), and such
physiological factors as the volume and turnover rate for both
tears and the aqueous humor.
The limitations of performing human pharmacokinetic
studies have led to the widespread use of animal models for
ocular studies.
ANIMAL MODELS
Rabbit Model
Because many anatomic and physiologic factors of the rabbit
and human eye are similar (Table 17.1) and because the animal
is relatively inexpensive, easy to handle, and has a larger eye
compared to other animals making it easier to perform clinical
studies, the rabbit is the animal model of choice in most ocular
experiments. In order to determine starting doses of ophthalmic
drugs for human preclinical studies, topical therapeutics should
be normalized to concentration (e.g., mg/area of application) or
amount of drug (mg) at the application site. Intraocular thera-
peutics should be normalized between rabbits and humans
according to the compartmental volumes and concentrations of
As useful as the rabbit model is, there are some differences
between the rabbit and human eye that can affect drug kinetics.
For example, the blink rate in humans (6–15 times/min) is higher
than in rabbits (4–5 times/h), which could allow the penetration
of drug through the cornea of the rabbits more than that of
humans because of a high drug concentration at the corneal
surface28,29 and low drug solution drainage (e.g., in the New
Zealand albino rabbit eye).30 Rabbits have a nictitating mem-
brane that humans do not possess, which may absorb many
substances and act as a depot, affecting pharmacokinetic mea-
surements. Although the albino has been used for most studies,
the absence of pigment will lead to differences in the
pharmacokinetics compared to the human eye. Consideration
should be given to using pigmented rabbits, especially for
drugs that work inside the eye. Moreover, rabbits appear to be
less sensitive than humans to moderate increases of vehicle
viscosity. For example, a suspension-type paraffin ointment
gives better results in humans than rabbits, probably because
shear effects facilitate drug release.20 Therefore, clinical trials in
humans must always be used to confirm data from rabbits.
Other Animals
Other animals besides rabbits are also used in ocular pharma-
cokinetic studies, but to a much lesser degree due to various
reasons. The eyes of rats and mice are too small for testing of
different delivery systems. Dogs, cats, tree shrews, and monkeys
are also used, but for ethical reasons, should only be used for
invasive ocular pharmacokinetic studies when necessary, and are

CHAPTER 17
the drug, as opposed to normalization between species based on thus in general practice limited to noninvasive kinetic measure-
body surface area (mg/m2) as typically done with systemically ments and pharmacodynamics.
administered drugs.27a

TABLE 17.1. Comparison of Pharmacokinetic Factors between Rabbit and Human Eye

Pharmacokinetic Factors Rabbit Human

Tear volume (mL) 5–10 7–30*

Tear turnover rate (mL/min) 0.5–0.8 0.5–2.2
†
Spontaneous blinking rate 4–5 times/h 6–15 times/min
Lacrimal punctum or puncta 1 2
Nictitating membrane‡ Present Absent
pH of lacrimal fluids 7.3–7.7 7.3–7.7
Turnover rate of lacrimal fluids (%/min) 7 16
Buffering capacity of lacrimal fluids Poor Poor
Milliosmolarity of tear (mOsm/L) 305 305
Initial drainage rate constant (/min) 0.55 1.6
Corneal thickness (mm) 0.35–0.45 0.52–0.54
Corneal diameter (mm) 15 11–12
2
Corneal surface area (cm ) 1.5–2.0 1.04
pH of aqueous humor 8.2 7.1–7.3
Aqueous humor volume (mL) 0.25–0.3 0.1–0.25
Aqueous humor turnover rate (mL/min) 3–4.7 2–3
Protein content of tears (%) 0.5 0.7
Protein content of aqueous humor (mg/mL) 0.55 30
Ratio of conjunctival surface to corneal surface 9 17
*Range depends on blinking rate and conjunctival sac volume.
†
Occurs during normal waking hours without apparent external stimuli.
‡
Significance of nictitating membrane from precorneal area is small relative to overall loss rate.
Data from references 4, 11, 13–15, 17, 20, 28, 30, 54, 55, and 136–141.
181
 PHARMACOLOGY AND TOXICOLOGY
PHARMACOKINETICS MODELS
Key Features
• The pharmacokinetics of topically applied ophthalmic drugs is
usually analyzed using compartmental modeling
• In multicompartmental modeling, the eye is divided into
kinetically homologous compartments divided by barriers that
do not necessarily correlate with anatomical compartments
• The pharmacokinetics of a drug following intravitreal injection
has been modeled based on Fick’s second law of diffusion and
assuming three major pathways for elimination
• Various models have been made in order to describe the
kinetics of drugs delivered by different types of systems,
including controlled-release devices and nanoparticle
preparations
A typical example of an aqueous humor drug concentration pro-
file for a topically applied drug such as pilocarpine is shown in
Figure 17.4. There are several important characteristics of this
figure. First, the drug disappears from the aqueous humor in
discrete steps and, in fact, the disappearance is triphasic. This
probably represents the distribution of the drug into various
anterior segment tissues that become reservoirs of the drug.
Over time, the loss of drug occurs with successively smaller
elimination rate constants. Second, the drug achieves Cmax in a
relatively short period of time – 20–40 min is typical – giving
the impression that the drug is rapidly absorbed across the
SECTION 4
cornea. In fact, the drug is typically not rapidly absorbed across
the cornea, and the early peak drug level is due to an unusual
constraint imposed by the kinetics of drug loss from the
precorneal pocket.
ONE- AND TWO-COMPARTMENT MODELS
The simplest pharmacokinetic model is to consider the eye as
one compartment (Fig. 17.5a).31,32 The equation describing drug
concentration in this model is dependent on absorption and
elimination rate constants. For systemically administered
drugs, absorption is generally the faster process, but for most
FIGURE 17.4. Aqueous humor concentration of pilocarpine versus
182 time profile after institution of 25 µL of 1 µ 10–2 M solution.
kabs kelim
a
kabs kelim
b
kloss
FIGURE 17.5. Schematic of two-compartment model without (a) and
with (b) the precorneal loss constant. kloss, precorneal loss constant;
kabs, absorption rate constant; kelim, elimination rate constant.
ophthalmic drugs, the true rate constant for absorption into the
eye is much smaller than the elimination rate constant, result-
ing in a scheme known as the ‘flip-flop’ pharmacokinetic model.
However, this is an oversimplification, and a scheme known as
a parallel elimination pathway more accurately describes the
pharmacokinetics of ocular therapeutics. In actuality there are
many factors that contribute to precorneal kloss. Thus, all rate
constants describing loss of the instilled dose from the tear film
are added together and the sum of these constants yields an
overall loss rate constant (kloss), and produces an apparent absorp-
tion rate constant (kabs) that is larger than the elimination rate
constant. In this model, the apparent absorption rate constant
kabs is described as:
Apparent kabs = kloss + true kabs
The magnitude of kloss is typically in the range of 0.5 min–1,
whereas true kabs is two to three magnitudes smaller. Therefore,
most topically applied drugs show an early peak drug level, and
the time of this peak level is essentially independent of proper-
ties of drug. Figure 17.5b shows the nature of the model taking
into account precorneal loss, and represents a two-compartment
model. These data suggest that to significantly improve ocular
drug bioavailability, it is necessary to make the kloss term smaller
by one to two orders of magnitude (for example, by using gels or
inserts to decrease loss through drainage) or to increase the true
kabs by one to two orders of magnitude (for example, by the
addition of penetration enhancers).
MULTICOMPARTMENT MODELS
A much more complicated model is needed to adequately describe
the pathway from precorneal application through the cornea and
into the aqueous humor followed by distribution into the surround-
ing tissues. The four-compartment model shown in Figure 17.6
was used to fit the drug concentration data for both cornea and
aqueous humor obtained after topical administration of pilo-
carpine to the albino rabbit eye. A mathematical derivation of this
pharmacokinetic model was also reported.32 However, the model
treated the cornea as a simple semipermeable membrane. In fact,
the cornea consists of an epithelium, stroma, and endothelium.
The results from pharmacokinetic studies demonstrated that
the lipophilic epithelium acts as a barrier to drug penetration by
hydrophilic drugs such as pilocarpine. Movement of water-
soluble drugs through the hydrophilic stroma is usually rapid.
Therefore, the corneal stroma and endothelium are kinetically
homogeneous with the aqueous humor. A four-compartment
model that treats the cornea as three separate tissues corrects
this deficiency (Fig. 17.6b and Table 17.2).31,32 In order to more
 Ocular Pharmacokinetics

kloss

kCabs

kahabs kcdist
kRdist

kahdist
ktarget
a b C

FIGURE 17.6. (a) Schematic of simplified four-compartment model. (b) Schematic of four-compartment model that considers corneal
stroma–endothelium and aqueous humor as one compartment. (c) Schematic of five-compartment model that considers the corneal
stroma–endothelium and aqueous humor as separate compartments. For description of parameters see Table 17.2.

TABLE 17.2. Parameters of Models Described in Figure 17.6

CHAPTER 17
Parameter Coefficient Associated With

kloss Elimination rate constant from the precorneal area

c ah
k abs, k abs Apparent absorption rate constants into the cornea and aqueous humor, respectively
c r ah
k dist, k dist, k dist Distribution rate constants into the cornea, reservoir, and aqueous humor, respectively
ktarget Absorption rate constant into the target area
Pp Transfer of drug between precorneal area and corneal epithelium
Pn Nonproductive loss
kd Drainage
QT(t) Tear flow
Pa 1. Transfer of drug between corneal epithelium and corneal stroma–endothelium–aqueous humor
2. Transfer of drug between corneal epithelium and corneal stroma–endothelium
Pm Drug loss via metabolism in or lateral diffusion from corneal epithelium
Pao Drug elimination from aqueous humor
Pr 1. Transfer of drug between corneal stroma–endothelium–aqueous humor and reservoir
2. Transfer of drug between aqueous humor and reservoir
Pro Drug elimination from reservoir
Ps Transfer of drug between corneal stroma–endothelium and aqueous humor
Pso Drug elimination from corneal stroma–endothelium

accurately model the pharmacokinetics of lipophilic drugs, such
as fluorometholone, the corneal stroma–endothelium and aqueous
humor are logically separated33 in a five-compartment model
(Fig. 17.6c and Table 17.2).
INTRAVITREAL INJECTION
Drugs that are introduced into the vitreous humor by intravitreal
injection spread through the vitreous humor and into the ante-
rior chamber at the same rate that they diffuse in free solution.13
Two pathways of exit from the vitreous chamber have been
predicted: (1) through the anterior hyaloid membrane into the
posterior chamber and out of the eye with aqueous drainage and
(2) directly across the retinal surface.
In one study, computer simulation was used to evaluate the
in vivo and in vitro pharmacokinetic correlation of dexametha-
sone sodium after intravitreal injection of m-sulfobenzoate in
rabbits.34 The mathematical model was developed based on
Fick’s second law of diffusion by assuming that the vitreous
body is a cylinder with three major pathways for elimination:
the posterior aqueous chamber, the retinal–choroid–scleral
(RCS) membrane, and the lens (Fig. 17.7). Results showed that 183
 PHARMACOLOGY AND TOXICOLOGY
FIGURE 17.7. Cylindrical model of the vitreous body of rabbits for
analyzing the pharmacokinetics of intravitreal drug delivery; the
surface of the vitreous body is divided into three areas of elimination
pathways: the posterior chamber, the RCS membrane, and the lens.
RCS, retina/choroid/sclera; H, effective height of vitreous body; R, b0,
effective radius of vitreous body and lens, respectively; x, y, horizontal
and vertical axes, respectively.
SECTION 4
the major route of elimination of the drug was through the pos-
terior aqueous humor because of an absence of barrier mem-
brane between the boundaries. By using the ratio of the product
of the diffusion coefficient and the effective area of the posterior
chamber, the RCS membrane, and the lens (50:4:0.1), the
authors concluded that after intravitreal injection, most hydro-
philic drugs are eliminated by the annular gap between the lens
and the ciliary body (i.e., from the posterior chamber and flow
into the anterior chamber), and the RCS membrane may act as
a major route of elimination of lipophilic drugs.
A recent ocular model, also based upon Fick’s second law of
diffusion, assumes a spherical, modified cylindrical eye, and can
predict the time course of the local tissue concentration in the
eye following a variety of ocular drug delivery systems including
topical, systemic, and transdermal administration as well as
vitreous injection and implantable delivery.35
MODELS DERIVED FROM DRUG DELIVERY
The five-compartment model (Fig. 17.8) was developed to study
the mechanism involved in transcorneal permeation of drugs
from delivery devices.36 The model consists of the tear film,
epithelium, stroma, endothelium, and aqueous humor, which
were assumed to be perfectly mixed and adequately represented
FIGURE 17.8. Schematic of five-compartment model that was
184 developed for drug delivery devices.
by plane sheet barriers of known physical thickness with con-
stant surface area. In this model, four routes of drug loss –
lacrimal drainage, conjunctival absorption, aqueous drainage,
and iris–ciliary body absorption – were included. The model was
validated by using the experimental in vivo data compared with
predicted aqueous humor drug concentrations from the model.
The results showed an excellent correlation, and it was also
possible to predict the amount of drug lost through each of the
four elimination pathways. This model was modified by adding
the compartments for the conjunctiva and the iris–ciliary body
in order to compare pharmacokinetic differences between ocular
inserts placed under the eyelid in the conjunctival fornix and
eye drops of timolol.37 The investigators observed increased
absorption into the iris–ciliary body and aqueous humor for
ocular inserts, and this is thought to partially be the result of
increased drug penetration across the conjunctiva and sclera.
Grass and Lee38 described and developed methods for con-
structing a pharmacokinetic model that can be used to predict
the effect of increasing drug retention in the conjunctival sac,
and varying the rate of release of the drug from a controlled drug
delivery device, on the ratio of drug concentration in aqueous
humor and plasma after topical dosing in rabbits. In addition, a
computer model was recently developed to describe the three-
dimensional convective–diffusive transport of drug released
from an intravitreal controlled release source.39
A multicompartment model was constructed to describe oph-
thalmic drug delivery with nanoparticle preparations.40 This
model was constructed from data that suggested that nanopar-
ticle preparations might be able to create a precorneal depot,41
thus enhancing drug penetration directly to its site of action,
the trabecular meshwork,40 through the scleral or noncorneal
pathway.42,43
FACTORS INFLUENCING BIOAVAILABILITY
Key Features
• Due to a number of anatomical and biological factors that exist
to protect the eye, the intraocular bioavailability of topically
administered medications is typically only 1–10%
• Smaller may be better: A smaller instilled eye drop may result
in decreased blinking, increased retention time, and greater
absorption
• A large portion of a topically instilled drop results in
nasolacrimal drainage and systemic absorption, which may
lead to adverse side effects
• The cornea is a potent barrier to drug absorption due to its
small surface area and its low permeability to both lipophilic
and hydrophilic drugs
• Some aspects of drug formulation that affect bioavailability
include hydrophilicity/lipophilicity, concentration, osmoticity,
pH, and viscosity
TOPICAL DELIVERY
There are several possible absorption pathways of a topically
delivered ophthalmic drug (Fig. 17.9). The primary ocular
absorption pathway for small lipophilic drugs is from the tear
film to ocular tissues, via the cornea and the aqueous humor.
After absorption into ocular tissues and systemic circulation,
the drug is eventually eliminated from the body. A substantial
portion of topically applied drug is lost due to drainage, and the
reduced amount of drug reaching systemic circulation because
of drainage is an important consideration in the dosage and
delivery of ocular drugs, as it is a major contributor of adverse
effects.

FIGURE 17.9. Typical profile of the fate of a topically applied drug.
Another important absorption pathway is through the con-
junctiva, the vascularized thin mucous membrane lining the
inside of the eyelids and anterior sclera. The conjunctiva has a
much larger surface area and greater permeability to water-
soluble compounds than the cornea. In fact, the conjunctiva
competes so effectively with the cornea for drug absorption that
it has been calculated that the conjunctiva is as important as
solution drainage loss in reducing the fraction of pilocarpine
available for corneal absorption.44
There is evidence that the absorption of ophthalmic drugs
into the sclera represents a significant pathway for large, hydro-
philic drugs. Ahmed and associates45 tested the scleral absorp-
tion of the lipophilic drugs propranolol, timolol, nadolol, and
penbutolol, and the hydrophilic compounds sucrose and inulin.
The results showed that resistance to penetration for all com-
pounds tested in the outer layer of the sclera is much less than
that of the corneal epithelium. The cornea offered substantially
more resistance to inulin (a hydrophilic drug) than did the sclera.46
However, the cornea and conjunctiva offered comparable resist-
ance against timolol (a lipophilic drug).45 In addition, Schoenwald
and co-workers47 have shown that the conjunctival–scleral
route of entry produced higher iris–ciliary body concentrations
of methazolamide analogs and 6-carboxyfluorescein, but not of
rhodamine B (a lipophilic dye). The explanation of this phe-
nomenon is that a hydrophilic drug is absorbed into the ciliary
body through vessel uptake into the sclera and deposits within
the ciliary body, whereas a lipophilic drug penetrates across the
cornea and diffuses through the pupil against aqueous flow to
enter the posterior chamber.48
Most ophthalmic medications are administered topically, a
route of delivery that has major advantages including localized
drug effects, avoidance of hepatic first pass metabolism, and con-
venience. In fact, it has been shown that topically administered
allergy drugs have greater efficacy in relieving symptoms as
compared to systemically or nasally administered drugs.49,50 On
the other hand, topically administered ocular drugs have the
disadvantage of low bioavailability to intraocular tissues due to
a number of anatomical and biological factors that exist to protect
the eye, and by consequence, the entry of ocular therapeutics. In
fact, it is estimated that the intraocular bioavailability of topi-
cally administered medications is typically only 1–10%.6,51 A
great deal of research has been dedicated to improving ocular
Ocular Pharmacokinetics
bioavailability. The two main approaches have been to alter or
supplement ophthalmic drug formulations in order to increase
absorption, or to improve upon or design new delivery systems.
The following is by no means a complete list of the recent
research in this field, but it will serve to highlight some of the
major areas of focus.
PREOCULAR RETENTION
One factor that influences ocular bioavailability after topical
delivery is the retention of the therapeutic in the preocular area.
The volume of liquid that the conjunctival sac can contain is
~20–30 mL52 and the volume of the tear film is 7 ± 2 mL.53 Due
to physical limitations of eye drop size when delivered from a
standard dropper, however, most bottles deliver 30–50 mL
instead of the ideal drop size of 10–20 mL.52 The delivery of this
larger volume causes reflex blinking, which increases the
drainage rate to the nasolacrimal canal, spilling on the cheeks
and splashing the excess of the solution to the eyelashes.54,55
This results in both wasted amounts of medication and possible
negative side effects due to high systemic absorption. It has been
found that a 50 mL drop has the same pharmacological activity
as a 20 mL drop of pilocarpine,56 and in fact, it has been pro-
posed that reducing the volume of the instilled drop of a drug
with low corneal permeability increases its bioavailability by
four times.57 Thus it appears as though a smaller instilled drop
may result in decreased blinking, increased retention time, and
thus greater absorption. The physical blockage of the lacrimal
CHAPTER 17
drainage system by punctal occlusion has also been studied as a
means for increasing ocular drug retention, but currently it is
unclear as to whether silicone punctal plugs provide any addi-
tional therapeutic benefit for topical antiglaucoma medica-
tions.58,59 A less invasive method of punctal occlusion is to press
down with a finger over the tear duct after administering eye
drops. This technique has been shown to improve efficacy and
results in safer usage of several antiglaucoma medications.60 A
pathological obstruction of the nasolacrimal duct may also
similarly alter bioavailability.
Tear film breakup also improves the absorption of topically
administered ophthalmic drugs. The tear film is a complex fluid
that covers the ocular surface, and functions to protect and
maintain the surface of the eye. The tear film consists of three
layers, a lipid, aqueous, and mucous layer. The tear film struc-
ture remains intact for a certain period of time before it begins
to break apart or rupture, exposing the ocular surface, at which
point, blinking is necessary to replenish this complex fluid. The
barrier function of the tear film makes it difficult for an ophthal-
mic agent to be effective by restricting the product’s interaction
with target receptors of the ocular surface. Additionally, the tear
film composition is responsible for diluting ophthalmic agents,
resulting in further reduced efficacy. Abelson and associates
have found that by having patients refrain from blinking, and
thus allowing tear film breakup to occur, for 6 s before drop
instillation, the efficacy of pilocarpine (1%) and tropicamide
(1%) in constricting and dilating the pupil, respectively, is
significantly improved.61
SYSTEMIC ABSORPTION
It has been calculated that, of an eye drop ~50 mL in volume,
~20 mL or 40% does not touch the cornea but goes directly to
the highly vascular drainage apparatus.16 The excess volume
from the standard eye drop is rapidly pumped by the lacrimal
puncta, passes into the lacrimal canaliculi, then successively
travels into the lacrimal sac, the nasolacrimal duct and finally
the nasal cavity. In the nasal cavity, the active ingredients are
absorbed by mucosal vessels and distributed into the general 185
 PHARMACOLOGY AND TOXICOLOGY
circulation. There are several examples of severe adverse sys-
temic effects correlated with topically administered ophthalmic
drugs. Used in the treatment of glaucoma, b-adrenoceptor anta-
gonists, or b-blockers, are the most frequent cause of systemic
adverse effects due to ophthalmic treatments. These topical
antiglaucoma treatments are known to be associated with pul-
monary, cardiac, and central nervous system effects. A body of
research is devoted to determining if cardioselective b-blockers
(such as betaxolol) have fewer or less severe systemic effects
than nonselective b-blockers.62 There have also been reports of
cardiac irregularities such as palpitations and tachycardia occur-
ring after topical ocular epinephrine administration, which is
used in the treatment of glaucoma and ocular hypertension.
Mydriatic drops, such as phenylephrine, applied either as a treat-
ment or as a diagnostic tool can also have potential adverse effects,
including severe cardiovascular and neurological disorders.63,64
CORNEAL ABSORPTION
The cornea is a potent barrier to drug absorption due to its small
surface area and low permeability, attributable to its anatomical
structure. The cornea is comprised of three layers: the lipophilic
outer epithelium; the hydrophilic stroma, which constitutes 90%
of the thickness of the cornea; and the inner endothelium, con-
sisting of a single layer of flattened epithelial-like cells. Because
the cornea has both hydrophilic and lipophilic structures, it acts
as an effective barrier against both lipophilic and hydrophilic
drugs. Thus, unique approaches have been taken in designing
SECTION 4
ocular drugs with increased corneal absorption, such as
prodrugs.
ADDITIVES
Various compounds can be added to topically administered oph-
thalmic drugs in order to increase corneal absorption, and most
fall into one of two categories: compounds that increase corneal
residence time, and compounds that increase corneal penetration.
Included in the category of increased corneal residence time are
those compounds that increase the viscosity of the therapeutic
or have mucoadhesive properties. These compounds will be
discussed later.
Preservatives such as benzalkonium chloride (BAK) and cetyl-
pyridinium (CPC), which act as surfactants, have been hypothe-
sized to increase absorption of ocular drugs across the cornea.
Other preservatives, such as thimerosal, chlorobutanol, chloro-
hexinide digluconate (CHD or CDG), hydrogen peroxide, sorbic
acid, sodium bisulfite, and EDTA, have also been shown to
increase corneal absorption. BAK has been accused of increasing
drug penetration as a result of its toxicity to the ocular surface.
However, these studies utilized exaggerated dosing regimens or
drug concentrations that contain unrealistically high levels of
BAK and were conducted in animal or in vitro cell models that
do not translate into relevant clinical information.65–69 Studies
using clinically applicable concentrations and dosing of BAK have
not shown toxicity to corneal epithelial cells and no significant
effects were observed with regard to corneal healing and epi-
thelial migration rates when BAK 0.01% was instilled qid.70–72
Recently, research has focused on the use of polymers as
penetration enhancers. The cationic polymer compound chitosan
hydrochloride (Ch-HCl) was shown to significantly enhance intra-
ocular drug penetration, thought to be due to increased corneal
permeability.73 Also, basic amino acid polymers such as poly-L-
arginine (PLA) appear to enhance the permeation of hydrophilic
compounds through the cornea, conjunctiva, and conjunctiva/
sclera composite, and thus may be used as permeation enhancers
for ocular drug delivery via both the corneal and noncorneal
186 pathways.74
SUSPENSIONS
Because the outer epithelium is such an effective barrier against
hydrophilic compounds, absorption of moderately lipophilic
compounds is favored. There are solubility issues associated
with formulating lipophilic drugs as ophthalmic eye drops,
therefore many compounds must be formulated as suspensions.
Topical ophthalmic suspensions have a few limitations
including particle distribution (they need to be shaken before
use) and sterility.6
As an alternative to conventional suspensions, water-soluble
derivatives have been used to enable drug formulation as aqueous
solutions.75,76 Though these formulations have decreased intra-
ocular absorption due to the lipophilic corneal epithelium, this
is offset by increased driving concentration resulting from the
increased aqueous solubility. Cyclodextrins and b-cyclodextrins
are being explored as a different formulation strategy with the
goal of increasing the aqueous solubility of compounds with low
water solubility. Cyclodextrins are cyclic sugars that have a hydro-
philic outer surface and a central lipophilic cavity. The lipophilic
cavity enables cyclodextrins to ‘hide’ a variety of compounds
with low water solubility in aqueous solution, while the hydro-
philic outer surface allows these complexes to remain water
soluble.77,78 A recent example of the use of cyclodextrins in
topical ophthalmic formulations is of methylated b-cyclodextrin
added to dorzolamide, a carbonic anhydrase inhibitor, used in
the treatment of glaucoma.79
It is important to keep in mind a caveat about suspensions:
the driving force for drug absorption is drug concentration, so a
10% solution is absorbed at a rate that is 10 times that of a 1%
solution. This is not so with a suspension. A 10% and a 1%
suspension have exactly the same amount of drug in solution,
and all additional drug is insoluble. Although suspension drug
particles that remain in the cul de sac can act as depot, the resi-
dence time of the solid in the precorneal pocket is only 2 min.
Undissolved drug is lost from the front of the eye and does not
contribute to ocular tissue drug levels. Thus, a 10% suspension
is rarely 10 times more bioavailable than a 1% suspension.
METABOLISM
Drug metabolism can affect bioavailability in a positive way by
utilizing endogenous enzymes in the corneal area. Some ocular
therapeutics are prodrugs, which can be chemically or enzyma-
tically converted to the active parent drug, either within the cornea
or after the corneal penetration. A classic example is dipivefrin,
an epinephrine-derived ester that is 600 times more lipophilic
than the native form of epinephrine. After passing through the
corneal epithelium, dipivefrin is hydrolyzed by esterases to yield
active epinephrine. The final bioavailability of dipivefrin is
17 times greater than that of an eye drop that contains native
epinephrine, and thus produces similar therapeutic properties in
the eye with fewer adverse effects.80,81 This is due to the addition
of pivaloyl groups to epinephrine to make dipivefrin, enhancing
its lipophilicity and thus its penetration to the anterior chamber.
The role of metabolism in ocular tissues continues to be an
important area of research, with numerous advances in ocular
prodrug therapeutics.82,83
DRUG FORMULATION
HYDROPHILICITY VERSUS LIPOPHILICITY
One inherent characteristic of an ocular therapeutic that can be
an important factor in determining the extent of its absorption
into ocular tissue is the drug molecule’s hydrophilicity or lipo-
philicity. For example, it has been shown that moxifloxacin, a

novel fourth-generation fluoroquinolone, has higher maximum
concentrations in ocular tissues in comparison to other fluoro-
quinolones. This is thought to be due to the unique structure of
moxifloxacin that combines high lipophilicity for enhanced cor-
neal penetration with high aqueous solubility at physiological
pH. The latter property creates a high concentration gradient at
the tear film/corneal epithelial interface, providing a driving
force for better ocular penetration for moxifloxacin.84,85
SOLUTION OSMOTICITY
Tears are slightly hypertonic (~330 mOsm). Hypertonic solutions
above 400 mOsm may be unpleasant to the eye and may induce
lacrimation, which in turn causes greater precorneal drainage loss.
In contrast, hypotonic solutions as low as 100 mOsm are still
comfortable in the eye and may actually lead to an increase in
the bioavailability of water-soluble drugs, presumably through a
solvent drag effect. For comparison, the osmolarity of the ocean
is ~1000 mOsm.
SOLUTION pH
For stability reasons, many eye drops are formulated at pH values
other than pH 7.4. The comfort zone of an ocular solution is
rather narrow and typically in the pH 6–8 range. Outside this
range, the solution can be uncomfortable and induce lacrima-
tion, resulting in drug loss. The pH boundary outside of which
actual tissue damage may occur is below pH 3 and above pH 10.
The ability of the eye to restore physiologic pH is very good, and
this occurs within a short time because of lacrimation, the high
turnover rate of tears (which is ~16%/min) and the tears’ buffer
system.53,54 With an ionizable drug, it is sometimes tempting to
adjust the pH either above or below the comfort range pH of 6–8
to convert the drug to a more favorable form for absorption (i.e.,
the undissociated form of the drug). When the pH is adjusted in
this way, any gain in drug bioavailability is generally negated by
precorneal loss owing to discomfort and lacrimation.
VISCOSITY
It is clear that the factor contributing the most to precorneal
drug loss is drainage. An increase in the viscosity of the solution
might appear to remedy this problem. It has been shown that
increased viscosity increases dwell time on the ocular surface,
but for formulations above 70 cp, there is an increased likelihood
of unwanted effects such as lid caking and blurring, and general
discomfort in the eye. Solutions of ~70 cp are able to maximize
residence time without these side effects. Of viscosity-enhancing
polymers, poly(vinyl alcohol) and poly(vinylpyrrolidone) are con-
sidered ideal because of their spreading characteristics the thick-
ness of the applied medication layer over the precornea area.86
Another approach to the precorneal residence time problem
is to employ a phase-change polymer. These systems are liquid
in the bottle, but when placed in the eye the polymers solidify
because of differences in temperature, pH, or mono/divalent ion
concentrations. Such phase-change solutions are typically better
accepted by the patient. A couple of examples of these gel-forming
systems are Timoptic-XE, used in the treatment of glaucoma and
ocular hypertension and Systane, which is used to treat dry eye.
Timoptic-XE contains timolol maleate, a nonselective b-blocker,
and Gelrite a purified gellan gum that forms a gel upon contact
with the precorneal tear film. Timoptic-XE was shown to reduce
systemic absorption as compared to a timolol maleate ophthal-
mic solution.87 Systane contains two demulcents (propylene
glycol, PEG-400) and a gelling agent, Hydroxypropyl (HP)-Guar.
Guar has a neutral pH of 7.0, and when it interacts with slightly
alkaline (pH ~7.5) human tears, it bonds with borate ions to
Ocular Pharmacokinetics
form a cross-linked lattice that helps the mucin layer to adhere
to the eye in desiccated areas. Systane was shown to protect the
cornea from desiccation in an in vivo rabbit model, and signifi-
cantly relieve dry eye symptoms in patients in comparison with
control solutions.88–90
NOVEL DRUG DELIVERY DEVICES
Key Features
• An ideal delivery system should be effective and consistent,
inexpensive, and comfortable for the patient
• Mucoadhesive polymers increase ocular drug bioavailability by
prolonging contact with the corneal layer
• Sustained release devices are able to reach near zero-order
kinetics, in which the level of administered drug remains
constant throughout the delivery period
• Liposomes are vesicles composed of phospholipid bilayers
and are able to accommodate both lipophilic and hydrophilic
drug molecules
• Particulate polymeric drug delivery systems such as micro-
and nanoparticles are stable, relatively comfortable in the eye,
and prolong ophthalmic absorption times
There are advantages and disadvantages to each of the topical
ocular delivery systems available. Improvements continue to be
made on these systems, and novel drug delivery devices are being
CHAPTER 17
researched. An ideal delivery system should be effective and
consistent, inexpensive, and comfortable for the patient.
METERED DELIVERY SYSTEMS
There are several new delivery systems being developed, includ-
ing UniDoser, Eye Instill, and Visine Pure Tears, that improve
upon the standard eyedropper by addressing drop size and
allowing for the delivery of a multidose medication without the
need for preservatives.
A new type of ocular drug delivery utilizes a system that
delivers a mist containing medication. For example, Kahn et al
are developing a small volume nebulizer system91 and Optimyst
Systems are working on a small, handheld device that uses
ultrasonic vibrations to create a fine mist.
MUCOADHESIVES
Poly(acrylic acid) and hyaluronic acid are two examples of muco-
adhesive polymers that can interact with the mucosal layer on
the cornea and sclera, increasing retention of the drug.92,93
Mucoadhesives can aid in the localized delivery of topical
ophthalmic drugs and increase bioavailability due to prolonged
contact with the corneal layer. After the mucoadhesive polymer
is administered, and after contact with water and subsequent
swelling, the polymer and mucin become physically entangled.
Un-ionized carboxylic acid residues on the polymer then form
hydrogen bonds with the mucin molecule.94
SUSTAINED RELEASE DEVICES
Zero-Order Kinetics
Ideal delivery of drugs would follow zero-order kinetics, in
which the level of administered drug would remain constant
throughout the delivery period. Zero-order release kinetics can
be calculated from data obtained from in vitro drug release studies:
the slope(s) of the log(percent drug release) versus log(time)
plots can be calculated from fitted linear regression lines. A
slope of 1.0 represents zero-order kinetics. 187
 PHARMACOLOGY AND TOXICOLOGY
There is interest in the development of devices with a con-
trolled and sustained release of ophthalmic drugs in order to
circumvent the inconvenience of frequent dosing. If dosages
could be sustained for extended periods of time, therapeutic
levels could be maintained for weeks or longer. These devices
represent the best hope of the drug delivery systems available
for reaching zero-order kinetics.
Delivery of most drugs follows what is known as first-order
kinetics, in which initially high levels of the drugs are attained
followed by an exponential decrease in concentration. There is
some difficulty in designing an optimal dosing regimen for drugs
that follow first-order kinetics. Drug concentrations in target
tissues need to be maintained above the minimum concentra-
tions for therapeutic effectiveness, but below toxic levels, and
staying in this range is tricky due to the rapid rise and fall in
drug concentrations.
INSERTS
Sustained release devices or inserts fall into two different cate-
gories: those that are insoluble or nonerodible, and those that are
soluble or erodible. An obvious advantage of erodible systems is
the fact that the delivery system does not have to be removed from
ocular tissues after the drug has been released. A disadvantage of
the erodible systems is that they are more likely to show patient-
to-patient variability in release kinetics due to different rates of
tear production/turnover and concentration of metabolic enzymes
in the tear film.
SECTION 4
Unfortunately, all of the insoluble ocular inserts on the
market have yet to gain wide acceptance. One example is
Ocusert Pilo, a pilocarpine-loaded, insoluble device placed in
either the upper or lower cul-de-sac for the treatment of glau-
coma. This device failed to achieve widespread use because of
its costliness and poor patient compliance due to ejection from
patients’ eyes. Other examples of insoluble sustained-release
devices in development include: presoaked hydrophilic contact
lenses;95 OphthaCoil, which consists of a drug-loaded adherent
hydrogel coating on a thin coiled metallic wire inserted into the
conjunctival sac;96 and a one-side-coated insert that releases
drug from only the uncoated side.97
A few examples of soluble or erodible ocular inserts include:
corneal collagen shields;98,99 and gel-forming erodible inserts for
the delivery of ofloxacin.100
IONTOPHORESIS
Iontophoresis, the process in which an electrical current drives
ions into cells or tissues, is not a new mode of drug delivery.
However, the method has been the focus of innovation, includ-
ing the use of hydrogel-containing probes.101,102 Recently, an
iontophoresis device has been shown to be safe and well tolerated
in a clinical setting for the management of active corneal graft
rejection,103 shows enhanced ocular absorption of small cationic
compounds such as carboplatin,104 and is being tested for its effi-
ciency in anterior delivery of Clonidine, an ocular hypotensive.
LIPOSOMES
Liposomes are microscopic vesicles composed of one or more
phospholipid bilayers. Due to the biphasic nature of liposomes,
both lipophilic and hydrophilic drug molecules are accommo-
dated, thus almost any type of drug can be encapsulated. Acting
as a drug carrier, liposomes bind to the cellular membrane and
facilitate the transport of drug across the membrane. The first
use of liposomes in ocular therapy was reported by Smolin et al,
who tested the effectiveness of liposome-associated idoxuridine
188 as compared to solution for the treatment of herpes simplex
keratitis in the rabbit eye.105 Recent studies on liposome ocular
therapeutics include: the encapsulation of acyclovir;106,107 disulfi-
ram, a potential anticataract agent;108 the mydriatic tropicamide;109
cyclosporine A;110 and antisense oligonucleotides, which could
potentially be used in the treatment of ocular viral infections.111
The efficacy of liposome-encapsulated O-palmitoyl prodrug
of tilisolol, a nonselective b-blocker, after both topical and intra-
vitreal injection has been studied,112 as well as liposomes carrying
plasmid DNA. Masuda et al showed that the delivery of these
liposomes resulted in efficient and stable transfer of the func-
tional gene to the cornea, iris, ciliary body, and retina of rats.113
MICROSPHERES AND NANOPARTICLES
While liposomes represent a promising avenue of ocular drug
delivery, they are less stable than particulate polymeric drug
delivery systems such as micro- and nanoparticles. The drugs
can be incorporated into or absorbed by the particles. This mode
of ocular drug delivery has been shown to increase drug absorp-
tion in the eye as compared to ophthalmic solutions, due to a
much slower elimination rate of the particles.114 Particles in the
micrometer size range (>1 mm) are called microparticles or micro-
spheres, whereas those in the nanometer size range (<1 mm) are
called nanoparticles. The upper size threshold for microparticles
for ophthalmic administration is ~5–10 mm. Patients experience
discomfort after application of particles above this size, and
generally, the smaller the particles, the better the patient tolera-
tion of the drug. Some recent examples of research into micro-
and nanoparticles in topical ocular delivery systems include:
5-fluorouracil microspheres;115 sodium ibuprofen-loaded polymeric
nanoparticle suspensions (Eudragit RS100);116 biodegradable
calcium phosphate nanoparticles (CAP) containing 7-hydroxy-
2-dipropyl-aminotetralin (7-OH-DPAT), an IOP-lowering agent;117
solid lipid nanoparticles (SLN) used in the delivery of tobra-
mycin;118 and chitosan (CS) nanoparticles for the delivery of
cyclosporine A.119 These particulates drain through the lacrimal
duct, but a certain percentage remains in the eyes for several
hours or even longer, thus an important characteristic of these
delivery systems is that the particulates are made of biodegrad-
able polymers.
VITREORETINAL DRUG DELIVERY
Key Features
• There is poor penetration of ocular therapeutics to the
posterior tissues of the eye due to the blood–ocular barrier and
tight junctional complexes in the retinal epithelium
• Microspheres, iontophoresis, and sustained release implants
are some of the delivery systems being developed to treat
vitreoretinal diseases
• Some treatments for AMD are being tested that target proteins
involved in angiogenesis, such as VEGF or PEDF
Lately there has been increased attention on the development of
ocular therapeutics for vitreoretinal diseases. A great deal of
research is focused on treatments for age-related macular degen-
eration (AMD), macular edema, diabetic retinopathy, and diabetic
macular edema. There is poor penetration of systemically admin-
istered ocular therapeutics to the posterior tissues of the eye due
to the blood–retinal barrier. The limited permeability of the
blood–retinal barrier results from the network of tight junc-
tional complexes (zonulae occludens) present in the retinal pig-
ment epithelium and the endothelial membrane of the retinal
vessels. Intravitreal injection is a relatively safe and easy method
of drug delivery to the posterior tissues but due to its invasive
 Ocular Pharmacokinetics

nature, there is the possibility of complications such as vitreous

hemorrhage, retinal detachment, and infection such as endoph-
thalmitis. In order to improve upon the delivery of drugs to the
posterior segments of the eye, alternative delivery routes, such as
subconjunctival injection, are being investigated, and less inva-
sive delivery systems, as well as advancements on injectable thera-
peutics, are in development for the treatment of vitreoretinal
diseases.

SUBCONJUNCTIVAL INJECTION
Subconjunctival injection refers to the injection of up to 0.5 mL
of a drug solution underneath the thin membrane lining the
eye, known as the conjunctiva. There are currently only a few
studies that have examined the pharmacokinetics of ocular
drugs delivered via this route, but it appears as though there is
greater absorption of drugs delivered by subconjunctival absorp-
tion than by systemic or topical administration. For example,
Weijtens et al120 measured the concentration of dexamethasone
in aqueous, vitreous, and serum in phakic patients following
subconjunctival injection of dexamethasone disodium phosphate
and compared the results to those following peribulbar and oral
administrations. It was found that the subconjunctival injection
was more effective than peribulbar and oral administration,120
as well as topical instillation.121 Some effects of subconjunctival
injection include backward drainage of solution along the FIGURE 17.10. Anecortave acetate is administered outside the globe
needle track, or diffusion across the conjunctiva,122,123 as well as with a curved, blunt-tipped cannula. The cannula is inserted between
Tenon’s capsule and the sclera and the drug forms a depot directly
considerable systemic absorption.120,124

CHAPTER 17
behind the macula where it is slowly released over 6 months. A
counter-pressure device (CPD; shown with a notch covering the
NOVEL VITREORETINAL DELIVERY SYSTEMS cannula) is used to prevent reflux of the suspension.

The use of drugs encapsulated in microspheres in the treatment

of vitreoretinal diseases has been studied by several groups. For
example, Moritera et al investigated the intravitreal injection of
poly(lactic acid) microspheres containing 5-fluorouracil,125 and
Saishin et al tested the periocular injection of microspheres
containing PKC412, a kinase inhibitor that has been shown to
inhibit ocular neovascularization in mice, as a potential treatment
for AMD.126
Other novel vitreoretinal delivery systems are being explored.
For example, it has been shown that transscleral Coulomb-
controlled iontophoresis (CCI) following intravitreal injection
enhances penetration of corticosteroids.127
An interesting potential delivery system is the Encapsulated Cell
Technology (NT-501) which uses encapsulated retinal pigment
cells, genetically modified to secrete ciliary neurotrophic factor,
for the treatment of glaucoma, retinitis pigmentosa, and AMD.128
INJECTABLE THERAPEUTICS
There are a variety of implants that are being tested for their use
in treating vitreoretinal diseases. Vitrasert is one of the initial
drug delivery devices for vitreoretinal disease. It is used in the
treatment of AIDS-related cytomegalovirus retinitis and is a device
that is surgically implanted into the vitreous, where it releases
the antiviral drug ganciclovir remaining active for approximately
seven and a half months.129 Retisert, which uses a similar tech-
nology as Vitrasert, is an intravitreal device approved to release a
constant amount of the steroid, fluocinolone acetonide, over a
treatment period of 30 months, with the potential to treat pos-
terior uveitis, diabetic macular edema, and AMD.130 Another
ocular insert with the potential to treat AMD is the I-vation
implant, developed for site specific delivery of the steroid triam-
cinolone acetonide (TA) into the posterior chamber of the eye
over a time period of 6 months to 2 years.131 Anecortave acetate,
marketed as Retaane 15 mg for the treatment of AMD, is a
synthetic analog of cortisol with angiostatic but not glucocor-
ticoid receptor mediated activity. Anecortave acetate is delivered
as a posterior juxtoscleral depot (PJD) onto bare sclera near the
macula (see Fig. 17.10).
Evidence is growing that targeting proteins involved in the
process of angiogenesis, such as VEGF or PEDF, might provide
specific and effective treatment of AMD. Abnormal regulation
of angiogenesis, or formation of new blood vessels from pre-
existing ones, has been implicated in the pathogenesis of several
disorders, including AMD. Vascular endothelial growth factor
type A (VEGF-A) is a stimulator of angiogenesis; its binding to
VEGF receptors has been shown to promote endothelial cell
migration and proliferation, two key features required for the
development of new blood vessels. Pegaptanib sodium injection
(Macugen) is a selective VEGF antagonist that requires repeated
injections into the vitreous cavity. Ranibizumab (rhuFab V2;
Lucentis), a humanized monoclonal antibody fragment against
VEGF, is also delivered by intravitreal injection.132 Budesonide
is capable of inhibiting VEGF expression through glucocorticoid
receptor activity. Kompella and colleagues showed that subcon-
junctivally administered budesonide-PLA nano- and micropar-
ticles sustain retinal drug delivery as compared with the
budesonide solution-treated group at the end of day seven of
their study.133 It is now believed that angiogenesis is regulated
by a balance between VEGF and PEDF (pigment endothelium
derived factor), as evidence is emerging that PEDF may inhibit
new blood vessel growth.134 AdPEDF.11 is an adenovector carry-
ing a progene for human PEDF, and is currently in clinical trials
as a genetic therapy via intravitreous injection for wet AMD.135
CONCLUSIONS
Quantitative understanding of the time course of drugs in the
eye through pharmacokinetic analysis provides mechanistic 189
 PHARMACOLOGY AND TOXICOLOGY
insight into the fate of drug disposition in this organ. It also aids
in the design of new, and improvement upon existing, ophthalmic
therapies either by enhancing efficacy or reducing toxicity, as
well as in the development of clinical strategies for how to opti-
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 CHAPTER

18 Anesthetics
Padma Gulur, David Weber, and Martin A. Acquadro

Anesthesia, over the years, has become very safe. The risk from The goals of preanesthetic medication include decreased
anesthesia when compared to the risk due to patient and anxiety, analgesia if preoperative pain is evident, and, if nec-
surgical factors is relatively low.1 This can be attributed in part essary, diminished airway secretions and diminished gastric
to better agents, medications, and advances in monitoring. acidity and volume. All these goals should be accomplished
The American Society of Anesthesiologists has stratified the without excessive sedation, which could compromise the
risk of patients undergoing anesthesia (Table 18.1). This has cardiopulmonary system.2–4
become a useful universal nomenclature. Modern practice of
this specialty strives for anesthesia, analgesia, amnesia,
areflexia, and autonomic stability. General anesthesia usually BENZODIAZEPINES
involves premedication, induction, maintenance, and recovery. Anxiolytics are usually used as preoperative medications.
Benzodiazepines (Table 18.2) are the most common of the
PREMEDICATION anxiolytic agents. When given in the usual doses, they produce
the greatest relief of anxiety with the least cardiopulmonary
Premedication for alleviation of anxiety is not a substitute for depression. These drugs are rarely implicated as a cause of
adequate preoperative discussion with the patient. A study nausea and vomiting. They can raise the threshold for central
comparing various techniques including, no preoperative visit nervous system (CNS) toxicity of local anesthetics5 and are not
or drug, preoperative discussion alone, premedications alone, analgesic, but they compound the anxiolytic effects of some
and preoperative discussion with premedication, demonstrated analgesics in small to moderate doses. Diazepam is usually
interesting results. The patients who displayed the most anxiety given orally. The solvent used in parenteral preparations can
were those who were premedicated without preoperative result in pain and phlebitis. Lorazepam can be given orally or
discussion or consultation. The patients with the least anxiety parenterally and often produces amnesia. It can also result in
were those who had both preoperative discussion and pre- prolonged sedation.4
operative medication. The patients who had only preoperative Midazolam has become popular because of its water solubility,
discussion, without any premedication, were not much more rapid onset and short duration of action, and reliability. It can
anxious, as a percentage of the population studied, than those be given intramuscularly or intravenously and often produces
who received both preoperative discussion and premedication.1 amnesia with few side effects. Mental function returns to
normal within 4 h, making midazolam a popular choice for
ambulatory surgery and regional anesthesia. Diazepam is more
TABLE 18.1. American Society of Anesthesiologists likely to produce cumulative effects than lorazepam or
Classification of Preoperative Risk midazolam.4
ASA Class Systemic Disturbance Mortality
NARCOTICS
1. Healty patient with no disease outside <0.03%
of the surgical process If the patient experiences preoperative pain, morphine is an
2. Mild to moderate systemic disease 0.2%
effective preoperative analgesic (Table 18.3). The choice of
caused by the surgical condition or narcotic is usually governed by the desired duration of activity.
by other pathological processes, Morphine’s clinical effects persist 4–6 h; fentanyl’s action lasts
medically well-controlled ~1–2 h. Urinary retention, wheezing, constipation, nausea,
3. Severe disease process which limits 1.2% and vomiting are not uncommon with opioid analgesics. The
activity but is not incapacitating respiratory depressant action of morphine may cause hypo-
ventilation and increased carbon dioxide tension with resultant
4. Severe incapacitating disease 8% increased intracranial pressure. Advantages and disadvantages
process that is a constant threat to life
need to be considered in the decision to use opioids in pre-
5. Moribund patient not expected to 34% anesthetic medication.2,4
survive 24 h with or without an Meperidine is used commonly as an intramuscular medica-
operation
tion. There is some concern that the metabolite of meperidine,
E. Suffix to indicate emergency surgery Increased normeperidine, may result in confusion, agitation, and seizures,
for any class particularly in the elderly, in patients with renal failure, and in
Adapted from Cohen MM, Ducan PG, Tate RB, JAMA 1988; 260:2859. children. This is more often a problem with long-term repeated
dosing. 193
 PHARMACOLOGY AND TOXICOLOGY

TABLE 18.2. Premedicants – Anxiolytics and Hypnotics4,7,10

Agent Dosage Metabolism Effects

Benzodiazepines
Midazolam IV: 0.03–0.07 mg/kg tE: 1–4 h
M: Liver
E: Kidney CNS depression,
amnesia, Ø seizure
Diazepam IV: 0.03–0.1 mg/kg tE: 7–10 h (2–8 days threshold, BP and
PO: 0.05–0.15 mg/kg for active metabolites) respiratory
M: Liver depression; may
E: Kidney cause paradoxical
Lorazepam IV: 0.05 mg/kg tE: 14 h CNS excitement
PO: 1–10 mg/kg M: Liver
E: Kidney
Barbiturates
Pentobarbital IV: 1 mg/kg up to tE: 20–50 h CNS depression; may
500 mg M: Liver cause depression of
IM: 100–200 mg E: Kidney (mostly), BP, hiccoughs,
PO: 100–200 mg liver laryngospasm,
respiratory
depression,
exacerbation of
porphyria; agents
cross placenta; may
antagonize oral
anticoagulants
BP, blood pressure; CNS, central nervous system; E, route of excretion; IM, intramuscularly; IV, intravenously;
M, site of metabolism; PO, per os; tE, elimination halflife.
SECTION 4

TABLE 18.3. Premedicants – Analgesics4,7,10

Agent Dosage Metabolism Opioid Effects

Meperidine IV: 0.5–1.0 mg/kg tE: 1.5–4 h Analgesic, CNS depression,

IM/SC: 0.5–1.0 mg/kg M: Liver euphoria, respiratory depression,
PO: 1 mg/kg q 2–4 h E: Kidney bronchospasm (rare), Œ blood
pressure, nausea, vomiting,
Morphine IV: 0.1 mg/kg t E: 2–4 h dysphoria, Œ biliary pressure, Œ
IM: 0.1 mg/kg M: Liver gastrointestinal/genitourinary
E: Kidney motility. Agents cross placenta.
Greater incidence of skeletal
Fentanyl IV: 10–100 mg t1/2: a: 1–2 min rigidity with fentanyl (accumulation
IM: 50–100 mg/kg t E: 4 h with frequent dosing). Narcotics,
M: Liver particularly Demerol, should be
E: Kidney avoided in patients taking MAO
inhibitors.
E, route of excretion; IM, intramuscularly; IV, intravenously; M, site of metabolism; PO, per os; SC,
subcutaneously; tE, elimination halflife; t1/2, halflife.

Remifentanil is an ultra-short-acting opioid, unique among
the other opioids, secondary to rapid metabolism rather than
redistribution. Rapid metabolism occurs from hydrolysis of a
methyl ester side chain by blood and tissue esterases. Because
of its short duration of action, it is generally administered by
continuous intravenous infusion.
ANTIEMETICS
Ondansetron hydrochloride is a commonly used selective
blocking agent of the serotonin 5-HT3 receptor, administered
orally or intravenously. These serotonin 5-HT3 receptors are
found centrally in the area-postrema-chemoreceptor trigger
zone, and peripherally on vagal nerve terminals. Although it is
194 not certain whether ondansetron’s effectiveness comes from
antagonism of central, peripheral, or both receptor sites, the
drug is effective against perioperative and chemotherapy-induced
emesis. The incidence of side effects is low when the drug is
given in normal doses to normal patients.6
BUTYROPHENONES
The most common butyrophenone is droperidol (Table 18.4). In
adults it is an antiemetic in very small doses, so that
cardiopulmonary stability is maintained. It should be noted
that droperidol does have a1-adrenergic-blocking activity and
must be given with caution if hypotension is already evident.
Restlessness and extrapyramidal dyskinesia may be noted.
Atropine is an effective antidote. A patient may exhibit
catatonia and appear outwardly calm though he or she is in fact
 Anesthetics

TABLE 18.4. Premedicants – Antiemetics4,6,7,10

Agent Dosage Metabolism Effects

Droperidol IV: 0.625–2.5 mg tE: ? Antiemetic, antipsychotic. May

IM: 2.5–10 mg M: Liver cause dysphoria, extrapyramidal
E: Kidney, liver effects, hypotension secondary to
a-blockade. Black box warning in
effect from FDA for QT
prolongation
Hydroxyzine IM: 25–100 mg tE: 3 h CNS depression, antiemetic
PO: 25–100 mg M: Liver effects, antagonism of histamine
E: Liver, kidney action on H1 receptors. May cause
dry mouth
Ondansetron IV: 4 mg slow M: Liver Antiemetic – chemotherapy and
PO: 8–16 mg, E: Kidney, liver postanesthesia N/V. Hypotension,
1 h before induction bradycardia, tachycardia, angina,
second degree heart block,
bronchospasm, extrapyramidal
effects, seizures
E, route of excretion; IM, intramuscularly; IV, intravascularly; M, site of metabolism; PO, per os; tE, elimination
half-life.

TABLE 18.5. Premedicants – Antagonists and Gastrokinetic Agents4,7,10

Agent Dosage Metabolism Effects

CHAPTER 18
Cimetidine IV/IM/PO: 300 mg q 6–8 h tE: 2 h May increase blood levels
M: Liver of propranolol or
E: Kidney benzodiazepines and
potentiate oral
anticoagulants; may cause
confusion
Ranitidine IV/IM: 50 mg q 6–8 h tE: 2–3 h Antagonizes histamine
PO: 150 mg q 12 h M: Liver action on H2 receptors with
E: Kidney decreased gastric acid
secretion
Metoclopramide IV/IM: 10 mg tE: 2–6 h Ø Gastrointestinal motility
PO: 10–15 mg M: Liver and Œ esophageal sphincter
E: Kidney tone; extrapyramidal
symptoms (rare)
E, route of excretion; IM, intramuscularly; IV, intravenously; M, site of metabolism; PO, per os; t E, elimination
half-life.

experiencing panic secondary to dysphoria produced by the
action of droperidol.4
ANTIHISTAMINES
Antihistamines are occasionally used as premedication because
these act as anxiolytics as well as H1 histamine receptor blockers.
Hydroxyzine and diphenhydramine are common agents.4
ALPHA-2 AGONISTS
Clonidine is a centrally acting alpha-2 agonist used as a
premedication for its sedative properties and for attenuation
of autonomic reflexes such as hypertension, tachycardia and
cathecholamine release associated with preoperative anxiety
and surgical stimulus. Caution is advised in patients on cloni-
dine for long periods due to risk of rebound hypertension with
its withdrawal. Dose is usually 2 mg/kg orally.
Dexmedetomidine is a more specific alpha-2 agonist that is
also gaining popularity as a premedication. Bradycardia and dry
mouth are possible side effects with this class of medications.
ANTICHOLINERGICS
This class of drugs is not routinely used as a premedication
in present practice. When used, these are chosen for their
antisialogogand sedative effects as well as for prevention of
reflex bradycardia with the latter being the most common
reason for their use as a premedication. This is especially true
of atropine and glycopyrrolate. Glycopyrrolate does not cross
the blood–brain barrier and therefore has the least of the unde-
sirable side effects of these drugs (central anticholinergic
syndrome). Physostigmine (15–60 mg/kg) is a specific treatment
for this syndrome due to atropine or scopolamine (Table 18.5).
Scopolamine in the form of a patch is gaining popularity as
an effective antiemetic agent. It is applied preoperatively and is 195
 PHARMACOLOGY AND TOXICOLOGY

worn for 72 h. Side effects of this class of drugs include central GENERAL ANESTHETICS
anticholinergic syndrome, tachycardia, lower esophageal
sphincter relaxation, body temperature increase, drying of air-
way secretions and an increase in physiologic dead space. Summary Box
Premedications
H2 HISTAMINE RECEPTOR ANTAGONISTS • The goals of preanesthetic medication include decreased
anxiety, analgesia if preoperative pain is evident, and, if
Cimetidine and ranitidine block H2 receptors and decrease
necessary, diminished airway secretions and diminished
gastric acid secretion. Ranitidine has become more popular,
gastric acidity and volume
because it appears to cause fewer cardiovascular and CNS side
• Decreased anxiety is the most common reason for
effects than cimetidine.4 Routine use of these medications is
premedication and benzodiazepines (midazolam in particular)
not recommended (Table 18.6). Instead, these are usually
are the commonly used class of medications. Midazolam is
reserved for patients at high risk for aspiration.
ideal based on its short duration of action and favorable
pharmacokinetics
ANTACIDS • Anticholinergics are used if reducing airway secretions is the
goal
Particulate and nonparticulate antacids effectively raise gastric
• For diminished gastric acidity and volume, H2 receptor
acid pH. If aspiration is a concern, a nonparticulate antacid is
antagonists, antacids (especially Bicitra which is a clear non-
preferred, because particulate antacids may cause more lung
particulate antacid), and gastrointestinal motility agents such
damage. Sodium citrate is a commonly used nonparticulate
as metaclopromide are used
antacid.4

GASTROINTESTINAL MOTILITY AGENTS

Metoclopramide, a dopaminergic antagonist, increases gastro-
intestinal motility and pyloric relaxation, thereby increasing the
speed of gastric emptying. Sodium citrate or anticholinergic
agents may interfere with the action of metoclopramide.4
SECTION 4
A patient under general anesthesia has no perception of any
sensation. This state, which allows surgical procedures to be
performed, can be induced with a wide variety of drugs, usually
used in combination. The objectives of a general anesthetic

Cisapride is another medication in this class of drugs. The
antibiotic erythromycin is being touted for use as a gastric
emptying agent to decrease risk prior to emergency anesthesia.
PHARMACOKINETICS
Among benzodiazepines, diazepam is metabolized by the liver,
with one-third of the metabolites being oxazepam. The active
metabolites are principally excreted by the kidneys. In general,
the benzodiazepines, barbiturates, and antihistamines are
metabolized by the liver and excreted by the kidneys, though
the amount of drug eliminated by the kidneys and liver varies
somewhat.4,7 Ondansetron and the butyrophenones are also
metabolized by the liver and excreted by the kidneys.6 Ten
percent of droperidol is excreted unchanged.4,7 Morphine is
metabolized by the liver and excreted by the kidneys, as are
the other opioids. Tables 18.2 to 18.6 list many of the drugs
commonly used.4,7
include analgesia, unconsciousness, and absence of movement
and autonomic stability.8–11
General anesthetics are commonly administered intravenously
or inhalationally. These routes are preferred over the intra-
muscular or oral route because of greater drug predictability and
reliability. Common inhalational and intravenous agents are
reviewed in this section.10
INHALATIONAL AGENTS
The common inhalational general anesthetic agents include
nitrous oxide and the halogenated agents like halothane,
enflurane, isoflurane, desflurane, and sevoflurane. Enflurane is
no longer commonly used due to risk of seizures. To compare
various inhalational agents and the concentrations in the
alveoli during steady state that produce equivalent levels of
anesthesia, the concept and definition of minimum alveolar
concentration (MAC) are necessary. The MAC of anesthetic at

TABLE 18.6. Premedicants – Anticholinergics4,7,10

Agent Dosage Metabolism Effects

Atropine IV/IM: 0.4–1.2 mg t1/2 a: 1 min Tachydysrhythmias, dry mouth,

tE: 2 h urinary retention; crosses
M: Minimal blood–brain barrier
E: Kidney and placenta
(some by liver)
Scopolamine IV/IM: 0.3–0.6 mg tE: 3 h Crosses blood–brain barrier
E: Kidney and placenta; may cause
excitement or delirium; superior
antisialogog
Glycopyrrolate IV/IM/SC: 0.1–0.2 mg E: Kidney Does not cross blood–brain
PO: 1–2 mg barrier or placenta; otherwise
similar to atropine
E, route of excretion; IM, intramuscularly; IV, intravenously; M, site of metabolism; PO, per os; tE, elimination
half-life.
196

Summary for Inhalational Agents
• Inhalational anesthetics form the mainstay of maintenance
when general anesthesia is administered. They may also be
used for induction in patients without intravenous access. This
practice is more common in children
• Commonly used Inhalational anesthetics today are isoflurane,
sevoflurane, and desflurane. Halothane has fallen out of favor
for its dysrhythmogenic potential and association with
halothane hepatitis. Enflurane is rarely used secondary to its
epileptogenic potential
• Sevoflurane is the most commonly used inhalational agent for
induction of anesthesia as it does not irritate the airways.
Nitrous oxide is usually used in conjunction with narcotics for
the maintenance of anesthesia
one atmosphere that produces immobility in 50% of patients or
animals exposed to a noxious stimulus is a useful measure of
potency of inhalational agents.5,10,12,13
Anesthetic potency is correlated with lipophilia. The more
potent the general anesthetic, the more lipophilic it is.10
Researchers cannot agree on one specific mechanism of action;
many believe that general anesthetics work at many different
levels and by a variety of mechanisms. This may explain why
diverse inorganic and organic compounds can bring on the state
of general anesthesia. The various theories of the mechanism of
action of general anesthetics are reviewed in references 14–16.
Although general inhalational anesthesia can start with
administration of oxygen, nitrous oxide, and an inhalation
agent, the more common technique is to administer a hypnotic,
such as propofol or thiopental sodium (Pentothal), intra-
venously.17 General inhalational anesthesia is often maintained
with oxygen, nitrous oxide, and a halogenated agent.10
Additional agents may include opiates or muscle relaxants.
The decisions to administer inhalational agents by mask or
endotracheal intubation, and to allow the patient to breathe
spontaneously or to control ventilation, are based on surgical
and anesthetic requirements.
Pharmacodynamics
Figure 18.1 shows the chemical structures of the general
inhalational halogenated anesthetic agents in common use.10
Enflurane and isoflurane are ethers with a difluoromethyl group
bonding to the one carbon via an ether bond. The newer
halogenated agents, desflurane and sevoflurane, are also ethers.
Desflurane is a fluorinated methyl ethyl ether, and sevoflurane
is a fluorinated isopropyl ether. For children and adults,
halothane and sevoflurane are far less irritating to breathe and
have a lower incidence of coughing, laryngospasm, and
F CI F F
F C C H H C C O
F Br CI F F
Halothane Enflurane
F H F F2C
F C C O C H
F F F F2C
Desflurane
Anesthetics
excitation during ventilatory induction when compared with
enflurane, isoflurane, or desflurane.6,10 The principal advantages
of sevoflurane and desflurane over halothane, enflurane, and
isoflurane are their low solubility in blood, which produces
rapid induction of anesthesia, and low tissue solubility, which
results in rapid elimination and awakening.6
The depth of anesthesia with the halogenated agents can be
judged by observing blood pressure, because they produce dose-
dependent reductions of arterial blood pressure principally
through peripheral vasodilation.6,10,17 There should be little
change of pulse rate or blood pressure and no body movement
in response to surgical stimulation. Following induction of
anesthesia with the halogenated agents halothane or sevoflu-
rane, or with hypnotic intravenous agents such as thiopental or
propofol, the clinician should start with a high-inspired
concentration of the inhalational agent. As maintenance of
anesthesia proceeds, the inspired concentration of anesthetic is
lowered, because the alveolar concentration increases during
maintenance.17 As a steady state is approached, based on
patient response to surgical stimulation, further appropriate
concentration adjustments of the inhalational agents can be
made rapidly.11,17
Halothane, Enflurane, Isoflurane, Desflurane, and
Sevoflurane
Cardiovascular system
With all five agents, blood pressure decreases by peripheral
vasodilation as the depth of anesthesia increases. Cardiac out-
CHAPTER 18
put with halothane decreases 20–50% from the baseline value.
The decrease in cardiac output is less with enflurane, desflu-
rane, and sevoflurane. Cardiac output is well maintained with
isoflurane. Heart rate decreases most with halothane, less
with enflurane, desflurane, and sevoflurane, and may increase
with isoflurane. This may explain why cardiac output is
maintained by use of isoflurane. All five agents diminish
baroreceptor reflex responses (tachycardia) to hypotension and
vasomotor reflex responses (increased peripheral resistance) to
hypovolemia, and they produce little change in the sympatho-
adrenal response and levels of catecholamines in the plasma.6,10
Inotropy and contractility diminish with all five agents, most
notably with halothane. Negative inotropy is less obvious and
similar with equipotent concentrations of isoflurane and
sevoflurane. Desflurane produces the least negative inotropy.
All five agents diminish sympathetic activity and increase vagal
predominance, particularly halothane. This is most common
when halothane is given to a child, especially in association
with manipulation of the airway.6,10
Like isoflurane, desflurane in typical clinical settings does not
sensitize the heart to catecholamines; in one study, however,
F F CI F FIGURE 18.1. Chemical structure of five
commonly used inhalational agents.6,10
C H F C C O C H
F H F
Isoflurane
H C OCH2F
Sevoflurane
197
 PHARMACOLOGY AND TOXICOLOGY
the ventricular arrhythmogenic threshold of sevoflurane was
between that of enflurane and isoflurane with submucosal
injection of epinephrine.6 Dysrhythmias are most common
with halothane. Reentrant tachycardia is common, because the
normal conduction pathway is slowed and the refractory period
of the conductive tissue is increased. Increased automaticity
also occurs with halothane, which is augmented by adrenergic
agonists. Exogenous epinephrine should be limited in local
anesthetics to a concentration of 1:100 000. No more than
0.1 mg of epinephrine in 10 min or 0.3 mg of epinephrine in 1 h
should be administered when halothane is used.9 With enflu-
rane, isoflurane, desflurane, or sevoflurane, three times this
amount may be permissible. Unlike isoflurane, the other halo-
genated agents – halothane, enflurane, desflurane, and probably
sevoflurane – do not cause coronary artery vasodilation that
may lead to coronary artery steal syndrome. With the exception
of isoflurane, the coronary circulation generally remains
responsive to myocardial demands for oxygen. With isoflurane,
coronary blood vessels are maximally dilated at ~1.5 MAC.
Blood flow is maintained despite decreased myocardial oxygen
demand. Some patients with ischemic heart disease have
narrowed blood vessels in some regions of myocardium. These
regions depend on collateral vessels for their blood supply.
Dilation of normal coronary vessels by isoflurane may result in
a steal of blood from the collateral vessels that exacerbates
ischemia.6,18
Pulmonary system
SECTION 4
The halogenated agents all cause increasing respiratory
depression as the concentration of the agent is increased. They
all cause a moderate (~20%) increase in PaCO2 that reflects an
increase in the rate of breathing, though, insufficient to offset a
decrease in tidal volume. Minute volume is reduced with all five
agents. Depression of ventilation reflects a direct depressant
effect on the medullary ventilatory center and perhaps
peripheral effects on intercostal muscle function. Bronchial
smooth muscle relaxation may be produced by a direct effect or
indirectly by reductions in afferent nerve traffic or central
medullary depression of bronchoconstriction reflexes.6 With
all five agents, respiratory depression is more evident when
opioids are used; assisted or controlled ventilation is usually
administered to avoid excessive hypercarbia. Hypercarbia in
relation to dysrhythmia potential can be more problematic with
halothane than with the other halogenated agents. With all five
inhalational agents, pulmonary exchange of oxygen becomes
less efficient, and an inspired oxygen concentration of 35% or
more is indicated. All produce blunting of hypoxic pulmonary
vasoconstriction, which can result in increased pulmonary
shunt flow of blood.
All five agents produce increase in secretions, coughing,
and laryngospasm, though halothane and sevoflurane are least
often problematic. This is why sevoflurane and the less costly
halothane are often employed in spontaneously ventilated
children and adult patients for induction of anesthesia. For
patients who tolerate an intravenous line at the start of
anesthesia, and no anticipated problems with endotracheal
intubation, intravenous induction is generally the method of
choice.6,10,19
Nervous system
Of the five agents, enflurane is associated with a higher
incidence of seizure activity. The seizures are short-lived and
self-limited and generally can be prevented by avoiding deep
anesthesia or hyperventilation. Interestingly, the drug does not
appear to aggravate seizures in epileptic patients, but avoidance
of enflurane is recommended for these patients. The halo-
198 genated agents have similar effects on the CNS. With the
halogenated agents, cerebral oxygen consumption is decreased.
There is also basal vasodilation, and cerebral blood flow is
increased whereas perfusion pressure remains constant. As a
result, intracranial pressure is increased. All effects are most
marked with halothane. The cerebrovascular system remains
responsive to carbon dioxide tension; with hyperventilation,
cerebral blood flow, metabolism, and intracranial pressure are
reduced.10,19
Muscular system
All five halogenated agents reduce the response of skeletal
muscle to nerve stimulation and enhance the neuromuscular
blocking effects of depolarizing and nondepolarizing muscle
agents. All five agents produce uterine vasodilation and a dose-
dependent decrease in uterine blood flow. The halogenated
agents have a direct muscle relaxing effect and appear to act
centrally as well as peripherally at the neuromuscular junction.
The halogenated agents potentiate muscle relaxants, and less
neuromuscular blocking agent is required. The least potentia-
tion occurs with halothane and nitrous oxide. Potentiation of
neuromuscular blocking drugs may involve desensitization of
the postjunctional membrane. Any of the three halogenated
agents can trigger malignant hyperthermia.6,10
Renal system
All five agents cause a dose-dependent reduction in renal blood
flow and glomerular filtration rate. The effects can be somewhat
attenuated by preoperative hydration and prevention of
hypotension. The changes in renal function are rapidly reversed
on conclusion of anesthesia and during recovery. The quantity
of fluoride released by metabolism is least with desflurane,
followed by isoflurane, and these agents are most frequently
used for patients with renal disease. Sevoflurane undergoes
oxidative metabolism in the liver with a serum fluoride con-
centration of ~22 mmol/L after a 1-MAC-hour exposure. The
magnitude of sevoflurane metabolism resembles that of enflu-
rane (peak plasma fluoride concentrations after a 2.5-MAC-
hour exposure to enflurane are ~20 mmol/L).6 When enflurane
is used in the presence of renal failure, concentrations of
fluoride ion decline rapidly after the anesthetic is discontinued.
It is postulated that much of the fluoride enters bone. It is
therefore probable that anesthesia with enflurane or sevoflurane
is safe for patients with renal disease.6,10,19
Gastrointestinal system
With halothane, enflurane, and isoflurane, and probably with
desflurane and sevoflurane, blood flow decreases with increasing
depth of anesthesia as systemic arterial pressure declines. There
is no evidence of direct ischemia. Hepatic necrosis has been
reported with repeated administration of enflurane. Hepatic
failure has not been reported with isoflurane. Isoflurane is less
metabolized by the liver when compared with enflurane and
halothane; this could be the reason why isoflurane is not linked
to hepatic failure. Halothane has been studied most extensively.
The diagnosis of halothane hepatitis is one of exclusion. The
pathologic appearance of hepatitis is similar whether the cause
is sensitivity to halothane, damage by some other hepatotoxic
drug, or transmission of hepatitis virus. The National Halothane
Study of 1966, a retrospective analysis of more than 850 000
administrations of anesthetics, suggested a small incidence of
hepatic necrosis in which there was no damage by some other
hepatotoxic drug, no transfusion of blood, and no evidence of
transmission of hepatitis virus or involvement of the liver by
some other disease process.
The incidence of halothane hepatitis appears to be low,
approximately one in 10 000 administrations for adults, and far
less for children. It often occurs after repeated administrations

of halothane over a short period. The unpredictable occurrence
of this syndrome may be the principal reason that halothane
use in adults has declined. More recent thinking indicates that
the inherent risks of the surgery involved, along with such factors
as major blood loss, major volume shifts, intraabdominal and
intrathoracic operations, and periods in which prolonged
hypotension may occur, may contribute to hepatic damage.
Furthermore, if hepatitis is caused by a halogenated agent, that
agent does not necessarily have to be halothane (enflurane and
isoflurane may also be involved). It is postulated that the
oxidative, and particularly the reductive, metabolites of these
inhalational agents are responsible for the hepatitis. A
chemically reactive or immunogenic product may result. This
excess of toxic product or metabolite may be capable of inducing
an immune response, which may be the main factor that leads
to hepatitis.6,10,19–22
Pharmacokinetics
Some 60–80% of halothane is exhaled in the first 24 h after it
is administered. Smaller amounts continue to be exhaled for
several days to weeks. Of the portion not exhaled, ~50% under-
goes biotransformation. The remainder is eliminated unchanged
via other routes. The cytochrome P-450 system of the endo-
plasmic reticulum of hepatocytes is responsible for the
biotransformation. Little fluorine is removed, but chlorine, and
to a lesser extent bromine, is removed. Analysis of the urine
shows the fluorine-containing compounds in the form of
trifluoroacetic acid.10
Approximately 80% of enflurane can be recovered unchanged
in expired gas. Of the remaining enflurane, 2–10% is metabo-
lized by the liver.23 A number of factors make enflurane, an
ether, different from halothane. The ether bond increases mole-
cular stability. The carboflurane bond is a higher-energy bond
than that between carbon and bromine or carbon and chlorine.
With the absence of bromine, and the presence of chlorine and
fluorine, the incorporation of the ether bond results in less
biotransformation of enflurane. Furthermore, because it is less
soluble than halothane in fatty tissue, enflurane leaves the fatty
tissue more rapidly in the postoperative period. This allows less
time for degradation of enflurane.10
Desflurane undergoes the least biotransformation, followed
by isoflurane, with 0.2% being metabolized.24 There is far less
liver metabolism than that for halothane, and less liver metabo-
lism than for enflurane and sevoflurane. The magnitude of
sevoflurane metabolism resembles that of enflurane. With less
biotransformation by liver metabolism, smaller quantities of
fluorine and trifluoroacetic acid are generated. This accounts for
hepatic and renal toxicity being lowest with desflurane and
isoflurane when compared with enflurane, possibly sevoflurane,
or halothane.6,10
Tables 18.7 and 18.8 summarize the advantages and disad-
vantages of the pharmacodynamic and pharmacokinetic
properties of the three halogenated agents.6,10
Nitrous Oxide
Nitrous oxide is a colorless and odorless gas with very low
solubility in blood. Nitrous oxide alone can predictably cause
surgical anesthesia only when given under hyperbaric con-
ditions. The MAC value is 105%, but variability among patients
is considerable. Analgesia can be induced with 20% nitrous
oxide; some patients lose consciousness when breathing 30%
nitrous oxide, and the majority do so with 80%. Using nitrous
oxide as a single agent at 80% concentration has risk of hypoxia.
Patients also often recall intraoperative events when nitrous
oxide is used alone. Even with nitrous oxide plus a narcotic,
intraoperative recall is not uncommon. If a combination of
narcotic, nitrous oxide, and muscle relaxant is used, the patient
Anesthetics
is immobilized and unable to communicate, but unconscious-
ness cannot be ensured. Because this can be unsettling to the
patient, frequently the clinician adds a potent inhalational
agent or intravenous drug such as a hypnotic or anxiolytic. The
main advantage of nitrous oxide is to reduce the needed
concentration of inhalational anesthetic. Smaller doses of
halogenated agents combined with nitrous oxide produce less
circulatory and respiratory depression and more rapid recovery.
The uptake of nitrous oxide is rapid, which has two beneficial
effects during the administration of anesthesiac: the concen-
tration effect and the second-gas effect.
When a very high concentration of an anesthetic is inhaled,
the partial pressure of the anesthetic in arterial blood increases
faster than if a smaller concentration of the anesthetic were
administered. As the anesthetic is rapidly taken up by the blood,
the gas administered by the anesthesia machine is rapidly drawn
into the alveoli, which continue to lose gas rapidly to the
passing blood. This is the advantage of using a high percentage
of nitrous oxide in the initial stage of anesthesia, and it makes
use of the concentration effect. The second-gas effect occurs
when a potent inhalational agent is combined with nitrous
oxide. As nitrous oxide is rapidly taken up by the blood from the
alveoli, and nitrous oxide in the alveoli is rapidly being replaced
by the anesthesia machine, the rate of delivery of halogenated
agent to the alveoli increases. Thus, the rise in arterial tension
of halogenated agents is more rapid.
To summarize, the concentration effect results from the
capacity of a rapidly absorbed gas to facilitate its own uptake.
CHAPTER 18
In the second-gas effect, a rapidly absorbed gas increases the
rate of uptake of the second anesthetic gas.19,25 During emer-
gence from anesthesia, the process is reversed. The possibility
of diffusional hypoxia is a concern because it can cause post-
operative hypoxemia, particularly if this is accompanied by
respiratory depression. As nitrous oxide rapidly comes out of
blood into the alveoli, oxygen concentration can be diluted. If
room air is used, nitrous oxide filling the alveoli from the blood
can bring the 21% oxygen concentration of room air down to
much lower levels, and hypoxia can result. This is why 100%
oxygen is administered during the emergence phase.10,19
In general, nitrous oxide has a sympathomimetic effect when
added to halogenated agents.10,26,27 The combined use of nitrous
oxide and halogenated anesthetic results in decreased amounts
of halogenated agents required and less hypotension.10 With
nitrous oxide combined with enflurane, activation of the sym-
pathetic nervous system is less marked than when nitrous oxide
is combined with halothane.27 When nitrous oxide is used alone
with narcotics, it does not displace sympathomimetic activity
but rather causes further cardiovascular depression. Nitrous
oxide has little effect on respiration when used alone, but it
further depresses respiration when combined with other
inhalational agents.10 Nitrous oxide has little effect on the CNS,
but response to hypoxia is diminished. Little, if any, skeletal
muscle relaxation occurs when nitrous oxide is used alone.10
There is no evidence that nitrous oxide triggers malignant
hyperthermia. The gastrointestinal, renal, and hepatic systems
show no effect from administration of nitrous oxide.10
Methionine synthetase, a vitamin B12-dependent enzyme, is
inactivated following prolonged administration of nitrous oxide,
which results in interference with DNA synthesis. This can
cause diminished bone marrow production of red and white
blood cells. Also, oxidation of the cobalt atom in vitamin B12 by
nitrous oxide can result in megaloblastic changes in the bone
marrow, with neuropathy. These changes do not normally occur
during clinical anesthesia for surgery.10
Nitrous oxide is excreted by the lungs, and there is little, if
any, biotransformation. Table 18.9 summarizes the advantages
and disadvantages of nitrous oxide.10 199
 PHARMACOLOGY AND TOXICOLOGY

TABLE 18.7. Pharmacodynamics of Inhalational General Anesthetics6,10

Organ System Effects Halothane Isoflurane Desflurane Sevoflurane

Cardiovascular
Peripheral vasodilation + ++ + +
Blood pressure – – – –
Inotropy – – – –
Heart rate – ++ =+ =
Cardiac output – = – –
Propensity for dysrhythmias ++ = = =
Catecholamines = = + =
Sympathoadrenal activity = = = =
Pulmonary
Bronchodilation + + + +
Response to hypoxia – – – –
End tidal CO2 + + + +
Shunt (Q/S) + + + +
Hypoxic pulmonary + + + +
vasoconstriction
Airway irritation + ++ ++ =
CNS
SECTION 4

Seizure activity = = = =
Cerebral blood flow +++ + + +
Cerebrospinal fluid pressure ++ = = =
Intracranial pressure ++ = + +
Cerebral metabolic rate – – – –
Muscle
Relaxation + + + +
Synergism with relaxants + + + +
Malignant hyperthermia + + + +
trigger
Renal
Renal blood flow – – – –
Glomerular filtration rate – – – –
Fluoride ion – + (minimal) + (minimal) + (minimal)
Hepatic/Gastrointestinal
Splanchnic blood flow – – – –
Hepatic cell function – – – –
Trifluoroacetic acid ++ + + –
=, no change; +, increase; – decrease.

TABLE 18.8. Pharmacokinetics of Inhalational General Anesthetics

Halothane Isoflurane Desflurane Sevoflurane

Metabolism 20% Liver 0.2% Liver <0.02% 5%

– – – – –
Ion concentration CL > Br > F F (minimal) F (minimal) F–
Elimination 60–80% via lung 99%+ via lung 99%+ via lung 95%
in first 24 h
200

TABLE 18.9. Advantages and Disadvantages of Inhalational Agents6,10
Agent Advantages
Nitrous oxide Nonirritating, colorless, odorless
Very rapid onset and recovery
Little or no toxicity with ordinary use
Excellent supplement with halogenated or opioid agents
(smaller doses of all agents and fewer complications)
Halothane Causes laryngospasm but is least irritating to airway
Bronchospasm uncommon
Controlled hypotension decreases blood loss
Isoflurane More rapid adjustment of anesthesia depth compared
with halothane
Cardiac output well maintained
Dysrhythmias less likely when used with epinephrine
compared with halothane
Potentiates muscle relaxants (lower concentration suffices)
Desflurane More rapid induction and emergence than isoflurane,
enflurane, or halothane
Minimal liver metabolism
No change in serum fluoride concentration
No coronary steal
Otherwise similar to isoflurane
Sevoflurane Less of an airway irritant; good for mask induction
More rapid induction and emergence than isoflurane,
enflurane, or halothane
No coronary steal
INTRAVENOUS AGENTS
Summary for Intravenous Agents
• Intravenous agents commonly used for induction of anesthesia
and on occasion may be used for maintenance of anesthesia
(total intravenous anesthesia)
• Commonly used agents are thiopental, propofol, etomidate,
and ketamine. Thiopental and Propofol are the most commonly
used agents for their ease of titration and favorable
pharmacokinetic profile
• Etomidate is favored for use in situations of hemodynamic
instability for its relatively low impact on hemodynamics. Its
prolonged use has been implicated in adrenocortical
suppression
• Ketamine has the advantage of maintaining spontaneous
respirations and is associated with hypertension
• Benzodiazepines are primarily amnestics and anxiolytics and
opioids are used primarily for analgesia in a balanced
anesthetic technique
Hypnotics
Barbiturates are not analgesics and may even increase sen-
sitivity to pain.2,10 Their main uses are induction of anesthesia
and induction of amnesia. The respiratory and cardiovascular
systems are depressed, and excessive doses may cause marked
hypotension and apnea. When barbiturates are used alone
without analgesia, it is not unusual to see tachycardia and other
sympathetic responses, including dilated pupils, tears, sweating,
tachypnea, and even movement or vocalization in response to
surgical stimulation (Table 18.10).10
When a barbiturate is administered for induction of general
anesthesia, coughing, laryngospasm, and bronchospasm can occur
Anesthetics
Disadvantages
No muscle relaxant activity
If used alone to achieve adequate anesthesia, can result in
hypoxia
Transient postanesthetic hypoxia may occur as large volume
is exhaled
Air pockets in closed spaces may expand in skull, chest,
abdomen
For proper analgesia, nitrous oxide or opioids usually must be
added
Relaxant drugs added for enhanced muscle relaxation
Visceral reflexes blunted with atropine
Transient dysrhythmias
Incidence of hepatic necrosis
More pungent odor than halothane
Increasing depression of cardiopulmonary function with
increasing depths of anesthesia
Coughing and excitement
Otherwise similar to isoflurane
CHAPTER 18
Serum fluoride concentration is similar to that of enflurane
upon mask ventilation or early attempts at laryngoscopy without
muscle paralysis. Saliva, insertion of an airway, obstruction by
soft tissues, and airway manipulation may trigger these responses.10
Thiopental is the most common induction agent used, fol-
lowed by methohexital sodium. Both cause a decrease in arterial
blood pressure and reduction of cardiac output. The clinician
must be careful when administering these agents in the
presence of hypovolemia, sepsis, or any kind of cardiovascular
instability, because a normal induction dose may result in
cardiac arrest.10
Extravascular injection may result in severe pain and tissue
necrosis. With intraarterial injection, the endothelium and deeper
layers of the arterial blood vessels can be immediately damaged
and endarteritis can follow. Associated thrombosis and arterial
spasm is common, which can result in vascular ischemia and
gangrene.10 Propofol is chemically unrelated to the barbiturates.
It is a propylphenol. The principal indication is amnesia and
unconsciousness, and the emergence from anesthesia is more
rapid with propofol than with thiopental. Emergence is
characterized by minimal postoperative confusion.10
Propofol can cause a 30% decrease in systemic arterial pressure
predominantly due to peripheral vasodilation. This can be of some
concern in the elderly, and one must be careful when adminis-
tering propofol in conjunction with opioids.10 There is some
pain at the site of injection, but phlebitis or thrombosis is rare.10
Benzodiazepines
Benzodiazepines can be used for induction, but these mainly
function as anxiolytics and amnestics. Larger doses of benzo-
diazepines can induce hypnosis and unconsciousness. Use of a
benzodiazepine as a sole agent is helpful when no analgesia is
required. The principal advantage of benzodiazepines is the 201
 PHARMACOLOGY AND TOXICOLOGY

TABLE 18.10. Intravenous General Anesthetics4,6,7,10

Agent Induction Dose Half-Life Organ Systems Effects Metabolism/Elimination

Thiopental 1–4 mg/kg t1/2 a: 3 min ŒCNS Liver/kidneys

tE: 5–10 h ŒCBF
ŒICP
ŒBP
ØHR
ŒRR
ØBronchospasm
Methohexital 1–2 mg/kg tE: 1–2 h ŒCNS Liver/kidneys
ŒRR
ŒCV
Midazolam 0.25–0.35 mg/kg tE: 1–4 h ŒCNS Liver/kidneys
Amnesia
ØSeizure threshold
Diazepam 0.1–0.5 mg/kg tE: 7–10 h; (active ŒCNS Liver/kidneys
metabolite: 2–8 days) Amnesia
ØSeizure threshold
Morphine 1–3 mg/kg tE: 2–4 h Analgesia Liver/kidneys
ŒCNS
Euphoria
ØRespiratory depression
Fentanyl 50–100 mg/kg T a: 1–2 min Similar to morphine, but chest wall Liver/kidneys
tE: 4 h rigidity more common with fentanyl
Ketamine IV loading dose t1/2 a: 10–18 min Poor visceral analgesia; good somatic Liver/kidneys
(LD): 1–3 mg/kg tE: 2.5 h analgesia
Maintenance dose: ØAirway reflexes
SECTION 4

1/3–1/2 LD ØHTN
ØIOP
ØCBF
ØCerebral metabolic rate
Propofol IV induction t1/2: 5–10 min ŒCNS Liver
2.0–2.5 mg/kg tE: 1–3 days ŒRR
IV maintenance ŒBP
100–200 mg kg–1 min–1
BP, blood pressure; CBF, cerebral blood flow; CNS, central nervous system; HR, heart rate; HTN, hypertension; ICP, intracranial pressure; IOP, intraocular pressure; RR,
respiratory rate.

TOTAL INTRAVENOUS ANESTHESIA

minimal depression of the cardiovascular system. Very large
doses, however, can cause a 20% decline in systemic arterial
blood pressure and vascular resistance. The stability of the
cardiovascular system with smaller doses has made these drugs
particularly attractive for use in monitored anesthetic care and
general anesthesia. One must be prepared for apnea, and
ventilatory support should be readily available. Benzodiazepines
generally have little effect on renal, hepatic, and gastrointestinal
systems. These do not produce neuromuscular paralysis, but
can be used to induce relaxation of spastic muscles.
CNS depression can be antagonized by physostigmine.
Physostigmine inhibits acetylcholinesterase. It crosses the blood–
brain barrier more easily than other acetylcholinesterase agents.
It is wise to consider administering atropine or glycopyrrolate
with physostigmine to prevent excessive salivation, abdominal
cramps, nausea and vomiting, and bradydysrhythmia.10
Opioids
Opioids are principally used for analgesia. In larger doses, opioids
can induce unconsciousness, but the common technique of
combining nitrous oxide and narcotic alone can result in
insufficient amnesia in some patients. Some patients become
hypertensive during surgical stimulation and may recall
202 intraoperative events. Table 18.9 reviews narcotic agents.7,10,28,29
With the advent of newer agents such as propofol, remifentanil,
and alfentanil, all of which have the desirable properties of
quick onset and short duration of action, traditional methods of
maintenance of anesthesia with inhalational agents has given
way to total intravenous anesthesia.
While the present cost of these drugs can be inhibitory, the
future is promising. Shorter stay in recovery and quicker
mobilization offset the initial costs. Also vaporizers and special
equipment for the delivery of these agents are not needed which
makes delivery of anesthesia outside of the OR more feasible.
LOCAL ANESTHETICS
Local anesthetics are a class of similar compounds that reversibly
block conduction in peripheral and central nervous tissue when
applied in appropriate concentrations. Local anesthetics cause
both sensory and motor paralysis in the innervated area by
blocking the generation and propagation of electrical impulses.
Nitrous oxide is usually used in conjunction with another
halogenated agent or in combination with narcotics for the
maintenance of anesthesia.
The era of local anesthesia commenced in 1864, when Koller
described the local anesthetic effect of cocaine and introduced it

for use in ophthalmology. Because cocaine, the alkaloid isolated
in 1860 from the leaves of an Andean mountain shrub,
Erythroxylon coca, has serious CNS toxicity and causes sloughing
of the corneal epithelium, its use in ophthalmology is limited.
This prompted the German chemical industry to seek less toxic
synthetic substitutes and resulted in the discovery in 1905 of
procaine, which became the prototype for current local anes-
thetics. The most widely used agents in ophthalmology today
are lidocaine, ropivacaine, and mepivacaine. Mepivacaine 2%
results in a good motor blockade, and can be used alone,
avoiding the toxic potential of the traditional mixture of lidocaine
and bupivicaine.
All clinically useful agents are either aminoesters or
aminoamides (Fig. 18.2). The amide or ester link contributes to
the anesthetic potency. The typical local anesthetic molecule,
exemplified by procaine and lidocaine, consists of a lipophilic
(hydrophobic) aromatic ring group joined to a more hydrophilic
base, the tertiary amine, by an intermediate band (Fig. 18.3).
Anesthetics
Increasing the size of the alkyl substitution produces com-
pounds that are more hydrophobic, thus increasing the duration
and potency of the agent.
MECHANISM OF ACTION
Local anesthetics block the generation and conduction of nerve
impulses. All excitable cells have ionic disequilibria across
semipermeable membranes, providing the potential energy for
impulse conduction. The Na+, K+-ATPase, the membrane-
bound enzyme, maintains the ionic disequilibrium in nerve
cells, pumping out three sodium (Na+) ions for every two of
potassium (K+) that are absorbed. During an action potential,
Na+ channels open briefly, allowing a small quantity of Na+ to
flow into the cell, causing depolarization. Local anesthetics block
impulses by inhibiting individual Na+ channels, thereby reduc-
ing the aggregate Na+ current, which may be modified by inhi-
bition of the recently discovered K+ channels.30–32 The interplay
FIGURE 18.2. Chemical structure of lidocaine
and procaine.
CHAPTER 18
FIGURE 18.3. Commonly used local
anesthetics in ophthalmology.
203
 PHARMACOLOGY AND TOXICOLOGY
between these competing channels determines the relative
potency of the various local anesthetics, whose pharmacologic
effects also depend on the temperature and pH of the medium.
Biochemical analysis of Na+ channels shows the presence of
one major glycoprotein with a molecular mass of ~200 000 Da,
with differing numbers of subunits of 40 000 Da, depending on
the tissue of origin. The Na+ channel is oriented with its
glycosylated groups of the glycoprotein on the outside surface of
the cell membrane.
Similarly, voltage-gated K+ channels make up a large molec-
ular family of membrane proteins involved in the generation
of nerve impulses. Like the proteins gating the Na+ channels,
these proteins span the cell membranes, forming K+-selective
pores that are rapidly switched open or closed, depending on the
membrane voltage. Recent cloning of the first K+ channel has
resulted in recombinant DNA manipulation of the K+-channel
genes, leading to a molecular understanding of K+-channel
behavior, especially toward elucidation of functional domains
responsible for channel gating and ionic selectivity. Local anes-
thetics act by several different mechanisms on ionic channels.
They may decrease the fraction of active channels by interfering
directly with activation; they may inhibit or alter the confor-
mational steps whereby channels change from an open form; or
they may reduce the ionic currents flowing through open
channels. In spite of various methods of detecting currents
through single-ion channels, the lack of general approaches for
crystallizing membrane proteins has prevented a direct view of
the structural complexities of their mechanisms.
SECTION 4
Recent work by Franks and Lieb33 suggests a more precise
theory of both local and general anesthetic action. Challenging
the well-entrenched ‘lipid hypothesis’, these authors suggest
that anesthetics operate not indiscriminately on membrane
lipids but precisely on certain sensitive membrane proteins
regulating ionic channels that govern the responses of nerve
cells. If the nerve cell’s anesthetic-sensitive proteins are isolated,
‘designer anesthetics’ could be synthesized to lock onto the sites
specifically in order to enhance an anesthetic’s sensitivity and
minimize its toxicity.
The chronology of local anesthetic action can be summarized
as follows:34
1. When local anesthetic molecules are deposited near the nerve,
partial removal of the molecules occurs by circulation, tissue
binding, and local hydrolysis of aminoester anesthetics. The
remaining molecules penetrate the nerve sheath.
2. After equilibrium is achieved inside the nerve axon’s
membranes, depending on the lipophilia of base and cation
species, Na+ channels are prevented from opening by
inhibition of conformational changes that occur with
channel activation.
3. The rates and onset of recovery from block are governed by
the slow diffusion of local anesthetic molecules in and out
of the nerve, not by the much faster binding and
dissociation from ionic channels.
CLINICAL PHARMACOLOGY
Successful ophthalmic anesthesia depends on knowledge of
the pharmacologic properties of commonly used local agents.
Aminoesters such as procaine are hydrolyzed in the plasma by
cholinesterase enzymes. The aminoamides, lidocaine and ropi-
vacaine, are extremely stable and undergo biotransformation
and enzymatic degradation in the liver. Allergic reactions to
aminoamides are extremely rare compared with reactions to
aminoesters.
For a local anesthetic to be successfully and safely used in
ophthalmic anesthesia, it must have potency, rapid onset of
204 action, long duration of sensory and motor block, and minimal
systemic toxicity. The individual profile of an agent is
determined mainly by its physicochemical characteristics.
In addition to the physicochemical properties, latency also
depends on the concentration. Lidocaine has a more rapid onset
of action than ropivacaine, and 0.75% ropivacaine causes a more
rapid anesthetic effect than 0.25% ropivacaine. Procaine has a
short duration of action, lidocaine an intermediate duration,
and ropivacaine the longest duration. Mixtures of local anes-
thetics, such as lidocaine and bupivacaine, have been popular
for ophthalmic anesthesia, because they combine the advantages
of rapid onset but short duration of action of lidocaine, and
slow onset but long duration of action of bupivacaine. For
example, a 2% solution of lidocaine mixed with equal parts of a
0.75% solution of bupivacaine produces anesthesia within 5 min
that lasts 3–4 h. At a concentration of 1:200 000, vasocon-
strictors such as epinephrine, mixed into the local anesthetic,
decrease the rate of vascular absorption and subsequent bio-
transformation. This allows more anesthetic agent to reach the
membrane receptors and prolongs the depth and duration of
anesthesia. With a judicious combination of lidocaine and bupi-
vacaine and a dilute vasoconstrictor such as epinephrine, the
duration of sensory and motor blockade is considerably
enhanced; this permits the ophthalmologist to perform compli-
cated intraocular procedures and minimize postoperative pain
and discomfort.
TOXICITY
The effectiveness and safety of local anesthetics depend on
proper dosage, correct administration, and preparedness for
emergencies. Systemic side effects, such as neurologic and
cardiac crises, are avoided by using the smallest effective anes-
thetic dose for a given procedure, thereby avoiding high plasma
levels and their associated effects. Unintentional intravascular
injection of local anesthetics can cause convulsions and respi-
ratory depression, and possibly arrest. Cardiovascular stimu-
lation or depression and cardiac arrest also may occur. Thus,
clinicians must be well versed in basic life support techniques
in order to manage toxic reactions due to local anesthetics. Ready
availability of oxygen and of cardiopulmonary resuscitative
drugs administered by a skilled anesthesiologist promotes rapid
and successful recovery.
Anesthetic solutions that contain epinephrine should be used
with extreme caution in patients with cardiovascular disease
such as hypertension, arteriosclerotic or cerebrovascular disease,
diabetes, heart block, or thyrotoxicosis. Patients taking medication
for systemic hypertension may also be more susceptible to
alterations in blood pressure.
DRUG INTERACTIONS
Cardiovascular arrhythmia may occur when local anesthetic
agents with epinephrine are used during general anesthesia with
halothane. Patients receiving monoamine oxidase inhibitors or
tricyclic antidepressants may experience severe and prolonged
hypertension with local anesthetics containing epinephrine,
thus vasoconstrictors are best avoided. CNS toxicity may occur
when local anesthetics are used in conjunction with narcotic
analgesics and phenothiazine-type compounds. In patients taking
echothiophate for control of glaucoma, inhibition of plasma
cholinesterases may result in increased plasma levels of local anes-
thetics and possibly cardiovascular and neurologic complications.
NERVE BLOCKS FOR OPHTHALMIC SURGERY
Retrobulbar block involves insertion of short-beveled needle
into the junction of the lateral and middle thirds of the inferior
 Anesthetics

orbital rim behind the globe. Complications include vasovagal

Summary for Local Anesthetics
reactions from fear and anxiety, ocular-cardiac reflex, retro-
• Local anesthetics cause sensory and motor paralysis in the
bulbar hematoma (most common), and direct trauma to the globe
innervated area by blocking the generation and propagation of
or optic nerve. Direct local anesthetic toxicity from intraarterial
electrical impulses
injection via the ophthalmic artery produces seizures.
• For a local anesthetic to be successful and safe when used in
Epidural/intrathecal injection via the optic sheath, produces a
ophthalmic anesthesia, it must have potency, rapid onset of
wide range of CNS side effects ranging from shivering,
action, long duration of sensory and motor block, and minimal
dysphagia, tachycardia, HTN, dilation of the contralateral
systemic toxicity
pupil, loss of consciousness, and respiratory/cardiac arrest.35
• The most widely used agents in ophthalmology are lidocaine,
Peribulbar block technique is performed by injection of
ropivacaine, and mepivacaine
local anesthetic above and below the orbit. Complications are
reduced as compared to the retrobulbar block, however, onset
time is slower, the incidence of incomplete anesthesia and
akinesia is greater, and globe perforation can still occur.

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anesthetist. JAMA 1963; 185:553.
2. Dripps RD, Eckenhoff JE, Vandam LD:
Premedication, transport to the operating
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Dripps RD, Eckenhoff JE, Vandam LD, eds.
Introduction to anesthesia: the principles of
safe practice. 6th edn. Philadelphia, PA:
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3. Firestone LL: General preanesthesic
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12. Eger EI, Saidman LJ, Brandstater B:
Minimum alveolar anesthetic concentration,
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Anesthesiology 1965; 26:756.
13. Eger EI: Anesthetic uptake and action.
Baltimore, MD: Williams & Wilkins; 1974.
14. Richter JJ: Mechanisms of general
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Stoelting RK, eds. Clinical anesthesia.
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25. Epstein RM, Rackow H, Salanitre E, Wolf
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Pergamon; 1990:269–284.
5. de Jong RH, Hearmer JE: Diazepam- and
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Anesthesiology 1973; 39:633.
6. Omoigui S: The anesthesia drugs
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Mosby; 1995:256, 296, 359–391.
7. Kofke WA, Firestone LL: Commonly used
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8. Nunn JF, Utting JE, Brown BR Jr:
Introduction. In: Nunn JF, Utting JE, Brown
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9. Calverley RK: Anesthesia as a specialty:
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10. Marshall BE, Longnecker DE: General
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11. Hickel RS: Administration of general
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17. Dripps RD, Eckenhoff JE, Vandam LD:
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CHAPTER 18
Nitrous oxide effects on the circulatory and
ventilatory responses to halothane.
Anesthesiology 1969; 31:250.
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205
 CHAPTER
19 Antibacterials
Harold G. Jensen, Henry D. Perry, and Eric D. Donnenfeld
FLUOROQUINOLONES
OVERVIEW AND MECHANISM OF ACTION
The fluoroquinolones are the newest class of antibacterials
available in the fight against microbes (Table 19.1). Fluoro-
quinolones are bactericidal agents that act by inhibiting DNA
replication. They have dual targets, topoisomerase II (DNA
gyrase) and topoisomerase IV, which are related but distinct
enzymes involved in DNA synthesis.1,2 By inhibiting bacterial
DNA gyrase and topoisomerase IV, DNA replication and tran-
scription are halted. Because DNA gyrase exists only in plant
and bacterial cells, fluoroquinolones have low toxicity in humans
relative to other antibacterial agents; the drugs will not affect
normal cell replication. The available topical agents include cipro-
floxacin, ofloxacin, levofloxacin, gatifloxacin, and moxifloxacin.
Ciprofloxacin and ofloxacin have been used in the treatment
of ocular infections for over 10 years. They are active against
most Gram-negative bacteria and some Gram-positive bacteria.
Levofloxacin is the L-isomer of ofloxacin and has demonstrated
increased activity against Gram-positive bacteria, but less potent
activity against Pseudomonas aeruginosa and certain Enterobac-
teriaceae. Gatifloxacin and moxifloxacin are the most recently
approved quinolones for ophthalmic infections. Earlier generations
of fluoroquinolones targeted only DNA gyrase, whereas the
newer fourth-generation agents, gatifloxacin and moxifloxacin,
target both DNA gyrase and topoisomerase IV.3
SPECTRUM OF ACTIVITY
Although all the quinolone agents have broad-spectrum activity,
gatifloxacin and moxifloxacin have demonstrated enhanced activ-
ity against Gram-positive organisms, especially Streptococcus
pneumoniae. Increased activity toward some of the atypical and
anaerobic organisms has also been shown with the newer fluoro-
quinolones. All the quinolones are generally active against enteric
Gram-negative rods such as P. aeruginosa, Haemophilus influen-
zae, and Neisseria gonorrhoeae. Gatifloxacin and moxifloxacin
have increased activity against most Staphylococcus aureus and
Staphylococcus epidermidis strains, but ciprofloxacin may have
slightly better activity against Pseudomonas aeruginosa. The beta-
hemolytic streptococcal and enterococcal sensitivities vary among
the older quinolones, but increased sensitivities are observed
with the newer agents.4 Bacterial resistance does not commonly
develop during treatment with quinolones for ocular infections;
however, it is still a possibility. Because quinolones target two
enzymes, bacterial resistance develops much slower with gati-
floxacin and moxifloxacin than with the older quinolones. Many
ciprofloxacin- and ofloxacin-resistant organisms are susceptible
to the two newer quinolones. Gatifloxacin and moxifloxacin are
more active than ciprofloxacin and ofloxacin against the atypical
Mycobacteria, including Mycobacterium avium-intracellulare,
Mycobacterium marinum, and Mycobacterium chelonei. Oflox-
acin and gatifloxacin have also shown activity against Chlamydia
trachomatis. In general, gatifloxacin and moxifloxacin have a
very comparable spectrum of activity against Gram-positive and
Gram-negative organisms. Gatifloxacin has minimally better
activity against Gram-negative bacteria, whereas moxifloxacin has
a minimally better spectrum of activity against Gram-positive
infections.
PHARMACOLOGY
The quinolones are well absorbed after oral or intravenous
administration and have variable pathways of metabolism and
excretion. The oral quinolones achieve systemic levels compar-
able to those of intravenous antibiotics because of their high
absorption and intrinsic solubility. After oral administration,
concentrations in serum peak after 1–2 h. The half-lives of
fluoroquinolones range from 3.5 h in ciprofloxacin to 20 h in
sparfloxacin, which allows for once- or twice-daily dosing. The
quinolones easily penetrate into phagocytes, thereby producing
concentrations within neutrophils and macrophages up to four-
teen times their concentration in the serum.5 This accounts for
their excellent in vivo activity against such intracellular patho-
gens as Listeria spp., Salmonella spp., and Mycobacterium spp.
Ofloxacin exhibits little or no in vivo metabolism, and is excreted
mainly (90%) via the kidneys. The other quinolones are cleared
by both hepatic and renal routes in various proportions, with
elimination occurring via the kidneys. Small amounts of these
drugs are also excreted in the bile.
OPHTHALMIC INDICATIONS
Ciprofloxacin, ofloxacin, and gatifloxacin are available in a 0.3%
commercial solution. Levofloxacin is available in both a 0.5%
and 1.5% solution, while moxifloxacin is available only as a
0.5% solution for ophthalmic use. Moxifloxacin is the only topical
solution without a preservative. All the ophthalmic quinolones
have labeled indications and have been shown to be effective for
the treatment of bacterial conjunctivitis.6–10 Ciprofloxacin,
ofloxacin, and levofloxacin have labeled indications for the treat-
ment of corneal ulcers and are particularly active against enteric
Gram-negative bacilli and quinolone-sensitive Pseudomonas spp.
In double-masked control clinical trials, ciprofloxacin and ofloxacin
were shown to be equivalent to fortified tobramycin and cefa-
zolin in the treatment of bacterial keratitis.11,12 Gatifloxacin has
shown excellent activity in both a human corneal ulcer study and
207
 PHARMACOLOGY AND TOXICOLOGY

TABLE 19.1 Available Ocular Antibacterials

Antibiotic Class Generic Name Trade Name(s) Effective Against

Fluoroquinolone Ciprofloxacin Cipro, Ciproxin Broad spectrum. All effective against Listeria spp.,
Ofloxacin Floxin Salmonella spp., and Mycobacterium spp.
Levofloxacin Levaquin
Gatifloxacin Tequin
Moxifloxacin Avelox
Tetracycline Chlortetracycline Aureomycin Certain Enterobacteriaceae, Vibrio spp.,
Oxytetracycline Terramycin Rickettsia spp., Mycobacterium marinum, and some
Doxycycline Ocular uses include Periostat, protozoans (e.g., Plasmodium spp., Entamoeba
Minocycline Vibramycin, Doryx histolytica)
Minomycin, Minocin,
Arestin, Akamin, Aknemin,
Solodyn, and Dynacin
Aminoglycoside Neomycin Maxitrol Staphyloccus aureus, Enterobacteriacae,
Gentamicin Garamycin P. aeruginosa, Acinetobacter spp.
Tobramycin Tobrex
Amikacin Amikin
Glycopeptide Vancomycin None – patent expired Methicillin-susceptible and resistant staphylococci,
Teicoplanin Targocid enterococci, Corynebacterium spp., Bacillus spp.,
Listeria monocytogenes, Clostridium spp.
Macrolides Erythromycin None – patent expired Gram + cocci, gram + bacilli, Neisseria spp.,
mycoplasmas, treponemes, rickettsiae, and
chlamydiae, Haemophilus influenzae,
Bartonella spp., Bacillus fragilis, Prevotella spp.,
Porphyromonas spp., Propionibacterium acnes,
Clostridium spp., M. Avium-intracellulare complex,
M. scrofulaceum, M. kansasii, M. chelonae
SECTION 4

Chloramphenicol Chloramphenicol Many; ocular uses include Chlamydia, mycoplasmas, rickettsia. Neisseria
Chloroptic and Chloromycetin meningitides, Haemophilus influenzae, most
Enterobacteriaceae. Many anaerobia bacteria are
inhibited at concentrations <10 µg/mL
Sulfonamides and Sulfonamide and Bactrim Active in vitro, but increasing resistance has limited
trimethoprim trimethoprim their efficacy. The combination of the two drugs
enhances their activity
Bacitracin and Bacitracin and None – patent expired Active against staphylococci and group A
gramicidin gramicidin beta-hemolytic streptococci. Some spirochetes,
Entamoeba histolytica, Actinomyces, and
Fusobacterium
Polymyxins Polymyxin Polysporin, Neosporin Pseudomonas spp., Serratia spp., Proteus spp.,
Providencia spp.

rabbit corneal ulcer studies against Pseudomonas aeruginosa and associated with crystal deposits in the cornea.17,18 This has not
Staphylococcus aureus. Moxifloxacin has also demonstrated been seen with the other topical quinolones, presumably due to
activity against these two organisms in a rabbit ulcer model.13–16 their higher solubility.

ADVERSE EVENTS TETRACYCLINES

Toxicity, fever, rash, and nausea occur in ~4% of patients given
oral quinolone therapy. On occasion, patients develop elevated
levels of liver enzyme. The drugs can crystallize in the urine,
especially in patients who are dehydrated. Interstitial nephritis
has been reported after high doses of ciprofloxacin. Insomnia
and restlessness have occurred in elderly patients taking fluoro-
quinolones. Studies with animals have shown quinolones to
cause irreversible cartilage erosions and skeletal abnormalities.
Therefore, although such effects have not yet been observed in
humans, quinolone use should be avoided in young children
until further research has been completed. There is no evidence
in humans for ocular toxicity with the new fluoroquinolones,
despite the fact that cataracts occurred in cats after months of
perfloxacin therapy, and macular bulla formation occurred in
patients with renal failure on flumequine (a quinolone used in
Europe). The topical administration of ciprofloxacin has been
208
OVERVIEW AND MECHANISM OF ACTION
Tetracyclines are broad-spectrum antibiotics that inhibit bacterial
protein synthesis by binding to the 30-S ribosomal subunit of the
bacteria. This prevents bacterial polypeptide synthesis. They are
bacteriostatic for most organisms. Various forms of tetracycline
are available, including chlortetracycline (topical), oxytetracycline,
doxycycline, and minocycline (see Table 19.1). Tetracyclines also
inhibit collagenase and polymorphonuclear leukocyte migration.
They also have an antilipase action, fostering the production of
long-chain fatty acids.19
SPECTRUM OF ACTIVITY
Tetracyclines are active against most Gram-positive organisms, cer-
tain Enterobacteriaceae, Vibrio spp. Rickettsia spp. Mycobacterium

marinum, and some protozoans such as Plasmodium spp. and
Entamoeba histolytica. They are not usually effective against
P. aeruginosa, Bacteroides species, or group B streptococci.
Organisms commonly acquire resistance to tetracycline via
plasmids, S. aureus resistance has climbed to ~40% in the
United States since the early 1990s. Due to increased resistance
rates, in vitro susceptibility testing is necessary to confirm the
activity of tetracycline against most organisms.
PHARMACOLOGY
Of the available tetracyclines, doxycycline has the best penetra-
tion into the eye. Ocular penetration of oxytetracycline and
chlortetracycline is hindered by the corneal epithelium and
therefore improved by the presence of a corneal defect. The
more lipophilic derivatives of tetracycline, such as minocycline,
appear to have better ocular penetration when administered
systemically than do derivatives such as chlortetracycline. These
drugs should be avoided in patients with renal failure, as they
are antianabolic and can speed the decline of renal function in
persons with chronic renal failure. Doxycycline is highly protein
bound, with a long half-life, so that it can be dosed once
daily. Doxycycline and minocycline can also be administered
intravenously.
OPHTHALMIC INDICATIONS
Topical tetracycline is indicated for the treatment of ocular tra-
choma and is used prophylactically for gonococcal ophthalmia
neonatorum. Oral tetracyclines are effective against several dis-
eases caused by chlamydia, including conjunctivitis, urethritis,
cervicitis, and pneumonitis. They may also be effective for Lyme
disease and nocardial infections. Minocycline has been used to
treat M. marinum infections. Tetracyclines have been shown to
be active in treating noninfectious corneal ulceration and acne
rosacea.20 Because of increased resistance to many of the common
Gram-positive and Gram-negative ocular pathogens, tetracycline
is not a common first-line antibiotic for most ocular bacterial
infections.
ADVERSE EVENTS
Tetracyclines have irritative effects on the upper gastrointestinal
tract, producing esophageal ulcerations, nausea, vomiting, and
epigastric distress. Hypersensitivity reactions are unusual, gen-
erally manifesting themselves as urticaria, fixed drug eruptions,
morbilliform rashes, and anaphylaxis. These drugs may cause
depression of bone growth, permanent discoloration of the teeth,
and enamel hypoplasia when given during tooth and skeletal
development.21 Therefore, these drugs are usually avoided in
children <8 years old and in women during pregnancy.
AMINOGLYCOSIDES
OVERVIEW AND MECHANISM OF ACTION
The aminoglycosides used in ophthalmology are generally lim-
ited to neomycin, gentamicin, tobramycin, and amikacin (see
Table 19.1). Aminoglycosides inhibit bacterial protein synthesis
by binding irreversibly to the bacterial 30S ribosomal subunit.
The aminoglycoside-bound bacterial ribosomes then become
unavailable for translation of mRNA during protein synthesis,
thereby leading to cell death. The aminoglycosides have a well
characterized ‘postantibiotic effect’, which means there is con-
tinued suppression of bacterial growth despite the decline of
antimicrobial concentration.
Antibacterials
SPECTRUM OF ACTIVITY
Aminoglycoside antibiotics are active primarily against aerobic
Gram-negative bacilli and Staphylococcus aureus. As a group,
they are particularly potent against the Enterobacteriaceae,
P. aeruginosa, and Acinetobacter spp. Although gentamicin and
tobramycin have similar antibacterial profiles, gentamicin is
more active in vitro against Serratia spp., whereas tobramycin is
more active against P. aeruginosa. Amikacin is often used as the
aminoglycoside of choice when resistance with gentamicin and
tobramycin is prevalent. In addition, amikacin is active against
many Mycobacterium spp. These agents are only moderately
active against Haemophilus spp. and Neisseria spp. They are
not active against anaerobes.
PHARMACOLOGY
Gastrointestinal absorption is low with oral aminoglycosides.
After intravenous administration they are freely distributed in
extracellular spaces, but do not penetrate well into the cerebro-
spinal fluid (CSF), vitreous, and biliary tract, even in the presence
of inflammation. In adults with normal renal function, the
aminoglycosides have half-lives in serum of ~2–3 h. The amino-
glycosides are excreted primarily by glomerular filtration. In
patients with renal failure, aminoglycosides accumulate and dosage
reductions are necessary.
CHAPTER 19
OPHTHALMIC INDICATIONS
Historically, the aminoglycosides have been a mainstay in the
treatment of ocular infections. However, increasing resistance
has limited their use in recent years. Gentamicin and tobramycin
are available as 0.3% topical solutions and ointments. Neomycin
is available only as a topical ointment and amikacin is not
available in a topical formulation. Either gentamicin or tobra-
mycin is often used as a fortified solution usually in addition to
one of the cephalosporins for the treatment of severe corneal
ulcers, especially those caused by Pseudomonas spp. The amino-
glycosides have shown a synergistic effect with the penicillins
and cephalosporins; however, penicillins may inactivate the
aminoglycosides if mixed together for topical application. Each
solution should be administered separately.
ADVERSE EVENTS
Nephrotoxicity and auditory or vestibular toxicity are the most
serious adverse events and are characteristic of all the amino-
glycosides. Neomycin is too toxic for parenteral administration,
and its use is limited to topical applications. Tobramycin and
amikacin are less ototoxic than gentamicin. Ototoxicity is a
result of selective destruction of the hair cells in the cochlea.
Approximately 2% of patients receiving systemic aminoglyco-
sides develop ototoxicity and half of these cases are irreversible.
Gentamicin and amikacin are more likely to be nephrotoxic than
tobramycin. Nephrotoxicity, which results from a high concen-
tration of aminoglycosides in proximal renal tubules, may pres-
ent as mild proteinuria to severe azotemia. As many as 26% of
patients receiving prolonged treatment with systemic amino-
glycosides develop evidence of mild renal impairment.22 The
likelihood of nephrotoxicity increases when cephalosporins or
other nephrotoxic drugs are coadministered with aminoglycosides.
Risk factors for nephrotoxicity and ototoxicity include long
duration of treatment, high aminoglycoside levels in the serum,
renal insufficiency and previous treatment with other ototoxic or
nephrotoxic drugs. Frequent dosing of fortified aminoglycoside
preparations used to treat bacterial keratitis can result in severe
209
 PHARMACOLOGY AND TOXICOLOGY
corneal epithelial toxicity. Occurrence of pseudomembranous
conjunctivitis is common with fortified topical gentamicin and
occasionally results from treatment with topical fortified
tobramycin.
GLYCOPEPTIDES
OVERVIEW AND MECHANISM OF ACTION
Vancomycin and teicoplanin are similar bactericidal antibiotics
which inhibit peptidoglycan synthesis in the bacterial cell wall
by complexing with cell wall precursors (see Table 19.1). Only
vancomycin is available in the United States, but teicoplanin is
available in many countries outside the United States.
SPECTRUM OF ACTIVITY
These are narrow spectrum antibiotics that are active primarily
against aerobic and anaerobic Gram-positive organisms, includ-
ing methicillin-susceptible and -resistant staphylococci, strepto-
cocci, enterococci, Corynebacterium spp., Bacillus spp., Listeria
monocytogenes, Clostridium spp., and Actinomyces spp. Teico-
planin is two- to fourfold more active than vancomycin against
most Gram-positive cocci.23 Increasing resistance of vancomycin
has been observed among clinical isolates of Enterococcus faecalis,
E. faecium,24 and coagulase-negative staphylococci.25 Cross-
resistance with teicoplanin is variable with these strains. Neither
vancomycin nor teicoplanin are active against Gram-negative
SECTION 4
organisms or mycobacteria.
PHARMACOLOGY
Although the glycopeptides can be administered orally or parent-
erally, the drugs are poorly absorbed after oral administration.
Because intramuscular administration is extremely painful, par-
enteral use is limited to intravenous administration. In patients
with healthy renal function, the glycopeptides are eliminated
from the body by glomerular filtration. The half-life of vanco-
mycin in serum is 6 h and ~45 h for teicoplanin. In patients
with severe renal insufficiency, their excretion may be prolonged
to ~9 days.
OPHTHALMIC INDICATIONS
Although there are no ocular formulations for vancomycin, it is
used for topical, subconjunctival, and intravitreal administration.
Vancomycin is frequently used for the treatment of infectious
corneal ulcers or endophthalmitis when Gram-positive organ-
isms are suspected. Initial treatment of serious ocular infections
with other antiinfectives is often changed to vancomycin when
culture results are proven to be a methicillin-resistant staphy-
lococcus or an Enterococcus spp. Intravenous, subconjunctival
and topical administrations do not result in detectable vitreous
levels. Intravitreal doses of 2 mg or less have been shown to be
nontoxic to the rabbit retina.26 To protect against increased bac-
terial resistance, use of vancomycin should be limited to sight-
threatening infections caused by Gram-positive organisms
resistant to other antibiotics.
ADVERSE EVENTS
The most frequent side effects of vancomycin are fever, chills,
and phlebitis at the site of infusion. Rapid infusion causes ting-
ling and flushing of the face, neck, and thorax, known as the red
man syndrome, as a result of histamine release by basophils
and mast cells.27 Vancomycin is also ototoxic and nephrotoxic
210
when given systemically. Ototoxicity is associated with high
serum levels of the drug and may result in permanent deafness.
The risk of ototoxicity and nephrotoxicity may be increased
when vancomycin is used in combination with aminoglycosides.
Hypersensitivity reactions, including fever, eosinophilia, urticaria,
and anaphylaxis may also occur.28 Subconjunctival injections
may cause conjunctival necrosis and sloughing. Topical admin-
istration has also been shown to retard epithelial wound healing
in rabbits.29
MACROLIDES
OVERVIEW AND MECHANISM OF ACTION
Until recently, erythromycin was the only macrolide formulated
for ophthalmic infections (see Table 19.1). However, clarithro-
mycin and azithromycin are derivatives that offer significant
advantages over erythromycin because of expanded antmicro-
bial spectra, improved pharmacokinetic parameters, and less
frequent and severe side effects. An ophthalmic formulation of
azithromycin 1.0% is currently being investigated for treating
bacterial conjunctivitis. Macrolides are generally bacteriostatic
agents that inhibit bacterial RNA-dependent protein synthesis.
They may be bactericidal in high concentration. The macrolides
bind reversibly to the 23S tRNA of the 50S ribosomal subunits
of susceptible organisms blocking peptide chain elongation.
SPECTRUM OF ACTIVITY
Macrolide antibiotics have a broad range of activity which includes
Gram-positive cocci, Gram-positive bacilli, Neisseria spp., myco-
plasmas, treponemes, rickettsiae, and chlamydiae. Clarithromycin
is more active against sensitive streptococci and staphylococci,
but cross-resistance does occur. Azithromycin is less active against
staphylococci and streptococci, and none of the three are active
against methicillin-resistant staphylococci. All are moderately
active against Neisseria gonorrhoeae; azithromycin is the more
active against Haemophilus influenzae. Both azithromycin and
clarithromycin are more active than erythromycin against Chla-
mydia spp. and are frequently used for systemic treatment of
the disease. The macrolides are among the most potent agents
against Bartonella spp., and have good activity against anaerobic
bacteria such as the Bacillus fragilis group, Prevotella spp.,
Porphyromonas spp., Propionibacterium acnes, and Clostridium
spp. Several of the atypical mycobacteria including M. avium-
intracellulare complex, M. scrofulaceum, M. kansasii, and
M. chelonae have also shown sensitivity to the macrolides.30,31
PHARMACOLOGY
Erythromycin is available in various topical, parenteral (lacto-
bionate and gluceptate), and oral (base stearate, ethylsuccinate,
and estolate) preparations. Erythromycin is rapidly inactivated
when administered orally, whereas the newer macrolides are stable
against acid degradation. Tissue distribution of macrolides is
excellent, with concentrations in various tissues 10- to 100-fold
higher than in serum. Erythromycin and clarithromycin are
metabolized by the liver and excreted primarily in the bile.
Azithromycin is excreted largely unchanged in the bile. Because
both azithromycin and clarithromycin have extended half-lives,
once a day dosing has been shown effective. Topical preparations
of erythromycin do not penetrate the cornea well, but are useful
for the treatment of conjunctivitis and blepharitis caused by
susceptible organisms. A topical ocular formulation of azithro-
mycin is being investigated within a delivery system (DuraSite,
Insite Vision, Alameda, CA) that remains in the eye for up to

several hours, which allows for sustained ocular penetration
and reduced dosing.
OPHTHALMIC INDICATIONS
The topical ocular formulation of erythromycin is used for con-
junctivitis and staphylococcal blepharitis. Because of its activity
against N. gonorrhoeae, erythromycin is used in many parts of
the world for prophylaxis of ophthalmia neonatorum. A primary
indication for oral erythromycin is for the treatment of Chlamydia
trachomatis infections in children. It is as effective as the tetra-
cyclines for chlamydial infections and is safer for pregnant women
and children under the age of eight. The topical ocular formula-
tion of azithromycin has been shown to be as safe and effective
as tobramycin based on clinical resolution and bacterial eradica-
tion in both pediatrics and adults with bacterial conjunctivitis.
In this study, azithromycin was dosed bid on days 1 and 2 and
qd on days 3–5, whereas tobramycin was dosed qid for 5 days.
Azithromycin in a single 1-g dose orally, or doxycycline at a dosage
of 100 mg orally twice per day for 7 days is recommended for
urogenital infections caused by chlamydia.32 Once-daily dosing
with azithromycin has also shown promising results in children
with ocular chlamydial infections in randomly selected
Ethiopian villages.33
ADVERSE EVENTS
Erythromycin is one of the safest antibiotics used. Side effects
are dose-related with gastrointestinal irritation including abdo-
minal cramps, nausea, vomiting, and diarrhea which occur pri-
marily with oral administration, but may also occur when given
intravenously. Side effects are similar with azithromycin and
clarithromycin; however, less nausea has been reported with the
newer drugs. Ototoxicity and reversible hearing loss may occur
with the use of large doses and high concentrations of the
macrolides.
CHLORAMPHENICOL
OVERVIEW AND MECHANISM OF ACTION
Chloramphenicol is a unique antibiotic molecule that contains
a nitrobenzene ring and was originally derived from Streptomyces
venezuelae (see Table 19.1). The drug is a bacteriostatic agent that
inhibits protein synthesis by binding reversibly to the peptidyl-
transferase component of the 50S ribosomal subunit and pre-
vents the transpeptidation process of peptide chain elongation.
SPECTRUM OF ACTIVITY
Chloramphenicol is active against many Gram-positive and
Gram-negative bacteria, chlamydia, mycoplasmas, and rickettsia;
however, the drug is often inactive against methicillin-resistant
Staphylococci spp. and is variably active against enterococci.
Neisseria meningitides, Haemophilus influenzae and most
Enterobacteriaceae are susceptible. Activity against Serratia and
Enterobacter isolates is variable and Pseudomonas spp. are usu-
ally resistant. Many anaerobic bacteria, including Bacillus fragilis
are inhibited at concentrations of <10 µg/mL.34
PHARMACOLOGY
Chloramphenicol is not absorbed in any significant amount
when applied topically, but it is rapidly and completely absorbed
from the gastrointestinal tract and peak serum levels are reached
in 2 h. It diffuses well into many tissues and body fluids, includ-
Antibacterials
ing CSF, where levels are generally 30–50% of the concentra-
tions in serum. Inactivated in the liver, active and inactive drug
are excreted rapidly in the urine.
OPHTHALMIC INDICATIONS
The use of chloramphenicol in the US has declined over the
past decades because of its potential for inducing severe systemic
adverse reactions, and the availability of newer antibiotics.
Outside the US, however, chloramphenicol drops continue to be
a commonly used and effective antibiotic for bacterial conjunc-
tivitis.35 Because of the risk of fatal idiosyncratic aplastic
anemia after topical administration,36,37 there should be careful
patient follow-up.
ADVERSE EVENTS
Bone marrow toxicity is the major complication of chloramphe-
nicol use. This may occur as either dose-related bone marrow
suppression or idiosyncratic aplastic anemia. Chloramphenicol
occasionally causes hypersensitivity reactions, including skin
rashes, drug fevers, and anaphylaxis. It should not be used with
other drugs known to produce hematologic side effects.
SULFONAMIDES AND TRIMETHOPRIM
OVERVIEW AND MECHANISM OF ACTION
CHAPTER 19
Sulfonamides competitively inhibit the bacterial modification of
p-aminobenzoic acid into dihydrofolate, and trimethoprim inhibits
bacterial dihydrofolate reductase. The sequential inhibition of
folate metabolism ultimately prevents the synthesis of bacterial
DNA. Because mammalian cells do not synthesize folic acid,
human purine synthesis is not affected by these agents. These
compounds act synergistically to enhance their spectrum of
activity.
SPECTRUM OF ACTIVITY
Sulfonamides are active in vitro against a variety of Gram-positive
and Gram-negative bacteria, actinomycetes, and chlamydia.
However, increasing resistance has limited their efficacy against
many of these organisms. Serratia marcescens, Pseudomonas
aeruginosa, enterococci, and anaerobes are usually resistant.
Trimethoprim is also active in vitro against many Gram-positive
cocci and Gram-negative bacilli. However, P. aeruginosa, most
anaerobes, Mycoplasma pneumoniae, Neisseria spp., Moraxella
catarrhalis, and mycobacteria are resistant. The combination of
these two drugs produces a synergistic effect greatly enhancing
the efficacy of either drug alone (see Table 19.1). Combinations of
trimethoprim with other agents such as rifampin, polymyxins,
and aminoglycosides have also demonstrated in vitro synergistic
antibacterial activity against many Gram-negative bacilli.
PHARMACOLOGY
Orally administered sulfonamides are absorbed quickly and com-
pletely from the gastrointestinal tract. They are metabolized in
the liver and excreted by the kidney. The sulfonamides are well
distributed with levels in the CSF and synovial, pleural, and
peritoneal fluids of ~80% of the levels found in serum. During
pregnancy, they cross the placenta and enter into the fetal
circulation. The antibacterial action of the sulfonamides can be
inhibited by blood, pus, and tissue enzymes, because the bac-
terial breakdown requirements of folic acid decrease in media
that contain purines and thymidine. Therefore, they should not
211
 PHARMACOLOGY AND TOXICOLOGY
be used for infections with marked purulent exudation. Orally
administered trimethoprim is absorbed almost completely in
the gastrointestinal tract. Peak levels are reached in the serum
in 1–4 h and are distributed widely in various body tissues,
including the kidneys, lungs, and prostrate. Its half-life in
serum is ~10 h in healthy subjects. About 80% is excreted in
the urine; the remaining 20% is excreted as inactive metabolites
by the kidneys or bile.
OPHTHALMIC INDICATIONS
A relatively high incidence of bacterial resistance has occurred
for individual use of the sulfonamides and trimethoprim, how-
ever a synergistic activity to each other and other antiinfective
compounds allow these combinations to be useful for ophthal-
mic infections. Sulfonamides are available in a topical formula-
tion as sulfacetamide. The formulation may also include a steroid
such as prednisolone for an antiinflammatory effect. Sulfisoxazole
can be used to treat chlamydial urethritis, and sulfacetamide
ophthalmic solution has shown efficacy for trachoma and inclu-
sion conjunctivitis. The sulfonamides are active against Nocar-
dia asteroides and show moderate activity against several of the
atypical mycobacteria, especially in combination with trimetho-
prim.38 Trimethoprim in combination with polymyxin B is used
as a broad-spectrum topical solution for adult and pediatric
bacterial conjunctivitis.24,39 The addition of polymyxin B to
trimethoprim makes the combination more effective against
Gram-negative organisms, especially Pseudomonas spp.
SECTION 4
ADVERSE EVENTS
The sulfonamides can produce a wide variety of side effects which
are common to the group. Crystalluria, hematuria, and anuria
have been associated as complications with systemic use of
these drugs. Anorexia, nausea, vomiting, and diarrhea are also
common side effects with systemic therapy. All routes of admin-
istration including topical may show hypersensitivity reactions,
including urticaria and rashes which may be accompanied by
pruritus and fever. Contact dermatitis is common with topical
application and have caused such problems as erythema nodosum,
erythema multiforme (Steven–Johnson syndrome), and exfolia-
tive dermatitis. Transient myopia has been known to occur with
systemic use.40 Myopia is usually bilateral, but the refractive
state usually returns to normal when the serum drug level
decreases. The most frequent adverse event from patients using
trimethoprim–polymyxin solution is local irritation, with tran-
sient burning or stinging, and itching following instillation.
Less than 2% of patients experience a hypersensitivity reaction
with this combination and there are no cross-allergic reactions
between trimethoprim/polymyxin B and the sulfonamides.
BACITRACIN AND GRAMICIDIN
OVERVIEW AND MECHANISM OF ACTION
Bacitracin and gramidicin are bactericidal peptide antibiotics
with similar activities against most Gram-positive organisms
(see Table 19.1). Bacitracin disrupts bacterial cell-wall synthesis
by inhibiting the dephosphorylation of a lipid pyrophosphate,
while gramicidin interferes more with cell membrane permea-
bility. Bacitracin may also act as a chelating agent.
SPECTRUM OF ACTIVITY
These drugs are primarily active against staphylococci and
group-A beta-hemolytic streptococci. Group C and G strepto-
212 cocci are less susceptible and group B is usually resistant.41
Neisseria spp. and Haemophilus influenzae may be susceptible
to bacitracin, but other Gram-negative organisms are resistant.
Some spirochetes, Entamoeba histolytica, Actinomyces, and
Fusobacterium have shown susceptibility to bacitracin.
PHARMACOLOGY
Bacitracin is limited to topical preparations for cosmetics, oph-
thalmic and cutaneous ointments, and solutions for wound
irrigation. Significant amounts of bacitracin are not absorbed
systemically when used as a topical preparation. There is poor
penetration through the cornea, which may be enhanced in the
presence of an epithelial defect. Large doses of bacitracin used
for wound irrigation may be associated with systemic toxicity.
OPHTHALMIC INDICATIONS
Bacitracin is generally combined with polymyxin B and zinc or
neomycin to provide a broad-spectrum antibiotic ointment for
ophthalmic infections. These ointments provide coverage for a
wide range of organisms implicated in conjunctivitis and staphy-
lococcal blepharoconjunctivitis. Although bacitracin is unstable
in solution, gramicidin is not and can be combined with poly-
myxin B and neomycin to have a similar broad-spectrum product
in solution form.
ADVERSE EVENTS
Systemic administration of bacitracin results in significant neph-
rotoxicity. Gramicidin is a potent hemolytic agent. Side effects
are rare when the drug is applied topically. It is generally non-
irritating to skin and mucous membranes, however recent
reports concerning allergic contact dermatitis and anaphylaxis
may limit its use as a dermatological antibiotic.42,43
POLYMYXINS
OVERVIEW AND MECHANISM OF ACTION
Polymyxins are a group of related cyclic basic polypeptides
originally derived from Bacillus polymyxa (see Table 19.1). Poly-
myxin E (colistin) was used to treat Pseudomonas spp. infections
prior to the advent of newer antibiotics, but now polymyxin B
is primarily used in formulations for ocular infections. These
bactericidal agents interact with the phospholipids of the bac-
terial cell membrane, which increases the cell permeability and
disrupts osmotic integrity. This process results in leakage of
intracellular constituents, leading to cell death.
SPECTRUM OF ACTIVITY
The polymyxins are active against most gram-negative bacilli,
especially Pseudomonas spp., Serratia spp., Proteus spp., and
Providencia spp. Gram-negative cocci, including Neisseria spp.,
are generally resistant, as are Gram-positive organisms. Cross-
resistance with other antibiotics has not been observed.
PHARMACOLOGY
The polymyxins may be administered parenterally, orally, or topi-
cally. They are not absorbed well when given orally or topically,
and intramuscular injections are usually painful. When used topi-
cally for ophthalmic infections they are poorly absorbed through
skin and mucous membrane surfaces. Polymyxin E is much less
irritating, but is about one-fourth as potent as polymyxin B. If
polymyxin B is used for irrigation of wound cavities or used in
subconjunctival injections, toxicity and necrosis may occur.

OPHTHALMIC INDICATIONS
Polymyxin B is generally used in combination products to pro-
vide the necessary Gram-negative coverage. Combinations with
trimethoprim, bacitracin, or neomycin are available commercially
for ophthalmic infections in either an ointment or solution
formulation. Combinations with an added antiinflammatory
agent are also available for more persistent ocular infections
such as staphylococcal blepharitis.
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2. Chen FJ, Lo HJ: Molecular mechanisms of
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Immunol Infect 2003; 36:1–9.
3. Blondeau JM: Expanded activity and utility
of the new fluoroquinolones: a review. Clin
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4. Gatifloxacin and moxifloxacin: two new
fluoroquinolones. Med Lett Drugs Ther
2003; 42:15–17.
5. Van der Auwera P, Matsumoto T, Husson M:
Intraphagocytic penetration of antibiotics.
J Antimicrob Chemother 1988; 22:185–192.
6. Gwon A: Ofloxacin vs tobramycin for the
treatment of external ocular infections.
Ofloxacin Study Group II. Arch Ophthalmol
1992; 110:1234–1237.
7. Power WJ, Collum LM, Easty DL, et al:
Evaluation of efficacy and safety of
ciprofloxacin ophthalmic solution versus
chloramphenicol. Eur J Ophthalmol 1993;
3:77–82.
8. Hwang DG, Schanzlin DJ, Rotberg MH,
et al: A phase III, placebo controlled clinical
trial of 0.5% levofloxacin ophthalmic
solution for the treatment of bacterial
conjunctivitis. Br J Ophthalmol 2003;
87:1004–1009.
9. Yee RW, Tepidino M, Bernstein P, et al: A
randomized, investigator-masked clinical
trial comparing the efficacy and safety of
gatifloxacin 0.3% administered bid versus
qid for the treatment of acute bacterial
conjunctivitis. Curr Med Res Opin 2005;
21:425–431.
10. Silver LH, Woodside AM, Montgomery DB:
Clinical safety of moxifloxacin ophthalmic
solution 0.5% (Vigamox) in pediatric and
nonpediatric patients with bacterial
conjunctivitis. Surv Ophthalmol 2005;
50(Suppl):S55–S63.
11. Hyndiuk RA, Eiferman RA, Delmar RC, et al:
Comparison of ciprofloxacin ophthalmic
solution 0.3% (Ciloxan) to fortified
tobramycin/cefazolin in the treatment of
bacterial corneal ulcers. Ophthalmology
1996; 103:1854–1863.
12. O’Brien TP, Maguire MG, Fink NE, et al:
Efficacy of ofloxacin versus cefazolin and
tobramycin in the therapy for bacterial
keratitis. Arch Ophthalmol 1995;
113:1257–1265.
13. Romanowski EG, Mah FS, Yates KA, et al:
The successful treatment of gatifloxacin-
resistant Staphylococcus aureus keratitis
with Zymar (gatifloxacin 0.3%) in a NZW
rabbit model. AJO 2005; 139:867–877.
14. Prajna V, Vajpayee R, Trocme S, et al:
Safety and efficacy of gatifloxacin 0.3% as
compared with ciprofloxacin 0.3% for the
treatment of acute bacterial, corneal ulcers.
ARVO 2006; B277 (poster #1916).
ADVERSE EVENTS
Systemic use of the polymyxins is limited due to possible severe
neural and renal toxicity. Dose-related renal dysfunction occurs
in ~20% of patients on appropriate therapy. Allergic reactions
including fever and skin rashes are rare, but may occur after
rapid intravenous infusion. Topical administration of polymyxin
B may cause hypersensitivity reactions, and chronic use may
lead to toxic conjunctivitis.
15. Jensen HG, Zerousala C, Carrier M, et al:
Comparison of ophthalmic gatifloxacin
0.3% and ciprofloxacin 0.3% in healing of
corneal ulcers associated with
Pseudomonas aeruginosa induced
ulcerative keratitis in rabbits. J Ocular
Pharm Ther 2005; 21:36–43.
16. Aliprandis E, Ciralsky J, Lai H, et al:
Comparative efficacy of topical
moxifloxacin versus ciprofloxacin and
vancomycin in the treatment of P.
aeruginosa and ciprofloxacin-resistant
MRSA keratitis in rabbits. Cornea 2005;
24:201–205.
17. Leibowitz HM: Clinical evaluation of
ciprofloxacin 0.3% ophthalmic solution for
treatment of bacterial keratitis. Am J
Ophthalmol 1991; 112:34S–47S.
18. Bower KS, Kowalski RP, Gordon YJ:
Fluoroquinolones in the treatment of
bacterial keratitis. Am J Ophthalmol 1996;
121:712–715.
19. Dougherty JM, McCulley JP, Silvany RE,
Meyer DR: The role of tetracycline in
chronic blepharitis inhibition of lipase
production in staphylococci. Invest
Ophthalmol Vis Sci 1991; 32:2970–2975.
20. Perry HD, Hodes LW, Seedor JA, et al:
Effects of doxycycline hyclate on corneal
epithelial wound healing in the rabbit alkali
burn model. Cornea 1993; 12:379–382.
21. Grossman ER, Walcheck A, Freedman:
Tetracycline and permanent teeth: the
relationship between doses and tooth
color. Pediatrics 1971; 47:567–570.
22. Smith CR, Lipshy JJ, Laskin OL, et al:
Double-blind comparison of the
nephrotoxicity and auditory toxicity of
gentamicin and tobramycin. N Engl J Med
1980; 302:1106–1109.
23. Gorzyski EA, Amsterdam D, Beam TR Jr,
Rotstein C: Comparative in vitro activities
of teicoplanin, vancomycin, oxacillin, and
other antimicrobial agents against
bacteremic isolates of gram-positive cocci.
Antimicrob Agents Chemother 1989;
33:2019–2022.
24. Eliopoulos GM: Vancomycin-resistant
enterococci: mechanism and clinical
relevance. Infect Dis Clin North Am 1997;
11:851–865.
25. Schwalbe RS, Stappleton JT, Gilligan PH:
Emergence of vancomycin resistance in
coagulase negative staphylococci. N Engl
J Med 1987; 316:927–931.
26. Plugfelder SC, Hernandez E, Fliesler SJ,
et al: Intravitreal vancomycin. Retinal
toxicity, clearance and interaction with
gentamicin. Arch Ophthalmol 1987;
105:831–837.
27. Polk RE, Healy DP, Schwartz LB, et al:
Vancomycin and the red-man syndrome:
pharmacodynamics of histamine release.
J Infect Dis 1988; 157:502–507.
Antibacterials
28. Kucers A: A good antimicrobial prescribing:
chloramphenicol, erythromycin,
vancomycin, tetracyclines. Lancet 1982;
2:425–429.
29. Petroutos G, Guimarres R, Pouliquen Y:
The effect of concentrated antibiotics on
the rabbit’s corneal epithelium. Int
Ophthalmol 1984; 7:65–69.
30. Barry AL, Jones RN, Thornsberry C: In vitro
activities of azithromycin (CP 62,993),
clarithromycin (A-56268, TE-031),
erythromycin, roxithromycin and
clindamycin. Antimicrob Agents Chemother
1988; 32:752–754.
31. Wallace RJ Jr, Swenson JM, Silcox VA,
Bullen MG: Treatment of non-pulmonary
infections due to Mycobacterium fortuitum
and Mycobacterium chelonei on the basis
CHAPTER 19
of in vivo susceptibilities. J Inf Dis 1985;
152:500–514.
31A. Protzko EE, Abelson MB, Shapiro AM; the
AzaSite Clinical Study Group: A randomized
trial assessing the safety and tolerability of
1.0% azithromycin ophthalmic solution vs
tobramycin in pediatric and adult subjects
with bacterial conjunctivitis. Presented at:
The association for research in vision and
ophthalmology annual meeting,
Ft. Lauderdale, FL, 30 Apr 30 4 May 4 2006.
31B. Abelson MB, Protzko EE, Shapiro AM; the
AzaSite Clinical Study Group. A randomized
trial assessing microbial eradication and
clinical efficacy of 1.0% azithromycin
ophthalmic solution vs tobramycin in
pediatric and adult subjects with bacterial
conjunctivitis. Presented at: The association
for research in vision and ophthalmology
annual meeting. Ft Lauderdale, FL, 30 Apr
to 4 May 2006.
32. Miller KE: Diagnosis and treatment of
Chlamydia trachomatis infection. Am Fam
Physician 2006; 73:1411–1416.
33. Chidambaram JD, Alemayehu W,
Melese M, et al: Effect of a single mass
antibiotic distribution on the prevalence of
infectious trachoma. JAMA 2006;
295:1142–1146.
34. Musial CE, Rosenblatt JE: Antimicrobial
susceptibilities of anaerobic bacteria
isolated at the Mayo Clinic during 1982
through 1987: comparison with results from
1977 through 1981. Mayo Clin Proc 1989;
64:392–399.
35. The Trimethoprim–Polymyxin B Sulfate
Ophthalmic Ointment Study Group:
Trimethoprim–polymyxin B sulfate
ophthalmic ointment versus
chloramphenicol ophthalmic ointment in
the treatment of bacterial conjunctivitis-a
review of four clinical studies. J Antimicrob
Chemother 1989; 23:261–266.
36. Fraunfelder FT, Bagby GC Jr, Kelly DJ:
Fatal aplastic anemia following topical
administration of ophthalmic 213
 PHARMACOLOGY AND TOXICOLOGY
chloramphenicol. Am J Ophthalmol 1982;
93:356–360.
37. Fraunfelder FT, Bagby GC Jr: Letter to the
editor. Ocular chloramphenicol and aplastic
anemia. N Engl J Med 1983; 308:1536.
38. Rodloff AC: In-vitro susceptibility test of
non tuberuculous mycobacteria to
sulphamethoxazole, trimethoprim, and
combinations of both. J Antimicrob
Chemother 1982; 9:195–199.
SECTION 4
214
39. Wagner RS: Results of a survey of children
with acute bacterial conjunctivitis treated
with trimethoprim–polymyxin B ophthalmic
solution. Clin Ther 1995; 17:875–881.
40. Rittenhouse EA: Myopia after use of
sulfanilamide. Arch Ophthalmol 1940;
24:1139–1143.
41. Finland M, Garner C, Wilcos C, Sabath LD:
Susceptibility of beta-hemolytic
streptococci to 65 antibacterial agents.
Antimicrob Agents Chemother 1976;
9:11–19.
42. Jacob SE, William JD: From road rash to
top allergan in a flash. Bacitracin Derm
Surg 2004; 30:521–524.
43. Dyck ED, Vadas P: Anaphylaxis to topical
bacitracin. Allergy 1997; 52:870–871.
 CHAPTER

20 Antivirals
Deborah Pavan-Langston and Thomas John

Viruses are obligate intracellular parasites that use the
metabolic processes of the invaded host cell. Therefore, a major
challenge in antiviral therapy has been formulating antiviral
drugs that do not interfere with the normal host-cell
metabolism by causing toxic side effects in the uninfected host
cells. Theoretically, antiviral drugs may be effective by
interacting directly with the virus, a virus-encoded enzyme or
protein, or a cellular receptor or factor required for viral repli-
cation or pathogenesis.1 To date, the most effective molecular
targets of antiviral treatment have been the viral enzymes and
proteins that play a role in the assembly of the virus. The
continuing search for new antiviral agents may result in the
development of drugs that are effective at one or more stages of
viral infection of the host cell, particularly the initial adherence
or adsorption of the virus to the host cells by electrostatic
interaction and receptors; viral penetration into the host cell
(e.g., by pinocytosis); release of viral nucleic acid by uncoating;
and replication, transcription, and translation of viral genome
within the infected host cell. The development of antiviral
drugs that are licensed currently for clinical use is the result of
an increased understanding of the molecular biology of viral
structures, enzymes, and replicative mechanisms and
virus–host-cell interactions. Although newer antiviral agents
are being introduced into the marketplace, continued research
in this field is required to provide better and safer antiviral drugs
in the future.
CLASSIFICATION OF VIRUSES
Viruses are made up of a nucleic acid core that contains either
ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) and is
surrounded by a protein-containing outer coat. The classi-
fication of a virus is based on the type of nucleic acid core (RNA
or DNA). Viruses can also be subdivided based on their
morphology, the site of viral multiplication (in the nucleus or in
the cytoplasm of the host cell), and serologic type.
The eye and adnexal structures may be directly infected by
RNA and DNA viruses or involved secondarily as part of a
systemic viral infection. Viral infections of ocular importance
are described in detail under ‘Viral Infections of the Cornea and
Anterior Segment’ and under ‘Retinitis’. The viral families are
outlined in Table 20.1.

TABLE 20.1. Virus Classifications

Classification Family Examples

RNA viruses Togaviridae Rubella virus (rubella, German measles)

Paramyxoviridae Measles virus (rubeola, measles); mumps virus (mumps, epidemic parotitis);
Newcastle virus (Newcastle disease)
Orthomyxoviridae Influenza virus (influenza)
Picornaviridae Enterovirus type 70 (acute hemorrhagic conjunctivitis, picornaviral hemorrhagic
conjunctivitis); coxsackie A24 virus (acute hemorrhagic conjunctivitis, picornaviral
hemorrhagic conjunctivitis)
Rhabdoviridae Rabies virus (rabies, hydrophobia)
Retroviridae Human immunodeficiency virus types 1 and 2 (HIV-1, HIV-2) (AIDS)
DNA viruses Herpesviridae Herpes simplex virus (HSV) types 1 and 2 (herpes simplex infection, ‘cold’ sores,
keratitis, genital infections, encephalitis); varicella-zoster virus (VZV) herpes virus 3
(chickenpox and shingles); Epstein–Barr virus (EBV) or herpesvirus 4 (infectious
mononucleosis, association with Burkitt’s lymphoma); cytomegalovirus (CMV) or
herpesvirus 5 (CMV disease, cytomegalic inclusion disease)
Adenoviridae Adenovirus types 3 and 7 (pharyngoconjunctival fever, acute follicular conjunctivitis);
adenovirus types 8, 19, and 37 (epidemic keratoconjunctivitis)
Poxviridae Molluscum contagiosum virus (molluscum contagiosum); vaccinia virus
(ocular vaccinia); variola virus (smallpox)

215
 PHARMACOLOGY AND TOXICOLOGY

UNITED STATES FOOD AND DRUG
ADMINISTRATION – APPROVED ANTIVIRAL
DRUGS
Twenty antiviral drugs are currently FDA approved for clinical
use. Half are for the treatment of HIV infections. The others are
for herpesvirus (e.g., herpes simplex virus (HSV), varicella
zoster virus and cytomegalovirus), hepatitis B virus, hepatitis C
virus, and influenza virus infections. Recent studies have
focused on antiviral therapies for viral infections that appear
amenable to antiviral drug treatment, as well as for viral
infections for which, to date, no antiviral drugs have been
approved (e.g., adenoviruses, human herpesvirus type 6, pox-
viruses, coronavirus, severe acute respiratory syndrome, and
hemorrhagic fever viruses).2 Gardasil (Merck), a vaccine against
human papilloma virus (HPV) was approved by the FDA in June
of 2006, and Glaxo-Smith-Kline is expected to see approval for
its HPV vaccine, Cervarix, in early 2007. Vaccines have also
been approved for human papilloma viruses related to cervical
carcinoma as well as for varicella (Varivax, Merck) and for
herpes zoster (Zostavax, Merck). The various types of antiviral
drugs are outlined in Table 20.2.3,4
This chapter reviews idoxuridine, trifluridine, vidarabine,
acyclovir, valacyclovir, and famciclovir, and discusses glan-
ciclovir, foscarnet, cidofovir, and bromovinyldeoxyuridine where
pertinent.5,6 The numerous anti-HIV agents include non-
nucleoside reverse transcriptase inhibitors, nucleoside/nucleotide
reverse transcriptase inhibitors, and protease inhibitors.
SECTION 4
HAART, which stands for highly active antiretroviral therapy.
HAART is extremely useful in boosting the body’s immune
response, thereby enhancing the efficacy of the antiviral defenses,
often without additional alternative antiviral therapy.
IDOXURIDINE (STOXIL, HERPLEX)
Idoxuridine (5-iodo-2„-deoxyuridine, IDU, Herplex), a
nucleoside analog of thymidine, was the first clinically effective
antiviral drug used as a topical ophthalmic preparation.7–10
Thymidine, a nucleoside found in DNA, has a methyl group at
the 5 position of the pyrimidine ring. In IDU the methyl group
is replaced by a single iodide substituent (Fig. 20.1). This
chemical substitution provides IDU with its antiviral property.
It replaces thymidine in the enzymatic step of viral replication.
Thus, IDU irreversibly inhibits the incorporation of thymidine
into viral DNA. This incorporation of the thymidine analog,
namely, IDU, into viral DNA renders the newly formed viral
particles noninfective.11,12 However, newer drugs (particularly
trifluridine) are more efficient and have higher efficacies;
therefore, idoxuridine is no longer commercially available.
TRIFLURIDINE (VIROPTIC)
Trifluridine (5-trifluoromethyl-2„-deoxyuridine, trifluorothy-
midine, F3T, Viroptic) is a fluorinated nucleoside analog of
thymidine. The methyl group at the 5„ position of the
pyrimidine ring of thymidine (see Fig. 20.1) is changed in F3T

Together, they are used in various combinations to make up such that each hydrogen of the methyl group is replaced by a
fluoride substituent (Fig. 20.1). This chemical change provides
F3T with its antiviral properties. TFT is a potent inhibitor of
Key Features: Virus Types and Antiviral Targets
thymidylate synthetase and therefore inhibits DNA synthesis.
• Viruses are classified based on their nucleic acid core: either
Trifluridine is incorporated into viral DNA directly, rendering
RNA or DNA. The core is surrounded by a protein outer coat
the viral particle noninfectious.13 However, its antiviral
and sometimes a triple membrane
mechanism of action is not fully known. In addition, F3T is also
• The most effective molecular targets of antiviral treatment are
incorporated into mammalian cells. It has exerted mutagenic,
the viral enzymes and proteins that play a role in viral assembly
DNA-damaging, and cell-transforming activities in various
• Twenty antiviral drugs are currently FDA approved for clinical
standard in vitro test systems. From a clinical standpoint, the
use, nine with proven efficacy in ocular viral disease:
significance of these test results has yet to be fully understood.
idoxuridine (IDU, Herplex), vidarabine (Ara-A, Vira A), trifluridine
Trifluridine is active against HSV types 1 and 2 and vaccinia
(TFT, F3T, Viroptic), acyclovir (ACV, Zovirax), famciclovir (FCV,
virus, both in vitro and in vivo. It also has an in vitro inhibitory
Famvir), and valacyclovir (VCV, Valtrex), and
effect against some strains of adenovirus.
bromovinyldeoxyuridine (BVDU, Brivudine). Ganciclovir
Trifluridine in a 1% solution is twice as potent and 10 times
(DHPG*, Cytovene), foscarnet (PFA, Foscavir), and HPMPC
more soluble than IDU.14–17 It is also lipid-soluble. The drug’s
(Cidovir). All but BVDU are FDA approved
biphasic solubility enhances corneal penetration by simple

TABLE 20.2. Antiviral Drugs

Antiviral Drug Abbrev. Brand Name Proven Efficacy in Specialized Roles FDA Approved in
Ocular Viral Disease One or More Forms3,4

Idoxuridine* IDU Herplex ✓ ✓

Vidarabine* ara-A Vira A ✓ ✓
Trifluridine TFT, F3T Viroptic ✓ ✓
Acyclovir ACV Zovirax ✓ ✓
Famciclovir FCV Famvir ✓ ✓
Valacyclovir VCV Valtrex ✓ ✓
Bromovinyldeoxy BVDU Brivudine ✓
uridine
Ganciclovir DHPG Cytovene ✓ ✓ ✓
Foscarnet PFA Foscavir ✓ ✓ ✓
Cidofovir HPMPC Vistide ✓ ✓ ✓
*No longer commercially available because other drugs offer greater convenience, or because of overlapping efficiencies.
216
 Antivirals

O O O
O NH3 FIGURE 20.1. Structures of thymidine and
N N antivirals.
N CH3 N I CF HN N
N From Pavan-Langston D: Ocular pharmacology of
H2N antiviral drugs. In: Tasman W, Jaeger E, Wilhelmus K,
O O N N N N
N N O N eds. Duane’s foundations of clinical ophthalmology.
HO
HOCH2 HOCH2 Philadelphia, PA: JB Lippincott; 2004:1–24.
HOCH2 HOCH2
O O O O

OH HO
PENCICLOVIR OH
OH H OH H OH H

THYMIDINE IDOXURIDINE TRIFLURIDINE VIDARABINE

O
N
N
5„-d-[G*C*G*T*T*T*G*C*T*C*T*T*C*T*T*C*T*T*G*C*G]-3„
sodium salt O H2N
O N N
N N
* - racemic phosphorothiate N N HOCH2
O
H2N H2N N N
N N
O O HOCH2
O HOCH2
HOCH2
HO P C OH O

OH
GANCICLOVIR
ACYCLOVIR OH H
FOSCARNET DEOXYGUANOSINE NH2

N
N
N
O O
N O
N H2N N
HN N
O HO O
CH3

CHAPTER 20
H2N P O
N N HO
O NM
CH2OCH2CH2OC C CH2(CH2)2 O CH3
OH
H
VALACICLOVIR O
CIDOFOVIR
FAMCICLOVIR
NH3
O H Br O H
N C C Br
C C
HN H H
MN
O
O N O
O N
O CH2
HO P CH2 CH
HO-H2C HO-H2C
OH O O
CH2 OH
HO

HPMPC OH HO
BV Ara U BVDU

diffusion.18 Trifluridine penetrates the intact cornea into the received F3T preoperatively.22 The passage of F3T through the
aqueous humor, and corneal penetration is further enhanced by human cornea without undergoing any significant metabolic
epithelial disruption. Experimentally, F3T is partly metabolized degradation has not been found to be therapeutically helpful in
to 5-carboxy-2„ deoxyuridine as the drug passes through the the treatment of herpes keratouveitis.
cornea, as evidenced by the presence of both F3T and 5-carboxy- Systemic absorption of F3T following therapeutic dosing
2„-deoxyuridine on the endothelial side. In a rabbit model of appears to be negligible. The half-life of F3T in serum is only
herpetic uveitis, topical F3T was shown to be effective because 12 min; therefore, it is ineffective as a systemic antiviral agent.
of its penetration into the anterior chamber.19 In another study The drug should not be used during pregnancy unless the
of rabbit herpes simplex keratouveitis, 1% F3T and 0.1% IDU potential benefits outweigh the potential hazards to the fetus.
had almost identical control of uveitis, keratitis, and Although it is unlikely that F3T is excreted in human milk after
conjunctivitis.20 The efficacy of topical 1% F3T was also ophthalmic use, it should not be prescribed for nursing mothers
demonstrated in rabbits with herpes simplex keratitis (HSK) unless the potential benefits outweigh the potential risks.
and may also be due to its intracorneal penetration property.21 TFT is used as an effective topical therapy for HSV
As in the experimental studies, intraocular penetration of keratitis.23,24 TFT not only interferes with the replication of
topical F3T has been shown to occur in humans.22 This HSV-1 and HSV-2, but also has an effect on vaccinia and certain
penetration of F3T into the aqueous humor may be enhanced in adenoviruses.25 TFT (0.2–1.7 mg/mL) inhibits the cytopathic
the presence of compromised corneal integrity and corneal effects of HSV-1 by 50% in plaque reduction assays.26 Plaque
stromal or uveal inflammation. However, unlike the in vitro formation was reduced by over 98% when HSV-1 grown into
results of ocular penetration of F3T, 5-carboxy-2„-deoxyuridine Vero cells was treated with 17 mg/mL TFT.27 TFT activity in
was not found in detectable concentrations within the aqueous vitro is comparable to IDU, and TFT is considerably more
humor at the time of penetrating keratoplasty in patients who active on a weight-for-weight basis than is vidarabine. As 217
 PHARMACOLOGY AND TOXICOLOGY
observed for both IDU and vidarabine, the strain of HSV-1
appears to be of major importance in determining the relative
antiviral efficacy. TFT was shown to inhibit five strains of
HSV-1 within a narrow range; however, the susceptibility of five
HSV-2 strains was variable, with two strains being insensitive
at the maximum nontoxic concentration.28
When TFT and IDU were compared with respect to their
ability to eradicate viruses from the preocular tear film, no virus
was recovered on days 2 and 4 of the 7-day treatment with TFT.
However, HSV-1 was present in IDU treated eyes throughout
the treatment regimen.29,30 Two days following discontinuation
of therapy, rebound virus shedding had occurred in both TFT
and IDU groups, with virus titers higher than those observed in
control, placebo-treated animals. These results indicate that a
critical time period exists in an acute herpetic infection during
which time continued presence of the antiviral is necessary to
control rebound virus shedding, even though infectious viruses
cannot be detected in the tear film.
Clinical studies comparing topical 1% TFT to 0.1% IDU
drops, to 3.0% vidarabine, and to acyclovir ointments have
shown that overall, the latter two drugs and TFT have efficacy
rates between 90% and 95%, even if steroids are in use.31–35 IDU
efficacy was only ~76%. There are several hypotheses for this
reduced efficacy: possible steroid use in some patients, perhaps
the agent has been in clinical use for so long that certain
organisms have become resistant, or patients may have become
allergic to the agent. While TFT had a slight edge over all other
drugs in the face of concomitant steroid therapy, no statistically
SECTION 4
significant difference could be shown.
The usual recommended dosage for infectious HSV dendritic
or dendrogeographic ulcers is nine times daily for 5 days and, if
the keratitis is improving, five times daily for a total of 2–3
weeks.3,5,6,24,36 This should produce a therapeutic response
within 2–4 days, and complete healing of more than 90% of
uncomplicated cases in 1–2 weeks. Atopic or immuno-
suppressed patients may take somewhat longer and need
combined oral and topical therapy. Patients with a history of
IDU treatment failure are usually responsive to TFT (87%).24,30
One study, however, demonstrated that IDU-resistant HSV is
cross-resistant to acyclovir, of intermediate resistance to TFT,
and fully sensitive to vidarabine and ganciclovir.
If healing has not occurred by 3 weeks, the possibility of a
toxic or trophic epithelial defect should be considered and
management should be changed. TFT should be stopped, and
lubricant antibiotic ointment, such as bacitracin or polymyxin-
bacitracin initiated TID. TFT’s toxic side effects may mimic
infectious disease; these side effects include follicular conjunc-
tivitis, superficial punctate keratitis (SPK), toxic epithelial ulcera-
tion, lacrimal punctal occlusion, anterior segment ischemia,
interference with wound healing, and true allergic blepharo-
dermatitis.33–35,37,38 Severe reversible ocular anterior segment
ischemia following topical F3T treatment for herpes simplex
keratouveitis has also been reported. Table 20.3 gives a more
complete listing of toxic reactions from topical antivirals. These
effects are rarely seen when the drugs are used for 2 weeks or
less and are reversible on cessation of their administration.39–41
Trifluridine is supplied as a 1% sterile ophthalmic solution
that should be refrigerated (2–8°C; 36–46°F). The preservative
in F3T 1% solution is thimerosal 0.001%.
VIDARABINE (VIRA-A)
Vidarabine (9-b-D-arabinofuranosyladenine) is a substituted
purine nucleoside previously known as adenine arabinoside
(Ara-A, Vira) (Fig. 20.1). Once widely available commercially for
topical and intravenous use, it is now available only through
218 compounding pharmacists for patients unable to use alternative
Key Features: Trifluorothymidine (TFT)
• TFT eyedrops not only interfere with the replication of HSV-1
and HSV-2 but also have an effect on vaccinia and certain
adenoviruses (DNA viruses)
• Clinical studies comparing topical TFT drops, vidarabine and
acyclovir ointments in HSV dendrogeographic keratitis have
shown that all three have efficacy rates between 90% and
95% regardless of whether steroids are in use
• If healing has not occurred by 3 weeks, the possibility of a toxic
or trophic epithelial defect should be considered. TFT should be
stopped and lubricant antibiotic ointment TID initiated
• TFT toxic side effects may mimic infectious disease; these include
follicular conjunctivitis, SPK, toxic epithelial ulceration, lacrimal
punctal occlusion, anterior segment ischemia, interference with
wound healing, and true allergic blepharodermatitis
TABLE 20.3. Topical Antiviral Ocular Toxicity
Site Toxicity
Cornea Fine punctate epithelial keratopathy
Filamentary keratitis
Indolent corneal ulceration
Perilimbal edema
Late superficial vascularization
Superficial stromal opacification
Conjunctiva Punctate staining with rose bengal or
fluorescein
Follicular conjunctivitis
Chemosis, congestion
Perilimbal edema
Conjunctival scarring
Lid margins Edema of meibomian gland orifices
Punctal edema and occlusion
Lids Ptosis
Allergic contact blepharodermatitis
Other Preauricular lymphadenopathy
antivirals. Along with TFT, it is recommended for therapy of
vaccinia blepharokeratoconjunctivitis.42
Vidarabine was the second antiviral drug developed for
human use.43 Researchers first synthesized the compound in
the early 1960s as a potential anticancer agent.44,45 It has
subsequently been obtained from fermentation cultures of
Streptomyces antibiotics.46 The mechanism of action of
vidarabine, although not fully established, appears to interfere
with the early steps of viral DNA synthesis and arrests the
growth of the viral deoxynucleotide chain. It is known that it is
not a completely selective antiviral agent. Although vidarabine
can affect normal cells, it is thought to be sufficiently safe for
systemic use. Vidarabine is rapidly deaminated to hypoxanthine
arabinoside (Ara-Hx). The principal metabolite, Ara-Hx,
possesses antiviral activity that is less potent than the parent
drug, vidarabine. Vidarabine is effective against herpes simplex,
varicella-zoster, and vaccinia (DNA viruses).47–49 It has a limited
range of activity against RNA viruses and no antiviral action
against adenovirus keratoconjunctivitis.50 Subepithelial corneal
infiltrates developed in both vidarabine-treated patients and
controls.
Because vidarabine is relatively insoluble, it is formulated as
a 3% ophthalmic ointment. The recommended dosage is five
times a day at 3-h intervals. Clinicians should consider other
forms of treatment if there is no clinical improvement after
1 week, or if complete corneal reepithelialization fails to occur
within 3 weeks. Following reepithelialization, an additional
week of treatment at a reduced dosage of twice daily should be

continued to prevent recurrence of infection. Vidarabine
treatment should not be continued for more than 3 weeks.
Vidarabine penetrates the aqueous humor better than IDU.
Two hours after topical application of 3% vidarabine in petro-
latum to rabbit eyes, aqueous levels of 6 mg/mL of the drug were
detected; 0.5% IDU failed to produce any detectable aqueous
levels.51 This is compatible with the clinical impression that
vidarabine treatment may be useful in herpetic uveitis.
Although vidarabine has been used intravenously in humans for
herpetic uveitis, this is not a popular mode of treatment.52
Vidarabine was also the first drug shown to be effective systemi-
cally in the treatment of herpetic encephalitis.53
Like other antiviral agents, vidarabine is not free from side
effects; a common one is corneal epithelial punctate kerato-
pathy.54,55 Other possible adverse reactions include foreign body
sensation, lacrimation, conjunctival hyperemia, burning,
irritation, pain, photophobia, sensitivity, and punctal occlusion.
Significant systemic absorption of vidarabine is not expected to
occur after topical ocular use. Animal trials have shown that
vidarabine is rapidly deaminated to its principal metabolite,
Ara-Hx, in the gastrointestinal tract. Although the chance of
fetal damage with ocular use of vidarabine during pregnancy is
remote, it is best avoided unless the potential benefit of therapy
justifies any potential risk to the fetus.
No significant difference was noted between vidarabine and
trifluridine in the treatment of herpes simplex dendritic corneal
ulcers.56,57 However, trifluridine was slightly more effective than
vidarabine in the treatment of herpes simplex geographic
corneal ulcers.56 A multicenter study involving 66 patients
compared the overall efficacy of 3% vidarabine ointment with
3% acyclovir ointment in the treatment of dendritic or geogra-
phic herpetic keratitis. No statistically significant difference
existed between the two medications with regards to healing rate,
the final visual acuity, the frequency of selected complications
such as punctate epithelial keratitis, or the development of
stromal keratitis.58 This is contrary to the earlier in vitro and
animal experiments, the results of which suggested that acyclovir
might be a more effective antiviral agent than vidarabine.59,60
Experimentally, vidarabine was compared with IDU to
evaluate which drug was less toxic to the corneal epithelium.61
The rate of rabbit corneal epithelial wound closure of 5- and
10-mm epithelial defects was not significantly different among
the eyes treated with 3% vidarabine, 0.5% IDU, and placebo
antibiotics, indicating that neither 3% vidarabine nor 0.5% IDU
retarded corneal epithelial wound healing.40,47,61 The quality of
the regenerated corneal epithelium as evaluated by slit lamp
was significantly better with vidarabine than with IDU.
However, 3% vidarabine and 0.5% IDU, and 1% TFT all
interfere with stromal healing to the same degree.
Vidarabine therapy may be useful in cases of IDU resistance.
In one study in which vidarabine 3% ointment was used to treat
56 cases of IDU-resistant HSK, 80% of epithelial herpes
keratitis cases and 52% of herpes stromal keratitis cases healed
within 2 weeks of treatment initiation.55 Others have also
Key Features: Vidarabine 3% Ointment
• Vidarabine is effective against herpes simplex, varicella zoster,
and vaccinia (DNA viruses)
• It is now available only through compounding pharmacists for
patients unable to use alternative antivirals
• In clinical trials no significant difference was noted between
vidarabine, trifluridine, or 3% acyclovir ointment in the
treatment of dendritic or geographic HSK
• Vidarabine side effects are generally mild but may include
corneal epithelial punctate keratopathy, punctal occlusion,
conjunctival hyperemia, irritation, photophobia, and lacrimation
Antivirals
shown vidarabine to be effective in many patients intolerant of
or resistant to IDU.62
ACYCLOVIR (ZOVIRAX)
Acyclovir (9-2-hydroxyethoxymethyl guanine, ACV, Zovirax), a
second-generation antiviral drug, is a synthetic purine
nucleoside analog derived from guanine. It differs from guanine
by the presence of an acyclic side chain.3,63,64 Acyclovir is used
against HSV and VZV in pill or liquid form, intravenously (IV),
and as a dermal ointment. It is also available as a 3%
ophthalmic ointment for HSV infections in Canada and Europe
and through compounding pharmacists in the United States.
There are multiple clinical uses of ACV. This important drug
is indicated or has been effective in the following conditions:
(1) primary genital HSV (PO or IV), (2) recurrent genital HSV in
immunocompetent patients (PO), (3) mucocutaneous HSV in
immunocompromised patients (PO or IV), (4) HSV encephalitis
(IV), (5) neonatal HSV (IV), (6) varicella in immunocompetent
(PO) or immunocompromised patients (PO or IV), (7) herpes
zoster in immunocompetent (PO) or immunocompromised
patients (IV or PO), and (8) possibly in EBV infections (PO). It
also has antiviral activity against EBV, herpes simiae (B virus),
and CMV but is infrequently used to treat these infections.
When used to treat HSV and VZV, acyclovir interferes with
DNA synthesis, thus inhibiting virus replication. In
herpesvirus-infected cells in vitro, the antiviral activity of
acyclovir appears to be dependent primarily on the intracellular
CHAPTER 20
conversion of acyclovir to acyclovir triphosphate. The
conversion of acyclovir to acyclovir monophosphate occurs
mainly via virus-coded thymidine kinase (TK). Acyclovir
monophosphate is phosphorylated to the diphosphate via
cellular guanylate kinase and to the triphosphate via other
cellular enzymes (e.g., phosphoglycerate kinase, pyruvate
kinase, phosphoenolpyruvate carboxykinase). In contrast,
acyclovir is only minimally phosphorylated by host cell
enzymes in uninfected cells in vitro. Because acyclovir has
antiviral activity against viruses that seem not to code for viral
TK (e.g., EBV and CMV), acyclovir is apparently converted to
acyclovir triphosphate by other mechanisms. However, research
suggests that acyclovir triphosphate is at least partially
produced within herpesvirus EBV- and CMV-infected cells; the
responsible cellular phosphorylating enzymes have not yet been
identified. The exact mechanisms of action against other
susceptible viruses are not fully understood.65–69
Acyclovir takes advantage of the subtle differences between
viral and cellular enzyme function in DNA synthesis. A slight
difference exists between the viral and cellular TK. Because
acyclovir is a nucleoside analog, it can function as a substrate
for viral TK but not for cellular TK. Therefore, acyclovir can
enter the sequence of DNA formation primarily in virus-
infected cells. The viral DNA polymerase more effectively
utilizes the acyclovir triphosphate than does the cellular DNA
polymerase. The viral DNA polymerase has a 10- to 30-fold
greater affinity in vitro for the acyclovir triphosphate than the
cellular a-DNA polymerase. When the acyclovir analog enters
the DNA chain, DNA synthesis is terminated. Thus, viral
DNA growth is more susceptible to acyclovir than the DNA of
uninfected host cells.70–73 Because of its poor uptake into these
cells, acyclovir has minimal pharmacologic effects in vitro on
the uninfected host cells; phosphorylation and intracellular
conversion to acyclovir triphosphate are minimal, and the
cellular a-DNA polymerase has a low affinity for acyclovir
triphosphate.
Acyclovir has been detected in the brain, kidney, saliva, lung,
liver, muscle, spleen, uterus, vaginal mucosa and secretions,
semen, cerebrospinal fluid, and herpetic vesicular fluid. 219
 PHARMACOLOGY AND TOXICOLOGY
Acyclovir diffuses into cerebrospinal fluid and crosses the pla-
centa. There is evidence that the drug is distributed into milk
via an active transport mechanism. Acyclovir is metabolized to
9-carboxymethoxymethylguanine (CMMG) and 8-hydroxy-9-
(2-hydroxyethoxymethyl) guanine. In in vitro herpesvirus-
infected cells, acyclovir is metabolized to acyclovir mono-, di-,
and triphosphate. The drug is excreted mainly in urine, via
glomerular filtration and tubular secretion.
Acyclovir is a crystalline white powder with a solubility of
1.3 mg/mL in water at 25°C. Commercially available acyclovir
sodium is a sterile, white, crystalline, lyophilized powder. At a
pH of 7.4 and 37°C, it is almost completely unionized and has
a maximum solubility of 2.5 mg/mL. Acyclovir capsules, pedi-
atric suspension, and the commercially available acyclovir sodium
sterile powder should be stored in tight, light-resistant containers
at 15–25°C. Reconstituted acyclovir sodium solution (50 mg
acyclovir/mL) is stable for 12 h at 15–30°C. Upon refrigeration,
a precipitate may form which will redissolve at room tempera-
ture. This precipitation and subsequent redissolution do not
appear to affect drug potency. Bacteriostatic water that contains
parabens should not be used for injection because this diluent
is incompatible with the drug and may cause precipitation.
Poirier and colleagues evaluated the intraocular penetration
of 3% acyclovir ointment, vidarabine monophosphate, and 1%
F3T drops following their administration to patients with normal
corneas before cataract extraction.74 The authors detected sub-
stantial levels of acyclovir in the aqueous humor, although only
meager levels of vidarabine monophosphate. In addition, no
SECTION 4
F3T was detected. Hence, 3% acyclovir may be superior to other
antiviral agents with regards to corneal penetration and in the
treatment of deep herpetic keratitis and uveitis. However,
acyclovir topical treatment did not significantly reduce the inci-
dence of stromal keratitis that developed with herpes simplex
epithelial keratitis.75
Three groups of rabbits with herpes simplex corneal infec-
tions were treated five times a day with 0.5% IDU, 3% vidara-
bine, or 3% acyclovir ointment. There was 50% less incidence
of severe iritis, epithelial loss, and conjunctivitis in the acyclovir
group compared with the other groups.76 Also, recoverable virus
levels on day 6 were much less in the acyclovir-treated rabbit eyes
compared with the other two groups. Acyclovir does not inter-
fere with corneal epithelial or stromal healing in rabbit eyes.
Pavan-Langston and associates compared the efficacy of acy-
clovir 3% ointment with vidarabine 3% ointment in the treat-
ment of patients with dendritic or geographic herpes keratitis.73
Within 2 weeks, more than 90% of the patients healed, with no
significant difference between the two drugs. However, herpes
dendritic corneal ulcers healed more rapidly when 3% acyclovir
was combined with debridement when compared with 3%
acyclovir alone (2 and 5 days, respectively).77 In its antiherpetic
effect, acyclovir is comparable to topical F3T.78
The use of oral acyclovir has had a revolutionary effect on the
treatment and prognosis of herpetic disease in every parameter
of the disease, in both immunocompetent and immunocom-
promised patients: genital herpes simplex infections, herpes
simplex encephalitis, acute herpes zoster (shingles), VZV
(chickenpox), and in mucosal or cutaneous herpes simplex
(HSV-1 and HSV-2) infections. In a study comparing oral acy-
clovir (400 mg five times daily) to 3% acyclovir ointment (five
times daily) in the treatment of herpes simplex dendritic corneal
ulceration, the authors found that healing occurred within
5 days in 89% of patients on oral acyclovir and in 97% of patients
on topical acyclovir ointment.79 Thus, oral acyclovir may be an
alternative to topical acyclovir ointment for the treatment of
herpes simplex dendritic lesions. In a controlled trial of oral
acyclovir versus placebo for 7 days with minimal wiping,
220 debridement in herpes simplex dendritic corneal ulcers was
carried out in 31 patients.80 At the end of treatment, the corneal
lesions had healed in 67% of patients receiving acyclovir and in
43% of patients given a placebo. Although there was no
significant difference in the proportion of corneal lesions that
healed in the two groups at 7 days, the rate of healing was
significantly faster in the acyclovir group.80,81 Jensen and
colleagues found that 3% topical acyclovir ointment was useful
both in epithelial and stromal herpes simplex corneal
infections. However, they also found that acyclovir ointment
was equally effective in herpetic keratitis in patients either
receiving debridement or no debridement.82
For ocular HSV, oral ACV 400 mg 5id is equivalent to topical
ACV in treating epithelial keratitis, with 90% of patients’ ulcers
healing in a mean of 5 days.75,79,83,84 Two hundred mg PO 5id
healed 18 of 19 patients with combined HSV epithelial and
stromal keratitis in 5–21 days.84 Other studies have confirmed
these findings.77–86 The therapeutic pediatric dosage is 20–
40 mg kg–1 day–1 for 7–14 days as a pediatric elixer (200 mg/tsp).83
A series of federally funded multicenter studies on the
efficacy of oral ACV and/or topical steroids on various forms of
ocular HSV were reported between 1994 and 2001.86–93 The
results may be briefly summarized as follows: (1) One year of
ACV 400 mg bid significantly reduced recurrence of herpetic
disease after resolution of any form of ocular HSV and without
rebound up to 6 months after ACV was stopped. (2) There was
no statistically significant benefit of ACV 400 mg 5µ/day for
10 weeks in treating active HSV stromal keratitis in patients
already on steroids and TFT. (3) Steroids were significantly
more effective than the placebo in resolving active stromal
keratitis, and postponing steroids slowed resolution, but had no
difference in outcome by 6 months. (4) Treatment of iritis with
ACV 400 mg 5µ/day for 10 weeks in patients already on steroids
and TFT may have some beneficial effect. (5) A 3-week course
of ACV 400 mg 5µ/day for epithelial keratitis in patients already
on TFT did not alter the subsequent incidence of stromal
keratitis or iritis. (6) ACV 400 mg bid for 1 year significantly
reduced the recurrence of HSV stromal or epithelial keratitis,
with greatest benefit in the stromal group. (7) Previous stromal
keratitis markedly increased the risk of the disease recurring.
Previous epithelial keratitis did not correlate with increased
recurrence. (8) Psychological stress, sun exposure, contact lens
wear, and systemic illness could not be shown to be triggers for
HSV reactivation. (9) The number of past episodes of either
epithelial or stromal keratitis was strongly associated with the
likelihood of a recurrence. (10) Long-term oral ACV signi-
ficantly lowers recurrence of either form of HSV keratitis, but is
more effective in preventing recurrence in stromal disease than
epithelial. It should be noted that prophylactic use of oral
antivirals is legitimate but defined as ‘off-label use’.
In a study of 105 HSK patients, Wu and Chen reported an
even more positive effect of prophylaxis on preventing recurrent
epithelial HSV in nonkeratoplasty patients than did the HEDS
study.94 Using low-dose ACV at 300 mg/day for 1 year, they
found a statistically significant difference between treated and
control groups: five recurrences of epithelial and one case of
stromal keratitis in the ACV group, and 14 cases of epithelial and
four cases of stromal keratitis in the untreated control group.
In their study on long-term oral acyclovir therapy in reducing
recurrences of dendritic or geographic HSK, Simon and Pavan-
Langston evaluated 13 patients with a history of frequently
recurring HSK (mean 27 months) during long-term oral
acyclovir.95 Eight were followed after the acyclovir was discon-
tinued. Treatment was given for an average of 34 months. During
treatment, the number of recurrences per month decreased from
0.15 to 0.03, and the average duration of relapses decreased
from 12.6 to 7.8 days (p < 0.01). Recurrences correlated with
daily doses of oral acyclovir (usually less than, but no more than

800 mg), intraocular surgery within 6 weeks of initiating
treatment, and discontinuation of therapy against medical
advice. This small study demonstrates that therapeutic doses of
oral acyclovir reduce both the rate and duration of recurrences
of infectious herpetic keratitis.
Additional indications for PO acyclovir in patients with
herpetic keratitis include use as an adjunct to topical antivirals
in patients with atopic disease or in immunosuppressed patients,
especially in AIDS patients. In AIDS patients, PO or IV therapy
is determined on the basis of severity of immunosuppression
(CD4+ helper lymphocytes less than 200 cells/mL, viral burden
> 105–7 plasma HIV RNA copies/mL).5,6,96–99 Dosage in atopy of
400 mg PO 5id for 2–3 weeks is generally quite effective.
Another indication for IV therapy is for patients who are unable
or unwilling to take topical antiviral agents for epithelial kera-
titis, such as those with crippling arthritis; children or uncoop-
erative adults; those whose occupation makes topical agents
difficult to use; and those with ocular toxic medicamentosa
from local antivirals.
Dosage, although not FDA approved for ocular use, in
nonatopic patients weighing over 50 pounds is 400 mg PO
tid–5id. For children who weigh less than 50 pounds, dosage is
20–40 mg/kg in divided dose for 7–10 days. Prophylaxis of HSV
epithelial recurrences in post-HSV keratoplasty patients with
PO ACV is effective and indicated.95,99,100 It is discussed in
further detail in the chapter on ‘Viral Infections of the Cornea
and Anterior Segment’ under ‘HSV Surgical Factors and
Management’.
ACV-resistant HSV strains have been isolated with greater
frequency from patients with AIDS. These strains do not
produce TK for drug activation.5,63,101,102 Alternative drugs for
treatment of ACV-resistant strains include vidarabine, which is
phosphorylated by cellular (rather than viral) TK, and foscarnet,
which requires no phosphorylation.103,104 Clinical experience is
limited with these alternatives, although some clinical success
has been reported.105 Sonkin and associates reported isolation of
an ACV-sensitive HSV with altered TK activity from a kera-
toplasty patient.106 However, the clinical course deteriorated on
treatment, and ACV-resistant HSV with deficient TK activity
was isolated. Despite foscarnet sensitivity, the graft eventually
failed. Foscarnet therapy of resistant HSV is discussed further in
the section titled ‘Foscarnet’.107
In a more recent study in the Netherlands, where ACV
is available over the counter, there were 542 isolates from 496
patients (410 HSV-1 and 132 HSV-2).108 Thirteen isolates
(8 HSV-1 and 5 HSV-2) from 10 patients (2%) were found
resistant to ACV. A single ACV-resistant strain was identified
among isolates from 368 immunocompetent patients (0.27%).
Resistant HSV was identified in nine isolates from 128 immu-
nocompromised patients (p < 0.05). None of the isolates were
resistant to foscarnet. This study indicates that the prevalence
of ACV resistance is low in immunocompetent patients
(0.27%), but relatively frequent in immunocompromised
patients (7%; p < 0.0001). Also, drug susceptibility monitoring
of HSV infections is essential in immunocompromised patients
with persisting infections, even with antiviral therapy.83 The
management approach to therapy of ACV-resistant HSV is
reviewed in the clinical section on ‘Ocular HSV in Immu-
nocompromised Patients’.
The role of ACV in herpes zoster ophthalmicus (HZO) is well
established for systemic use; however, because this drug is now
a second-line agent, famciclovir and valaciclovir are currently
the drugs of choice. Data are mixed for topical therapy, with
some studies reporting 5% acyclovir ointment 5id was highly
effective in resolving zoster epithelial keratitis and in
significantly reducing the incidence of recurrent disease. Other
studies showed topical steroids were useful in the management
Antivirals
of the inflammatory aspects of zoster ophthalmicus, but
showed no clear benefit of topical acyclovir ophthalmic
ointment when used alone.109 Zaal et al have reported that at
3 months post onset of HZO, patients who received 3% topical
ACV had longer duration of periocular lesions and significantly
more visual loss compared to the group receiving oral ACV, and
that all patients on combined topical ACV and dexamethasone
drops developed chronic disease.110
Because of the high complication rate in HZO, several
studies have been conducted to compare ACV to placebo or
other antiviral therapy. ACV 800 mg PO 5id for 7–10 days
induces prompt resolution of rash and pain, induces more rapid
healing, reduces the duration of viral shedding and reduces the
duration of new vesicle formation. There is also a significant
reduction in the incidence and severity of acute dendritiform
keratopathy, scleritis, episcleritis, and iritis. The incidence (but
not the severity) of corneal stromal immune keratitis, and the
incidence of late-onset ocular inflammatory disease (e.g.,
episcleritis, scleritis, iritis) was also reduced.111–115 The effect on
post herpetic neuralgia (PHN) was variable, with some reports
showing no efficacy, and others showing a notable decrease in
severity and incidence.111,114–117 As noted below, both
famciclovir and valaciclovir are superior in their effect on PHN.
Gastrointestinal upset, particularly diarrhea, is a distressing
side effect of ACV. This appears to be due to lactose intolerance,
which is relatively common in North American adults: ~75%
of Native Americans and blacks, 51% of hispanics, and 21% of
whites are lactose intolerant.118 ACV tablets contain lactose;
CHAPTER 20
intolerance to lactose is a common cause of intolerance to milk
and milk products in people who lack the intestinal enzyme
lactase. Manka has reported reversal of this oral ACV side-effect
by administration of oral lactase in the form of one Lactaid
caplet five times daily PO. Other systemic side effects of oral
acyclovir include nausea, vomiting, and headache. Less
common adverse reactions include dizziness, anorexia, fatigue,
edema, skin rash, leg pain, inguinal adenopathy, taste
perversion, and sore throat.
Acyclovir has also been used intravitreally experimentally
and clinically.119–121 Two patients with acute retinal necrosis
were treated with intravitreal infusion of acyclovir, vitrectomy,
and prophylactic scleral buckles; both patients had an
uneventful postoperative course and recovered visual acuity.
Key Features: Acyclovir (ACV, Zovirax)
• Oral ACV 400 mg 5id resolves 90% of infectious HSV
dendrogeographic corneal ulcers in a mean of 5 days.
Recommended effective dosages range from 400 mg PO tid to
5id with higher doses usually for immune-altered patients such
as excema or HIV+ patients
• The HEDS study key findings include: (1) 1 year of ACV 400 mg
bid significantly reduced recurrence of herpetic disease after
resolution of epithelial or stromal keratitis; (2) no effect on
active stromal keratitis and equivocal effect on iritis;
(3) previous stromal, but not epithelial, keratitis markedly
increased the risk of recurrent similar disease in the future; and
(4) the number of past episodes of either epithelial or stromal
keratitis was strongly associated with the likelihood of a
recurrence
• For zoster ophthalmicus ACV 800 mg PO 5id for 7–10 days,
induces significant resolution of rash, pain, new vesicles and
viral shedding, lower incidence and severity of acute and late
dendritiform keratopathy, scleritis, episcleritis, iritis, and the
incidence but not severity of stromal keratitis
• Poor effect on postherpetic neuralgia makes this drug now a
second choice for zoster infections, famciclovir or valaciclovir
being the drugs of choice
221
 PHARMACOLOGY AND TOXICOLOGY
VALACICLOVIR (VCV, VALTREX)
Valaciclovir (L-valine 2-(guanin-9-ylmethoxy)ethyl ester, VCV,
Valtrex) is a prodrug of acyclovir synthesized to enhance GI
uptake of ACV. It is hydrolyzed back to ACV, increasing the
bioavailability to five times that of ACV.122,123 Studies on the
ocular penetration of acyclovir and its prodrugs following sys-
temic administration revealed that the plasma bioavailability
for acyclovir, valacyclovir, and val-valacyclovir were similar, but
that anterior segment area under curve values were 53.70
(±35.58), 139.85 (±9.43), and 291.05 (±88.13) min µ mmol/L,
respectively. The mean residence time values were 46.47
(±24.94), 76.30 (±7.24), and 188.39 (±80.73) min, respectively.
This indicates that the valine and valine-valine ester prodrugs
of ACV penetrated the anterior segment of the eye significantly
better than acyclovir alone, probably via a carrier mediated
transport mechanism. Vitreous levels of the prodrugs were not
measurable.124 Viral susceptibility is similar to that of ACV.
Clinical studies comparing valaciclovir 1.0 g PO tid with
acyclovir 800 mg PO 5 µ day for 7 or 14 days in 1141
immunocompetent zoster patients (35 with HZO) revealed
drug-equivalency in acceleration of dermal healing and
reduction of duration of viral shedding. VCV was significantly
better in acute pain resolution and reduced duration of PHN
through 1 year of follow up.125,126 Data from 14 days of
treatment did not differ from that of 7 days. Studies on PHN
revealed that the median time to pain resolution was 38 days
with VCV and 51 days with acyclovir (p < 0.03). Other studies
SECTION 4
support the high efficacy of valaciclovir in herpes zoster,
particularly if started within 72 h of rash onset.127–131
In a study by Sozen et al of 30 eyes in 28 ocular HSV
patients, patients were randomized to receive either topical
ACV ointment or oral VCV. The corneal lesions healed
significantly faster in the oral VCV-treated eyes than in the
topical ACV-treated eyes. Symptoms were also lower in the
VCV group.132
Only one ocular study compared ACV with VCV. In 121
immunocompetent patients with acute HZO; an incidence of
keratitis, uveitis, and episcleritis was reported that was similar
in both groups.133 Neither group had any incidence of
neurotrophic keratitis or scleritis, and acute pain was noted in
~66% of each group. It was concluded that VCV was a valid
alternative to ACV in treatment of HZO, but as with
famciclovir (see further ahead), was superior in acute and long-
term pain inhibition and in patient compliance with only tid
dosing. The absence of neurotrophic keratopathy is in marked
contrast to this author’s experience, however.
For ocular HSV, VCV dosages have been adopted from genital
HSV data.3 Recommendations for acute genital HSV are VCV
1 g PO bid for 7–10 days. For recurrent genital HSV, VCV therapy
is 500 mg PO bid for 5 days and for suppression of recurrent
Key Features: Valaciclovir (VCV, Valtrex)
• VCV is a prodrug of acyclovir with enhanced GI uptake. It is
hydrolyzed back to ACV resulting in five times the
bioavailability of the latter drug. VCV is very effective clinically
in HSV-1, HSV-2, and VZV infections
• For ocular HSV VCV dosages have been adapted from genital
HSV data. Author-suggested doses for acute infections are
somewhat higher
• Clinical studies comparing valciclovir with acyclovir in
treatment of zoster ophthalmicus revealed drug-equivalency
but also that VCV was significantly better in acute pain
resolution and reduced duration of postherpetic neuralgia, thus
making it one of the two drugs of choice for zoster
222
episodes of HSV 500 mg to 1 g PO qd up to 1 year. The author
recommends 250 mg PO bid – tid for acute or recurrent epi-
sodes to add therapeutic leeway. Tolerance to valaciclovir, like
its active metabolite acyclovir, is generally good. Central neuro-
logical toxicity may be observed with high doses, but regresses
on withdrawal.134 Some severely immunocompromised HIV
patients have developed thrombocytopenic purpura/hemolytic
uremic syndrome, with a few deaths. As a result, this drug is
not FDA approved for use in immunocompromised patients,
but is approved for therapy of varicella zoster and genital HSV.
FAMCICLOVIR (FCV, FAMVIR)
Famciclovir (2-(acetyloxymethyl)-4-(2-aminopurin-9-yl)-butyl
acetate, FCV, Famvir), a third-generation nucleoside, is the
orally bioavailable diacetyl ester of the active antiviral,
penciclovir. It is similar to ACV in mechanism of action and
antiviral activity against HSV-1 and HSV-2, VZV, and
Epstein–Barr virus but superior in GI absorption: 77% versus
only 30% for ACV.135–139 FCV is metabolized to penciclovir
intracellularly, where it is active 10–20 times as long as ACV.
It inhibits viral DNA polymerase-mediated chain elongation.
The drug is FDA approved for treatment of herpes zoster
infection at doses of 500 mg tid for 7 days. It is preferable to
start treatment within 72 h of onset of rash. Clinical studies
indicate that famciclovir accelerates healing time as well as
ACV, but with less frequent dosing.140–144
For therapy of ocular HSV, VCV dosages have been adopted
from genital HSV data.3 For acute first episodes, it is 250 mg PO
tid for 7–10 days. For recurrent episodes, 125 mg bid for 5 days
is recommended. For the eye, however, 7–10 days at 250 PO
bid–tid is recommended by the author.
The efficacy and safety of long-term randomized, double-
blind, placebo-controlled famciclovir for suppression of
recurrent genital HSV (> 6 recurrences per year) revealed that
with doses of 250 mg PO BID for 52 weeks, a significantly
greater proportion of famciclovir-treated patients (151/191,
79%) were free from HSV recurrences at 6 months than placebo
recipients (48/184, 26%) (p < 0.001); efficacy was maintained at
12 months. Furthermore, the median time for the first clinically
confirmed lesional episode was significantly prolonged for the
famciclovir group (more than 1 year) compared with
the placebo group (59 days; p < 0.0001). Famciclovir was well
tolerated, with an adverse-experience profile comparable to
placebo.145
Prophylaxis of herpesvirus infections using any of the oral
antivirals usually involves preventing the recurrence of HSV,
but also on rare occasions to prevent complications of herpes
zoster in immunocompetent patients. In immunocompromised
patients, prophylaxis is used to prevent opportunistic virus re-
activation by HSV or VZV. The effectiveness of acyclovir 400
mg twice daily in preventing the recurrence of HSV eye disease
in immunocompetent patients has been well demonstrated in
HEDS. The issue of treatment duration for patients with highly
recurrent ocular herpes remains unresolved.146 In the author’s
personal experience ACV, VCV, and FCV are all comparable in
their efficacy as prophylactic antivirals.
FCV is a very effective drug in acute zoster. As with VCV, pre-
vention of PHN has occurred with antiviral therapy (famciclovir
500 mg PO tid or valaciclovir 1 g PO tid µ 7 days), started
within 72 h of onset of the rash, and with analgesic treatment.
However, the best adjunct for minimizing or even preventing both
acute zoster and PHN is a zoster vaccine (Zostavax, Merck)
recently approved by the FDA. In a double-masked study of
more than 37 000 patients, there was significantly lower inci-
dence (>55%) and severity (>60%) of both zoster and PHN.147

Key Features: Famciclovir (FCV, Famvir)
• FCV, the diacetyl ester of an ACV relative, penciclovir, is similar
to ACV in mechanism of action and antiviral activity against
HSV-1 + HSV-2, and VZV but superior in GI absorption and
intracellular half-life
• For ocular HSV FCV dosages have been adopted from genital
HSV data. Author-suggested doses for acute infections are
somewhat higher
• Progress in prevention of PHN has been made with FCV
antiviral therapy started within 72 h of onset of the rash, and
analgesic treatment
GANCICLOVIR (DHPG, GCV, CYTOVENE)
Ganciclovir (9-(1,3-dihydroxy-2-propoxy(methylguanine)),
DHPG, GCV, Cytovene), a synthetic purine nucleoside analog
of guanine, is structurally and pharmacologically related to
acyclovir. It differs from acyclovir only by a second terminal
hydroxymethyl group at C-2 of the acyclic side chain on the
ribose ring.148 This structural difference contributes to the
substantially increased antiviral activity of ganciclovir against
CMV and in less selectivity for viral DNA. Although ganciclovir
has antiviral activity both in vitro and in vivo against various
herpesviridae (herpes simplex types 1 and 2, human herpesvirus
type 6, EBV, and VZV), its main clinical use has been against
human CMV.
The exact mechanism of action of ganciclovir is not fully
known. It appears to exert its antiviral effect on human CMV
and other human herpesviruses by interfering with DNA
synthesis via competition with deoxyguanosine for incor-
poration into viral DNA, and by incorporation into growing
viral DNA chains.149–151 The formation of ganciclovir
monophosphate appears to be the rate-limiting step in the
formation of ganciclovir triphosphate. In contrast to acyclovir,
which is only minimally phosphorylated by cellular (host cell)
enzymes, ganciclovir seems to be more susceptible to phos-
phorylation by enzymes in uninfected cells, especially in rapidly
dividing cells (e.g., bone marrow). This phosphorylation in
uninfected cells can range from less than 10% to being equal to
that in virus-infected cells. Unfortunately, this also makes the
drug more toxic to the bone marrow, causing a significant
neutropenia in more than 50% of patients treated. Other less
frequent side effects include nausea, neurotoxicity, hepatic
dysfunction, fever, and local rash or phlebitis (DHPG = pH 11).
DHPG is also carcinogenic, teratogenic, and induces azoo-
spermia.
The phosphorylated form of ganciclovir that is active can
competitively inhibit viral DNA polymerase and can also be
incorporated into growing DNA chains as a false nucleotide.
This results in the termination of DNA synthesis and in the
formation of a mutant DNA chain, and thus inhibition of viral
replication. Although the drug inhibits cellular a-DNA poly-
merase, it requires a higher concentration than that required to
inhibit viral DNA polymerase. The increased antiviral effect of
ganciclovir against CMV compared with acyclovir has been
attributed to slower catabolism of ganciclovir triphosphate by
intracellular phosphatases. The drug does not code for TK and
is, therefore, of use in TK-resistant HSV and VZV strains.
As with all other antivirals, ganciclovir is virostatic rather
than virucidal.5 Because it is only virostatic, continuous therapy
with the IV drug is necessary to prevent viral breakthrough in
the immunosuppressed patient. However, despite careful
management, ~40% of patients ultimately experience reactiva-
tion of disease. Experimentally, when the drug is removed from
culture medium in vitro, previously inhibited viral DNA
Antivirals
synthesis resumes with restored viral replication. Additional
data supporting ganciclovir as virustatic come from histo-
pathologic studies of enucleated globes from patients who died
while receiving ganciclovir therapy.152,153 These studies showed
that ganciclovir does not eliminate CMV from the retina, nor
does it suppress expression of all viral genes.
Because ganciclovir is poorly absorbed from the gastro-
intestinal tract, intravenous administration is preferred.
Ganciclovir is 1–2% bound to plasma proteins. Although the
tissue distribution of ganciclovir is not fully known, autopsy
studies on patients who received intravenous ganciclovir
suggest that the drug concentrates mainly in the kidneys with
lower concentrations in the liver, lung, brain, and testes.154 The
drug appears to have good ocular distribution following
intravenous administration; concentrations in the aqueous and
vitreous humors 2.5 h after intravenous administration were,
respectively, 0.4 and 0.6 times the simultaneous plasma
concentration of the drug.155 Ganciclovir crosses the blood–
brain barrier. It is unknown whether ganciclovir is distributed
into human milk; however, no drug is present in the milk of lab
animals. It also crosses the placenta in lab animals. The
primary route of excretion is in urine, and it appears to be
mainly via glomerular filtration. Except for intracellular phos-
phorylation of the drug, it is not significantly metabolized in
humans and is mainly excreted unchanged in the urine.
As noted, the primary clinical use of ganciclovir is in the
treatment of CMV retinitis in immunocompromised patients,
especially those with AIDS. The safety and efficacy of the drug
CHAPTER 20
have not been established for congenital or neonatal CMV
disease, for the treatment of other cytomegaloviral infections,
such as pneumonitis or colitis, or for use in nonimmuno-
compromised individuals. The intravenous route of ganciclovir
therapy has been shown to be effective in the treatment of
cytomegaloviral retinitis in immunocompromised patients.156–162
However, because ganciclovir is only suppressive against CMV
– it does not result in increased immunocompetence – the
retinitis will recur or progress following cessation. After induc-
tion therapy with ganciclovir for CMV retinitis and discon-
tinuation of the drug, relapse of CMV usually occurs within
4 weeks in immunosuppressed patients. Hence, for the dura-
tion of the patient’s immunosuppression, long-term main-
tenance therapy and intermittent induction therapy seem to be
necessary. The advent of HAART for the treatment of AIDS
itself, however, has greatly reduced the number of cases of CMV
retinitis in the past few years.163
The most common dose-limiting adverse effect of ganciclovir
is neutropenia (absolute neutrophil count < 1000/mm3), which
is potentially fatal. Usually, interruption of ganciclovir therapy
or a decrease in dosage results in increased neutrophil counts.
Thrombocytopenia (platelet count < 50 000/mm3) can also
result from a direct, dose-dependent effect of the drug. Less
commonly, anemia and eosinophilia can occur. Ocular side
effects include rhegmatogenous retinal detachment as a result
of ganciclovir-induced resolution of retinitis. As a result,
ganciclovir has also been administered intravitreally in patients
with CMV retinitis.164–169 It was found to be effective and safe
both as an alternative to intravenous ganciclovir therapy in
myelosuppressed patients and as a supplement to intravenous
therapy in uncontrolled CMV retinitis.170
Ganciclovir may also have a topical therapeutic role. Two
randomized HSV clinical trials have been carried out in Africa
and Europe comparing ganciclovir 1.5% gel with 3% ACV
ointment in treating herpetic keratitis in 107 patients.171 There
was no statistically significant difference between the treatment
groups, although the group receiving 0.15% ganciclovir gel had
healing rates of 85% compared with 72% in the group receiving
223
 PHARMACOLOGY AND TOXICOLOGY
acyclovir ointment. Local tolerance was superior with the gel
formulation of ganciclovir with fewer complaints of discomfort
(stinging, burning) or blurred vision after application. Systemic
absorption of the drug was low and no hematologic changes
were detected.
The drug should be stored at room temperature and should
not be exposed to temperatures greater than 40°C. Recon-
stituted ganciclovir sodium solution with sterile water for
injection (ganciclovir 50 mg/mL) is stable for 12 h at 15–30°C
and should not be refrigerated, as a precipitate may form. To
avoid precipitation, bacteriostatic water for injection containing
parabens should not be used to reconstitute ganciclovir sodium.
Oral ganciclovir and ganciclovir implants are effective
alternative routes of drug administration. Oral ganciclovir is
valganciclovir (Valcyte), which has a much higher GI absorption
than its prodrug form. As a result, it may be given in
therapeutically effective doses for treatment of CMV retinitis.
Dosage is 900 mg PO bid for 3 weeks, then 900 mg PO qd.
Myelosuppression and CNS or liver toxicity are potential side
effects.172
The ganciclovir implant (Vitrasert) reflects an alternative
approach to treating CMV retinitis by providing local
concentrated therapy to the infected retina without the risks of
systemic toxicity associated with other routes of admin-
istration.173 Additionally, the sustained intravitreal release of
ganciclovir negates the need for repeated injections. The
implant is placed surgically in the vitreous cavity, and can
provide therapeutic levels of up to 8 months depending on the
SECTION 4
rate of drug release.174,175 Although the ganciclovir implant has
been shown to be effective in treating CMV retinitis, there was
the increased risk of CMV retinitis developing in the fellow eye
and of systemic involvement in the patients who received
implants compared with patients who received the drug
intravenously. To decrease this risk, these patients may be given
oral ganciclovir.176 On the whole, intravitreal therapy has been
well tolerated, and local reactions (such as foreign-body
sensation, small conjunctival or vitreous hemorrhage, con-
junctival scarring, and scleral induration) have been noted only
occasionally in patients receiving multiple intravitreal
injections (see Table 20.3). Because of the high pH of the
ganciclovir infusion solution, inflammation, phlebitis, and pain
at the site of intravenous infusion can occur.
Key Features: Ganciclovir (DHPG, Cytovene)
• Ganciclovir, a synthetic purine nucleoside analog of guanine, is
structurally and pharmacologically related to acyclovir. It is
poorly absorbed from the gastrointestinal tract, necessitating
IV or intravitreal administration
• In contrast to acyclovir, which is only minimally phosphorylated
by cellular (host cell) enzymes, ganciclovir seems to be more
susceptible to phosphorylation by enzymes in uninfected cells
thus making it more toxic
• DHPG has good antiviral activity against HSV-1 and HSV-2,
VZV, EBV, and HHV-6, its clinical use is in CMV retinitis
primarily in immunocompromised patients. Because ganciclovir
is only suppressive against CMV; without improvement in
immunocompetence, the retinitis will recur or progress
following cessation of drug
• Oral ganciclovir is valganciclovir (Valcyte) which has a much
higher GI absorption than its prodrug form. As a result, it may
be given in therapeutically effective doses for treatment of
CMV retinitis
• The ganciclovir implant (Vitrasert) provides local concentrated
therapy to the infected retina without the risks of systemic
toxicity
224
FOSCARNET (PFA, FOSCAVIR)
Foscarnet (phosphonomethanoic acid, phosphonoformic acid
trisodium, PFA, Foscavir), an organic analog of inorganic
pyrophosphate, is structurally unrelated to other available an-
tiviral drugs. Following intravenous administration of foscarnet,
it is not metabolized to any significant extent, and therefore
does not cause any major interference with the host cellular
processes.177 The drug is excreted renally. It is active against
herpesviruses (CMV, HSV, EBV, VZV), and the retrovirus HIV. It
inhibits herpesvirus DNA polymerases and HIV-1 reverse
transcriptase. Foscarnet directly affects the pyrophosphate
binding site of DNA polymerase and, therefore, does not require
phosphorylation to activate. Because it does not need
phosphorylation by TK to be activated, it is of use (and superior
to vidarabine) in treatment of ACV-resistant (and presumably
famciclovir or valaciclovir-resistant) HSV and VZV, which is
most commonly seen in AIDS patients. It is FDA approved for
treatment of CMV retinitis. In a rapid screen test for
susceptibility to acyclovir and foscarnet in 320 clinical HSV
isolates (16% type 1, 84% type 2), 60% were resistant to ACV
and only 5% were resistant to foscarnet. This correlated closely
with clinical response.178–181
Like other antivirals, foscarnet is virustatic. It may be
administered intravenously or intravitreally (Table 20.4) to treat
CMV retinitis. Foscarnet has poor oral absorption, and
gastrointestinal side effects are common; therefore, it is not
used orally. Foscarnet should also not be administered by rapid
or bolus intravenous injection because the toxicity may be
increased by excessive drug levels in the plasma. An infusion
pump must be used.
Foscarnet, like ganciclovir, is considered a drug of choice to
treat CMV retinitis in patients with AIDS. It is especially useful
in those patients who are intolerant to (or unresponsive to)
ganciclovir therapy. Because foscarnet does not cause myelo-
suppression, it can be used in conjunction with zidovudine and
other antiretroviral agents. Foscarnet can be administered
intravenously in combination with ganciclovir in patients with
CMV retinitis that is resistant to one drug. This combination
therapy reduces the dosage of the individual drug, appears to be
fairly well tolerated, and has prolonged sight in patients with
CMV retinitis.182 In the initial treatment of CMV retinitis in
patients with AIDS, foscarnet seems to be as effective as
ganciclovir.183,184 However, to prevent recurrent CMV retinitis,
chronic maintenance therapy is required with foscarnet, as with
ganciclovir.185 Foscarnet is more effective than ganciclovir in
prolonging the lives of AIDS patients, which may be the result
of its anti-HIV effect, and because it can be used with
zidovudine.186
Foscarnet is not tolerated as well as ganciclovir by patients.
Side effects include fever and gastrointestinal upset, including
nausea, vomiting, diarrhea, anorexia, and abdominal pain. The
most significant side effect with foscarnet is renal impairment.
It is necessary to monitor the serum creatinine levels and adjust
TABLE 20.4. Intravitreal Antivirals
Drug Dosage
Ganciclovir (Cytovene) 200–400 mg/0.1 mL
Foscarnet (Foscavir) 1200 mg/0.05 mL
Cidofovir (Vistide) 20 mg/0.1 mL
Prusoff WH, Bakhle YS, McCrea JF: Incorporation of 5-iodo-2„-deoxyuridine into
the deoxyribonucleic acid of vaccinia virus. Nature 1963; 199:1310.
 Antivirals

the drug dosage accordingly.187 Because foscarnet can alter
plasma electrolyte levels and cause seizures, patients treated
with foscarnet should be monitored.181,188–192
The current foscarnet induction dose recommendations are
either 60 mg/kg three times a day or 90 mg/kg twice a day for
a 2–3-week period. Subsequent maintenance therapy is required
with foscarnet, and the dosage range suggested is 90–
120 mg kg–1 day–1. Some doctors recommend the higher dosage
of 120 mg kg–1 day–1 to obtain a better response when treating
CMV retinitis without significantly increasing toxicity.
Intravitreal foscarnet has been used to treat CMV retinitis in
patients with AIDS. This route is especially useful for patients
in whom ganciclovir is contraindicated as a result of acyclovir
allergy, and in whom intravenous foscarnet is contraindicated
because of renal failure. Foscarnet is passed through a 0.22-mm
filter, and 1200 mg (0.05 mL) is injected intravitreally.193 The
recommended dose is two injections of foscarnet as induction
therapy once per week for 3 weeks, followed by a maintenance
dose of one injection per week (see Table 20.4).193
Key Features: Foscarnet (PFA, Phosphonoformate,
Foscavir)
• Foscarnet, an organic analog of inorganic pyrophosphate, is
structurally unrelated to other available antiviral drugs but
effective against CMV, HSV, VZV, and EBV but used primarily
for CMV retinitis or ACV-resistant HSV. It may be administered
intravenously or intravitreally
• As with ganciclovir, to prevent recurrent CMV retinitis, chronic
For immunocompromised patients of any age, restoring
immunity inhibits or prevents herpesvirus disease, as demon-
strated for cytomegalovirus (CMV) in AIDS patients receiving
HAART (highly active antiretroviral therapy).201 Specific
antiviral therapy during the initial period after transplantation
could prevent reactivation of HSV or CMV in seropositive
recipients. Whether preemptive therapy or prophylaxis with
ganciclovir is the optimal approach against CMV remains
controversial, and the relative merits and limitations of each
approach may guide the choice. In stem cell transplantation,
preemptive therapy with foscarnet avoids the neutropenia and
related complications associated with ganciclovir. In renal
transplant recipients, universal prophylaxis of CMV infection
with valaciclovir has the same efficacy as ganciclovir. Although
it is relatively toxic, cidofovir should be further evaluated
because of its in vitro activity against most DNA viruses.202
Key Features: Cidofovir (HPMPC, VISTIDE)
• HPMPC, another derivative of phosphonoformic acid, does not
require activation by TK and persists intracellularly up to 65 h
• It is effective against HSV-1 + HSV-2, VZV, EBV, DHPG-
sensitive and -resistant CMV as well as several adenoviruses
but used clinically as IV therapy for CMV retinitis
• It has been used intravitreally to treat CMV retinitis in patients
with AIDS
BROMOVINYLDEOXYURIDINE (BVDU,

maintenance therapy is required with foscarnet
• Intravitreal foscarnet has been used to treat CMV retinitis in
patients with AIDS. This route is useful for patients in whom
ganciclovir is contraindicated as a result of acyclovir allergy, and
intravenous foscarnet is contraindicated because of renal failure
CIDOFOVIR (HPMC, VISTIDE)
Cidofovir ((1-(4-amino-2-oxo-pyrimidin-1-yl)-3-hydroxy-pro-
pan-2-yl) oxymethylphosphonic acid, HPMPC, Vistide,
Forvade), another derivative of phosphonoformic acid, does not
require activation by TK. It works by DNA polymerase in-
hibition and resists degradation, thus persisting intracellularly
up to 65 h.194,195 It is effective against HSV-1 and HSV-2, VZV,
EBV, DHPG-sensitive and -resistant CMV, as well as several
adenoviruses. The drug is FDA approved for IV treatment of
CMV retinitis but has significant toxic ocular side effects.196 It
has been used intravitreally to treat CMV retinitis in patients
with AIDS (see Table 20.4). Ocular side effects include
decreased intraocular pressure and mild uveitis.197
Cidofovir is also of interest as a broad-spectrum anterior
segment antiviral. In preclinical trials, it has been shown to be
therapeutically effective as a topical 0.2% drop against
adenovirus 5 and to be as effective as TFT against HSV-1.198,199
In a clinical case report of HSV-1 and HSV-2 infection in an
AIDS patient, topical HPMPC on the skin was therapeutically
effective when foscarnet and ACV had failed.200
CHAPTER 20
BRIVUDINE)
This antimetabolite ((E)-5-(2-bromovinyl)-2„-deoxyuridine,
BVDU, Zostex, Zerpex, Zonavir, Brivudine) is a highly potent
and selective inhibitor of HSV-1 and VZV infections. The high
selectivity of BVDU, like ACV, VCV, and FCV, depends
primarily on a specific phosphorylation of BVDU by the virus-
encoded TK. It is a highly effective topical treatment of herpetic
keratitis, of recurrent herpes labialis, and of the systemic (oral)
treatment of herpes zoster.203 In studies on its efficacy in acute
zoster, there was equivalent efficacy of brivudin and famciclovir
regarding the prevention of chronic pain and the resolution of
symptoms. Compared with famciclovir, brivudin provides
equivalent efficacy and safety at a more convenient once-daily
dose schedule of 125 mg qd.204 Compared to ACV, BVDU was
significantly better in resolution of PHN.205 The drug is
available throughout Europe, but has not yet been reviewed for
approval in the United States.206
Key Features: (E)-5-(2-Bromovinyl)-2„-Deoxyuridine
(BVDU, Brivudin)
• This antimetabolite, activated by virus-encoded TK, is a highly
potent and selective inhibitor of HSV-1 and VZV infections
• It is highly effective topical treatment of herpetic keratitis and
recurrent herpes labialis and the systemic (oral) treatment of
herpes zoster

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229
 CHAPTER

21 Antifungal Agents
Eduardo C. Alfonso, Jorge Cantu-Dibildox, Terrence O’Brien, and Darlene Miller

The choice of an antifungal agent in ophthalmology depends
on several variables, including the primary site of infection, the
route of administration, the organism involved, and the sensi-
tivity data available.1–5 The major classes of antifungals used in
ophthalmology are polyenes, imidazoles, and pyrimidines
(Table 21.1).6 Other compounds have been tried as antifungals,
but the clinical experience is very limited.7,8 These include rose
bengal, salicylic acid, benzoic acid, thimerosal, gentian violet,
silver nitrate, zinc, copper sulfate, boric acid, potassium, iodide,
and iodine. A great number of experimental compounds are
described in the literature.9–12 For most of these, sufficient data
on the treatment of human mycoses are lacking.13–16
POLYENE ANTIBIOTICS
Polyene antibiotics are produced from a Streptomyces species.17,18
Their chemical configuration gives them their basic classifica-
tion based on the number of double bonds as well as the number
of carbon atoms (group I <30 atoms; group II >30 atoms).19
They interact with cell membrane sterols, primarily ergosterol,
which causes increased permeability that leads to cell lysis.20 It
is the binding to mammalian cell membrane cholesterol that
accounts for their toxicity. Two mechanisms of action of the
polyene antibiotics are known and depend on the size of the
antifungal molecule.21 Small molecules such as natamycin
work by an all-or-none mechanism of action. They bind to the
esterols in the fungal cell wall, forming ‘blisters’ and causing
lysis of the cell. This action is not concentration dependent.
The larger molecules, such as amphotericin, work by creating
‘pores’ in the cell wall, allowing small ions such as potassium
to leak out and causing imbalances in the osmotic gradient and
eventual cell lysis. This mechanism of action is concentration
dependent and may be altered by changes in the osmotic envi-
ronment.22 Other factors have been implicated in the interac-
tion of the polyenes with cell membranes.23 The most widely
used of the polyenes are amphotericin B and natamycin.24
AMPHOTERICIN B
Amphotericin B is most commonly used in ophthalmology as
a topical preparation for keratitis and scleritis, intraocularly for
endophthalmitis, and systemically for these conditions and
for scleritis, dacryocystitis, and cellulitis.25–29 The spectrum of
organisms and in vitro sensitivities identified in the published
literature and in our laboratory is presented in Tables 21.2 and
21.3, respectively.30–33 Dosages for antifungal agents are given
in Table 21.4.
For the treatment of keratitis and scleritis, a topical concen-
tration of 2.5–10 mg/mL given every 30–60 min for the first
48–72 h appears to deliver the optimal dose.34,35 Higher concen-
trations may cause surface toxicity.36,37 This concentration is
achieved by mixing the powdered amphotericin with sterile

TABLE 21.1. Classification of Antifungals

Polyenes Imidazoles Triazoles Pyrimidines Others

†§ § *† *
Amphotericin B Clotrimazole Fluconazole Flucytosine Pradicimicins||
Amphotericin B methyl ester† Miconazole†§ Itraconazole*† Cispentacin||
Natamycin‡ Econazole§ Terconazole§ Jasplakinolide||
*§ *§
Ketoconazole Vibunazole Terbinafine||
Thiabendazole* Alteconazole*§ Nystatin§
Bifonazole§ Voriconazole*† Caspofungin†
§ *†
Butoconazole Posaconazole
§
Croconazole Ravuconazole*†
Fenticonazole§
*Oral.
†
Intravenous.
‡
Ocular.
§
Dermatologic.
||
Not available.
231
 PHARMACOLOGY AND TOXICOLOGY

TABLE 21.2. Antimicrobial Activity of Antifungal Agents Based on Published Reports

Antifungal Alternaria Aspergillus Candida Cephalo- Clado- Curvularia Fusarium Paecilomyces Penicillium
Agent sporium sporium

Polyenes
Amphotericin S S S S S S R S
Nystatin S S S
Natamycin S S S S S R
Imidazoles
Clotrimazole S S S S S I S
Miconazole S S S I I
Econazole S I S S I
Ketoconazole I S S S S
Triazoles
Itraconazole S S R S
Fluconazole S S S S
Pyrimidines
Flucytosine R S S R R I
Abbreviations: S, susceptible; I, variable susceptibility; R, resistant.
SECTION 4

humans.1,40–44 Concurrent surgical management of the vitreous

TABLE 21.3. Ten-Year Summary of Sensitivity Testing of is often necessary to control the infection.45,46
Clinical Isolates at the Microbiology Laboratory of the Bascom
Palmer Eye Institute*
For intravenous use, a test dose of 1 mg of amphotericin in
150 mL of 5% dextrose in water is given.47,48 Once this test
Antifungal Fusarium Candida Aspergillus Curvularia dose is tolerated, 1–5 mg is given over 4–6 h. The dose is
(n = 40) (n = 10) (n = 15) (n = 6) increased by 5 mg daily until the desired dose of 0.5–1 mg kg–1
Amphotericin day–1 is reached. If chills, fever, nausea, or hypertension develops
with the test dose, the patient may require concomitant use
Range 0.078–5.0 0.08–5.0 0.01–2.5 0.04–0.31 of 25–30 mg of hydrocortisone intravenously.49 Also, aspirin,
Mean 1.2 (S) 2.7 (S) 1 (S) 0.16 (S) diphenhydramine, or prochlorperazine may be required. Other
potential side effects are a decrease in the glomerular filtration
Natamycin
rate to 20–60% of normal, which may be restored to normal after
Range 0.15–5.0 0.31–5.0 0.62–25.0 0.62–2.50 cessation of therapy for ~5 days.50 Hypokalemia may require
Mean 1.5 (S) 2.5 (S) 2 (S) 1.4 (S) potassium supplements. A drop in the platelet count and hema-
tocrit may also be observed during therapy. Hepatic damage
Ketoconazole occurs rarely. The water-soluble semisynthetic methyl ester
Range 0.78–50.0 0.10–1.6 0.78–250 0.20–12.50 derivative of amphotericin B has been shown in animal models
to carry fewer side effects than the parent compound.51–53
Mean 10.9 (I) 0.71 (S) 4 (S) 2.7 (S)
Miconazole
NATAMYCIN
Range 0.78–50.0 0.78–62.0 0.20–3.10 0.05–3.1
Natamycin (pimaricin) is a small semisynthetic tetraene and is
Mean 14.21 (I) 2 (S) 1.2 (S) 1.3 (S) considered the least toxic, the least irritating, and the most
Flucytosine stable of the polyenes.23 It has been available for topical use
as a 5% suspension since its approval by the US Food and
Range 0.05–100.0 0.05–3.10 25–100
Drug Administration in the late 1970s.54,55 It has a broad
Mean 921 (R) 1.2 (S) 68 (R) spectrum of sensitivities, especially to Fusarium species, as
Abbreviations: S, susceptible; I, variable susceptibility; R, resistant. shown in Table 21.3.56,57 It has decreased penetration through
*Ranges and means in micrograms per milliliter. an intact epithelium, and surface debridement may be desir-
able during therapy,58,59 although experiments have shown that
its penetration on intact epithelium is greater than ampho-
tericin B.60 Since natamycin is used as a suspension, it can
dry on the ocular surface and cause irritation.56 Lavage with a
water.38 The mixture should be stored in a dark bottle and saline solution of the lid margins is often necessary. Natamycin
refrigerated to maintain drug stability. Subconjunctival injection can be toxic to the corneal and conjunctival epithelium, causing
of amphotericin is not recommended because of severe hyperemia and epithelial defects.59 As with amphotericin,
toxicity.7,39 topical therapy is given every 30–60 min for the first 48–72 h,
For endophthalmitis, intravitreal injection of 5 µg of and treatment is usually continued on a tapering fashion for
232 amphotericin in 0.1 mL appears to be safe and effective in 3–6 weeks depending on the activity of the keratitis.60
 Antifungal Agents

TABLE 21.4. Antifungal Dosages

Antifungal Agent Topical Subconjunctival Intravitreal Intravenous Oral

Amphotericin B 2.5–10.9 mg/mL 750 mg/mL every 5–10 mg Maintenance dose

other day 1 mg kg–1 day–1
refrigerated
Clotrimazole 1% Suspension 5–10 mg 60–150 mg kg–1 day–1
1% Solution (0.5–1 mL) (adults)
Econazole 1% Suspension 30 mg kg–1 day–1 200 mg t.i.d.
1% Ointment
Fluconazole 2% Suspension 400 mg/day initial dose
1% Solution 200 mg/day maintenance dose
Itraconazole 2% Suspension 200 mg/day
Ketoconazole 1% Suspension 200–400 mg/day
Miconazole 1% Suspension 5–10 mg 0.25 mg 600–3600 mg/day
(0.5–1 mL) divided into three doses
1% Solution
(10 mg/mL)
Voriconazole 1% Solution (not 3 mg kg–1 h–1, 200 mg/12 h oral
available commercially) over 1–2 h IV
Natamycin 50 mg/mL
Nystatin Ointment 100 000 U/g
Thiabendazole 4% Suspension 25 mg kg–1 day–1
–1 –1
Flucytosine 10 mg/mL 50–150 mg kg day

CHAPTER 21
at 6-h intervals
Caspofungin 70 mg/day 1, followed
by 50 mg/day IV

Subconjunctival and intravitreal administration are not
recommended because of significant toxicity.61,62 Systemic
intravenous use of natamycin does not render significant levels
in the eye, and oral preparations are not well absorbed.63,64
However, natamycin is considered to be the mainstay of topical
therapy for most fungal keratitis.64a
NYSTATIN
Nystatin has been studied experimentally in ophthalmology,
and cases have been reported in which it has been used in
external ocular infections caused by Candida.40,65 It has been
used as a dermatologic ointment, which has a concentration
of 100 000 U/g, and at a frequency of application every 4–6 h.
Subconjunctival injections show marked toxicity, and experi-
mental intravitreal injection of 0.1 mL of a concentration of
2000 U/mL did not cause a significant reaction and cured an
experimental case of Aspergillus endophthalmitis.18,66
AZOLES
IMIDAZOLES
The imidazoles possess a broad spectrum of antifungal activity,
but in contrast to the polyenes, they are relatively resistant to
light, hydrolysis, and pH changes and are soluble in organic
substances.67 A number of compounds are available as approved
preparations for systemic use.
The imidazoles have a combination of mechanisms for
antimycotic activity.68–70 At low concentrations, miconazole,
econazole, and ketoconazole affect the formation of ergosterol
needed by the cell membranes.71 At high concentrations, clotri-
mazole and miconazole can disrupt lysosomes, causing direct
cell membrane damage. In addition, most imidazoles inhibit
catalase and cytochrome C peroxidase intracellulary, causing
accumulation of hydrogen peroxide and leading to cell death.
There also appears to be a triggering mechanism of host defense
cells by the imidazoles. When ketoconazole is added in vitro to
polymorphonuclear leukocytes and macrophages, it has the
ability to eradicate both the yeast and the mycelial forms of
Candida, in the absence of polymorphonuclear leukocytes and
macrophages.72 One can see that because of these combined
mechanisms of action, most of the imidazoles can be fungistatic
and fungicidal.73,74
CLOTRIMAZOLE
Clotrimazole has a wide spectrum of activity against numerous
fungi, but poor results have been obtained with Fusarium.
Most strains are inhibited at concentrations of 2–4 mg/mL,
which can be readily achieved with topical and oral adminis-
tration (see Table 21.3).75,76 It is poorly absorbed parenterally.77
The topical preparation of clotrimazole is made by dilution
in arachis oil to a 1% solution. It has been applied hourly for
2–3 days, then tapered over 8–12 weeks.78 Oral administration
in a dosage range of 60–150 mg kg⫺1 day⫺1 can be given with an
achievable serum concentration of 0.4–5.5 mg/mL. No com-
mercial oral dosage forms are available in the United States.
Clotrimazole has been recommended by several authors as
the drug of choice for Aspergillus infections of the eye.78–80 Side
effects of the systemic administration of clotrimazole may
include anorexia, nausea, hallucinations, confusion, and
epigastric pain. It should not be given in the first 3 months of
pregnancy or to patients with a history of hypersensitivity, 233
 PHARMACOLOGY AND TOXICOLOGY
adrenal, or liver problems. Liver enzyme level elevations are
normal with the use of clotrimazole, and these tend to return to
normal once the drug is withdrawn.81
MICONAZOLE
Miconazole is a phenethylimidazole that is very stable in
solution.82 Its mechanism of action is similar to that of the
other imidazoles.70 It has a broad spectrum of activity against
Cryptococcus, Aspergillus, Curvularia, Candida, Microsporum,
Paecilomyces, and Trichophyton (see Table 21.3).83–86
Miconazole may be given intravenously in dosages ranging
from 200 to 3600 mg/day in three divided doses. In children,
a dose of 15 mg/kg per infusion should not be exceeded.82 It
may also be used as a topical, subconjunctival, or intravitreal
preparation.87 For topical use, a 1% solution in arachis oil or
a 10 mg/mL commercial solution (Monistat IV) is well toler-
ated. It is also available as a 2% dermatologic ointment, but
this may cause some irritation to the eye.88 For subconjunctival
injections, 10 mg/day may be used. For intravitreal injections,
0.25–0.50 mg may be used.86,89,90
After intravenous administration of miconazole, reported
side effects may be a rash with pruritis, chills, nausea, and
vomiting. These side effects may be minimized by the con-
comitant administration of antihistamines and antiemetics.91,92
Reports also mention a possible decrease in sodium levels and
the hematocrit, with aggregation of erythrocytes and thrombo-
cytosis.85 Topical use of miconazole may cause surface toxicity
SECTION 4
after prolonged use.90,93,94
KETOCONAZOLE
Ketoconazole is a synthetic acetylchichlorophenyl imidazole.
It dissolves in water with a resultant pH of ~3.95 Its mechanism
of action is similar to that of the other imidazoles.68,96 This
drug has a broad spectrum of activity in vitro (see Table 21.3).97
Ketoconazole is available for oral administration. It is well
absorbed from the gastrointestinal tract and bound to albumin,
and high therapeutic blood levels are maintained.68 Ninety
percent of the drug is excreted by the liver and the remainder
by the kidneys.95 Ketoconazole is available in 200-mg tablets
with a recommended daily dose of 200–400 mg. A topical pre-
paration may be formulated in a 1–5% concentration by dis-
solving in arachis oil.98,99 Ketoconazole may also be dissolved in
polyethooxylated castor oil67 or in 4.5% boric acid.7,100
Systemic side effects associated with the use of ketoconazole
have been minor and usually reversible. Pruritus, nausea,
vomiting, diarrhea, cramps, gynecomastia,101 and elevations in
liver enzyme levels have been reported after oral adminis-
tration.101 Topical use of ketoconazole shows minimal reversible
toxicity in animals.102 Ketoconazole can affect the efficacy and
concentration of cyclosporine, warfarin, phenytoin, and
theophylline.103
In ophthalmology, topical ketoconazole has been used clini-
cally and experimentally for the treatment of keratitis.99,104,105
Oral ketoconazole has been used in both experimental35 and
human keratitis.106 In experimental endophthalmitis, keto-
conazole was effective if started 24 h after injection.107 It has
been suggested that oral ketoconazole may augment topical
natamycin therapy.25,108
THIABENDAZOLE
Thiabendazole is a thiazolyl benzimidazole. Its primary clinical
use for many years has been in the treatment of roundworm
infections.108 Its mechanism of action is similar to that of the
234 other imidazoles.68 It has been shown to be active against ocular
isolates of fungi, but poor results have been obtained against
Candida and Aspergillus species (see Table 21.3).85,99
Oral thiabendazole may be given at a dose of 25 mg/kg two
times per day with a maximal daily dose of 3 g. Its peak serum
concentration is in 1–2 h, and 90% is excreted in the urine.68
Topical application of a 4% thiabendazole suspension has been
reported in the treatment of Aspergillus flavus keratitis.109 Side
effects have been few, the major ocular side effects being surface
irritation and dryness and mild reversible hepatic disease.18
Clinical experience with thiabendazole in ophthalmology is
limited, and this drug has been reserved for cases unresponsive
to conventional treatment.110
ECONAZOLE
Econazole is a deschlorophenethylimidazole.23 Its mechanism
of action is similar to that of the other imidazoles.68 The
spectrum of activity is similar to that of the other imidazoles,
with increased activity against Aspergillus, Fusarium, and
Penicillium. It has less activity against Candida.111
Econazole is available as a dermatologic ointment. For topical
use, a 1% suspension may be prepared in arachis oil.112 For oral
use, 200 mg of econazole three times a day may be used. For
intravenous use, 30 mg kg⫺1 day⫺1 is recommended.112 The sys-
temic preparation is not commercially available in the United
States.
The clinical use of econazole in ophthalmology is very
limited,112 although some studies suggest that it could be as
effective as natamycin for a broad spectrum of fungal
keratitis.113 However, there appears to be no synergism between
concurrent use of econazole and natamycin as topical treat-
ments for fungal keratitis.114
TRIAZOLES
The triazoles – fluconazole, itraconazole, terconazole, and
others (see Table 21.1) – were developed in order to increase the
spectrum of activity and reduce the side effects of their
predecessors, the imidazoles.
FLUCONAZOLE
Fluconazole is perhaps the most widely used member of the
triazoles because of in vitro studies that have shown a very
wide spectrum of activity against many pathogens.115 The
in vivo activity has not followed its laboratory spectrum of acti-
vity. It has been used for the treatment of Candida species.116
Unlike amphotericin B, fluconazole is capable of penetrating
intact corneal epithelium, due to its lower molecular weight.117
It has also been used for the treatment of experimental endo-
phthalmitis in its oral form16 and in the treatment of experimental
Candida albicans keratitis in a topical solution.16,118 Animal
studies suggest efficacy in both topical and oral form against
Aspergillus fumigatus.119
Oral fluconazole can be given in a dose of 50–40 mg/day,
with the usual adult dose being 200 mg/day. A topical 1%
solution in sterile water can be made. The 2 mg/L aqueous
solution for intravenous use can also be applied topically.120
Human studies on the subconjunctival use of fluconazole
have given promising results in the treatment of severe non-
responding fungal keratitis121, and some animal studies demon-
strate peak concentrations in the central cornea at 2 h after
subconjunctival injection.122 Further studies need to be done on
this alternative to evaluate safety, dosage, and efficacy.
Systemic side effects of fluconazole include gastrointestinal
upset, headaches, rash, hepatotoxicity, anaphylaxis, Stevens–
Johnson syndrome, and thrombocytopenia. Fluconazole can

increase cyclosporine’s serum concentration and decrease the
metabolism of warfarin. Rifampin can increase the metabolism
of fluconazole.123
ITRACONAZOLE
Itraconazole also has, like fluconazole, a wider spectrum of
activity than the imidazoles. Its spectrum of activity includes
excellent in vitro activity against Aspergillus. Its broad spectrum
of antifungal activity includes Candida species, Paecilomyces,
Paracoccidioides, and Coccidioides.124 It has not been very
effective against Fusarium.125
It has had a very limited use in clinical ophthalmology. In
an experimental model of Candida endophthalmitis, it was
shown to be as effective as fluconazole and ketoconazole.16
There is a published report of successful treatment of
Aspergillus scleritis with oral itraconazole after cataract
surgery.126 The oral administration of itraconazole appears to
have less penetration than other triazoles into the cornea,
aqueous, and vitreous.16
Itraconazole has been used in its oral preparation as an
adult dose of 200 mg/day. Side effects include gastrointestinal
upset, hypertriglyceridemia, and hypokalemia.127 Although
natamycin continues to be the treatment of choice for fila-
mentous fungal keratitis, in its absence topical itraconazole
therapy should be considered, specially if the infection is due to
Aspergillus.128
PYRIMIDINES
The pyrimidines are a group of antimetabolites with known
antifungal activity. The main drug in this group is
flucytosine.129
FLUCYTOSINE
Flucytosine (5-FC) is a fluorinated pyrimidine that is soluble in
water and alcohol. Several mechanisms of action have been
described.130 It may alter fungal RNA and DNA synthesis. It
enters the cytoplasm by the action of cytosine permease and is
then deaminated by cytosine deaminase into 5-fluorouracil. It
is then phosphorylated and incorporated into RNA. In the
nucleus, 5-FC forms 5-fluoro-2’-deoxyuridylic acid (FdUMP),
which inhibits thymidilate synthetase and thus DNA
synthesis.131
Flucytosine has a limited spectrum of activity, and resistance
may be acquired at low doses (see Table 21.2).48,132 The limited
activity and resistance of 5-FC are due to the fungal cell’s
inability to transport the drug into its cytoplasm and incor-
porate it into its RNA or insufficient FdUMP synthesis to
inhibit DNA formation.130 The spectrum of activity may be
enhanced and the emergence of resistance may be reduced by
concomitant administration of amphotericin B.2,32,132,133
Both topical and oral preparations of 5-FC may be used.134 It
is available for oral administration in 250- and 500-mg
capsules. It is water soluble and rapidly absorbed from the
gastrointestinal tract. The recommended dose of 5-FC is
50–150 mg kg–1 day–1 at 6-h intervals. The drug is excreted
unchanged in the urine, and thus the dosage should be adjusted
according to the creatinine clearance.135
A topical preparation of 1% 5-FC may be formulated; it has
limited penetration and thus is primarily effective for surface
infections (conjunctivitis, blepharitis, and canaliculitis) and
anterior stromal keratitis.136
Most side effects reported with 5-FC have been minimal and
reversible.133 Reversible elevations in levels of liver enzymes,
Antifungal Agents
aspartate aminotransferase, and alkaline phosphatase may be
seen. Anemia, leukopenia, and thrombocytopenia have been
reported in patients with other severe underlying disorders who
are taking 5-FC. Two patients with intestinal perforations have
been reported.
In ophthalmology, 5-FC has been used to treat primarily
surface infections such as blepharitis, conjunctivitis, cana-
liculitis, and anterior keratitis.108 The topical preparation of
5-FC is preferred, since subconjunctival injections offer little
enhancement of penetration and are associated with toxicity
and discomfort.134 Its primary use has been in cases of Candida
keratitis that have not responded clinically to amphotericin B,
in which 5-FC is added to the topical regimen.137
New Agents
Voriconazole
Voriconazole is a new triazole antifungal agent derived from
fluconazole with activity against various fungi resistant to fluco-
nazole. It can be used orally and intravenously. Its bioavail-
ability is 96%, and reaches peak plasma concentration 2–3 h
after oral dosing. Its intraocular penetration in oral dosage has
been found to be 1.13 ± 0.57 mg/mL and 0.81± 0.31 mg/mL in
aqueous and vitreous respectively.138 Animal studies have
demonstrated that up to 25 mg/mL of intravitreal injection of
voriconazole causes no ERG changes or histologic abnormali-
ties in the retina.139 The most common side effect is photopsia,
followed by skin rashes. As with other azole agents, hepatic
enzyme elevations can occur. In vitro studies from nonocular
CHAPTER 21
isolates have shown voriconazole to have broad spectrum of
fungistatic action against most yeast and many filamentous
fungi. It has been approved for treatment of invasive
aspergillosis, and infections from P. boydii, S. apiospermeen,
and Fusarium infections in patients intolerant or with
refractory infections to other agents. Its role in ocular infections
needs to be studied further.140
Under current development in this drug group are new agents
such as posaconazole (a second-generation triazole), with fungi-
cidal activity against Aspergillus, and ravuconazole, a fungicidal
with a long half-life (100 h), structurally similar to voriconazole.
Although some studies suggest high effectiveness of these
agents, further studies are awaited to determine safety and any
possible ophthalmologic application.141
Caspofungin
Caspofungin acetate is a parenteral antifungal for the treatment
of invasive aspergillosis in patients intolerant or refractive to
other antifungal agents. It is a member of a new class of
echinocandins, whose mechanism of action is distinct from
other antifungals, in that it inhibits synthesis of B(1,3)-D-
glucan, a component of fungal cell wall. It has demonstrated in
vitro antifungal activity against Aspergillus, Candida albicans,
C. glabrata, C. parapsilosis, and other Candida species. Some
intermediate activity has been found against Histoplasma
capsulatum and Blastomyces dermatitides. Cryptococcus
neoformans and Fusarium spp. have demonstrated resistance to
caspofungin in vitro. The dosage in patients with normal
hepatic function is 70 mg intravenously on day 1, followed by
50 mg daily. Adverse effects include fever, phlebitis, and
headaches.142
Studies in animal models suggest that topical caspofungin
0.5% can be as effective as amphotericin B 0.15% for the
treatment of Candida keratitis.143 There is also evidence of
possible clinical efficacy of intravenous use of caspofungin for
treatment of endophthalmitis by Candida glabrata.144 Further
studies are necessary to determine its clinical usefulness in
ophthalmology.
235
 PHARMACOLOGY AND TOXICOLOGY
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 CHAPTER

22 Antiparasitics
Savitri Sharma, Virender S. Sangwan, and Nalini A. Madiwale

INTRODUCTION PARASITES AFFECTING THE EYE

Parasitology as a science has grown dramatically in the recent
years, particularly owing to the parasitic diseases that have
found prominence in patients with compromised immune
systems. The rapidity of modern international travel has only
added to the spread of parasitic diseases from endemic to
nonendemic areas.
Parasitic infections of the eye may be a manifestation of
generalized systemic disease or a localized phenomenon.
Considerable ocular morbidity and blindness can be caused by
parasites, some of them confined to geographical areas and
some of them widespread. While onchocerciasis is common in
Africa, Acanthamoeba infections have been reported from all
over the world.
The control and eradication of parasitic infections require a
multifaceted approach that includes vector control, health edu-
cation, and improved sanitation. Nevertheless, chemotherapy
remains the most efficient and effective means of control of
parasitic diseases. Chemotherapy with antiparasitics is required
to deal with these infections that may be important causes of
morbidity and mortality.
This chapter describes chemotherapeutic agents, currently
used for the treatment of ocular parasitic infections, along
with brief description of the parasite and accompanying ocular
manifestations.
Key Features
• Classification
• Nomenclature
• Relationship with intermediate host
Whittaker in 1969 proposed five kingdoms for all living organ-
isms: Monera, Protista, Fungi, Plantae and Animalia. Protozoa
are eukaryotic unicellular organisms belonging to the kingdom
Protista and helminths are eukaryotic multicellular organisms
and are placed in the kingdom Animalia.1 Table 22.1 lists the
parasites that have been reported to affect the eye.
Parasitic infections may originate from a large number of
sources, contaminated water and soil being the commonest.
Other sources include fresh water fishes, crabs, undercooked/
raw beef or pork, blood sucking insects, housefly, pet animals,
etc. In most cases, the definitive host is the mammalian host in
which either the most developed form of the parasite occurs or
the sexual reproduction of the parasite takes place. Table 22.2
outlines the relationship of some of the common parasites to
the intermediate host, which harbors the larval or sexual stage
of the parasite. The modalities of chemotherapy often depend
on the stage of the parasite occurring in the human host.

TABLE 22.1. Classification of Parasitic Eye Infections Caused by Protozoa, Helminths and Arthropods

Protozoa Helminths Arthropods

Nematodes Cestodes Trematodes

Toxoplasmosis Toxocariasis Cysticercosis Schistosomiasis Ophthalmomyiasis

Acanthamoebiasis Ascariasis Echinococcosis Paragonimiasis
Entamoebiasis Onchocerciasis Coenurosis
Malaria Loiasis Sparganosis
Giardiasis Dirofilariasis
Leishmaniasis Filariasis
Trypanosomiasis Dracunculiasis
Pneumocystosis Thelaziasis
Microsporidiosis Gnathostomiasis
Angiostrongyliasis
Trichinosis
239
 PHARMACOLOGY AND TOXICOLOGY
TABLE 22.2. Relationship of Common Parasites to Their Intermediate Host
No Intermediate Host
Protozoa Helminths
Acanthamoeba Trichuris trichiura
Microsporidia Ascaris lumbricoides
Giardia Ancylostoma duodenale
Entamoeba Necator americanus
ANTIPARASITICS FOR PROTOZOAL
INFECTIONS
SECTION 4
TOXOPLASMOSIS
Key Features
• Geographical distribution
• Life cycle
• Ocular manifestations
• Treatment
Toxoplasmosis is a common parasitic infection in humans. It is
estimated to infect at least 10% of adults in northern temperate
countries and more than half of adults in Mediterranean and
tropical countries. Toxoplasmosis is caused by Toxoplasma
gondii, an obligate intracellular protozoan of cosmopolitan
distribution. The domestic cat is the definitive host. Oocysts
excreted in cat feces have been shown to survive in soil for long
periods of time. Human infection can occur after ingestion of
either tissue cysts (bradyzoites) or oocysts (sporozoites).
Transmission occurs by contact with contaminated feces,
ingestion or handling of infected meat, or drinking of con-
taminated water. Transplacental spread causes a congenital
infection. On entry into the host, the cyst wall is disrupted,
releasing actively replicating, invasive tachyzoites. The host’s
immune response then transforms the tachyzoites into slowly
dividing bradyzoites in tissue cysts. The life cycle is completed
only when the cat ingests infected uncooked meat.
Acute focal retinochoroiditis, papillitis, papilledema, vitritis,
and recurrent retinitis are commonly seen ocular mani-
festations. A granulomatous anterior uveitis is sometimes seen.
In the immunocompetent host, toxoplasmosis is a self-limiting
disease. In the immunocompromised host the retinochoroiditis
takes on a severe necrotizing form and may occur in conjunc-
tion with life-threatening systemic infection.
The goal of medical therapy is to prevent damage to the
retina and optic nerve, thereby preventing permanent vision
loss. The management of ocular toxoplasmosis in immuno-
competent adults must consider various factors such as: self-
240 limiting nature of the active phase of the disease, retinal
One Intermediate Host
Parasite Intermediate
Host
Taenia solium Pig
Taenia saginata Cow
Echinococcus granulosus Man
Plasmodium Man
Trypanosoma cruzi Reduviid bug
Wuchereria bancrofti Mosquito
Brugia malayi Mosquito
Schistosoma Snail
Leishmania Sandfly
Trypanosoma Tsetse fly
Loa loa Chrysops
Onchocerca volvulus Simulium fly
necrosis due to proliferation of organisms, damage to the intra-
ocular tissues due to immune response to the organisms, and
inability of the current drugs to eliminate tissue cysts and
prevent recurrence.
In 1991 Engstrom and associates conducted a survey of all
the physician members of the American Uveitis Society to
determine the current practices in the management of ocular
toxoplasmosis.2 Among the respondents, only 6% treated all the
active lesions, regardless of ocular findings. The majority of
respondents felt that the lesions should be observed without
treatment if the visual acuity remained 20/20 in the affected eye
and lesions were located in the far periphery of the retina.
Majority of the respondents agreed that the following factors
were indications for medical therapy: any decrease in visual
acuity, macular or peripapillary lesions, lesions greater than one
disk diameter in size, lesions associated with moderate to severe
vitiritis, presence of multiple active lesions, persistence of active
lesions for more than a month, and any ocular lesions asso-
ciated with recently acquired infection. Various drugs used for
treatment of ocular toxoplasmosis are listed in Table 22.3.
Systemic corticosteroids should be used either concomitant
with antimicrobials or after 24–48 h of antimicrobial therapy.
The combination of pyrimethamine and sulfadiazine is
probably most effective against toxoplasmosis and therefore
recommended as the treatment of choice for sight-threatening
ocular toxoplasmosis.3 Quadruple therapy, consisting of
clindamycin, pyrimethamine, sulfonamides, and prednisone,
has been claimed to represent an even more effective alter-
native, but no comparison between the triple and quadruple
therapy is available. Some of the newer antimicrobial agents,
including atovaquone and azithromycin, reduce the number
of tissue cysts in animal models.4 Rothova and associates found
a relationship between treatment with pyrimethamine/
sulfadiazine and reduction of lesion size.5
ACANTHAMOEBIASIS
Acanthamoeba is an important cause of microbial keratitis. It is
a free-living ubiquitous protozoa and is an opportunistic
pathogen. It exists in nature as a dormant cyst, which under
favorable conditions turns into active trophozoite. First
described in 1973, the reported incidence of Acanthamoeba
 Antiparasitics

TABLE 22.3. Drugs Used in the Treatment of Ocular Toxoplasmosis

Drug Dosage

Pyrimethamine Adults: 100 mg loading dose, followed by 25 mg/day for 30–60 days
Children: 4 mg/kg loading dose followed by 1 mg/kg divided dose
Newborns should be treated daily for first 6 months and then 3 times a week for rest of life
Dosage: 1 mg/day divided into 2 doses
Sulfadiazine Adults: 2 g loading dose followed by 1 g every 6 h for 30–60 days
Children: 100 mg kg⫺1 day⫺1 divided every 6 h
Newborns should be treated daily for their first year of life. Dosage: 100 mg kg⫺1 day⫺1 divided into
two doses
Folinic acid 5–20 mg/day during pyrimethamine therapy, depending on neutrophil and platelet count
Azithromycin 500–1000 mg/day for 3 weeks
Trimethoprim/Sulfamethoxazole 160/800 mg (one tablet) twice-daily for 30–40 days
Atovaquone 750 mg every 6 h 4–6 weeks
Clindamycin 300 mg every 6 h for 30–40 days
Children: 16-20 mg kg⫺1 day⫺1 divided every 6 h
Spiramycin Adults: 500–750 mg every 6 hour for 30–40 days
Children: 100 mg kg⫺1 day⫺1 divided every 6 h
Tetracycline 500 mg every 6 h loading dose, followed by 250 mg every 6 h for 30–40 days
Minocycline 100–200/day for 30–40 days
Clarithromycin 1 g every 12 h loading dose followed by 500 mg every 12 h for 4 week
Prednisone Adults: 40–100 mg/day
Children: 1–2 mg kg⫺1 day⫺1

keratitis increased in 1980s in association with the rising
popularity of contact lens wear in UK 6 and many other
countries in Europe and USA. The disease has been reported
from almost all parts of the world.7,8
Before the development of potent amoebicidal drugs in the
mid 1980s and early 1990s, the prognosis of Acanthamoeba
keratitis was generally poor. Successful use of topical pro-
pamidine isethionate (Brolene) and neomycin–polymyxin–
bacitracin (Neosporin) has been reported by many.9,10 An
extensive review on in vitro efficacy of a large number of drugs
against Acanthamoeba was reported by Wright et al in 1985.11
Remarkable clinical and visual improvement were reported by
Larkin et al by using topical (0.02%) polyhexamethylene
biguanide (PHMB) in six cases of Acanthamoeba keratitis
refractory to multiple antiamoebic agents.12 Chlorhexidine and
PHMB are potent cysticidal drugs and at 0.02% concentration
are safe to the ocular surface. Although their mode of action
is similar (cationic antiseptic) they have been shown to be
synergistic in vitro.13 Commercial eye drops of these medica-
tions are not available and they need to be made in local
pharmacy. Currently, a combination of topical propamidine
isethionate with PHMB or chlorhexidine is considered effica-
cious in the treatment of Acanthamoba keratitis.14,15 Combina-
tion therapy with PHMB and chlorhexidine has also shown to
be efficacious.7
The dormant Acanthamoeba cysts in the cornea may contri-
bute to chronic disease with propensity to recur. Over 25% of
patients were shown to have at least one recurrence in a review
of 20 patients reported recently.16 All patients had received
topical PHMB with propamidine isethionate and some had in
addition received chlorhexidine or neosporin. A wide range of
treatment duration (5–72 months) was seen in this study.
Surgical intervention (penetrating keratoplasty) was required in
30% of the cases, however, no patient lost the eye. The role of
topical steroid therapy has been debated inconclusively.17 The
pathogenicity of Acanthamoeba cysts and trophozoites has
CHAPTER 22
been shown to increase with dexamethesone both in vitro
and in vivo.18
ENTAMOEBIASIS
Early reports of ocular amoebiasis associated with Entamoeba
histolytica are based on circumstantial evidence, i.e., eye lesions
were present along with intestinal amoebiasis responding to
antiamoebic therapy but the organism was not isolated from
ocular samples. Although amoebic choroidosis was described
with excellent documentation19 E. histolytica is believed to
rarely affect the eye. Case reports of cutaneous amoebiasis
affecting the eyelid are available.20 The treatment of amoebiasis
depends on the stage of the disease and general health of
the patient. Symptomatic intestinal amoebiasis is treated with
a combination of metronidazole and diiodohydroxyquin,
750 mg three times per day for 10 days of the former and
650 mg three times per day for 20 days of the latter. For liver
abscess treatment, a combination of metronidazole and
dehydroemetine or emetine is preferred.
MALARIA
Ocular manifestations in malaria include retinal hemorrhage or
exudates, usually in cerebral malaria and indicate a poor
prognosis.21 Retinopathy after chloroquine treatment has
also been reported.22 Other rare findings in malaria include
malarial amaurosis, optic neuritis, oculomotor paralysis, and
cortical blindness. Oral therapy of malaria consists of
chloroquine phosphate and in cases with chloroquine - resistant
Plasmodium falciparum (CRPF) infections, quinine sulfate
with pyrimethamine and sulfadiazine. For patients sensitive to
pyrimethamine or sulfadiazine, the preferred drug is quinine
sulfate with tetracycline for the treatment of CRPF. In
emergencies, intravenous use of quinine dihydrochloride or
quinine gluconate is recommended. Chloroquine phosphate, 241
 PHARMACOLOGY AND TOXICOLOGY
500 mg once a week, beginning 1 week before travel to an
endemic area and continuing until 6 weeks after return, is
recommended by CDC for chemoprophylaxis of malaria.
Mefloquine is the drug of choice for travelers at risk of infection
with CRPF.
GIARDIASIS
Giardiasis is a waterborne infection caused by Giardia lamblia,
a binuclear flagellate protozoan that affects the upper part of the
gastrointestinal (GI) tract. Water supply contaminated with
cysts is the usual source of infection. An increased prevalence
among homosexual males has been documented. Iridocyclitis,
choroiditis, and a hemorrhagic retinopathy can coexist with
both latent and overt systemic infections. The basis of the
ocular involvement is thought to be immunologic.23
Quinacrine hydrochloride, 100 mg three times a day for
5 days, and metronidazole, 250 mg three times a day, for 5 days
are equally effective. Concurrent ocular steroids are needed to
control the exacerbation of inflammation that occurs after
initiation of treatment.
LEISHMANIASIS
Mucocutaneous leishmaniasis is caused by Leishmania
braziliensis. About 10–20% of the cases show ocular involve-
ment. The extracellular flagellate, and promastigote forms are
injected into the skin through the bite of the phlebotomus
SECTION 4
mosquito. The parasites proliferate as aflagellate amastigotes
within macrophages and endothelial cells of capillaries. Lysin
of the amastigotes by host macrophages and lymphocytes
causes an open ulcer. During a mosquito bite, the amastigotes
enter the vector and transform into promastigotes that are
transmitted to the next human through the saliva of the
infected vector.24
Ocular manifestations include granular or nodular conjunc-
tivitis, interstitial keratitis, nodular keratitis with heavy pannus
formation, and ulcerative keratitis.25 Cutaneous leishmaniasis
generally involves eyelids, most often on the external corner.26
Eyelid lesions are usually ulcerative, with occasional spread to
conjunctiva and lacrimal ducts.
Sodium stibogluconate (Pentostam) is the drug of choice
for the treatment of leishmaniasis. A single course consists
of 10 mg/kg to a maximum of 600 mg intramuscularly or
intravenously for 6–10 days. A maximum of three courses of
treatment can be repeated at 10-days intervals. However,
amphotericin B, 0.5–1.0 mg kg⫺1 day⫺1 intravenously for up
to 8 weeks is used when antimonials are ineffective or
contraindicated.
TRYPANOSOMIASIS
Sleeping sickness or African trypanosomiasis is caused by
Trypanosoma brucei gambiense and Trypanosoma brucei
rhodesiense and the vector is tsetse fly. The ocular mani-
festations of this disease are generally mild and may be
associated with congestion of the eyes, edema of the lids, diffuse
corneal opacification or interstitial keratitis.27 Unilateral
anterior uveitis with or without corneal involvement may be
present. In the terminal stage, papilledema, ophthalmoplegia,
ptosis, papillitis and optic neuritis may be present, especially
with rhodesiense infections.
Treatment depends on the stage of the disease. During the
early stages suramin is given intravenously. At first a test dose
of 100–200 mg is given followed by one gram intravenously on
days 1,3,7,14, and 21. Pentamidine isethionate may be given
242 intramuscularly in the dosage of 4 mg kg⫺1 day⫺1 for 10 days.
In CNS involvement the standard drug is melarsoprol
(Mel B), a trivalent arsenic compound that may cause severe
reactive arsenic encephalopathy.28 An alternative and safer
drug is eflornithine (a-difluoromethylornithine) in the dosage
of 400 mg kg⫺1 day⫺1 intravenously for 14 days followed by
300 mg kg⫺1 day⫺1 orally for 21–28 days.29 This is effective and
safer than melarsoprol.
American trypanosomiasis or Chagas’ disease is caused by
Trypanosoma cruzi and is transmitted by reduviid bugs.
The most important ocular manifestation is unilateral
palpebral edema which is a pathognomonic feature of Chagas’
disease. Granulomatous uveitis, with the presence of T. cruzi
in the infiltrate has been reported.27 The drug of choice for
Chagas’ disease is nifurtimox given 8–10 mg kg⫺1 day⫺1 orally
in four divided doses for 120 days. Alternatively, benznidazole,
5–7 mg kg⫺1 day⫺1 may be given for 30–120 days.
PNEUMOCYSTOSIS
Pneumocystis carinii is considered a protozoan although one
study indicated it to be closer to fungi than protozoa.30 The
organism has three development stages; precyst, cyst and
trophozoite. Pneumocystosis was originally described as an
epidemic form of interstitial plasma cell pneumonitis in
children following the second world war in Europe. Since 1979,
P. carinii pneumonia (PCP) is being reported in patients with
acquired immunodeficiency syndrome, which is probably a
reactivation of latent subclinical infections.
Manifestations in the eye probably occur when there is
disseminated infection. P. carinii choroidopathy has been
documented.31,32 The drug of choice is a combination of trime-
thoprim and sulfamethoxazole with the dosage of the former
being 20 mg kg⫺1 day⫺1 and the latter 100 mg kg⫺1 day⫺1, either
oral or intravenous in four divided doses, for 14 days. Alter-
native therapy with pentamidine isethionate has been reported.
MICROSPORIDIOSIS
Microsporidia are obligate intracellular parasites belonging to
the phylum Microspora. Multiple genera are involved in a wide
range of clinical diseases. The most common infection involves
the GI tract and others include encephalitis, sinusitis, myositis,
ocular infections and disseminated infection. Two forms of
microsporidial infection of the cornea have been described,
stromal or interstitial keratitis in immunocompetent33 and
superficial keratoconjunctivits seen in immunosuppressed 34 or
immunocompetent individuals.35 Various therapeutic agents
have been used, however, there are no defined guidelines for the
optimal treatment of microsporidial infections. Costa and
Weiss have described antimicrosporidial drugs in an extensive
review recently.36 Table 22.4 describes the drugs that have been
used for the treatment of ocular microsporidiosis.
TABLE 22.4. Drugs Used in the Treatment of Ocular
Microsporidiosis36
Drug Microsporidial Species
Albendazole Encephalitozoon cuniculi
Encephalitozoon hellem
Encephalitozoon intestinalis
Fumagillin Encephalitozoon cuniculi
Encephalitozoon hellem
Encephalitozoon intestinalis
Itraconozole Encephalitozoon cuniculi

The superficial corneal lesions in microsporidial kerato-
conjunctivitis have been reported to have resolved following
débridement and oral itraconazole.37 Administration of alben-
dazole (400 mg twice-daily for 2–4 weeks) has led to resolution
of symptoms in patients with AIDS and symptomatic Encepha-
litozoon intestinalis infection. Fumagillin, an antiangiogenic
agent derived from Aspergillus fumigatus, inhibits replication of
E. cuniculi in vitro and has been used topically to treat ocular
infections due to E. hellem and E. intestinalis.38,39 Fumidil B, a
purified fumagillin, has been used as topical drops in the
treatment of microsporidial keratoconjunctivitis.
ANTIPARASITICS FOR HELMINTHIC
INFECTIONS
Key Features
• Geographical distribution
• Life cycle
• Ocular manifestations
• Treatment
TOXOCARIASIS
Toxocariasis is caused by dog ascarid, Toxocara canis and less
frequently by Toxocara cati, the cat ascarid. Infection of man by
these organisms leads to persistent larval migration in various
viscera (visceral larva migrans) including the eye (ocular larva
migrans). The latter is usually seen in older children and young
adults and may manifest as unilateral, solitary painless lesion
located posteriorly close to optic nerve and disk. Diethyl-
carbamazine (DEC), thiabendazole and mebendazole are useful
in the treatment of systemic toxocariasis.
ONCHOCERCIASIS
Onchocerciasis is caused by the nematode Onchocerca
volvulus. It is widely distributed across the African continent
and South America.
Humans are the only known reservoir of onchocerciasis. The
female Simulium fly is the intermediate host and vector that
ingests the microfilariae on biting an infected person during a
blood meal. The larvae then transform into infective forms that
may enter a new host when the simuliid takes another blood
meal. The larvae migrate in the body for ~1 year before they
settle in a nodule, which is most frequently subcutaneous.
Here, the male and female mate and produce numerous
microfilariae that migrate to various parts of the body.
Ocular manifestations of onchocerciasis include punctate
keratitis surrounding dead microfilariae, sclerosing keratitis,
anterior uveitis with secondary cataract and glaucoma,
chorioretinitis, and papillitis with severe constriction of the
visual fields.40
For several years DEC and suramin were the only two drugs
available for the treatment of onchocerciasis. DEC is effective
against microfilariae but causes an initial aggravation of the
ocular disease and has several troublesome side effects. Suramin
is active against adult worms but has a very high intrinsic
toxicity. These two drugs were at best suboptimal for mass
treatment regimen and consisted of decreasing doses of DEC
over 18 days followed by suramin intravenously, 1 g/week for
5 weeks.
In recent years, ivermectin has revolutionized the treatment
of onchocerciasis and has largely replaced DEC and suramin.
Numerous double-blind placebo-controlled studies have
demonstrated the efficacy and safety of ivermectin, its
Antiparasitics
suitability for mass therapy, and its superiority over DEC.41
Community-based treatment with ivermectin has been shown
to reduce the transmission of onchocerciasis. Ivermectin is
usually given in a single, annual, oral dose of 150 mg/kg. This
dosage seems to be adequate for all except the intensely infested
patients with severe ocular involvement in hyperendemic areas.
LOIASIS
Loiasis is caused by Loa loa and is transmitted by mango flies
of genus Chrysops. It is endemic in Central and West Africa.
The clinical disease mainly results from the migration of the
adult worms in the subcutaneous tissues called Calabar or
‘fugitive’ swelling. The worms may migrate across the bulbar
conjunctiva. Loa loa-induced retinopathy, uveitis, and
migration of the worm in the eyelid, the vitreous and the
anterior chamber have been documented.42 The drug of
choice for treatment of loiasis is DEC in a complex dosage
schedule.43
DIROFILARIASIS
Dirofilariasis is caused by Dirofilaria immitis, D. tenuis,
D. repens, D. ursi or D. subdermata. Primarily seen in dogs, the
disease has been reported in humans from almost all parts
of the world. It is transmitted by mosquitoes of genera Aedes,
Anopheles and Culex. Ocular form of dirofilariasis is less
common than pulmonary and subcutaneous forms. Eyelids are
CHAPTER 22
commonly involved followed by orbit, subconjunctival tissue
and intraocular tissues.44 The larvae inoculated by mosquitoes
migrate and mature in the subcutaneous tissues. Most
infections consist of single worm and its surgical removal
achieves complete cure. DEC, in dosage similar to loaiasis, is
commonly used.
FILARIASIS
Bancroftian filariasis is caused by Wuchereria bancrofti and
brugian filariasis by either Brugia malayi or Brugia timori. The
adult worms live in lymphatic systems and the infection is
transmitted by mosquitoes. The ocular manifestations may be
caused by either the adult worms or microfilariae. The
treatment of choice has been DEC given orally for 21 days. Oral
ivermectin can be used alternatively. Recurrence has been
reported following therapy with either drug.
DRACUNCULIASIS
Dracunculus medinensis, also known as guinea worm, causes
dracunculiasis or dracunculosis. Man acquires infection by
drinking contaminated water containing infected cyclops.
The disease is endemic in Africa and Asia. After primary
infection, the gravid female worm forms swellings in the lower
extremity and releases larvae when in contact with water.
Orbital involvement is described in early literature and the only
case describing a swelling of 4 mm diameter on the bulbar
conjunctiva is from India.45 Mechanical removal of the worm
accompanied with medical treatment with metronidazole or
thiabendazole is the usual mode of therapy.
TRICHINOSIS
Trichinosis is caused by larvae of Trichinella spiralis. Eating
infected pork is the commonest mode of infection. The larvae
parasitize skeletal muscles where they encyst. Ocular signs and
symptoms may be the first in early phase of muscle invasion.
The earliest sign may be bilateral palpebral edema which is 243
 PHARMACOLOGY AND TOXICOLOGY
due to invasion of extraocular muscles and concomitant
systemic allergy due to the parasite. Patient may have sub-
conjunctival hemorrhage, photophobia, diplopia, visual hallu-
cinations, etc. Fundus examination may reveal hyperemia,
papillary edema, retinal hemorrhages, optic neuritis or
neuroretinitis. The effective drug of choice is thiabendazole,
25 mg/kg twice-daily for 5 days during the intestinal phase.
During the muscular invasion phase mebendazole should be
used. Albendazole may also be effective in tissue phase.
Anthelminthic therapy is usually combined with topical
corticosteroids for relief of pain and swelling.46
CYSTICERCOSIS
Cysticercosis is caused by larvae of tape worms Taenia solium
or Taenia saginata, the larvae of the former being called
Cysticercus cellulosae and that of latter Cysticercus bovis. In
taeniasis, man is the definitive host, the adult tape worms
residing in the intestine. In cysticercosis, man acts as the
intermediate host. Most commonly the infection is contracted
by ingesting eggs in contaminated food or water. It can occur in
patients with taeniasis, either by fecal–oral auto infection or by
reverse peristalsis of proglottids into the stomach.
Ocular involvement is very common in cysticercosis
(13–46%) and it is the most common helminthic ocular
infection in man.47 Posterior segment of the eye is involved in
more than 70% of reported ocular cases. In subcutaneous
cysticercosis, the lesions are numerous, firm, elastic, round,
SECTION 4
painless nodules or papules which may become caseated or
calcified. Cysticercosis of the extraocular muscles is not
uncommon.48
The recommended treatment for neurocysticercosis includes
praziquantel therapy, however, effect of this drug in ocular
cysticercosis is not known.49,50 Metrifonate, 75 mg/kg daily for
5 days, repeated six times at 2-week intervals, is reported to be
successful in the treatment of ocular as well as cerebral and
subcutaneous cysticercosis. Treatment with a combination of
oral albendazole and prednisolone was reported to be effective
in a series of 26 cases of ocular myocysticercosis from southern
India.48 Similar combination therapy was found effective by
these authors in a series of orbital cysticercosis.50
SCHISTOSOMIASIS AND PARAGONIMIASIS
Schistosomiasis or bilharziasis is caused by fluke species
Schistosoma japonicum, Schistosoma mansoni and
Schistosoma haemotobium. Man gets infected through skin on
contact with water contaminated with schistosomal cercariae.
Adult worms grow in liver veins and migrate to mesenteric or
vesical veins and the damage to liver or urinany bladder is
caused by the eggs deposited in the vessels. Damage to the eye
is caused in a similar manner. Egg granulomas may be located
in the conjunctiva, lacrimal gland or in the choroid. Adult
S. mansoni worm bas been reported from the anterior chamber
and superior ophthalmic vein.51
Praziquantel is the drug of choice, the dosage for S. mansoni
being 40 mg/kg in two doses for 1 day. The other recommended
drug is oxamniquine in single dose of 15 mg/kg. Metrifonate
has been used for the treatment of S. haemetobium infection.
Paragonimiasis is caused by a lung fluke; Paragonimus
westermani. Man gets infected by eating infected crustacean
hosts such as crabs or crayfish. Ocular manifestations of uveitis
is mainly due to migration of the immature worm in the ocular
tissues. There may be associated retinal hemorrhage, vitreous
hemorrhage, exudative inflammation and secondary glaucoma.
The parasite is susceptible to praziquantel at a dosage of
244 25 mg/kg body weight three times daily for 2 days.
PHARMACOLOGY OF ANTIPARASITIC
AGENTS
Key Features
• Systemic agents
• Topical agents
• Dosage
• Efficacy
• Toxicity and side effects
SYSTEMIC AGENTS
DEC
DEC was discovered in 1947 as a result of an intensive search
for antifilarials. It is a piperazine derivative with the following
structural formula:
Diethylcarbamazine
O C2H5
H3C—N N—C—N
C2H5
It is used as a citrate salt that is highly soluble in water.
The mechanism of action of DEC is twofold, consisting of;
(1) decrease in the muscular activity of the microfilariae and
their immobilization, probably by virtue of the hyperpolarizing
effect of the piperazine moiety; and (2) change in the surface
membranes of the microfilariae, rendering them more sus-
ceptible to the host defense mechanisms. DEC is effective
against adult worms and microfilariae of Loa loa and only
microfilariae of O. volvulus.
DEC is rapidly absorbed from the GI tract. Peak plasma levels
of 1.6 mg/mL are achieved 1–2 h after a single oral dose of
200 mg. The minimum effective blood level appears to be
0.8–1.0 mg/mL. It rapidly equilibrates with all tissues except
fat and does not have a cumulative effect. Over 50% of the drug
is excreted unchanged in acidic urine.
DEC is a drug with low intrinsic toxicity. Anorexia, nausea,
headache, and less frequently vomiting and skin rash occur and
subside in a few days despite the continuation of treatment.
The drug appears to be safe in pregnancy. The major adverse
effects of DEC are a direct or indirect result of the death of
the microfilariae and adult worms. A severe encephalitis may
be induced in Loa loa-infected patients. Patients with oncho-
cerciasis typically manifest the Mazotti reaction, which occurs
in a few hours after the first dose and lasts 3–7 days. It consists
of itching, skin rash, painful lymphadenopathy, fever, tachy-
cardia, arthralgia, and headache. Higher doses can be tolerated
after this reaction subsides. In the eye, it produces migration of
microfilariae into the cornea, straightening and immobility of
the microfilariae, reaction around dead microfilariae, globular
limbal infiltrates of uncertain (probably immunologic) origin,
and worsening of eye lesions in heavily infected patients.
Retinal pigment epithelial changes also are known to occur. The
beneficial effects of DEC, namely, a decrease in skin and corneal
microfilariae, are short lived, making it an unsuitable agent for
the prophylaxis or mass treatment. DEC is well absorbed on
topical ocular and skin application but neither preparation has
any added advantage over oral administration.

Itraconazole
Itraconazole is an investigational triazole antifungal agent.
Its mode of action against Acanthamoeba remains to be
elucidated. It has been used in the treatment of microsporidial
keratoconjunctivitis.37
Itraconazole is closely related to ketoconazole. Its absorption
from the GI tract is enhanced when given with food. The mean
plasma level of a single dose of 100 mg is 132±67 ng/mL. The
plasma levels rise in the first 13 days, with a half-life of 36 h
after 15 days of dosing. Active drug is not detectable in the urine
or cerebrospinal fluid (CSF).
Itraconazole is well tolerated. Ten to 15% of patients com-
plain of nausea and vomiting. Rash, pruritus, dizziness, vertigo,
pedal edema, paresthesia, decreased libido, and impotence have
been reported occasionally.
Ivermectin
Ivermectin is a member of a new class of semisynthetic macro-
cyclic lactones called avermectins. It has a broad spectrum of
antiparasitic activity. It is now the drug of choice for oncho-
cerciasis. It is absorbed through the GI tract and is mainly
concentrated in the liver and adipose tissue. Peak plasma levels
are achieved in 4 h after oral administration. Its half-life is
~10 h. Animal studies indicate nearly all ivermectin is excreted
in the feces unchanged. Extremely low levels of the drug are
found in the brain. Not much is known about the pharmoco-
kinetics of ivermectin in the eye. It can be speculated that
because the drug is a macrocyclic lactone, it has poor ocular
penetration and therefore does not achieve microfilaricidal
concentrations in the eye. This would cause microfilarial move-
ment out of the eye along a concentration gradient.
The exact mode of action of ivermectin is unknown. It
modifies the release of the neurotransmitter g-aminobutyric
acid (GABA) but the relationship of this property to the
microfilaricidal activity is unclear. The microfilaricidal action
of ivermectin is slow, unlike that of DEC, and hence there is
no exacerbation of ocular inflammation. Ivermectin is neither
macrofilaricidal nor embryotoxic. It causes an initial increase
followed by a decrease in embryogenesis. There is a seques-
tration of normally developed embryonic forms in the uterus of
the adult female worms. The failure of microfilariae to be
released explains the lack of build-up of microfilariae after
single-dose treatment of ivermectin is continued for the life
span of the adult worm (10–15 years). It can interrupt trans-
mission and provide clinical prophylaxis and treatment of
ocular onchocerciasis.
Systemic side effects of ivermectin are mild and transient,
consisting of headache, and painful glands lasting a few hours;
skin rash lasting a few days, an asymptomatic and intermittent
increase in the pulse rate, a decrease in the blood pressure,
an increase in temperature, and electrocardiographic (ECG)
changes.
Ivermectin therapy is not associated with exacerbation of
ocular inflammation and this is an overwhelming advantage
over medications previously used in the treatment of oncho-
cerciasis. The hematologic changes associated with the admin-
istration of ivermectin are a transient decrease in hemoglobin,
neutrophil leukocytosis, and lymphocytopenia and an initial fall
followed by a steady rise in the eosinophil count.52
Metronidazole
Metronidazole is a nitroimidazole with a broad spectrum of
antiprotozoal and antimicrobial activity. It has the following
structural formula:
Metronidazole is directly effective against trophozoites of
Giardia lamblia at concentrations of 1–50 mg/mL in vitro.
Mechanism of action is linked to the ability of the nitro group
Antiparasitics
Metronidazole
H
NH COOCH3
C
N
N
O
to trap electrons from electron transport proteins and divert
them from normal energy-yielding pathways. Studies with
mammalian DNA reveal that reduced metronidazole can cause
the loss of helical structure and strand breakage of DNA.
Metronidazole is completely and promptly absorbed from the
GI tract and therapeutic plasma levels are observed 1 h after
oral administration of a single dose of 500 mg. The half-life of
the drug is 8 h. Ten percent of the drug is bound to plasma
proteins. It shows good penetration into body tissues and fluids.
Metronidazole crosses the blood–brain barrier. Greater than
50% of the systemic clearance occurs in the liver. Phase I
biotransformation by oxidation yields active metabolites.
Conjugation with glucuronides also occurs.
The most common side effects associated with metronida-
zole are headache, nausea, dry mouth, and a metallic taste.
Occasionally, vomiting, diarrhea, and abdominal pain occur.
Neurotoxicity in the form of dizziness, ataxia, convulsions,
encephalopathy, and sensory neuropathies occur. These necessi-
CHAPTER 22
tate prompt withdrawal of the drug. Temporary and reversible
leukopenia can occur. Metronidazole has a well-documented
disulfiram-like effect. Patients should therefore be cautioned
against alcohol. Active CNS disease is a contraindication and
severe hepatic or renal dysfunction necessitate reduction in
dosage. Metronidazole and its metabolites have mutagenic activity
and hence should not be used in the first trimester of pregnancy.
Pentosam (Sodium Stibogluconate)
Pentosam is a pentavalent antimonial that interferes with the
glycolysis and oxidation of fatty acids in the organelles called
glycosomes within the amastigotes of Leishmania brasiliensis.
Nonspecific binding of antimony to the sulfhydryl groups in the
amastigote protein may be another mechanism of action.
Pentosam is rapidly absorbed when given intramuscularly or
intravenously and is eliminated in two phases: The first rapid
phase has a half-life of 2 h and a second slow phase has half-life
of 33–76 h.
Pain at the site of intramuscular injection, GI disturbance,
muscle pain, joint stiffness, and a reversible increase in hepatic
transaminases are relatively mild side effects of pentosam
administration. However, reversible T-wave flattening and
increase in QT interval may precede serious arrhythmias.
Pyrimethamine
Pyrimethamine is a diaminopyrimidine with the following
structural formula.
It is a competitive antagonist of folic acid by virtue of its pre-
ferential inhibition of dihydrofolate reductase of the parasites.
This prevents the reduction of dihydrofolate to tetrahydrofolate
that is necessary for synthesis of purines and pyrimidines. Pyri-
methamine is synergistic to sulfas by virtue of this sequential
inhibition and hence is almost always used with sulfonamide.
It is only active against actively proliferating Toxoplasma organ-
isms. Pyrimethamine is slowly and completely absorbed after
oral administration. It accumulates in the kidney, lung, liver,
and spleen. Elimination is slow, with a half-life of 80–95 h.
Occasional skin rash and decreased hematopoiesis are associ-
ated with the use of pyrimethamine. Large doses of pyrimethamine 245
 PHARMACOLOGY AND TOXICOLOGY
Pyrimethamine
CH3 CH2 N NH
N occur on the basis of hypersensitivity to the sulfonamides.
NH2
C mechanism of action of suramin is not clear. Its interference
for a long period of time can cause a megaloblastic anemia that
is readily reversible by discontinuing the drug or administration
of folinic acid. A severe reversible thrombocytopenia as a result
of hematologic depression is an important side effect of pyrime-
thamine therapy and necessitates discontinuation of the drug.
Quinacrine
Quinacrine is an acridine derivative previously used as an
antimalarial but currently being used only for the treatment of
giardiasis. It is readily absorbed from the GI tract and is slowly
eliminated. Quinacrine has a cumulative effect. Its metabolism
and its mode of antiparasitic action are not well understood.
Headache, dizziness, and vomiting are frequent side effects
SECTION 4
associated with quinacrine use. Blood dyscrasias, urticaria,
exfoliative dermatitis, yellow pigmentation of the skin, and blue
or black pigmentation of the nails may also occur. Occasionally,
ocular toxicity resembling that of chloroquine occurs.
Quinacrine should be administered with caution in patients
with psoriasis, because it can cause a severe exacerbation.
Sulfonamides
Sulfonamides are structural analogs and competitive anta-
gonists of para-aminobenzoic acid (PABA). They act by the
inhibition of dihydropteroate synthetase, which is the enzyme
responsible for the incorporation of PABA into dihydropteroic
acid, the immediate precursor of folic acid. Sulfonamides are
synergistic to other antifolates such as pyrimethamine and
trimethoprim. The structural formula of sulfadiazine is as
follows:
Sulfadiazine
N conjunctival injection, chemosis, follicular conjunctivitis,
H2N SO2HN
N
Sulfadiazine in combination with pyrimethamine is the
treatment of choice for toxoplasmosis.
Sulfonamides are rapidly absorbed from the GI tract. After a
single dose, peak plasma levels are reached in 3–6 h and thera-
peutic concentrations occur in the CSF in 4 h. They readily
cross the placental barrier. Sulfonamides are metabolized in the
liver and excreted mainly by the kidneys in the acetylated and
the free form. The excretion of both forms is accelerated by the
administration of alkali, which decreases tubular reabsorption.
The acetylated form of sulfonamides loses the antimicrobial
activity while retaining the toxicity of the parent compound.
The most common side effects associated with the use of
sulfonamides are fever, urticaria, and GI disturbances. Urinary
246 tract disturbances such as crystalluria and hematuria are
associated with deficient hydration and acidic or neutral urinary
pH. Hemolytic anemia, especially in patients with a glucose-6
phosphate dehydrogenase (G-6PD) deficiency; reversible agra-
nulocytosis; and an irreversible aplastic anemia are rarely seen.
The Stevens–Johnson syndrome, exfoliative dermatitis, serum
sickness, and sometimes, a fatal acute necrosis of the liver can
Suramin
Suramin is the only drug effective against adult Onchocerca
volvulus. It is mirofilaricidal to a lesser extent. Suramin is an
organic urea compound with high intrinsic toxicity and hence
needs to be administered under close supervision. The exact
with DNA and RNA metabolism may be the basis of its
antiparasitic action. Suramin acts mainly on female worm,
causing its death and degeneration in 5 weeks.
Suramin can only be administered intravenously. It binds
firmly to plasma proteins. After intravenous administration,
the plasma concentration of suramin drops rapidly in the first
few hours and then stabilizes in a few days. It has a half-life of
48 h. Suramin is a large polar anion that does not enter cells
readily. It does not cross the blood–brain barrier. Suramin is not
metabolized to any extent and is excreted unchanged, mainly by
the kidney.
Suramin therapy is usually associated with significant
morbidity due to systemic side effects such as malaise, nausea,
nervous fatigue, fever, arthralgia, myalgia, peripheral neuro-
pathy, and the worsening of ocular signs and symptoms that
occur in the initial phases of treatment. Optic atrophy has also
been reported. Rarely, circulatory shock and coma can occur as
an immediate reaction to suramin. Other serious reactions such
as agranulocytosis, renal shutdown, hemolytic anemia, and
jaundice are fortunately rare. Fatal reaction to suramin therapy
has been reported. Suramin has largely been replaced by
ivermectin in the treatment of onchocerciasis.
TOPICAL AGENTS
Dibromopropamide Isethionate and Propamidine
Isethionate
Dibromopropamidine isethionate and propamidine isethionate
are both aromatic diamidines with a broad spectrum of anti-
bacterial and antifungal activity. They are marketed in England
as Brolene ointment (0.15%) and drops (0.1%). They are not
available in the United States. Intensive use of the ointment
causes local irritation and similar use of drops causes increased
punctate corneal lesions which are reversible and do not
necessitate discontinuation of medication.11,53
Miconazole
Miconazole is an imidazole antifungal agent that also has
antiamoebic activity. All imidazoles can be made into a 1%
suspension in arachis oil or a 10-mg/mL solution for topical
use. Foster et al have shown that in rabbits miconazole reaches
high levels in the cornea and aqueous humor after topical
or subconjunctival administration.54 It was also shown to
readily penetrate the blood–aqueous barrier after intravenous
administration. Ocular side effects include superficial punctate
keratitis and stinging.
Cationic Antiseptics
Chlorhexidine and PHMB are two important cationic anti-
septics that are topically used in the treatment of Acantha-
moeba keratitis. While chlorhexidine is a biguanide, PHMB is a
polymeric biguanide. Both act by compromising the integrity of
 Antiparasitics

the mucopolysaccharide plug that seals the ostiole of the
Acanthamoeba cyst. Irreversible loss of essential cellular com-
ponents through the damaged plasmalemma results in cell
death. Corneal epithelial toxicity (clinically) is minimal for
chlorhexidine and PHMB at a concentration of 0.02%.55 Both
chlorhexidine and PHMB have amoebicidal and cysticidal
activity.56 PHMB is manufactured principally as an industrial
grade sterilant. It is used in cosmetics and soaps as preserva-
tives, as an algastatic compound in swimming pools and a
constituent of contact lens disinfecting fluids. In early 1990,
PHMB was found to be highly effective in killing both cysts and
trophozoites in in vitro studies.57 Larkin et al reported its
successful clinical use at a concentration of 0.02%.12 Lam et al
reported that topical PHMB monotherapy leads to persistence
of infection and hence suggested use of combination therapy in
treatment of Acanthamoeba keratitis.58 PHMB has advantages
over propamidine in having high consistent cysticidal activity
and no toxicity.

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 CHAPTER

23 Corticosteroids in Ophthalmic Practice

Mark B. Abelson and Salim Butrus

Key Features formulations. In 1952, ocular penetration studies of steroids

• Corticosteroids are 21-carbon structures synthesized naturally
started to surface. By that time, modification of chemical struc-
or synthetically through adenocorticotropic hormone-controlled
tures of cortisone and hydrocortisone led to a series of com-
conversion of cholesterol
pounds with better penetration and bioavailability and more
• Although their mechanism of acitgon is still enigmatic, it is
potent antiinflammatory effects. In 1959, 0.1% Decadron eye
known that corticosteroids work at both molecular and cellular
drops were introduced for treating ocular inflammation.6 In 1956,
levels
it became clear that inflammation in anterior ocular structures
• The effectiveness of a corticosteroid is largely determined by
is best treated with steroid drops and posterior uveitis by oral
its ability to penetrate the cornea
therapy. It was quickly recognized that topical therapy mini-
• In ophthalmic practice, corticosteroids are most frequently
mized systemic side effects, but its ocular side effects began to
used to control post-surgical inflammation. They are also used
be appreciated.
to treat symptoms of immune hyperreactivity and to treat
diseases with immune and infectious processses. CHEMICAL PROPERTIES AND
• The practioner must be vigilant for the onset of ocular side STRUCTURE–ACTIVITY RELATIONSHIPS
effects, which can occur with prolonged steroid use.Side
effects most frequently involve the anterior segment, and can
Cortisone, the first steroid used therapeutically for antiinflam-
include glaucoma, cataracts, and enhanced bacterial infection.
matory effect, is a 21-carbon four-ringed structure (Fig. 23.1).
They can also inhibit corneal epithelial and stromal healing
Modification of this structure at different sites changes its bio-
logic potency, transcorneal penetration, and, thus, effectiveness
and side effects.5
Corticosteroids (glucocorticoids and mineralocorticoids) are Different sites of alterations (Fig. 23.1) result in different
21-carbon structures that are synthesized by adrenocorticotropic antiinflammatory potency and duration of action of these dif-
hormone (ACTH)-controlled conversion of cholesterol in the ferent compounds (Table 23.1). These modifications and alter-
adrenal cortex. They can take the form of cortisol, cortisone, ations can be summarized as follows:
corticosterone, or aldosterone. They can also exist in synthetic 1. Prednisone and prednisolone have, in addition to the basic
forms such as prednisone, methylprednisolone, dexamethasone, nucleus, a 1,2 double bond in ring A (Fig. 23.1b). This
triamcinolone, betamethasone, medrysone, fluorometholone
(FML), and others.
In 1930, Swingle, Pfiffner, Hartman, and co-workers prepared
adrenocortical extracts that had a reasonable degree of activity.
In 1935, Kendall first isolated and characterized cortisone in
the laboratory. In 1942, Reichstein and Shoppee identified the
chemical and crystalline structure of steroids.1 The first
advantageous clinical result of steroids was reported by Hench
and co-workers in 1949.2 They observed the dramatic effects of
a b
cortisone and ACTH in the treatment of rheumatoid arthritis
and subsequently provoked the interest of many investigators
with remarkable therapeutic applications that extended to other
diseases.
In 1954, Stone and Hechter established that ACTH actually
controls the enzymatic conversion of cholesterol to steroids in
the adrenal cortex through cleavage of the side chain of the cho-
lesterol molecule.3 Later, Haynes took a further step by demon-
strating that this conversion is mediated by adenosine 3„,5„-cyclic
monophosphate (cAMP).4,5 c d
Corticosteroids and ACTH were first introduced into ocular
therapy by Gordon and McLean in 1950. It was not until 1951, FIGURE 23.1. The cortisol nucleus (a). Note the sites where different
with the introduction of topical and systemic use of cortisone, chemical groups are added to form compounds with different
that cortisone acetate was prepared in eye drop, ointment, and antiinflammatory potency. Prednisolone (b); dexamethasone (c);
subconjunctival, retrobulbar, and anterior chamber-injection triamcinolone (d). 249
 PHARMACOLOGY AND TOXICOLOGY

TABLE 23.1 Classification of Glucocorticoids

Biologic Antiinflammatory
Half-Life (h) Effect (h)

Natural Steroids
Cortisol 8–12 1
Cortisone 8–12 0.8
Corticosterone 8–12 0.3
Synthetic Steroids
Prednisone 12–36 4
Prednisolone 12–36 4
6-Methylprednisolone 12–36 5
Triamcinolone 12–36 5
9-Fluorocortisol 12–36 10
Paramethasone 36–72 10
Betamethasone 36–72 25
Dexamethasone 36–72 25
FIGURE 23.2. Binding of corticosteroid to a receptor and subsequent
entry into the cell cytoplasm and nucleus. This leads to the synthesis
of specific proteins and specific target cell responses.

modification increases their carbohydrate-regulating

potency and prolongs their metabolism compared with cortisol and prednisolone. Glucocorticoid receptors have been
SECTION 4

cortisol.
2. Methylation of carbon 6 in ring B leads to 6a-methyl
prednisolone. This compound has slightly greater
antiinflammatory effect than prednisolone.
3. Fluorination at a 9a-position in ring B, as in
fluorocortisone (9a-fluorocortisol) enhances its
antiinflammatory property.
4. 11-Desoxycortisol has an oxygen function at the C-11 site
of ring C, augmenting its antiinflammatory activity.
5. Methylation or hydroxylation at site 16 in ring D
eliminates the sodium-retaining effects and has only a
slight effect on the antiinflammatory potency.
6. In ring D, 17a-hydroxylation is present in most of the
antiinflammatory steroids.
7. Most of the active synthetic analogs and all natural
corticosteroids have the hydroxyl group attached to carbon
21 in ring D.
MECHANISM OF ACTION, SITE OF
ACTIVITY, AND OPHTHALMIC INDICATIONS
Corticosteroids have numerous effects on many stages of inflam-
mation and arms of the immune response. Despite widespread
use, their precise mechanism of action is not well understood.
There is consensus that they work at two levels: molecular and
cellular. At the molecular level corticosteroids freely penetrate cell
membranes and bind to a specific steroid-binding protein receptor
in the cytoplasm, forming a steroid–receptor complex.7–18 This
complex then moves into the nucleus and binds to chromatin,
signaling the production of messenger RNA and coding for
enzymes and proteins that determine the response of that par-
ticular cell to the hormone (Fig. 23.2).5,19
The cytoplasmic steroid-binding receptor has binding sites
that exhibit high affinity for glucocorticoids (e.g., the naturally
occurring cortisol and corticosterone) and synthetic corticosteroids
(e.g., prednisolone, dexamethasone, and triamcinolone).20 In con-
trast, these receptors have a low affinity for estrogens, androgens,
cortisone, and prednisone. Hence, cortisone and prednisone are
250 inactive compounds that are activated when transformed to
identified in the iris, ciliary body, cornea, sclera, trabecular mesh-
work, and Schlemm’s canal.21–23
These molecular and cellular changes result in steroid-induced
inhibition of all the cardinal signs of inflammation, such as pain,
heat, redness, and edema.13,24 This is achieved through inhibition
of: (1) leukocyte chemotaxis, (2) production of potent chemical
mediators, and (3) function of immunocompetent cells. Corti-
costeroids have the dual characteristics of being both anti-
inflammatory and immunosuppressant.25 They accomplish their
antiinflammatory activity through the following mechanisms:
1. Constriction of blood vessels and reduction of vascular
permeability induced by acute inflammation. This
minimizes leakage into the target site of fluid, proteins,
and inflammatory cells.26
2. Stabilization of intracellular lysosomal membranes and
inhibition of the expression of various damaging enzymes;
polymorphonuclear (PMN)-cell degranulation is also
significantly inhibited.
3. Stabilization of mast cell and basophil membranes is
important in inhibiting the process of degranulation and
subsequent release of histamine (vasoactive amines),
bradykinin, platelet-activating factor (PAF), proteases, and
eosinophilic chemotactic factors (ECFs).
4. Mobilization of PMNs from the bone marrow results in
neutrophilic leukocytosis (Fig. 23.3).27 Corticosteroids
simultaneously prevent adherence of PMNs to the
vascular endothelium, making them less mobile and less
accessible to the site of inflammation.28
5. Suppression of lymphocyte proliferation and lymphopenia.
In small- to moderate-sized doses, corticosteroids more
significantly affect T lymphocytes. In larger doses,
B lymphocytes are affected as well. Corticosteroids do
not destroy T lymphocytes but rather affect their
redistribution into circulation, concentrating them in the
bone marrow (Fig. 23.4).29–31
6. Reduction of circulating eosinophils and monocytes.
7. Inhibition of macrophage recruitment and migration.32,33
Steroids also interfere with the ability of macrophages to
process antigens.

FIGURE 23.3. Schematic effects of corticosteroids on bone marrow
and circulating neutrophils.
Adapted from Nussenblatt RB, Palestine AG: Uveitis: fundamentals and clinical
practice. Chicago, IL: Year Book; 1989.
8. Suppression of fibroplasia.34
9. Depression of the bactericidal activity of monocytes and
macrophages.
10. Steroids inhibit phospholipase A2, via a protein called
macrocortin, resulting in inhibition of arachidonic acid
degradation and subsequent synthesis of prostaglandins
and leukotrienes by cyclooxygenase and lipoxygenase
pathways (Fig. 23.5).35–39
ABSORPTION RATE AND EXCRETION
AFTER OPHTHALMIC DELIVERY
Corticosteroids are readily absorbed by the cornea, conjunctiva,
and sclera. Corneal penetration is a limiting factor for their anti-
inflammatory effect. The penetration of corticosteroids through
the normal cornea is a complex process in which multiple factors
determine the rate of penetration. In general, these factors are
similar to those governing penetration (i.e., relative solubility in
water and lipid).40,41 Other factors include viscosity, concentra-
tion, hydrogen ion concentration (pH), tonicity, condition of the
Corticosteroids in Ophthalmic Practice
CHAPTER 23
FIGURE 23.4. Schematic effects of corticosteroids on lymphocytes.
Adapted from Nussenblatt RB, Palestine AG: Uveitis: fundamentals and clinical
practice. Chicago, IL: Year Book; 1989.
corneal epithelium, size of particles in suspension, and addition
of other compounds or vehicles, such as preservatives or methyl-
cellulose. Part of the topically applied corticosteroid can go
through the upper and lower puncti and then through the nasal
mucosal blood vessels into the circulation, where it binds to
globulin and albumin. Eighty percent of circulating cortisol is
bound to a-globulin as transcortin (corticosteroid-binding glo-
bulin), an inactive transport complex. A smaller portion is bound
to albumin, and this portion can diffuse into the extravascular
fluid and bathe tissue cells. Synthetic analogs of cortisol do not
compete with it for binding to transcortin. In addition, synthetic
analogs are less bound to albumin, enabling them to diffuse more
completely into the extravascular tissue than cortisol.25
Tritiated dexamethasone applied topically to rabbit eyes was
traced and found in plasma, kidneys, urine, and liver. Systemic
absorption of topical dexamethasone phosphate is considerable:
as much as 20–35% of the drug was found systemically in rabbits
24 h after instillation.42,43 Reduction of the double bond in the
1,5-position in the liver and kidney renders the corticosteroid 251
 PHARMACOLOGY AND TOXICOLOGY
FIGURE 23.5. Corticosteroids prevent formation of prostaglandins
and leukotrienes through inhibition of phospholipase A2 and release of
arachidonic acid.
SECTION 4
inactive. All synthetic analogs of cortisol are metabolized more
slowly by the liver, owing to chemical modifications of the
steroid molecule (Fig. 23.1) and the rapid equilibration in blood
and peripheral tissues.
PHARMACOKINETICS
Four factors regarding ophthalmic corticosteroids must be con-
sidered:20 (1) ocular penetration of the corticosteroid through
the cornea; (2) antiinflammatory potency, topically and once in
the aqueous humor; (3) duration of action; and (4) side effects.
Different routes by which corticosteroids are delivered into the
eye include topical, periocular, oral, parenteral, and intravitreal.
The penetration of corticosteroids is dependent on the cornea
and on the physical and chemical properties of the corticos-
teroid. The ideal steroid should be biphasic in polarity, because
the cornea contains both hydrophobic and hydrophilic layers.44
Removal of the corneal epithelium reduces the hydrophobic
properties and allows greater penetration by hydrophilic prep-
arations. Particle size may also affect the bioavailability of cor-
ticosteroids.45,46 Results suggest that ophthalmic dexamethasone
suspensions can be optimized for bioavailability by using sus-
pensions with the smallest particle possible. Particle size for
prednisolone acetate (<5 mm and 5–10 mm in diameter), however,
did not affect the degree of corneal penetration. Both fractions
of prednisolone acetate achieved comparable levels in the
aqueous humor.
Topical preparations can take the form of solutions, suspen-
sions, or ointments. Phosphate and hydrochloride preparations
are relatively hydrophilic and thus are water soluble. Acetate
and alcohol derivatives are hydrophobic and fat soluble. Alcohol
preparations possess intermediate hydrophobicity between phos-
phates and acetates.47 Owing to the respective polarities, phos-
phates are generally formulated as solutions, whereas acetates are
generally formulated as suspensions and ointments. Acetates,
owing to their hydrophobic nature, appear to penetrate the cornea
to a greater extent than do phosphates.48–51
Corticosteroids can also be released from a drug depot placed
252 on the ocular surface or by iontophoresis. Examples of drug
depots are cotton pledgets52 and collagen shields.53 One advan-
tage of drug depots is the steady, sustained, and slow release of
the corticosteroid over the ocular surface.
Dexamethasone phosphate penetrates into the cornea and
aqueous humor within 10 min. It reaches a peak within 30–60 min
and remains inside the eye from several hours to 24 h.42 The
corneal tissue concentration of tritiated dexamethasone alcohol
(Maxidex) reaches 14.79 mg/g of cornea 7.5 min after instillation,
then declines to 1.86 mg/g at 4 h.54
One percent prednisolone phosphate (Inflamase) is a highly
soluble compound with limited lipid solubility. Thus, it tradi-
tionally was thought that this compound had limited solubility
through an intact cornea. Its corneal level, however, reaches
10 mg/g, while the aqueous humor concentration reaches 0.5 mg/g,
30 min after instillation. When the corneal epithelium is removed,
the corneal concentration reaches 235 mg/g and in aqueous
humor 17 mg/g.55
It has been shown that 1.1% tritiated dexamethasone
phosphate instilled into rabbit eyes reaches the aqueous humor.
Its major metabolite in the anterior chamber is 9a-fluoro-11b-
hydroxy-16a-methyl-1,4-androstadiene-3,17dione. Ocular pen-
etration of corticosteroids is better when they are injected
subconjunctivally than when they are instilled. Hydroxycor-
tisone is found in the anterior chamber almost immediately after
subconjunctival injection. Its degree of penetration is not related
to external factors such as lid movements or tear volume. It is
usually injected near the site of inflammation to obtain maximal
antiinflammatory benefits.
ANTIINFLAMMATORY EFFECTS OF
TOPICAL OPHTHALMIC
CORTICOSTEROIDS
Any attempt to compare the inflammatory potency of different
ophthalmic corticosteroids should take into account the fol-
lowing considerations: (1) type of corticosteroid, (2) formulation,
(3) concentration, and (4) what model of inflammation is used.
Models of ocular inflammation in animals and humans are dif-
ficult to design and standardize, and some do not reflect clinical
action in humans. Furthermore, some current data on corti-
costeroids have been extrapolated from previous studies con-
ducted on organ systems other than the eye.56
Studies by Leibowitz, Kupferman, and Cox involved measuring
decreased radioactivity of radiolabeled neutrophils in a rabbit
keratitis model induced by injection of clove oil.57–67 This research
focused on the comparison of the sodium phosphate, alcohol,
and acetate derivatives of dexamethasone and prednisolone
(Table 23.2). Data indicated that after a given period, corneal
drug concentration administered with the corneal epithelium
intact was highest with prednisolone acetate, followed by pred-
nisolone sodium phosphate and dexamethasone alcohol suspen-
sion; no dexamethasone sodium phosphate was absorbed. With
a denuded epithelium the highest concentration was achieved
with the prednisolone sodium phosphate solution, followed by
the dexamethasone sodium phosphate solution, prednisolone
acetate, and last, the dexamethasone alcohol suspension. With
intact and denuded epithelium, the drug concentrations in the
aqueous humor followed the same pattern. The results with
denuded epithelium may more accurately represent the clinical
situation in keratitis.57–60
With an intact epithelium but in the presence of intraocular
inflammation (i.e., experimentally induced anterior or posterior
uveitis), prednisolone acetate concentration in the cornea was
highest with the sodium phosphate solutions of prednisolone and
dexamethasone equivalent, and was least with the dexametha-
sone acetate and alcohol. Concentrations of prednisolone acetate
suspension and the sodium phosphate solution were equivalent

TABLE 23.2 Comparison of Different Topical Corticosteroids in Suppressing Rabbit Corneal Inflammation
Corneal Epithelium Intact % Decrease
Prednisolone acetate 1% 51
Dexamethasone alcohol 0.1% 40
Prednisolone sodium phosphate 1% 28
Fluoromethalone alcohol 0.1% 31
Dexamethasone sodium phosphate 0.1% 19
Dexamethasone sodium phosphate ointment 0.05% 13
in the aqueous humor in the eye inflamed with uveitis, followed
by the dexamethasone solution, and last, the dexamethasone
alcohol suspension. Thus, with intraocular inflammation, for
which the highest concentration of drug is most desirable in the
aqueous humor, it is interesting that there was no difference
between the prednisolone acetate suspension and the sodium
phosphate solution.64
Leibowitz and Kupferman also evaluated these steroid deriva-
tives for antiinflammatory potency in a model of corneal inflam-
mation. A significant increase in antiinflammatory effect was
noted with prednisolone acetate compared with the sodium phos-
phate solution when evaluated with the corneal epithelium intact.
When the corneal epithelium was absent there was no significant
difference between the two64,66 or dexamethasone alcohol. Thus,
when a break in the corneal epithelium is associated with corneal
inflammation, the greater absorption of the sodium phosphate
solution equilibrates their relative potency. The dexamethasone
sodium phosphate solution was clearly significantly inferior with
the epithelium intact or absent.64,66
Changing the concentration and dosing frequency of a par-
ticular steroid obviously changes its antiinflammatory potency.
Increasing the concentration of prednisolone acetate from 0.125%
to 1% produces a significant increase in its corneal concentration58
and antiinflammatory effectiveness.63,64
The concentrations in the cornea and aqueous humor of the
corticosteroid, and thus their antiinflammatory potency, depend
to a large extent on the frequency of instillation. For example,
hourly instillation of 1% prednisolone acetate produces much
more effective suppression of corneal inflammation than does
instillation every 4 h (Table 23.3).65
The ocular bioavailability of topical prednisolone preparations
has been further investigated. One criticism of the clove oil model
used by Leibowitz and others is that the oil alters the absorption
of water-soluble drugs in favor of water-insoluble drugs because
TABLE 23.3 Dosage Schedules and Antiinflammatory
Effectiveness of Topical Prednisolone Acetate 1%
Total Doses Decrease in Corneal
Regimen Delivered (No.) Inflammation (%)
1 drop q 4 h 6 11
1 drop q 2 h 10 30
1 drop q 1 h 18 51
1 drop q 30 min 34 61
1 drop q 15 min 66 68
1 drop each eye 90 72
for 5 min every h
Corticosteroids in Ophthalmic Practice
Corneal Epithelium Absent % Decrease
Prednisolone acetate 1% 53
Dexamethasone alcohol 0.1% 42
Prednisolone sodium phosphate 1% 47
Fluorometholone alcohol 0.1% 37
Dexamethasone sodium phosphate 0.1% 22
of the oil barrier in the stroma after injection. A pharmaco-
kinetic model of absorption of water-insoluble drugs, such as
prednisolone acetate, and water-soluble drugs, such as pred-
nisolone phosphate, was used to compare the drug elimination
rate in the precornea and anterior chamber, the rate of drug dis-
solution, the rate of drug penetration in the cornea, and the rate
of drug transport into the aqueous humor. In this mathematical
model, the two forms of prednisolone had similar absorption
capacity.55 Similar bioavailability was also found in a rabbit eye
model in vivo when prednisolone phosphate, acetate, and their
metabolite, prednisolone, were directly quantitated in aqueous
humor by reverse-phase high-performance liquid chromatography
(HPLC).68,69
CHAPTER 23
In light of the fact that the acetate and phosphate forms may
actually be equivalent under optimal conditions of dissolution,
the drawbacks of using a suspension in clinical practice may be
the deciding factor in determining which is superior. Suspensions
need to be shaken, and if particles are not evenly distributed,
incorrect doses may be removed from the bottle. Patient com-
pliance for shaking suspension eye drops has been reported to
be poor.70 The risks of incorrect dosing and sudden cessation of
steroid administration are well known.71,72 The difficulty of pre-
dicting a steroid concentration in suspension drops suggests that
the consistent dosing provided by solutions may be superior.
Two weaker topical corticosteroids are also available for ocular
use. FML 0.1% and 0.25% suspensions have much less corneal
penetration73 than prednisolone but do have moderate anti-
inflammatory effects.74 Surprisingly, the lower concentration of
0.1% FML acetate has a therapeutic effect comparable to 1% pred-
nisolone in alleviating corneal (but not intraocular) inflammation.
Lower ocular levels are required to produce a substantial thera-
peutic effect in the cornea. FML has mildly hydrophilic prop-
erties, concentrating in the corneal epithelial layer and reaching
saturation levels before passing on through the hydrophilic layers
of the stroma. This may explain why FML penetrates the cornea
in comparatively low concentrations, yet produces moderate but
effective suppression of corneal inflammation.74
Medrysone (HMS) is another relatively weak corticosteroid.
It comes in a 1% suspension and, owing to its weak effect on the
cornea, is used only for minor conjunctival inflammation.
Loteprednol etabonate is a novel ‘soft’ steroid that was designed
to improve the benefit/risk ratio of topical corticosteroid therapy.
Its molecular structure is a modification of prednisolone (see
Fig. 23.1b), where a labile ester function occupies the 17-position
and a stable carbonate group occupies the 17-position. The ‘soft
drug’ undergoes rapid hydrolysis in the anterior chamber to the
inactive 17-carboxylic acid derivative after it penetrates the
cornea.75 In animals it was shown to retain its antiinflammatory
effects in the cornea,76 and in one study in humans, it was shown
to be useful in treating giant papillary conjunctivitis.77 In recent
years, loteprednol ophthalmic solution has been investigated for
use in treating inflammation due to keratoconjunctivitis sicca 253
 PHARMACOLOGY AND TOXICOLOGY

in patients with delayed tear clearance78 and has been approved
for treating seasonal and perennial allergic conjunctivitis.79 It is
also used to treat inflammation after cataract surgery.80
Rimexolone is another ‘soft steroid’ with decreased propensity
to raise IOP.81 The corticosteroid is indicated for the treatment
of postoperative inflammation following cataract surgery and
for treatment of anterior uveitis, and is commercially available
as a 1% ophthalmic suspension (Vexol). In a study consisting of
197 patients who had undergone cataract extraction, rimexolone
1% was significantly more effective than placebo in reducing
postoperative inflammation.82 The degree of improvement with
rimexolone was comparable to that of bethamethasone.83
Corticosteroids are also available as ointments. Although oint-
ments increase contact time between the drug and the ocular
surface, it has been shown that dexamethasone phosphate oint-
ment allows less drug absorption in the cornea and anterior
chamber than the solution form. This may be because the oint-
ment forms a barrier, preventing rapid release of the drug into
the tears.66 In the case of FML, it was shown that FML crystals
suspended in water or ointment both produced similar concen-
trations of drug in the aqueous humor, possibly because the tear
film is oversaturated by microcrystals of the dissolved drug.84
Corticosteroids may also be injected into parts of the eye.
Supratarsal injection of corticosteroids has been investigated to
treat refractory vernal keratoconjunctivitis.85 All patients experi-
enced dramatic symptomatic relief within 1–5 days, regardless
of type of corticosteroid injected.
OPHTHALMIC INDICATIONS FOR
CORTICOSTEROID THERAPY86
Since corticosteroids were first reported effective in the treatment
of rheumatoid arthritis more than 50 years ago, they have become
the most widely used antiinflammatory and immunosuppressant
agents in medicine and ophthalmology. It is estimated that more
than 5 million patients are treated with corticosteroids yearly.
The antiinflammatory and antiallergic activities of corticosteroids
are the most important reason for their clinical use in ocular
disease. Table 23.4 lists the ophthalmic indications for corti-
costeroid treatment as primary or adjunctive therapy. Some of
these indications are isolated inflammatory conditions and some
are part of a multisystem process. It must be remembered that
the antiinflammatory and immunosuppressive qualities of corti-
costeroids are nonspecific, palliative, and never curative.
The use of steroids in clinical ophthalmic practice may be
divided into three classes of therapy: (1) posttraumatic control

TABLE 23.4 Some Indications for the Use of Corticosteroids in Ocular Disease

Conjunctivitis Uvea
SECTION 4

Allergic (hay fever, vernal, atopic GPC) Iridocyclitis

Viral (EKC, herpes zoster) Posterior uveitis
Chemical burns Sympathetic ophthalmia
Cicatricial pemphigoid Vogt–Koyanagi–Harada syndrome
Mucocutaneous inflammation Pars planitis
(Stevens–Johnson, graft vs host disease, Endophthalmitis
toxic epidermal necrosis)
Keratitis Retina
Herpes zoster Vasculitides
Disciform herpes simplex Choroiditis
Interstitial keratitis (syphilis, herpes simplex) Retinitis
Immune infiltrates (Staphylococcus, herpes, Cystoid macular edema
varicella, contact lens, EKC, leukemia)
Acute retinal necrosisOptic
Peripheral ulcerative (connective tissue
disease, e.g., Wegener’s granulomatosis, Nerve
polyarteritis nodosa) Optic neuritis
Mooren’s ulcer Temporal arteritis
Reiter’s, Lyme disease, sarcoid Orbit
Corneal graft rejection Graves’ orbitopathy
Post-refractive surgery (DLK) Pseudotumor
Dry eyes Extraocular Muscles
Trauma and Postsurgery Myositis
Juvenile xanthogranuloma Myasthenia gravis
Hemangioma Sclera
Lids Epscleritis
Blepharitis Scleritis
Atopic dermatitis
Discoid lupus
Chalazion
254
 Corticosteroids in Ophthalmic Practice

of inflammation after surgery; (2) abnormalities of excessive

TABLE 23.5 Systemic Complications of Corticosteroid Therapy
immunoreactivity; and (3) for diseases that have combined
immune and infectious processes. Control of postoperative Musculoskeletal
inflammation is certainly the indication for which steroids are
Myopathy
used most.
The second group of conditions for which steroids are used is Osteoporosis, vertebral compression fractures
disorders of immune hyperreactivity. The immune system can Aseptic necrosis of bone
cause damage with overzealous defense mechanisms which can
lead to permanent tissue impairment. These disorders include Gastrointestinal
iritis, posterior uveitis, immune infiltrates, allergic disorders, such Peptic ulcer (often gastric)
as allergic conjunctivitis, atopic and vernal keratoconjunctivitis,
Gastric hemorrhage
and graft rejection.
The third class of disorders treated with steroids may originate Intestinal perforation
with an infectious process. Disorders such as disciform herpes Pancreatitis
and bacterial corneal ulcers are treated very cautiously and judi-
ciously with steroids, whereas the infection is treated or con- Central Nervous System
trolled with antibiotics. It must be recognized that even in the Psychiatric disorders
absence of an infectious agent, whenever complete immuno-
Pseudotumor cerebri
suppression is established by the use of steroids, prophylactic
antimicrobial therapy should be considered. The sensitivity of Ophthalmic
treating such serious problems with steroids must be empha- Glaucoma
sized, because often only certain phases of these diseases respond
to steroids, and in other phases steroids may be contraindicated. Posterior subcapsular cataracts
For a complete discussion of medical treatments the reader Cardiovascular and Renal
should refer to specific diseases.
Hypertension
In general, steroids are at first administered in medium- or
large-size doses to adequately suppress inflammation. The dose Sodium and water retention edema
is then tapered gradually to prevent rebound inflammation. Often

CHAPTER 23
Hypokalemic alkalosis
the physician can gain insight into the amount and severity of
inflammation by observing the patient’s response to steroids. Metabolic
The potential usefulness of prophylactic therapy with Precipitation of clinical manifestations, including ketoacidosis,
steroids or of a loading, pretreatment period needs to be diabetes mellitus
established. These are commonly recommended courses of Hyperosmolar nonketotic coma
treatment with systemic steroids. We have shown in the
allergen challenge model that a 48-h loading period was needed Hyperlipidemia
to achieve efficacy in inhibiting the signs and symptoms of Centripetal obesity
ocular allergy.87 Loading periods are considered the standard for
Endocrine
nonsteroidal antiinflammatory agents, yet steroids are not
commonly used like this in the preoperative period. Further Growth failure
investigation is needed to clarify this issue. Secondary amenorrhea
Suppression of hypothalamic–pituitary–adrenal system
SIDE EFFECTS OF TOPICAL
CORTICOSTEROID THERAPY Inhibition of Fibroplasia
Impaired wound healing
Corticosteroid-induced side effects are either systemic or ocular,
or both. Systemic side effects are most often associated with Subcutaneous tissue atrophy
oral or parenteral corticosteroid therapy. It has been shown that Suppression of the Immune Response
6 weeks of treatment with topical 0.1% dexamethasone sodium
phosphate caused suppression of the adrenal cortex, reflected in Superimposition of a variety of bacterial, fungus, and viral
infections in steroid-treated patients
a decrease in serum cortisol levels. Systemic absorption of steroids
after topical treatment is actually considerable, and, if given to
a patient with hay fever, it may improve systemic symptoms and
decrease the blood eosinophil count. Potential systemic com- TABLE 23.6 Ocular Side Effects of Corticosteroid Therapy
plications of corticosteroid therapy88 are included in Table 23.5.
Since topical corticosteroids are the most widely used drug in Cataracts
the treatment of many ocular conditions, their ocular toxicity Glaucoma
and side effects should always be recognized. The patient must
be aware of these side effects, particularly if corticosteroids are Secondary infection
to be used for an extended period. Ocular side effects involve Retardation of wound healing
mainly the anterior segment, including the cornea, conjunctiva,
Uveitis
trabecular meshwork, anterior chamber, and iris (Table 23.6).
Topical corticosteroids may cause glaucoma or cataracts, Mydriasis
enhance secondary herpetic or bacterial infections of the ocular Ptosis
surface, or inhibit corneal epithelial and stromal healing,
resulting in further corneal melting and perforation. All of these Exophthalmos
potential ocular complications of prolonged corticosteroid Pseudotumor cerebri
therapy can be devastating and threaten vision. 255
 PHARMACOLOGY AND TOXICOLOGY

The generalized effect of steroids on the delay of wound heal-
ing is important to consider, both in postoperative therapy and
in association with epithelial and stromal defects. The steroid’s
effect on the fibroblast results in delayed collagen synthesis,
which can cause or exacerbate corneal melting.34,72
CATARACT INDUCTION BY
CORTICOSTEROIDS
Several years after corticosteroids became widely used for rheu-
matoid arthritis, Black and co-workers89,90 reported the develop-
ment of cataracts in patients receiving long-term systemic therapy.
The dosage and duration of steroid therapy correlated with the
incidence of posterior subcapsular cataract (PSC) formation.
Seventy-five percent of patients who receive more than 16 mg/day
of prednisone develop cataracts. If the dose is decreased to
10 mg/day for 1 year, the chance of PSC formation is minimal.
Individuals who have undergone prolonged topical corticosteroid
therapy, such as for vernal or atopic keratoconjunctivitis or
those who received corneal transplantation for keratoconus, are
under the threat of developing PSCs. Donshik and co-workers
have shown that 28 eyes of 86 transplanted for keratoconus
developed PSCs after 1 year of 0.1% dexamethasone therapy.91
It seems that PSC formation is significantly related to the total
cumulative steroid dose and the total time that steroids were
administered. Once PSCs have developed, cessation of corti-
costeroids does not resolve the opacity. It is also important to
consider the overall status of the patient, because factors such
SECTION 4
Steroids such as FML, which has limited intraocular bioavail-
ability, have been shown to have less tendency toward induction
of ocular hypertension.104–108 In a corticosteroid provocative
test, Akingbehin found that 15 of 24 eyes treated with 0.1%
dexamethasone showed a rise in intraocular pressure of more
than 5 mmHg, whereas only two of the 24 eyes treated with 0.1%
FML showed such an increase.104 In a study of 14 steroid
responders to 0.1% dexamethasone, 13 were not affected by sub-
sequent treatment with 0.1% FML.105 Also, the time to an evoked
ocular hypertension in known steroid responders was sig-
nificantly longer (4 weeks) for 0.1% FML actetate than for 0.1%
dexamethasone sodium phosphate.106 Cantrill and associates
showed that 0.1% dexamethasone had more than three times
the ocular hypertensive effect of 0.1% in corticosteroid
responders.107 Mean intraocular pressure increases were also
significantly lower with twice the concentration of FML (0.25%)
compared with 0.1% dexamethasone sodium phosphate-treated
eyes in known steroid responders who took the drugs four times
daily for as long as 6 weeks.104
There has been much investigation into the development of
steroids that do not elicit ocular hypertension and glaucoma.
Lodoprednenolol, a steroid developed using the soft drug con-
cept, is an inactive compound that is activated locally in the eye
and is degraded in the bloodstream, thus limiting systemic
activity.76,77 It has been proposed that the side chain responsible
for the steroid ocular hypertensive response is absent from this
compound; however, most research into the structure–activity
relationships of steroids has shown that a steroid’s antiinflam-

as diabetes appear to increase susceptibility to these complica-
tions of topical steroid administration. The pathogenesis of
corticosteroid-induced cataract formation has not been fully
explained. One theory holds that corticosteroids enter the lens
and bind to its fibers, leading to biochemical changes and protein
aggregation in the cells.
STEROIDS AND GLAUCOMA
Corticosteroids have been shown to produce increased intra-
ocular pressure when applied topically to the eye92–99 or given
systemically.100,101 This elevation in intraocular pressure is usu-
ally reversible but can lead to optic nerve damage and visual
field changes similar to those seen in patients with chronic
open-angle glaucoma. The genetic basis for this predisposition
is probably a recessive homozygous gene. Although the exact
mechanism of corticosteroid-induced glaucoma is not clear, there
is evidence of mucopolysaccharide deposition in the trabecular
meshwork.102 Identifying the effects of topical application of
0.1% dexamethasone has no predictive value.103
matory activity is closely related to its ocular hypertensive
activity.109
INFECTIONS ENHANCED BY
CORTICOSTEROIDS
For bacterial, viral, and protozoal ocular infections, use of corti-
costeroids should always be given careful consideration. Corti-
costeroids substantially suppress the activation and migration
of leukocytes, which is a major part of the cellular host defense
against invading microorganisms and infection. Secondary infec-
tions caused by corticosteroids can take the form of bacterial
conjunctivitis and keratitis, viral keratitis, or more serious vision-
threatening infections, such as fungal keratitis, fungal endoph-
thalmitis, and toxoplasmic chorioretinitis. Management of these
complications involves tapering, and eventually stopping, the
corticosteroid and initiating therapy with appropriate antiinfec-
tive agents. Prophylactic coverage with appropriate antiviral or
antibacterial agents should be considered.110

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 CHAPTER
24 Nonsteroidal Antiinflammatory Drugs
King W. To, Mark B. Abelson, and Arthur H. Neufeld
Key Features
• NSAIDs affect the cyclooxygenase pathway of the arachidonic
acid cascade, and offer varying degrees of anti-inflammatory
and analgesic effects through inhibition of prostaglandins.
• Ophthalmic NSAIDs tend to be associated with fewer adverse
events than systemic NSAIDs. The mechanisms through which
the undesired effects appear are uncertain, and may be linked
to concurrent conditions, rather than directly to the NSAID.
• Topical NSAIDs can reduce intraoperative miosis during ocular
surgery, thereby increasing the surgeon’s visualization and
decreasing the risk of complications. Pre-operative use is key
to achieving the NSAID’s full effect.
• Given the more favorable side effect profile of NSAIDs, they
are being increasingly used over corticosteroids to control
inflammation after cataract surgery.
• NSAIDs are gaining off-label attention for their ability to
prevent and treat cystoid macular edema, which can arise as a
complication of cataract surgery.
Prior to the development of corticosteroids, aspirin was used to
treat intraocular inflammation.1 Salicylic acid (ortho-
hydroxybenzoic acid) or aspirin (acetylsalicylic acid) was intro-
duced over a century ago as an antipyretic and for the treatment
of rheumatic fever. Aspirin reduces inflammation primarily by
inhibiting the cyclo-oxygenase involved in the production of
prostaglandins2,3 although additional antiinflammatory actions
are probably involved. Prostaglandins (PGs) are 20-carbon,
unsaturated fatty-acid derivatives with a cyclopentane ring; these
biologically active lipids have a diverse spectrum of actions,
including the control of the inflammatory response, pain, body
temperature, intraocular pressure, blood coagulation, lipid and
carbohydrate metabolism, and cardiovascular, respiratory, and
renal physiology. The PGs are eicosanoids, which are a family
of molecules derived from arachidonic acid. The mechanism of
TABLE 24.1. Ocular Effects of PGs
Prostaglandin Effect
D Stimulates vasodilation and chemosis
E1, E2 Increase inflammation
Increase intraocular pressure
Increase capillary permeability
Stimulate vasodilation
Stimulate miosis
F2 Reduces intraocular pressure
Has minimal effect on inflammation
Has minimal effect on miosis
action of PGs is not well understood. Some PGs act anta-
gonistically with one another, whereas individual PGs can have
different effects on different tissues. In addition, responses to
certain PGs can vary significantly in different animal models
and human studies. The ocular effects of PGs that have been
isolated from the eye are summarized in Table 24.1.
In the past 20 years, research has led to the development of
useful aspirin-like, nonsteroidal antiinflammatory drugs
(NSAIDs). NSAIDs are among the most commonly prescribed
drugs. Their most useful application is in the management of
inflammation in diseases such as osteoarthritis, rheumatoid
arthritis, and ankylosing spondylitis. This chapter provides an
overview of NSAIDs and their ophthalmic applications.
CHEMICAL PROPERTIES
The NSAIDs, a heterogeneous group of compounds, all have
some degree of antiinflammatory, antipyretic, and analgesic
properties; however, their therapeutic properties differ signifi-
cantly. Because PGs have such a diverse range of actions,
NSAIDs, which inhibit the production of PGs, also possess a
broad range of pharmacologic properties. Systemic NSAIDs at
therapeutic doses can produce adverse changes in the gastro-
intestinal, respiratory, hepatic, endocrine, coagulation, and renal
systems.4 The NSAIDs can be divided into the following groups:
salicylates, fenamates, and derivatives of indole, pyrazolone,
propionic acid, phenylacetic acid, and oxicam (Table 24.2). Only
the derivatives of indole, propionic acid, and phenylacetic acid
are commercially available as topical ophthalmic agents.
Indocid, a commercial form of ophthalmic indomethacin
solution, currently is not yet available in the United States. Six
Food and Drug Administration (FDA)-approved NSAID topical
ophthalmic agents are currently available (Table 24.3).
MECHANISMS OF ACTION
Arachidonic acid is the primary precursor of PGs, leukotrienes
(LTs), and related compounds (Fig. 24.1). Arachidonic acid may
be ingested or derived from dietary linoleic acid. Arachidonic
acid is bound to phospholipids in the plasma membrane; its
release by phospholipases is closely regulated by a wide variety
of chemical, physical, and hormonal factors. The blockage of
PG biosynthesis by NSAIDs is primarily due to the inhibitory
effects of NSAIDs on cyclo-oxygenase, which is responsible for
the conversion of arachidonic acid to endoperoxides (PG G2,
PG H2) in ocular and nonocular tissues.5 Endoperoxides are
precursors of all other PGs. The inhibitory activity of NSAIDs
on cyclo-oxygenase demonstrably correlates with its antiinflam-
matory activity.3 Experimental studies have shown that certain
PGs are potent mediators of ocular inflammation.6,7 Topical 259
 PHARMACOLOGY AND TOXICOLOGY

TABLE 24.2. Classes of NSAIDs Available in the United States

Generic Name Trade Name

Salicylates
Aspirin Multiple names and manufacturers
Fenamates
Mefenamate Ponstel
Meclofenamate Meclomen
Indole Derivatives
Indomethacin Indocin
Ketorolac Toradol, Acular*
Sulindac Clinoril
Tolmetin Tolectin
Pyrazolone Derivatives
FIGURE 24.1. Structure of arachidonic acid cascade; synthesis of
Phenylbutazone Butazolidin prostaglandins and related compounds.
Propionic Acid Derivatives
Fenoprofen Nalfon application of arachidonic acid or certain PGs produces dilation
Flurbiprofen Ansaid, Ocufen*
Ibuprofen Advil, CoAdvil, IBU-TAB Medipren, Motrin,
of conjunctival vessels with chemosis, changes in intraocular
Nuprin, Children’s Motrin, Rufen pressure, and miosis.8 PG levels are elevated in the aqueous
Ketoprofen Orudis humor following argon laser iridectomy,9 cataract surgery,10 and
Naproxen Naprosyn trauma.11 By inhibiting cyclo-oxygenase, NSAIDs have been
Suprofen Profenal* shown to reduce the de novo synthesis of PGs.11–13 Unlike
Phenylacetic Acid Derivatives NSAIDs, corticosteroids affect both the cyclo-oxygenase and
lipoxygenase pathways by preventing the release of arachidonic
Diclofenac Voltaren, Voltaren Ophthalmic*
SECTION 4

acid.14,15 However, NSAIDs do not inhibit lipoxygenase and

Oxicam Derivatives may lead to an increase in the production of LTs by increasing
Piroxicam Feldene
the amount of arachidonic acid available to be metabolized by
lipoxygenase. The additional inhibition of leukotriene formation
*Ophthalmic topical agents. may be partially responsible for the greater antiinflammatory
activity of corticosteroids. Other sources provide detailed
discussion on the broad spectrum of actions of the PGs
TABLE 24.3. Topical Ophthalmic Suspension NSAIDs Available systemically16 and in the eye.17
in the United States

Generic Name and Trade Name Indication(s) for Use PHARMACOKINETICS

Solution Concentration (Manufacturer) Approved by the FDA
In general, orally ingested NSAIDs are rapidly absorbed and
Ketorolac 0.4% Acular 1. Seasonal allergic distributed throughout most body tissues. The NSAIDs are
Acular, 0.5% LS (Allergan) conjunctivitis bound extensively to plasma proteins, and concentrations peak
2. Intraocular
inflammation after in blood 1–2 h after administration. Biotransformation occurs
cataract surgery primarily in the hepatic endoplasmic reticulum and mito-
3. Reduction of pain chondria. The unchanged NSAID and its metabolic products
after corneal are then eliminated in the urine. Therefore, patients with
refractive surgery underlying liver or kidney dysfunction are at significant risk for
Flurbiprofen 0.03% Ocufen (Allergan) 1. Minimizing the development of a wide range of toxic effects from normal
Suprofen 1% Profenal (Alcon) intraoperative doses of systemic NSAIDs.
miosis during
cataract surgery
COMPLICATIONS
Diclofenac 0.1% Voltaren 1. Intraocular inflam-
(CibaVision) mation following Oral NSAID therapy is associated with a variety of complica-
cataract surgery tions. Only the most common and clinically significant adverse
2. Reduction of pain effects are addressed here. The most common undesirable effect
and photophobia
after cataract
is gastrointestinal irritation, which can lead to nausea, vomiting,
surgery cramps, and gastric or intestinal ulceration.18,19 Gastrointes-
tinal ulceration can lead to significant blood loss and anemia. In
Nepafenac 0.1% Nevanac (Alcon) 1. Reduction of pain addition to the local irritative effects of the NSAIDs on the
and inflammation
associated with
gastrointestinal mucosa, inhibition of certain key gastric PGs
cataract surgery (E2, I2) that normally protect against erosion may contribute to
this side effect. The NSAIDs also increase the bleeding time by
Bromfenac 0.09% Xibrom (Ista) 1. Treatment of inhibiting platelet production of thromboxane A2, a potent
postoperative
inflammation aggregating agent.20 Although NSAIDs do not significantly
following cataract affect renal function in healthy young patients, these aspirin-
surgery like drugs can produce acute renal failure in patients with
260 chronic renal disease, congestive heart failure, cirrhosis with

ascites, volume depletion secondary to diuretics, and hypo-
tension secondary to hemorrhage. PGs protect the kidneys in
disease states when renal perfusion is compromised by stimu-
lating vasodilation and maintaining renal perfusion. NSAIDs
block this PG-mediated compensatory response.21 Therefore, it
is not surprising that NSAIDs may produce renal compromise
in the elderly,22 which is important because the prevalence of
rheumatic disease, in which the treatment of choice is NSAIDs,
increases with age. Stevens–Johnson syndrome has been
reported in association with rofecoxib (Vioxx).23
Topical NSAIDs generally appear to be significantly safer than
oral NSAIDs. Application of these topical agents sometimes
causes a stinging sensation. The benefits of greater comfort
cannot be overemphasized, because comfort is clearly an
important factor in a patient’s adherence to a therapeutic
regimen. Topical NSAIDs should be avoided in patients with a
history of aspirin or NSAID sensitivity. Bronchospastic exacer-
bation was caused by topical ketorolac in a patient with asthma
and nasal polyps.24 Rare corneal complications such as corneal
melting after topical NSAID use have been reported.25–29 Topical
diclofenac, ketorolac, and bromfenac have all been associated
with corneal ulceration. The exact mechanisms remain unclear,
but this rare side effect reminds us to carefully observe all our
patients on topical NSAIDS.
Some have suggested that the increased bleeding of ocular
tissues (including hyphemas) in the setting of surgery and
impairment of wound healing is associated with topical NSAID
use.30 In our clinical experience, the potential for increased
bleeding and impairment of wound healing with topical NSAID
use does not seem to be a problem. Whether topical NSAIDs
may be used safely in the presence of fungal, bacterial, or viral
infections remains unclear.
PREVENTION OF INTRAOPERATIVE MIOSIS
Miosis is a well-known complication of surgical trauma. In an
effort to identify the agent responsible for stimulating miosis as
part of the ocular response to trauma, researchers isolated a
substance called irin more than 40 years ago.31,32 Irin, which
was isolated from extracts of iris tissue, was found to produce
miosis when introduced into the anterior chamber of animal
eyes. PGs were later identified in these iris extracts. Although
the mechanism of the PG-mediated miotic response, as well as
what other compounds in irin may be involved remains to be
determined, topical application of cyclooxygenase blockers
appears to help minimize the amount of intraoperative miosis.
For many years, topical flurbiprofen 0.03% (Ocufen) has been
used in preventing intraoperative miosis. Miosis during eye
surgery, a common occurrence, can severely limit the surgeon’s
visualization and potentially increase the complication rate of
the procedure. Surgical trauma that stimulates the production
of PGs appears to play an integral role in the development of
intraoperative miosis. PGs have been observed in the aqueous
humor of traumatized eyes and appear to induce miosis
independent of cholinergic mechanisms.33 By inhibiting PG
synthesis by blocking the cyclooxygenase pathway,34 0.03%
flurbiprofen, when administered every 30 min beginning 2 h
preoperatively, has limited intraoperative miosis during anterior
segment surgery in animal35,36 and human eyes.37 Preoperative
treatment is the key, because once the PGs are released, topical
flurbiprofen does not block the PGs’ effect on the iris.
Some cataract surgeons have suggested that flurbiprofen may
retard the reversal of the mydriasis by agents such as intra-
cameral acetylcholine and carbachol, which potentially increase
the chances of such complications as intraocular lens pupillary
capture. Theoretically, flurbiprofen should have no effect on
intracameral acetylcholine or carbachol; there is no known
Nonsteroidal Antiinflammatory Drugs
pharmacologic basis for any such interaction.38 A possible
explanation may be that some surgeons tend to rub the end of
the cannula on the iris as the intraocular solution of acetyl-
choline or carbachol is injected to hasten the development of
the miosis. Such a maneuver in eyes not dosed wtih
flurbiprofen would likely stimulate the iris to produce PGs and
induce miosis; eyes previously treated with flurbiprofen would
not show similar effects.38
Topical flurbiprofen, however, does not appear to be as
effective in minimizing miosis during vitreoretinal surgery.39,40
Whether this is because surgical manipulation is generally
greater with vitreoretinal surgery than with anterior segment
surgery and, therefore, more PGs are released, leading to miosis,
remains to be determined. Another topical NSAID, suprofen,
has been demonstrated to also be effective in reducing pupillary
constriction during cataract surgery.41 The relative efficacy of
flurbiprofen and suprofen remains to be determined. Addi-
tionally, although topical diclofenac is only approved by the
FDA for treatment of uveitis following cataract surgery, this
drug can also minimize intraoperative miosis.42
The mechanisms involved in surgical miosis are complex.
Although certain PGs have been associated with producing
miosis, no single PG possesses a miotic effect in all species or
is potent enough of a miotic to completely account for surgical
miosis.43,44 The specific mechanism of action of cyclooxygenase
blockers such as flurbiprofen may well have a variety of biologic
effects that cannot be satisfactorily explained by inhibition of
PG synthesis alone.
CHAPTER 24
POSTSURGICAL INFLAMMATION AND
DISCOMFORT
A number of topical NSAIDs have been tested as potential
substitutes for topical corticosteroids for the treatment of
postoperative inflammation. Because steroid use after cataract
surgery may be associated with increased intraocular pressure
and glaucoma, increased risk of infection, and inhibition of
wound healing, a topical NSAID has been sought for the treat-
ment of postsurgical inflammation. Because intraocular inflam-
mation is associated with the breakdown of the blood–aqueous
barrier, investigators have used the leakage of fluorescein into
the anterior chamber after systemic administration to indirectly
gauge the amount of inflammation.45,46 It has been suggested
that a reduction in the leakage of fluorescein with NSAID
treatment is an indication of a reduction in inflammation. The
breakdown of the blood–aqueous barrier, assessed by fluoro-
photometry or slit-lamp examinations after cataract surgery,
appears to be reduced by several topical NSAIDs, including keto-
rolac tromethamine, diclofenac sodium, and flurbiprofen.47–52
Randomized, controlled studies to compare the antiinflam-
matory actions of 0.5% ketorolac tromethamine versus 0.1%
dexamethasone47 and 0.01%, 0.05%, or 0.1% diclofenac sodium
versus 1% prednisolone sodium phosphate49 demonstrated that
topical NSAIDs were superior to the topical steroids in reducing
breakdown of the blood–aqueous barrier as measured by fluoro-
photometry. These preliminary studies suggest that topical
NSAIDs are a useful substitute for topical corticosteroids in the
management of postoperative inflammation.
The only topical NSAIDs currently approved by the FDA
for the treatment of inflammation following cataract surgery
are diclofenac sodium 0.1% (Voltaren), ketorolac 0.4% (Acular
LS), bromfenac 0.9% (Xibrom), and nepafenac 0.1%
(Nevanac).52–54 In addition to reducing trauma-induced inflam-
mation, topical nepafenac, an amide analog of the NSAID
amfenac, has also been shown to inhibit inflammation-
mediated retinal edema and ocular neovascularization in
animal models.54,55 261
 PHARMACOLOGY AND TOXICOLOGY

Topical diclofenac and ketorolac are approved for use after
refractive surgery. The frequent occurrence of pain after
refractive surgery, such as photorefractive keratectomy or radial
keratotomy, has been reduced with the administration of topical
ketorolac or diclofenac.56–62 Topical diclofenac has also been
shown to be a suitable replacement for topical steroids in
managing postoperative inflammation following strabismus
surgery.63
OCULAR INFLAMMATORY DISORDERS
Few areas in ophthalmology have received more attention than
cystoid macular edema (CME). Although CME still remains
poorly understood, most researchers would agree that inflam-
mation is important to its pathogenesis. While there is no FDA-
approved treatment of CME following cataract surgery, preli-
minary studies involving topical or systemic NSAIDs have been
encouraging.54,64–69 These studies suggested that NSAIDs may
be useful in the prophylaxis and treatment of CME following
cataract surgery. In our clinical practice, we initially start our
CME patients on intensive topical steroids (eight times a day)
and topical NSAIDs (four times a day) for 2–4 weeks. If there is
no response or if the CME worsens, topical NSAIDs are
discontinued and the use of systemic NSAIDs is considered.
The NSAIDs have also been evaluated in the treatment of
inflammatory diseases of the sclera. When taken orally, flur-
biprofen may be effective in treating scleritis and episcleritis;70
however, the topical form does not appear to be useful in the
SECTION 4
Traditional NSAIDs were once thought to have some promise
in the management of allergic disorders of the eye. Topical
flurbiprofen (0.03%) and suprofen (1%) have been used in the
treatment of allergic conjunctivitis78 and vernal conjunctivitis79
respectively. However, topical ketorolac remains the only FDA-
approved topical NSAID for seasonal allergic conjunctivitis. Its
approval was based on two fairly small studies,80 and other
agents, such as antihistamine/mast cell stabilizers, are generally
preferable.80,81 NSAIDs have little, if any, place in the treatment
of ocular allergy.80,81
While bromfenac 0.09% (Xibrom) is only FDA approved for
treatment of inflammation after cataract surgery, like other
NSAIDS, bromfenac may prove useful in managing other forms
of ocular inflammation. Topical bromfenac is unique due to its
twice-a-day dosing and may improve patient compliance.
Oral aspirin has been shown to be useful as both primary82
and adjunctive83 therapy with steroids in the relief of
conjunctival and episcleral redness and in the resolution of
keratitis and limbal infiltrates in vernal keratoconjunctivitis
(VKC). Patients with VKC have shown improvement after
treatment with up to 1 g of oral aspirin daily for 6 weeks.
Because of the relatively high dose, the clinician should closely
monitor any patient during an aspirin therapy regimen, and
should be aware of all contraindications to aspirin use. Aspirin
has many other properties we have yet to define, and clinically,
aspirin is of interest as a potential ocular therapeutic agent,
particularly if the barriers to developing a safe method of topical
ocular aspirin delivery can be overcome.

management of episcleritis.71 Oral NSAIDs also may be useful

as an adjunct in the management of chronic iridocyclitis in
childhood.72 When children with idiopathic iridocyclitis or
iridocyclitis in association with juvenile rheumatoid arthritis
were treated with oral NSAIDs, both inflammation in the
anterior chamber and the need for topical and systemic steroids
were reduced.72
Another potentially useful application of NSAIDs is in sup-
pressing the inflammatory response associated with ocular
infections. It is well known that topical steroid use can
exacerbate viral, bacterial, and fungal infections of the eye. The
effect of topical NSAIDs on corneal epithelial herpes simplex
viral infections remains controversial; two experimental studies
have found that topical NSAIDs did not worsen herpes simplex
viral infections of the cornea,73,74 whereas an earlier study
suggested that the exacerbation of ocular herpes simplex viral
infections by topical flurbiprofen is similar to that of topical
dexamethasone.75 Preliminary studies have found that topical
NSAIDs have no adverse effect on either bacteria76 or fungal77
ocular infections.
PREVENTION OF CATARACT FORMATION
Although corticosteroid use is associated with cataract forma-
tion, aspirin84,85 and other NSAIDs86,87 may protect against
cataracts. The mechanism for this apparent protective effect
remains nebulous; however, it may be related to aspirin’s
acetylation of the lens proteins, which protects these proteins
from a variety of chemical insults.88,89 In addition, the lowering
of blood glucose levels in diabetics and nondiabetics associated
with NSAIDs may play a role in preventing cataracts.87 Nearly
half of all patients with cataracts have been estimated to
have abnormal glucose tolerance.90 Because diabetes is clearly
associated with cataracts, perhaps the glucose-lowering effect
of NSAIDs serves to favorably affect these patients with
chronic elevation of glucose levels. Other observational
studies91–93 and a randomized study94 did not find that aspirin
lowered the incidence of cataracts. It seems that aspirin or
aspirin-like agents neither prevent nor slow cataract formation,
although a small benefit cannot be ruled out.94

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71:497–499.
83. Abelson MB, Butrus SI, Weston JH: Aspirin
therapy in vernal conjunctivitis. Am J
Ophthalmol 1983; 95:502–505.
84. Cotlier E: Aspirin and senile cataract in
rheumatoid arthritis. Lancet 1981;
1:338–339.
85. Cotlier E, Sharma YG, Niven T, et al:
Distribution of salicylate in lens and
intraocular fluids and its effects on cataract
formation. Am J Med 1983; 74:83–90.
86. van Heyningen R, Harding JJ: Do
aspirin-like analgesics protect against
cataract? A case-control study. Lancet
1986; 1:1111–1113.
87. Harding JJ, van Heyningen R: Drugs,
including alcohol, that act as risk factors
for cataract, and possible protection
against cataract by aspirin-like analgesics
and cyclopenthiazide. Br J Ophthalmol
1988; 72:809–814.
88. Crompton M, Rixon KC, Harding JJ: Aspirin
prevents carbamylation of soluble lens
proteins and prevents cyanate-induced
phase separation opacities in vitro: a
possible mechanism by which aspirin could
prevent cataract. Exp Eye Res 1985;
40:297–311.
89. Rao GN, Lardis MP, Cotlier E: Acetylation
of lens crystallins: a possible mechanism
by which aspirin could prevent cataract
formation. Biochem Biophys Res Commun
1985; 128:1125–1132.
90. Dugmore WN, Tun K: Glucose tolerance
tests in 200 patients with senile cataract.
Br J Ophthalmol 1980; 64:689–692.
91. Siegel D, Sperduto RD, Ferris F: Aspirin
and cataracts. Ophthalmology 1982;
89:47A.
92. Klein BK, Klein R, Moss S: Is aspirin use
associated with lower rates of cataracts in
diabetic individuals? Diabetes Care 1987;
10:495–499.
93. West SK, Munoz BE, Newland HS, et al:
Lack of evidence for aspirin use and
prevention of cataracts. Arch Ophthalmol
1987; 105:1229–1231.
94. Seddon JM, Christen WG, Manson JE,
et al: Low-dose aspirin and risks of
cataract in a randomized trial of U.S.
physicians. Arch Ophthalmol 1991;
109:252–255.
 CHAPTER
Antihistamines and Mast Cell Stabilizers in Allergic
25 Ocular Disease
Gregg J. Berdy, Andrea Leonardi, and Mark B. Abelson
Ophthalmologists frequently see allergic diseases of the eye.
They may be the most common clinical problems involving the
external ocular adnexa. Approximately 20% of the US popula-
tion (~60 million people) is affected with these disorders.
Although allergic ocular diseases may affect the skin and
subcutaneous tissues of the eyelids, it is the conjunctiva, the
mucous membrane of the eye, which is more commonly and
severely affected. In certain cases, the eye may be the only organ
system involved. In most of these patients, however, the ocular
tissues participate as part of a systemic allergic response to
exogenous or intrinsic antigens.
Allergic conjunctivitis is observed more frequently in indus-
trialized countries as a consequence of the deviation of the
immune system toward a T helper cell lymphocyte (Th2-type)
immune response favored by a reduction in infection, air
pollution, and modern lifestyles. This disease ranges in severity
from mild forms, which still interfere significantly with quality
of life, to severe cases characterized by potential impairment of
visual function.
Ocular allergy encompasses a spectrum of diseases character-
ized by the IgE- and Th2-mediated hypersensitivity responses.
The most common ocular allergies are seasonal and perennial
allergic conjunctivitis (SAC and PAC), the ocular counterpart of
allergic rhinitis. Exposure to environmental allergens such as
pollens, animal dander, and dust causes the symptoms and
signs of ocular hay fever in sensitized persons. An acute attack
is characterized by conjunctival injection, chemosis, tearing,
eyelid swelling, burning, and ocular and periocular itching.
The chronic allergic ocular diseases, vernal keratoconjuncti-
vitis (VKC), atopic keratoconjunctivitis (AKC), and giant papillary
conjunctivitis (GPC) are relatively rare but clinically well charac-
terized. Mast cells, T-cell lymphocytes, eosinophils, and their
mediators all play major roles in the clinical manifestation of
these diseases. Typical Th2-type cytokines, IL-4, IL-5, and IL-13,
as well as other proinflammatory cytokines, chemokines, growth
factors, and enzymes are overexpressed in the conjunctiva of
patients with chronic allergic diseases. Each of these diseases
has specific clinical features in terms of diagnosis and treatment.
MAST CELL AND PATHOPHYSIOLOGY
Knowledge of the pathogenesis of ocular allergic disease is
critical to understanding the role of therapeutic medications
used in the treatment of these diseases. SAC is the prototype of
this group of diseases and begins as an antigen–IgE antibody
interaction on the surface of conjunctival mast cells.1 Exposure
of sensitized IgE-coated mast cells to specific allergen causes the
cross-linking of membrane-bound IgE receptors (FCeRI), the
activation of mast cells, and the release of preformed and newly
formed mediators.
Mast cell populations in humans demonstrate heterogeneity
in different organ systems, and the neutral protease content of
the mast cell cytoplasmic granules has provided one basis for
subclassification. Mast cells containing tryptase alone (mucosal
type mast cells or MCT) are found most frequently at mucosal
sites. Those mast cells containing tryptase and chymase (con-
nective type mast cells or MCCT) are more characteristic of
connective tissue sites.2 Immunohistochemical phenotyping of
mast cells in the normal human conjunctiva has demonstrated
that the MCCT phenotype is predominant, similar to the findings
in human skin.3 In addition to chymase, MCCT also contain
cathepsin-G and carboxypeptidase-A, both of which are absent
from MCT.4
The exogenous allergen binds to two separate IgE molecules,
creating a dimer formation that initiates a chain of reactions in
the mast cell plasma membrane.5,6 It is thought that the bridging
of mast cell IgE molecules (cross-linking) induces activation
of membrane-associated enzymes, leading to an increase in
the uptake of calcium.7 Enzymes identified with intracellular
calcium mobilization and initiation of the biochemical process
of histamine release are membrane-associated proteolytic
enzymes,8 methyltransferases,9 and adenylate cyclase.10,11 In
addition, the cross-linking of membrane-bound IgE molecules
induces the activation of phospholipase A2 with subsequent
release and metabolism of arachidonic acid.12 This 20-carbon,
unsaturated fatty acid serves as a precursor for newly synthe-
sized substances, such as prostaglandins, leukotrienes,13 and
platelet-activating factor,14,15 that have been implicated as
important mediators of clinical allergic disease.16
The FceRI on mast cells consists of an a-chain, a b-chain,
and two g-chains. The a-chain is responsible for IgE binding,
while the b-chain promotes stability and enhances the signaling
capacity. Monomeric IgE binding to the a-chain does not result
in conformational changes, but enhances mast cell survival and
growth. The dimer of the g-chain is shared by other Fc receptor
complexes and carries two immunoreceptor tyrosine-based
activation motifs (ITAMs) for downstream signaling. The signal
for mast cell degranulation is aggregation of FceRI and the
minimal signal only requires dimerization. Maximal degranu-
lation of mast cells and basophils is associated with distinct
aggregation of both b- and g-chains, but g-chain aggregation
alone can result in suboptimal stimulation. The process of FceRI
cross-linking (mediated through interaction of antigen with
receptor-bound IgE) results in phosphorylation of the ITAMs (of
the b and g subunits) by the Src family tyrosine kinase lyn
(probably under regulation of the phosphatase, CD45) and
recruitment of the protein tyrosine kinase Syk. Syk amplifies
the signal as it targets multiple proteins for activation (inclu-
ding phospholipase Cg (PLCg, the guanine nucleotide exchange
factor Vav1, and adaptor molecules SH2 domain-containing 265
 PHARMACOLOGY AND TOXICOLOGY
leukocyte protein of 76 kDa (SLP-76), and linker for activation
of T cells (LAT)). This promotes activation of various kinase
cascades, which overlap and share signaling components. The
phospholipase C (PLC) – inositol pathway is primarily involved
in degranulation, which is the result of Ca2+ mobilization and
cytoskelatal changes and culminates in immediate release of
stored mediators.17
At the ultrastructural level, it has been demonstrated that
human lung mast cells, once stimulated, show swelling of indi-
vidual granules, with the subsequent fusion and formation of
interconnected chains of altered granules. These intracellular
cytoplasmic channels eventually fuse with the plasma membrane
of the mast cell, thereby releasing their contents into the extra-
cellular space.18–20 These secretory granules contain several
preformed mediators, including biogenic amines (histamine),
neutral proteases (chymase, tryptase), proteoglycans (heparin),
and acid hydrolases, that initiate and promulgate the allergic
response. The downstream signaling stimulates a number of
transcription factors, leading to activation of genes regulating
the release of newly formed mediators such as prostaglandin
(PG)D2, leukotriene (LT)C4, and cytokines (e.g., TNFa). These
transcription factors include nuclear factor of activated T cells
(NF-AT), nuclear factor kappa B (NFkB), signal transducer and
activator of transcription (STAT)-6, activator protein-1 (AP-1),
c-fos, and c-jun.17
In combination with the other mast cell serine protease,
chymase, tryptase may be implicated in the activation of other
proteases, such as collagenase (MMP-1), gelatinases A and B
SECTION 4
(MMP-2, MMP-9), and stromelysin (MMP-3), which are all
involved in extracellular matrix degradation and inflammatory
cell infiltration.
Conjunctival mast cells have been shown to be a source of
several cytokines and growth factors. Interleukin (IL)-4, IL-5,
IL-6, tumor necrosis factor alpha (TNFa), transforming growth
factor beta (TGF-b)-1, or (FGF) and stem cell factor were local-
ized to mast cells in normal and allergic conjunctiva.21,22 The
pattern of cytokine expression in the two mast cell subtypes
showed that IL-4 and IL-13 were preferentially associated with
the MCCT subset, whereas IL-5 and IL-6 were associated to the
MCT subset, suggesting that differences in protease phenotype
may also reflect functional differences manifested by different
patterns of cytokine distribution.23 These cytokines appeared to
be stored within the cytoplasmic secretory granules, suggesting
that they may be rapidly released upon IgE- and non-IgE-
mediated mast cell activation.
Mast cell degranulation releases proinflammatory mediators
and cytokines which induces the activation of epithelial cells
and vascular endothelial cells leading to the expression of
chemokines (e.g., RANTES, MCP-1, IL-8, eotaxin) and adhesion
molecules (e.g., ICAM-1, VCAM, and p-selectin).24 These
factors initiate the recruitment phase of inflammatory cells in
the conjunctival mucosa.
Several cytokines can be found in tears of allergic and non-
allergic subjects, however, the cellular source of these cytokines
is difficult to determine. Altered ratios of proinflammatory
cytokines could reflect differences in the patterns of TH2 versus
TH1 and proinflammatory versus antiinflammatory cytokines
between nonallergic and allergic tears.25
Great advances in conjunctival mast cell biology and function
were gained from a series of studies using in vitro cultured mast
cells derived from human cadaveric conjunctival tissues.
Stimulated conjunctival mast cells have been shown not only to
express mRNA for TNFa but also to release TNFa protein,
consequently upregulating ICAM-1 expression on conjunctival
epithelial cells.26 A subsequent paper demonstrated that
mast cells express functional receptors such as ICAM-1, c-kit,
266 and FceRI have surface bound IgE.27 The expression of these
surface markers was modified by stimulus with TNFa and
IL-4, showing that cytokines may modify mast cell functions.
In vitro mast cell behavior may also be considered a model for
studying the effects of antiallergic drugs.
ROLE OF HISTAMINE
The sentinel role of histamine in the acute allergic response has
been well established. Histamine was first synthesized in 1907
and discovered to be an imidazolylethylamine.28 In 1910, the
biologic activity of this amine was discovered when it was
detected as a uterine stimulant in extracts of ergot. Later that
year, Dale and Laidlaw29 observed bronchospastic and vasodi-
lator activity in animals with the intravenous administration
of histamine. In 1919, these authors observed that histamine
applied locally produced redness, swelling, and edema. In
addition, they noted that large doses of intravenous histamine
produced a symptom complex that was identical to that of a
systemic anaphylactic reaction.30 Eight years later, investigators
deduced that histamine was a humoral mediator involved in
acute allergic reactions.31
In 1953, the presence of histamine was noted in mast cells
taken from human skin.32 This discovery spurred the interest of
many researchers, leading to the elucidation of histamine’s
synthesis, secretion, metabolism, and biologic activity.33,34 It is
the biologic activity of histamine that creates the signs and
symptoms of the acute allergic reaction in ocular hay fever.
The physiologic and pharmacologic effects of histamine are
mediated by specific receptor subtypes present on effector cell
surfaces. Four distinct histamine receptors have been charac-
terized to date and it is generally accepted that the H1 receptor
plays the greatest role in allergic disease. In 1966, Ash and
Schild35 identified specific receptors that were blocked by the
antihistamines known at that time and labeled them H1
receptors. These authors discovered that only certain responses
to histamine were blocked by the histamine antagonist mepyra-
mine, and these responses were defined as being mediated by
H1 receptors. Six years later, Black et al36 identified a second
histamine receptor subtype, H2, by using specific antagonists
that blocked only the H2 receptors. They demonstrated that
histamine-induced hypotension that was only partially relieved
by mepyramine was totally blocked by the addition of the
H2-receptor antagonist burimamide. H2 and H3 receptors play
critical roles in a variety of tissues including the central and
peripheral nervous systems, gastrointestinal tract, and heart.
The H4 receptor is the most recently discovered of the hista-
mine receptors.37,38 This novel receptor is highly expressed in
peripheral blood leukocytes and to the greatest extent on
eosinophils. Organ specificity of expression demonstrated high
levels of mRNA in several organs that are critical to immune
regulation such as bone marrow, spleen, and thymus. It is
speculated that the H4 receptor may become an important
future therapeutic target for regulation of immune function,
particularly with respect to allergy and asthma.
Histamine receptors belong to the large family of seven trans-
membrane G-protein coupled receptors (GPCR). G-proteins
derive their name from a high affinity for guanine nucleotides.
The binding of a ligand molecule to a GPCR in the plasma
membrane stimulates the trimeric G-protein resulting in
initiation of the PLC-inositol pathway. Generation of diacyl-
glycerol (DAG) and inositol 1,4,5-triphosphate (IP3) results in
activation of a Ca2+-dependent protein kinase (PKC) and Ca2+
mobilization from the endoplasmic reticulum, respectively.
Ca2+ functions as a ubiquitous intracellular messenger. When
activated, PKC phosphorylates specific serine or threonine
residues on target proteins, such as MAPK and IkB-NFkB, leading
to increased transcription of specific genes. Furthermore,
 Antihistamines and Mast Cell Stabilizers in Allergic Ocular Disease
DAG can be cleaved to release arachidonic acid, which acts
as a messenger as well as a substrate in the synthesis of
eicosanoids. It should be noted that there is a great deal of
overlap between the target proteins involved in FceRI and H1
receptor mediated activation.17 Therefore, while mast cell
mediators, particularly histamine, released upon ocular expo-
sure to allergen can initiate all of the symptoms associated with
ocular allergy, it is the subsequent effect of histamine on other
ocular surface cells that is thought to perpetuate the inflam-
matory response.
Identification of the H1 and H2 receptors has permitted
investigators to better understand histamine’s role in human
allergic disease. Owen and co-workers39 concluded that the
vasodilator response to histamine was mediated by both H1 and
H2 receptors; however, the increase in vascular permeability was
mediated solely by H1 receptors. When injected intradermally,
histamine causes a localized triple response. The initial compo-
nent is the development of erythema immediately surrounding
the injection site as the result of vasodilation mediated by both
H1 and H2 receptors.40,41 A second component is the cutaneous
flare that occurs as an indirect response to stimulation of
histamine receptors on afferent nonmyelinated nerve endings.
Antidromic nerve conduction initiates a reflex arc that
culminates in the release of various neuropeptides, including
substance P and calcitonin gene-related peptide, which directly
affect arteriolar vasodilation.42 The wheal results from exuda-
tion of plasma through gaps between vascular endothelium of
postcapillary venules and is mediated by H1 receptors.43 Addi-
tionally, intradermal injection of histamine causes a sensory
response that is manifested as the sensation of itching.
Allergic conjunctivitis can be characterized as ocular
anaphylaxis occurring when a sensitized person is exposed to a
specific aeroallergen. Abelson et al44 demonstrated the presence
of mast cell-derived mediators in subconjunctival tissues and
precorneal tear film in patients with ocular atopic diseases. This
is not unexpected, because the human conjunctiva contains
large numbers of mast cells subjacent to the epithelium.45–47
Previously, Abelson and co-workers48 demonstrated the
presence of histamine in the tear film of normal humans at
concentrations of 5–10 ng/mL whereas tear samples of patients
with active VKC contained significantly higher levels of
histamine. It has been calculated that a single conjunctival
mast cell contains 4.6 pg of histamine,49 signifying that the
total potential amount of histamine that can be released with
massive mast cell degranulation is 23 ng/mm3.50 The increased
levels of histamine in tears of VKC patients may be related not
only to a massive mast cell degranulation but also to a reduced
activity of histaminase enzymes.51 Inducing an acute reaction
by challenging allergic patients with specific allergen, tear
histamine levels are significantly increased compared with base-
line. These levels are even higher when histaminase enzymes
are inactivated.52 Using this procedure, increased histamine
tear levels have also been found during the late phase reaction.53
The topical instillation of histamine produced the itching
and redness associated with allergic conjunctivitis in a dose-
dependent fashion.54 Identification of specific histamine
receptors on the ocular surface has made it possible to selec-
tively identify the pathologic effects of histamine. Stimulation
of H1 receptors with the highly selective H1-receptor agonist
2-(2-aminoethyl) thiazoledihydrochloride elicited symptoms of
ocular itching.55 On the other hand, selective stimulation of H2
receptors by dimethylaminopropylisothiourea, a highly selective
H2-receptor agonist, produced vasodilation of conjunctival
vessels without itching.56
Histamine stimulates PI turnover and Ca2+ mobilization in
the human conjunctival epithelium inducing the release of
cytokines (IL-6, IL-8, GM-CSF) in these cells. Similar effects
can be induced in human conjunctival fibroblasts. In both types
of cells, blockage of H1 receptor activation using selective H1
receptor antagonists, antagonizes these events. The effects of
histamine are not significantly blocked by the H2 and H3 anta-
gonists cimetidine and thioperamide. Furthermore, histamine-
mediated activation of epithelial cells and fibroblasts increases
the permeability of the epithelium, the expression of adhesion
molecules and cytokines resulting in increased permeability of
macromolecules, such as allergens, and increased recruitment
and survival of inflammatory cells that is observed in both acute
and chronic ocular allergic inflammation.
THERAPEUTIC OPTIONS
Treatment of allergic ocular diseases, specifically allergic con-
junctivitis, may be approached in the same manner as one
would treat allergic rhinitis. Ideally, removing the offending
allergen or modifying the patient’s environment would be most
effective. However, this is not always practical. Systemic
medications such as oral antihistamines may be employed, but
these agents do not reliably relieve ocular symptoms, and their
soporific effects may mitigate their use. In most cases,
treatment with topical medications in the form of eye drops has
provided symptomatic relief without systemic side effects.
Topical corticosteroid preparations, such as fluorometholone,
prednisolone 0.125%, and loteprednol etabonate 0.2% are
extremely effective in providing relief of itching, chemosis, and
mucous discharge. These drugs should be used only in cases
CHAPTER 25
that do not respond to other forms of therapy, because they have
been associated with the development of elevated intraocular
pressure, cataract formation, and secondary bacterial, fungal,
and viral infections.57
Mast cell stabilizer preparations have been purported to
stabilize the mast cell plasma membrane, thereby preventing
subsequent degranulation and release of inflammatory
mediators. The ophthalmic literature has debated the thera-
peutic value of disodium cromoglycate in allergic conjunctivitis.
Several studies have demonstrated a salutary effect,58,59 whereas
others have shown no effect.60,61 A second-generation prepa-
ration, lodoxamide 0.1%, has shown salutary effects in patients
with VKC.62,63
Mast cell modulation is thus a fundamental target for anti-
allergic treatment. In fact, most of the ocular antiallergic drugs
have been designed as mast cell stabilizers. A decrease of calcium
influx into the cytoplasm is reported to be the mechanism of
the most widely used ocular mast cell stabilizers: sodium cromo-
glycate, lodoxamide, nedocromil and pemirolast. An advance-
ment in the treatment of ocular allergy comes from newly
designed ocular antihistamine compounds, such as olopatadine,
ketotifen, azelastine, and epinastine.64 These drugs have a dual
activity as antihistamines and mast cell stabilizers, probably
due to their effect on calcium mobilization or on phospholipid
cellular membrane. In fact, mast cell stabilizers inhibit
degranulation by interrupting the normal chain of intracellular
signals resulting from the cross-linking and activation of the
high-affinity IgE receptor (FceRI) by allergen.17 This promotes
activation of various kinase cascades, resulting in Ca2+ mobil-
ization, cytoskeletal changes and culminating in immediate
release of stored mediators.
The drugs most commonly used to treat ocular hay fever are
topical antihistamines. Their mechanism of action is com-
petitive inhibition with histamine for the histamine receptors
on effector cells. Currently, the only antihistamine preparations
available are H1-receptor antagonists. These agents reliably
relieve the symptoms of itching found in allergic conjunctivitis;
however, several preparations have little effect on chemosis and
redness.65 As such, these drugs are manufactured in combination 267
 PHARMACOLOGY AND TOXICOLOGY
with a vasoconstrictor agent that helps to relieve ocular
injection. Recently, several H1-selective receptor antagonists
have been introduced that relieve both the itching and the
redness associated with allergic conjunctivitis.66,67
ANTIHISTAMINES
In 1927, Lewis68 described the wheal-and-flare response seen in
human skin and suggested that histamine could be released
from intracellular stores by local injury. Armed with this
information, investigators began the search to develop
pharmacologic methods to blunt histamine’s profound effects.
In 1937, Bovet and Staub69 fortuitously noted that a compound
that they had been screening for adrenergic-blocking activity
also possessed some antihistaminic activity. This compound,
2-isopropyl-5-methyl-phenoxyethyldiethylamine, when admin-
istered to guinea pigs protected them from lethal doses of
histamine, antagonized histamine-induced smooth muscle
contraction, and diminished the systemic symptoms of
anaphylaxis. Unfortunately, this substance was too toxic for
clinical use, but it led to the discovery of phenbenzamine
(Antergan), a dimethylamine derivative that was the first anti-
histaminic compound to be used in humans.70 In 1944, Bovet
and co-workers71 discovered another clinically effective
compound, pyrilamine maleate (Neo-Antergan), which is still
used today.
The first description of topical antihistamine use in the eye
was published in 1946 by Bourquin.72 He observed satisfactory
SECTION 4
results with the use of antazoline (Antistine) in patients with
vernal catarrh, phlyctenular conjunctivitis, conjunctivitis asso-
ciated with hay fever, and scleritis. Two years later, in the
American literature, Hurwitz73 reported favorable results with
the same drug. Since the discovery that topical antihistamines
could alleviate symptoms of allergic conjunctivitis, several
authors have published results demonstrating that topical H1
antihistamines were clinically effective.74,75
Topical antihistamines are the first line in the treatment of
ocular allergy.76 These drugs are H1 receptor competitive
antagonists of varying specificity, potency, and duration of
action. The first-generation antihistamines, pheniramine and
antazoline, have a long safety record, but are known for their
burn upon instillation, their rapid onset and disappearance
of their effects, and their limited potency. These are still
available in over-the-counter products, particularly in asso-
ciation with vasoconstrictors. The newer antihistamines are
still H1 antagonists, but have a longer duration of action
(4–6 h), and are better tolerated then their predecessors. These
include levocabastine hydrochloride (Livostin, 0.5%) and
emedastine difumarate (Emadine, 0.05%). Both drugs are
effective and well tolerated also in pediatric subjects with
allergic conjunctivitis.
The clinical efficacy of ophthalmic levocabastine was shown
in numerous studies,66,77 while the newer emedastine appears
to be stronger and more selective. In fact, in a direct comparison
with levocabastine, emedastine proved significantly more
effective in alleviating chemosis and lid swelling.78 In two in
vitro studies, emedastine, and to a much lesser degree levo-
cabastine, blocked histamine-stimulated proinflammatory
cytokines (IL-8 and IL-6) release from conjunctival epithelial
cells and fibroblasts.79,80
MAST CELL STABILIZERS
Several mast cell stabilizers are available for use in the eye:
cromolyn sodium 4%, nedocromil sodium 2%, lodoxamide
tromethamine ophthalmic solution 0.1%, spaglumic acid 4%,
268 and pemirolast potassium ophthalmic solution 0.1%. All these
drugs inhibit mast cell degranulation, the release of histamine
and the other preformed mediators and the arachidonic acid
cascade.
Cromolyn sodium (DSCG), a derivative of khellin, a
chromone found in Ammi visnaga, an eastern Mediterranean
plant, was first synthesized in 1965. The drug is thought to act
on the mast cell plasma membrane via control of trans-
membrane calcium flux. The effect of DSCG is to stabilize the
membrane, thereby preventing degranulation and release of
inflammatory mediators.81,82 Thus, DSCG must exert its effect
prior to allergen binding or, at least, before the mast cell
membrane is altered with subsequent mediator release.
Since its discovery, investigators have shown DSCG to have
salutary effects in patients with allergic asthma and other IgE-
mediated diseases.83–90 In 1984, the US Food and Drug Admin-
istration (FDA) granted approval of DSCG for ocular use in
patients with VKC on the basis that the drug alleviated
symptoms and signs of the disease and allowed a reduction in
the frequency of steroid use in these patients.91,92 However,
the ability of DSCG to suppress ocular allergic symptoms in
environmental studies has yielded conflicting results.93–97 To
date, results of studies evaluating DSCG in allergic conjunc-
tivitis have been encouraging, but the effectiveness of the drug
in this condition remains controversial. However, both 4%
DSCG and 0.1% lodoxamide have been shown to be effective in
controlling the signs and symptoms of VKC.62,91,98
Nedocromil appears to be more potent than cromolyn, and is
approved for two times daily dosing.99,100 Nedocromil was
shown to stabilize both connective tissue and mucosal mast
cells, as opposed to cromolyn, and appears to inhibit by a
common pathway mast cells, eosinophils, epithelial cells, and
sensory nerves. It has been shown to be superior to placebo and
cromolyn in trials of seasonal and PAC, and other ocular allergic
disorders.101
Lodoxamide has been available since 1993 in the United
States and Europe for the treatment of VKC; however, it has
also been shown effective against allergic conjunctivitis. Its
mechanism of action is thought to be similar to that of
cromolyn, since it was shown to prevent histamine release.
Inhibition of eosinophil activation and degranulation is the
proposed mechanism for its efficacy against corneal signs such
as keratitis and shield ulcers in severe allergic disease.102
Lodoxamide was shown superior to placebo,103 cromolyn104
and N-acetyl aspartyl glutamic acid105 for treatment of
VKC, and equal or superior to cromolyn for the treatment
of allergic conjunctivitis.106 Its recommended dosing is four
times daily.
Pemirolast is another mast cell stabilizer that has been shown
to alleviate the signs of allergic conjunctivitis.107 Previous in
vitro and in vivo studies have demonstrated the efficacy of
pemirolast in inhibiting the antigen-induced release of inflam-
matory mediators (e.g., histamine, leukotriene C4, D4, E4)
from human mast cells and subsequently in preventing signs
and symptoms associated with allergic conjunctivitis. Pemiro-
last is currently approved for a four times daily (QID) dosing
regimen.108
Dipeptide N-acetyl-aspartyl glutamic acid (NAAGA) 6% has
been widely used in Europe as topical eye drops in the treatment
of allergic conjunctivitis, VKC, and GPC.105,109 NAAGA is
known to inhibit leukotriene synthesis, histamine release by
mast cells, and complement-derived anaphylatoxin production.
This antiallergic compound was also shown to directly inhibit
leukocyte adhesion to endothelial cells induced by proinflam-
matory stimuli, and abrogates TNFa-induced expression of
adhesion molecules on granulocytes and endothelial cells.110
These pharmacological properties confer a potential antiinflam-
matory activity to NAAGA.
 Antihistamines and Mast Cell Stabilizers in Allergic Ocular Disease
DUAL ACTION ANTIHISTAMINE/MAST CELL
STABILIZERS
This new category of drugs with dual mechanism of action
includes molecules that inhibit both mediator release from
mast cells (mast cell stabilizing effect) and, competitively,
histamine binding to H1 receptors (antihistaminic effect). In
this class are included olopatadine, ketotifen, azelastine, and
epinastine. The advantage offered by these molecules is the
rapidity of symptomatic relief given by immediate histamine
receptor antagonism, which alleviates itching and redness,
coupled with the long-term disease-modifying benefit of mast
cell stabilization.
Olopatadine has been shown in numerous in vitro, in vivo,111
and clinical studies112 to effectively and potently inhibit con-
junctival mast cells in allergic patients with seasonal and PAC
and allergic symptoms associated with contact lens wear.112
Olopatadine 0.1% was shown to reduce the levels of histamine,
the cellular infiltrate, and ICAM expression compared with
placebo after conjunctival allergen challenge, indicating that it
reduced the release of mast cell-derived mediators in humans.113
Olopatadine was demonstrated more effective and comfortable
than ketotifen in seasonal studies114–116 and more effective than
the mast cell stabilizer, nedocromil,117 and the antiinflam-
matory agent, ketorolac, in the conjunctival allergen challenge
model in allergic subjects.118
Ketotifen has been shown to inhibit the release of inflam-
matory mediators from mast cells, basophils, and neutrophils,
to inhibit the production and release of LTC4 and LTB4, platelet
activating factor (PAF) production by normal human neutrophils
and eosinophils, and eosinophil chemotaxis.119 Clinically, it has
been shown effective in the allergen challenge model, superior
to both placebo120 and cromolyn,121 and a safe treatment option
for children with allergic conjunctivitis.122
Azelastine, available in the past for rhinitis, has been approved
for ocular itching associated with allergic conjunctivitis.
Azelastine was shown to reduce ICAM-1 expression on con-
junctival epithelium, and inflammatory cell infiltration during
both early and late phase allergic reactions.123 In placebo-
controlled environmental and antigen challenge clinical trials,
azelastine was demonstrated to be significantly effective in
adults and children of at least 4 years of age, and to be at least
as effective as levocabastine.124–127 The duration was shown to
be at least 8 h. The most significant side effect with azelastine
is an unpleasant taste following instillation.
Epinastine is a new generation histamine H1-receptor anta-
gonist with mast cell stabilizing activity and no effect on
muscarinic receptors.128 Epinastine was shown to suppress
allergic inflammation not only through its strong antihistamine
and antimediator effects, but also by inhibiting eosinophilic
chemotaxis and the expression of adhesion molecules involved
in chemotaxis.129,130 Its safety and efficacy have been investi-
gated in the clinical conjunctival allergen challenge model, and
in patients with active seasonal allergy, where it was shown to
rapidly and significantly inhibit hyperemia, chemosis and lid
swelling for at least 8 h.131,132
HISTAMINE H1-RECEPTOR ANTAGONISTS
CHEMISTRY
Histamine receptors were defined pharmacologically by the
actions of their agonists and antagonists. Histamine H1-
receptor antagonists pharmacologically compete with histamine
at the H1-receptor site on effector cells and have been classified
by their chemical structures into six groups: ethylenediamines,
ethanolamines, alkylamines, phenothiazines, piperazines, and
piperadines (Table 25.1). The H1-receptor antagonist com-
pounds can be described by the general structure shown in
Figure 25.1. These compounds are composed of one or two
aromatic (heterocyclic) rings connected via a nitrogen, carbon,
or oxygen atom (X) to the ethylamine group. The nitrogen atom
of the ethylamine group is tertiary – that is, it has two sub-
stituents. The H1-receptor antagonists are structurally similar
to histamine in that they both contain an ethylamine group.
However, histamine consists of a single heterocyclic ring, in this
case imidazole, which is connected directly to the ethylamine
group. Unlike that of the H1-receptor antagonists, the nitrogen
atom of the ethylamine group is primary or unsubstituted.
STRUCTURE–ACTIVITY RELATIONSHIP
The H1-receptor antagonists possess two chemical moieties
that determine the pharmacokinetic properties of this group of
drugs and thereby confer pharmacologic activity (Table 25.2).
The H1 antihistamines contain multiple aromatic rings, which
make these compounds very lipophilic and contribute to
receptor site binding via hydrophobic forces.133 The second
functional moiety is the positively charged side chain, which is
usually an ammonium group. Both histamine and the H1-
receptor antagonists share an amino group that is believed
to be important for H1-receptor recognition.134 Table 25.2
demonstrates the chemical structural similarities and
differences between histamine and the H1- and H2-receptor
antagonists.
CHAPTER 25
MECHANISM OF ACTION
The H1-receptor antihistamines act by occupying H1 receptor
on effector cells. Binding of antagonists to the receptor site does
not initiate a response in the effector cell; rather, it prohibits
histamine from binding. Therefore, histamine is unable to
cause an effector cell response. The binding of the H1-receptor
antagonist is a reversible, competitive equilibrium reaction and
is determined by the relative concentrations of histamine and
H1-receptor antagonist in the area of the receptor site. To ensure
effective blockade of the H1-receptor, the antihistamine con-
centration should be sufficiently high to compete with tissue
histamine levels created by local mast cell degranulation.
PHARMACOKINETICS: ABSORPTION,
DISTRIBUTION, BIOTRANSFORMATION, AND
ELIMINATION
The majority of H1-receptor antagonists are chemically stable
and do not contain labile ester or amide moieties. The equili-
brium constant of the base and its conjugate acid of the anti-
histamine compounds is greater than 8.0. Thus, at physiologic
pH, all of the compounds would be at least 90% protonated
and water-soluble. As a result of their basic properties, the
H1-receptor antihistamines may be administered orally.
Following oral administration, the drugs are rapidly absorbed
and render symptomatic relief beginning within 15–30 min.
The duration of action usually is 3–6 h. The H1 receptor-
blocking agents are widely distributed in body tissues and
cross the blood–brain barrier. The compounds are metabolized
in the liver and excreted in the urine within 24 h of an oral
dose.135,136
Little information is available on the pharmacokinetics of
topically applied ocular H1 antihistamines. These drugs are
administered to the ocular surface via application of water-
soluble salts; maleate salts and phosphoric acid are most
commonly used in ocular preparations. Currently, only three H1
antihistamines are approved for use in the eye; these include 269
 PHARMACOLOGY AND TOXICOLOGY

TABLE 25.1 The Six Major Groups of Classic H1 Antihistamines

Linkage Atom General Class Other Members General Comments

N Ethylenediamines Antazoline Relatively weak CNS effects, but drowsiness may occur
Methapyrilene in some patients; gastrointestinal side effects
Tripelennamine common
O Ethanolamines Bromodiphenhydramine Significant antimuscarinic activity; CNS depression
(aminoalkyl ethers) Carbinoxamine common in about half of the patients using
Clemastine members of this group; relatively low incidence
Dimenhydrinate of gastrointestinal side effects
Diphenylpyraline
Doxylamine
Phenytoloxamine
C Alkylamines Brompheniramine Cause less CNS depression than members of other
(propylamine derivatives) Dexbrompheniramine groups; some CNS stimulation possible; best classic
Dexchlorpheniramine group of antihistamines for daytime use
Dimethindene
Pheniramine
Pyrrobutamine
Triprolidine
N (in phenothiazine Phenothiazines Methdilazine Sedative effects very prominent with this class; most
ring) Trimeprazine have pronounced antimuscarinic activity; usually used
primarily as antiemetics
N (in piperazine Piperazines Buclizine Degree of sedation and antimuscariniceffects produced
ring) Chlorcyclizine by this class is relatively mild; buclizine, cyclizine,
Hydroxyzine and meclizine are used for treating motion sickness;
Meclizine hydroxyzine is used as sedative, tranquilizer, and
antiemetic
SECTION 4

N (in piperidine Piperidines Azatadine Sedative potential is comparable to that of the

ring) Phenindamine ethylenediamine class; drowsiness is most common
side effect
From Trzeciakowski JP, Mendelsohn N, Levi R: Antihistamines. In: Middleton E, Reed CE, Ellis EF, et al, eds. Allergy principles and practice. 3rd edn. St Louis, MO: CV
Mosby; 1988.

TABLE 25.2 Chemical Differentiation between Histamine and

Its Respective Receptor Antagonists

H2 Antagonist Histamine H1 Antagonist

Imidazole for Imidazole Aryl rings

analogous ring
Hydrophilic Hydrophilic Lipophilic
Thiourea or guanidine Ammonium Ammonium (or similar
group)
Preferably uncharged Charged Charged
From Ganellin CR: Chemistry and structure–activity relationship of H2-receptor
antagonists. In: Rocha e Silva M, ed. Handbook of experimental pharmacology.
Histamine II and antihistaminics: chemistry, metabolism, physiological, and
pharmacological actions. New York: Springer; 1978.

FIGURE 25.1. A comparison of the chemical structure of histamine
(top) and of H1-receptor antagonists (bottom).
pheniramine maleate, antazoline phosphate, and pyrilamine
maleate. These preparations are well distributed in the preocular
tear film and seem to have excellent penetration into the con-
junctival epithelium and substantia propria. Systemic absorp-
tion occurs via drainage through the nasal lacrimal duct with
subsequent absorption by the nasopharyngeal and oropharyn-
270 geal mucosal surfaces.
PHARMACOLOGIC PROPERTIES
The pharmacologic actions of the H1-receptor antagonist
subclasses are similar: They block the effects of histamine
mediated by the H1 receptors on effector cells. The effects of
histamine on the vascular system are mediated by both H1 and
H2 receptors.137 Stimulation of H1 receptors causes systemic
vasodilation as well as localized cutaneous erythema due to
capillary dilation.41 However, when H1 receptor-blocking agents
are administered alone, the systemic hypotension caused by
histamine-induced vasodilation is only partially blocked. When
H1- and H2-receptor blockers are given concurrently prior to
histamine challenge, the fall in blood pressure is negated. Cuta-
neous capillary permeability is increased after local injection of
histamine, resulting in the formation of edema.39 H1-receptor
 Antihistamines and Mast Cell Stabilizers in Allergic Ocular Disease
antihistamines antagonize this action of histamine and inhibit
the egress of plasma through capillary walls.
Histamine has a direct constrictor action on smooth muscle.
In humans, histamine-induced bronchoconstriction of respira-
tory smooth muscle can be blocked with prophylactic adminis-
tration of H2-receptor antagonists.138 In animal species, in vivo
experiments have demonstrated histamine-induced contraction
of gastrointestinal smooth muscle. The guinea pig ileum model
had been used to provide early evidence for the effects of
histamine and to document the presence of specific histamine-
receptor subtypes. In addition, this animal model had been used
to test various types of H1-receptor antihistamines as these
agents were developed.
In the eye, topical application of histamine induces ocular
itching and conjunctival vasodilation. It has been demonstrated
that the H1 receptors mediate the symptoms of itching, whereas
conjunctival vasodilation is mediated by both H1 and H2
receptors.55,56,65 Pretreatment with topical H1 antihistamines
blocks the histamine-induced itching and decreases the amount
of conjunctival hyperemia.
Many of the H1 antihistamines possess pharmacologic pro-
perties unrelated to H1-receptor blockade. These agents possess
varying degrees of anticholinergic activity that is dose dependent
and varies among the subclasses. The anticholinergic action has
been used in treating several diseases, including motion
sickness, vertigo resulting from vestibular disorders, and rigidity
associated with Parkinson’s disease.
Several H1-receptor antagonist compounds have been demon-
strated to possess local anesthetic action.139 However, this effect
occurs only with concentrations several orders of magnitude
greater than the pharmacologic dosages employed to block the
H1 receptor. In eyes pretreated with antazoline phosphate,
itching was blocked after topical histamine challenge, whereas
corneal sensation was shown not to be decreased by
anesthesiometry.65
ADVERSE EFFECTS
SYSTEMIC ADMINISTRATION
Therapeutic doses of oral H1 antihistamines may be associated
with mild systemic side effects; however, occasionally the unto-
ward responses may necessitate drug withdrawal. The most
common adverse effect observed with H1-receptor antagonists is
sedation, which varies between the drug subclasses and indi-
vidual patient response.140 Although sedation may not be
problematic when medication is administered upon retiring
for the night, this soporific effect may lead to potentially
life-threatening accidents in patients who drive or operate
heavy automated machinery. Other central nervous system
(CNS) side effects include disturbed coordination, dizziness,
fatigue, and difficulty in concentration, which result from a
generalized depression of the CNS. Paradoxically, patients
may also experience euphoria, nervousness, insomnia, and
tremors.
Gastrointestinal adverse effects occur less frequently and
include loss of appetite, nausea, vomiting, epigastric distress,
and constipation or diarrhea. Occasionally, these symptoms are
diminished by administering oral H1 antihistamines with meals.
Several less-frequent side effects of the H1-receptor antagonists
are attributable to their anticholinergic properties. Patients may
note dryness of the mucous membranes of the oropharynx and
the appearance of dry eye symptoms that may lead to contact
lens intolerance or frank keratoconjunctivitis sicca. Other
atropine-like effects include mydriasis that could precipitate an
attack of acute angle-closure glaucoma in untreated, predisposed
persons. Ciliary muscle paresis with an associated decrease in
accommodation may account for visual difficulties experienced
by some patients.
Systemic H1 antihistamines should be used judiciously in
young children; acute poisoning may result from an inability to
metabolize the drugs rapidly and may produce dangerously high
blood concentrations. The CNS effects of the H1-receptor anta-
gonists constitute the greatest danger to children, and the con-
stellation of signs and symptoms are related to anticholinergic
activity as evidenced by excitement, nervousness, irritability,
incoordination, insomnia, and tremors.141 Other signs asso-
iated with cholinergic blockage are fixed and dilated pupils,
facial flushing, and elevated body temperature.
Safety in pregnancy for humans has not been established for
systemic H1 antihistamines.142 However, the piperazine com-
pounds may have teratogenic effects.
The use of systemic antihistamines for the treatment of
ocular allergy is controversial. Ocular allergy is a topical disease
with typical anatomical and pharmacological conditions for
convenient local delivery.143 Topical antiallergy eyedrops provide
faster relief of ocular symptoms compared with oral agents,
because the former are delivered directly onto the target tissue
at a higher concentration. In contrast, allergic rhinitis is an
equally frequent condition generally treated with systemic anti-
histamines, which have been proven effective in relieving nasal
signs and symptoms. First generation oral H1-receptor anta-
gonists may provide some relief of ocular itching, but are
sedating and possess anticholinergic effects such as dry mouth,
dry eye, blurred vision, and urinary retention. The second
CHAPTER 25
generation oral H1 antihistamines offer the same efficacy as
their predecessors, but with a low-sedating profile and lack of
anticholinergic activity. These drugs attentuate the early phase
and some of the features of the late phase ocular response,
including swelling and redness. Second generation antihista-
mines include acrivastine, cetirizine, ebastine, fexofenadine,
loratadine, and mizolastine. Desloratadine and levocetirizine
are considered a further evolution of the second generation
agents. Nevertheless, in a recent study, the most successful
systemic antihistamine, loratadine, was shown to be inferior to
local, topical antiallergy therapy in alleviating the signs and
symptoms of allergic conjunctivitis.144
TOPICAL OCULAR ADMINISTRATION
Topical administration of H1-receptor antagonists in the eye has
been associated with a low incidence of systemic adverse effects.
However, these agents are available only in combination with
sympathomimetic decongestant agents that have been asso-
ciated with systemic side effects. Ocular medications gain
access to the systemic circulation via absorption through the
nasal and oropharyngeal mucosae. Therefore, combination drugs
should be used with caution in patients with poorly controlled
hypertension, cardiovascular disease with arrhythmias, and
poorly controlled diabetes mellitus. Additionally, patients using
monoamine oxidase inhibitors for hypertensive disease may
suffer a hypertensive crisis if administered a topical sympa-
thomimetic decongestant agent.145,146
Pupillary mydriasis may be induced by either component of
an H1-receptor antagonist/decongestant combination and may
trigger an attack of acute angle-closure glaucoma. The combina-
tion drugs have not been evaluated for safety during pregnancy.
PREPARATIONS AND DOSAGES
Currently, both prescription and over-the-counter H1-receptor
antagonist antihistamine agents are available to treat disease.
Three over-the-counter H1-receptor antagonist antihistamines
are available for topical ocular administration and are produced 271
 PHARMACOLOGY AND TOXICOLOGY

TABLE 25.3 Antihistamine–Decongestant Combinations TABLE 25.4 Ocular Decongestants, Decongestant–Astringents,

and Decongestant–Antibacterials
Generic Name Commercial Recommended
Preparations Dosage Generic Name Commercial Recommended
Preparations (drops) Dosage (7–14 Days)
Antazoline PO4 (0.5%) Albalon A 1–2 drops/eye q3–4h
Naphazoline Vasocon A or less to relieve Decongestants
HCl (0.05%) symptoms
Naphazoline Albalon (OTC) 1 drop/eye q3–4h or
Pheniramine maleate AK-Con A 1–2 drops/eye q3–4h (0.12% [Rx]; Clear Eyes (OTC) less to relieve
(0.3%) or less to relieve 0.012% [OTC]) Degest-2 (OTC) symptoms
symptoms Opcon (OTC)
Naphcon (OTC)
Naphazoline HCl Opcon A Naphcon Forte (Rx)
(0.025%) Naphcon A Vasoclear (OTC)
Pyrilamine maleate Prefrin-A 1–2 drops/eye q3–4h Vasocon Regular
(0.1%) Prefrin-A or less to relieve (OTC)
symptoms Phenylephrine AK-nephrine (OTC) 1 drop/eye q3–4h or
Phenylephrine (0.12%) (0.12%) Prefrin (OTC) less to relieve
Relief (OTC) symptoms
From Pavan-Langston D, Dunkel EC: Handbook of ocular drug therapy and
ocular side effects of systemic drugs. Boston, MA: Little, Brown; 1991. Tetrahydrozoline Collyrium (OTC) 1 drop/eye q3–4h or
HCl (0.05%) Murine PLUS (OTC) less to relieve
Visine (OTC) symptoms
Decongestant–Astringents

only as combination antihistamine/decongestant preparations. Naphazoline Vasoclear A (OTC) 1–2 drops/eye up to

(0.02%) 4 times daily
The H1 antihistamines are 0.5% antazoline phosphate, 0.3% Zinc SO4 (0.25%)
pheniramine maleate, and 0.1% pyrilamine maleate and are
found in combination with either 0.025–0.05% naphazoline Phenylephrine HCl Visine AC (OTC) 1–2 drops/eye up to
hydrochloride or 0.012% phenylephrine (Table 25.3). Each of (0.12%) 4 times daily
Zinc SO4 (0.25%)
SECTION 4

the three H1 antihistaminic agents is efficacious in reducing the

chemosis and itching associated with allergic conjunctivitis.
The decongestant agents are included for their vasoconstrictor
properties and are efficacious in relieving conjunctival injection.
The recommended dosage is one to two drops instilled in the
eye up to four times daily as needed to control symptoms.
New H1-selective receptor antagonist agents have been
introduced that block both the itching and redness associated with
allergic conjunctivitis. Levocarbastine (Livostin) is a potent new
topical ocular H1-receptor antagonist that has been demonstrated
to effectively control the symptoms of allergic conjunctivitis.147 A
topical preparation of 0.05% levocarbastine hydrochloride
administered prior to conjunctival histamine challenge effectively
prevented itching, conjunctival injection, and chemosis.148 In
conjunctival antigen challenge (CAC) studies, 0.05%
levocarbastine hydrochloride has been shown to be more effective
than placebo and 4% DSCG in inhibiting itching, hyperemia,
eyelid swelling, chemosis, and tearing after allergen challenge.66,149
A second H1-selective receptor antagonist agent, 0.1% olopa-
tadine (Patanol), has been added to the armamentarium to treat
allergic conjunctivitis. In CAC studies, 0.1% olopatadine has been
shown to be more effective than placebo in inhibiting itching and
redness after antigen challenge.150 Additionally, the recommended
dosing schedule of 0.1% olopatadine is twice daily, and it has
been approved for use in children at least 3 years of age.
In addition to the antihistamine/decongestant preparations,
several over-the-counter and prescription decongestant prepara-
tions and decongestant/astringent combinations are available
(Table 25.4). These agents may be used in circumstances of
mild ocular irritation or allergic conditions and are effective in
reducing conjunctival injection and clearing mucus from the
ocular surface.
HISTAMINE H2-RECEPTOR ANTAGONISTS
CHEMISTRY
The H2-receptor antagonists were born of the idea to develop
272 compounds that would block those responses induced by
Tetrahydrozoline Zincfrin (Rx) 1–2 drops/eye up to
HCl (0.05%) 4 times daily
Zinc SO4 (0.25%)
Decongestant–Antibacterials
Phenylephrine HCl Vasosulf (Rx) 1–2 drops/eye q4h
(0.12%)
Sulfacetamide 1–2 drops/eye q4h
Na (15%)
Modified from Pavan-Langston D, Dunkel EC: Handbook of ocular drug therapy
and ocular side effects of systemic drugs. Boston, MA: Little, Brown; 1991.
histamine that could not be blocked by the currently available
H1-receptor antagonists. The H2-receptor antagonists were
synthesized by a series of modifications of the histamine
molecule and therefore have a structural relationship to hista-
mine (Table 25.5). The first selective H2-receptor antagonist,
burimamide, was synthesized in 1969 by substituting bulkier,
uncharged side chains to the imidazole ring.36 Subsequently,
two imidazole ring congeners – metiamide,151 a thione-containing
compound, and cimetidine,152 a cyanimino compound – were
developed. More recently, ranitidine, a furan derivative, has
become available.153 Each of these compounds contains a polar
heterocyclic ring in its side chain.
STRUCTURE–ACTIVITY RELATIONSHIP
The H2-receptor antagonists bear a closer structural relation-
ship to histamine than do the H1-receptor antagonists.
Burimamide, metiamide, and cimetidine have an imidazole ring
and are polar, hydrophilic compounds similar to histamine (see
Table 25.2). It appears that the imidazole or another hetero-
cyclic, side chain ring is critical for H2-receptor site recognition
and plays a role in determining drug activity.133 The H2-receptor
antagonist compounds have similar equilibrium constants (pKa
values of ~14). These drugs are weak bases and highly water-
soluble; thus, they exist in the uncharged form in aqueous
solutions under physiologic conditions (pH of 7.4).133
 Antihistamines and Mast Cell Stabilizers in Allergic Ocular Disease

minor.155 As previously mentioned, systemic administration of

TABLE 25.5 Representative Histamine H2-Antagonists
histamine caused vasodilation and severe hypotension that
Compared with Histamine
were completely blocked only by the concurrent use of both H1-
Structure and H2-receptor antihistamines.
and Name Relative Antagonist In the eye, stimulation of H2-receptors with a selective H2
Ring Type Potency Reference agonist produced diffuse conjunctival vasodilation.56 Cimeti-
Imidazole 0.001 Durant et al dine has been the only H2-receptor antagonist to be formulated
Durant et al into an ophthalmic preparation. Studies have shown that the
Ganellin addition of an H2-receptor antagonist to a classic H1 anti-
Imidazole 0.1 Black et al histamine reduced the amount of conjunctival vasodilation in
Durant et al response to histamine challenge.75
Ganellin
Imidazole ~1 Black et al ADVERSE EFFECTS
Durant et al
Forrest et al SYSTEMIC ADMINISTRATION
Ganellin
The H2-receptor antagonists are generally well tolerated when
Imidazole 1 Brimblecombe et al
Durant et al taken systematically. The side effects of cimetidine are minor,
Ganellin seldom posing a serious problem, and include headaches, fatigue,
myalgias, constipation, and skin rashes. The CNS-depressive
Furan 3–5 Brittain and Daly
effects seen with the H1 antihistamines are not seen with the H2-
Imidazole 1–4 Blakemore et al receptor blockers, because these compounds are hydrophilic and
Mills et al penetrate the blood–brain barrier poorly. However, cimetidine has
From Trzeciakowski JP, Mendelsohn N, Levi R: Antihistamines. In: Middleton E, been associated with confusion, delirium, and convulsions, usually
Reed CE, Ellis EF, et al, eds. Allergy principles and practice. 3rd edn. St Louis, occurring in patients with concurrent liver or kidney disease.
MO: CV Mosby; 1985.
Cimetidine possesses weak antiandrogenic effects and has
been responsible for reports of gynecomastia in men and
MECHANISM OF ACTION galactorrhea in women. These effects have occurred in patients

The H2-receptor antagonists work in a manner similar to that
of the H1-receptor antihistamines. These agents bind reversibly
and competitively to the histamine H2 receptors on effector
cells. When bound to the receptor site, the H2-receptor anta-
gonist agents do not elicit a tissue response and block the effect
of histamine.
PHARMACOKINETICS: ABSORPTION,
DISTRIBUTION, BIOTRANSFORMATION, AND
ELIMINATION
Cimetidine, the prototype of the H2-receptor antagonist drugs, is
well absorbed after oral administration. After an oral dose, peak
blood concentrations are reached in ~60–90 min with good tissue
distribution throughout the body.136 The one exception is the
CNS; cimetidine penetrates the CNS poorly because the
compound is poorly lipophilic. The drug has been found to
cross the placental barrier and is excreted in breast milk.136
The majority of an oral dose of cimetidine is excreted in
the urine, with a minor portion handled in the bile and by hepatic
microsomal biotransformation. In patients with normal renal
function, the plasma half-life (t1/2) is ~2 h. However, the t1/2
increases in patients with impaired hepatic or renal function.154
PHARMACOLOGIC PROPERTIES
Cimetidine and the other H2-receptor antagonist antihista-
mines are selective in their action and block the effects of
histamine mediated through the H2-receptor. The most note-
worthy systemic effect is the ability of these agents to inhibit
gastric secretion induced by histamine, gastrin, or pentagastrin
in humans.155–157 Cimetidine inhibits all phases of physiologic
secretion of gastric acid. In humans, a single 300-mg dose
decreases the fasting secretion of gastric acid and decreases the
amount of acid induced by food or via vagal stimulation.136
When given intravenously in high doses, cimetidine may
cause bradycardia and hypotension. However, when given to
normal volunteer subjects, the cardiovascular changes were
CHAPTER 25
treated for an extended length of time. Cimetidine has been
demonstrated to release prolactin when given in large intra-
venous doses.158,159 There have been sporadic reports in the
literature of bone marrow suppression associated with cimeti-
dine therapy. Patients have experienced leukopenia, thrombo-
cytopenia, and hemolytic anemia, which seems to be an
idiosyncratic reaction.160
Cimetidine is metabolized partially by the hepatic microsomal
enzyme system and therefore may impair the elimination of
drugs that are catabolized in this manner. These drugs include
oral anticoagulants,161 theophylline,162 benzodiazepines,163 and
propranolol.164 Additionally, the pharmacokinetics of calcium
channel blockers are altered by cimetidine.165
TOPICAL OCULAR ADMINISTRATION
Currently, no H2-receptor antihistamines are approved for ocular
use. However, it is conceivable that combination drops con-
sisting of H1 and H2 antagonists have a place in the treatment
of ocular allergic disorders. Studies have shown that combina-
tion drops have a synergistic effect in reducing conjunctival
vasodilation and chemosis when compared with the individual
agents alone. Topical epinastine, a dual acting molecule with
mast cell stabilizing and antihistaminic effect, has also an H2
antagonism.
PREPARATIONS AND DOSAGES
Studies evaluating cimetidine as a topical ocular preparation
have found concentrations of 0.1%, 0.5%, and 1.0% to be well
tolerated and efficacious in reducing ocular symptoms induced
by histamine challenge.
MAST CELL STABILIZERS
CHEMISTRY
The first mast cell-stabilizing compound was developed in the
late 1960s from khellin, a chromone (benzopyrene) derived from 273
 PHARMACOLOGY AND TOXICOLOGY
FIGURE 25.2. The chemical structure of disodium cromoglycate
(cromolyn sodium) (1,3-bis(2-carboxychromon-5-yloxy)-2-
hydroxypropane).
Ammi visnaga, an eastern Mediterranean plant.166 Successive
modifications in structure yielded several bis-chromone com-
pounds, one of which was DSCG. DSCG is the disodium salt of
1,3-bis(2-carboxychromon-5-yloxy)-2-hydroxypropane (Fig. 25.2).
The compound is composed of two chromone rings joined by a
flexible carbon chain, with each ring possessing a polar carboxyl
group. The compound is an odorless, white, dehydrated crystal-
line powder that is moderately soluble in water but practically
insoluble in alcohol.167 The drug was first discovered to have
antiasthma properties when Altounyan demonstrated on him-
self that cromolyn could afford protection against an asthmatic
attack induced by bronchial provocation with pollen antigens.83
STRUCTURE–ACTIVITY RELATIONSHIP
SECTION 4
Disodium cromoglycate forms complexes with divalent cations,
including magnesium (Mg2+), calcium (Ca2+), strontium (Sr2+),
barium (Ba2+), zinc (Zn2+), and manganese (Mn2+) when placed
in organic solvents. These complexes are formed by an electro-
static interaction between the two carboxyl groups of DSCG
and the divalent cations with a 1:1 stoichiometry.168 Although
cromolyn has been associated with reduced calcium flux across
the mast cell membrane, chelation of calcium by cromolyn does
not fully account for the drug’s ability to inhibit mast cell
degranulation. It has been demonstrated that DSCG interacts
with a membrane-bound cromolyn receptor, which is a calcium-
transporting protein necessary for the secretion of histamine.
This interaction requires the presence of calcium ions in order
to proceed.169
MECHANISM OF ACTION
It had been thought that DSCG possessed membrane-
stabilizing features in that the drug somehow modified the mast
cell membrane to prevent histamine release in the presence of
IgE antibody. When cromolyn was discovered, little was known
of its mechanism of action. However, since the 1980s, evidence
from research has shed light on the interaction between this
drug and the mast cell. In 1980, Mazurek and co-workers
identified a binding site on mast cells and basophils for
DSCG.169 The authors identified the cromolyn receptor as a
membrane-binding protein that required the presence of
calcium ions for the interaction to proceed. The evidence from
the experiments suggests that the cromolyn-binding protein is a
calcium-transporting protein that is necessary for the secretion
of histamine after stimulation by an IgE antibody–antigen inter-
action.170 It is theorized that the membrane-bound cromolyn-
binding protein interacts with the Fc receptors for IgE in such a
way that cross-linking of the Fc receptors does not occur upon
antigen binding to the IgE molecule.171
Also in 1980, other researchers examined the association
between DSCG and protein phosphorylation in the activation
274 and regulation of histamine secretion in mast cells. Theoharides
and associates demonstrated that cromolyn induced phospho-
rylation of a 78 000-Da mast cell protein.172 These authors
presented compelling data suggesting that DSCG and phospho-
rylation of the membrane-bound protein are intricately involved
in the regulation of histamine secretion. The concentration
range over which DSCG induced phosphorylation of proteins
was similar to that for cromolyn-induced inhibition of hista-
mine release stimulated by compound 48/80. Additionally, both
activation of phosphorylation and inhibition of secretion by
DSCG demonstrated tachyphylaxis – that is, a second exposure
to cromolyn failed to induce phosphorylation in mast cells that
were pretreated with the drug. Lastly, dephosphorylation after
cromolyn-induced phosphorylation of the 78 000-Da protein had
a time course identical to that of the loss of sensitivity of mast
cells to the inhibition of histamine release caused by cromolyn.
The mechanism of protein phosphorylation has not been
elucidated; however, it has been shown that cyclic guanosine
monophosphate can phosphorylate the same 78 000-Da mast
cell protein as cromolyn does. Thus, it has been theorized that
cromolyn may act via a cyclic guanosine monophosphate-
dependent protein kinase.173 This is not surprising in that
DSCG has been identified as an inhibitor of cyclic nucleotide
phosphodiesterase.174
PHARMACOKINETICS: ABSORPTION,
DISTRIBUTION, BIOTRANSFORMATION, AND
ELIMINATION
DSCG is poorly absorbed from the gastrointestinal tract after
oral administration. Therefore, it is available as an inhalant
that can be administered via the nasal or respiratory tract.
When given as an inhaled dose, ~8% is absorbed systemically
through the bronchial tree.175 The half-life (t1/2) of the compound
is ~80 min, with more than 98% being eliminated within
24 h.136 Cromolyn is not metabolized and is excreted unchanged
in the urine and bile.
Little information is available on the pharmacokinetics of
topically applied ocular disodium cromoglycate. Cromolyn is
administered to the ocular surface via application of a water-
soluble solution.
Currently, two mast cell-stabilizing drugs are approved for
use in the eye: 4% DSCG (Crolom) and 0.1% lodoxamide
tromethamine (Alomide). Both preparations are well distributed
in the preocular tear film and seem to adequately penetrate the
conjunctival epithelium and substantia propria. When
administered to normal volunteer subjects, ~0.03% of DSCG
was absorbed following an ocular dose.
PHARMACOLOGIC PROPERTIES
The pharmacologic actions of mast cell stabilizers result from
the ability of the drug to bind to membrane-bound protein
receptors on mast cells. This interaction inhibits histamine
release when IgE-primed mast cells are challenged with antigen.
Mast cell stabilizers do not interfere with the binding of IgE to
the Fc receptors on mast cells or the interaction between mast
cell-bound IgE and antigen. Mast cell stabilizers have no bron-
chodilator, antiinflammatory, or anticholinergic activity; rather,
they suppress the mast cell secretory response to antigen. Thus,
the drug is effective only when given prophylactically prior to an
antigen–IgE antibody interaction.
Inhaled DSCG is recognized as an effective prophylactic drug
for the treatment of asthma.84–86 Cromolyn has also been
demonstrated to have salutary effects in patients with food
allergy,87 systemic mastocytosis,88 and seasonal allergic
rhinitis.89,90 However, it should be noted that the effectiveness
of the drug in these conditions remains controversial.
 Antihistamines and Mast Cell Stabilizers in Allergic Ocular Disease
In the eye, 0.1% lodoxamide tromethamine62,63 and DSCG91,92
have shown effectiveness in relieving the signs and symptoms
of VKC. The latter helped to reduce the frequency of steroid use
in patients with VKC. When these two drugs were compared in
a multicenter, double-masked, parallel-group clinical study,
0.1% lodoxamide was found to be statistically superior to 4%
cromolyn in alleviating itching, tearing, foreign body sensation,
and discomfort in patients with VKC.62
Likewise, clinical studies have demonstrated encouraging
results with DSCG in acute allergic conjunctivitis. In general,
investigators have reported satisfactory results with DSCG in
acute allergic conjunctivitis. Greenbaum and associates93 con-
ducted the first environmental study evaluating 4% DSCG in a
double-blind, placebo-controlled fashion and reported that eye
symptom scores for patients receiving DSCG were significantly
lower when compared with the previous year’s ragweed season.
In a double-masked, placebo-controlled, parallel-group pro-
spective environmental study, Friday et al94 demonstrated
that 4% DSCG was a safe and effective method of controlling
the symptoms of ragweed conjunctivitis in patients with
serum IgE levels less than 100 ng/mL. Patients with serum IgE
levels greater than 100 ng/mL did not experience a significant
improvement in symptoms. Leino and Tuovinen95 evaluated
DSCG in 33 patients with VKC, allergic conjunctivitis, or
chronic conjunctivitis in a prospective uncontrolled study.
The authors reported a beneficial effect associated with the
use of DSCG; however, regression of the signs and symptoms
varied widely.
Two well-designed, double-blind, placebo-controlled, compa-
rative environmental studies reported that DSCG suppressed
allergic eye symptoms in specific groups of patients identified by
serum IgE antibody levels. However, the results were contra-
dictory. Welsh et al96 showed that 4% DSCG caused a signifi-
cant reduction in eye itching and irritation in subjects whose
preseasonal IgE ragweed antibody level was less than 99 ng/mL;
patients with IgE levels exceeding 100 ng/mL did not experience
the same benefit. Kray et al97 stratified their subjects by radio-
allergosorbent (RAST) scores to IgE antibodies, including
ragweed. These investigators noted a significant suppression of
eye symptoms in subjects with class 3 or 4 RAST scores (higher
antibody level). Subjects with classes 0, 1, and 2 RAST scores
noted no significant difference between DSCG and placebo.
ADVERSE EFFECTS
SYSTEMIC ADMINISTRATION
Therapeutic doses of DSCG are well tolerated by patients. Most
adverse reactions are mild and are associated with a direct irritant
effect of the powder on the bronchial tree, including bronchospasm,
wheezing, cough, sneezing, nasal congestion, and pharyngeal
irritation.136 Other adverse effects have been documented in
case reports and consist of dermatitis, gastroenteritis, myositis,
urethral burning, and pulmonary allergic granulomatosis.176–179
DSCG has no known effect on pregnancy in laboratory
animals; however, safety for human use during pregnancy has
not been established, and no controlled human studies have
been performed.136 It is not known whether the drug is excreted
in human breast milk, and the safety and efficacy of cromolyn
have not been established in children younger than 4 years.
TOPICAL OCULAR ADMINISTRATION
Topical administration of mast cell stabilizers in the eye has
been associated with a low incidence of systemic adverse effects.
Ocular side effects are common but usually mild and self-
limited. Ocular administration of cromolyn has been associated
with transient stinging and conjunctival injection. Other local
adverse reactions include chemosis and ocular and periocular
itching and irritation.
PREPARATIONS AND DOSAGES
Currently, the only mast cell-stabilizing drugs formulated for
topical ocular use are cromolyn sodium 4%, nedocromil sodium
2%, lodoxamide tromethamine ophthalmic solution 0.1%, spag-
lumic acid 4%, and pemirolast potassium ophthalmic solution
0.1%. Four percent disodium cromoglycate contains 40 mg of
DSCG in purified water with a preservative and is a clear, color-
less, sterile solution with a pH of 4.0–7.0. The recommended
dosage is one to two drops instilled in the eye four times daily.
One drop of the solution contains ~1.6 mg of DSCG. The 0.1%
lodoxamide tromethamine contains 1.78 mg of lodoxamide
tromethamine in purified water with EDTA, benzalkonium
chloride, and other inactive ingredients. This preparation has
been shown to be 2500 times more potent than DSCG180 and
has demonstrated satisfactory results in patients with AKC and
GPC.181 The recommended dosage is one drop applied four times
daily, although patients have been able to use 0.1% lodoxamide
tromethamine twice daily and still remain asymptomatic.
Because therapy with mast cell stabilizers is prophylactic, it
is advisable to initiate treatment before the onset of allergic
symptoms. It is not unexpected that symptomatic response to
treatment may take up to 2 weeks with DSCG and up to 4 days
with 0.1% lodoxamide tromethamine. Once therapy has
CHAPTER 25
commenced, it should be continuous and maintained even after
symptomatic improvement.
NEW THERAPY FOR OCULAR ALLERGY
Since the 1980s, researchers have explored different methods to
block the allergic response in type I hypersensitivity reactions.
It was discovered that Fc fragments from IgE antibody could
competitively inhibit IgE binding to effector cells and block the
Prausnitz–Küstner reaction when preinjected into skin.182
HEPP (pentigetide), a synthetic pentapeptide derived from the
Fc region of human IgE, was developed and consisted of an
amino acid sequence of aspartyl-seryl-aspartyl-prolyl-arginine.
In tests on atopic persons, HEPP blocked the Prausnitz–Küstner
reaction;183 however, its mechanism of action remains unknown.
In a double-blind, randomized, parallel study, 0.5% pentigetide
(Pentyde) ophthalmic solution was compared with 4% DSCG in
patients with allergic conjunctivitis.184 After a 2-week com-
parison, patients treated with 0.5% pentigetide experienced
significant improvement in conjunctival hyperemia, chemosis,
tearing, and itching. With further study, this drug may prove to
be a useful adjunct in the treatment of allergic conjunctivitis.
As mentioned previously, other mediators of inflammation
contribute to and help perpetuate the ocular allergic response.
Several classes of pharmacologic agents have demonstrated
efficacy in blocking the effects of these mediators of inflam-
mation, and hence possess antiallergic properties when used as
ocular preparations. In CAC studies, topical nonsteroidal anti-
inflammatory drugs such as 0.5% ketorolac tromethamine
(Acular), 0.03% flurbiprofen sodium (Ocufen), and 0.1% diclo-
fenac sodium (Voltaren) and topical corticosteroid agents such
as 0.5% loteprednol etabonate (Lotemax) and 1% rimexolone
(Vexol) have demonstrated effectiveness in controlling the signs
and symptoms of allergic conjunctivitis. Researchers are actively
investigating compounds that blunt the response to or inhibit
the action of these inflammatory mediators. In the future, we
expect to have available topical medications such as antiplatelet
activating factor and leukotriene inhibitors to add to our list of
antiallergic drugs. 275
 PHARMACOLOGY AND TOXICOLOGY
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279
 CHAPTER
26 Tear Film and Blink Dynamics
Mark B. Abelson, Marshall G. Doane, and George Ousler
DYNAMICS OF THE BLINKING PROCESS
Blinking is usually performed as a nonconscious closing of the
eyelids and serves to carry secreted tear fluid from the superior
and inferior marginal menisci over the anterior surface of the
eye, continuously reestablishing the tear film over the cornea.
Also, the blinking action wipes debris and particulate matter
from the surface of the cornea and sclera into the inferior
marginal tear meniscus. As we shall see, once in the meniscus,
such debris is effectively directed toward the medial canthal
region by subsequent blinks and usually drained with ‘used’ tear
fluid via the punctal openings. This constant drainage via the
puncta is necessary to allow for the removal of used tear fluid,
but it also removes instilled tear substitutes from the menisci,
therefore limiting their effective residence time and requiring
frequent reinstillation of such products.
Although the normal blink rate is often given as 12–15 per
minute, this is, at best, an average of a greatly variable para-
meter, and it is strongly influenced by external events. A loud
noise or bright flash of light, of course, immediately elicits a
blink by reflex action, but more subtle events such as a visually
intensive task (e.g., reading, watching a computer monitor)
reduces the blink rate, thus increasing the length of interblink
periods and minimizing how often vision is blocked by the
closing of the eyelids. A person’s tear film stability also can
influence the blink rate; because discomfort is usually asso-
ciated with the breakup and drying of the tear film on the
cornea, this can stimulate blinking. Thus, many dry-eyed
patients tend to have shortened blink intervals (i.e., high blink
rates) as a result of the decreased tear film breakup time during
interblink periods. A major goal of any tear substitute is to
increase the stability of the tear film layer, usually by incor-
porating surfactants and viscosity agents, as described in the
following section.
The details of the actual motion of the blinking eyelids occur
too rapidly to see. With a high-speed motion picture or video
camera, the recorded images can be replayed at a slower speed
and the details of the motion accurately determined. Truly
nonconscious blinks can only be recorded if the subject is not
aware that such blinks are being measured or indeed that blink-
ing is a subject of interest. Self-conscious blinks are invariably
forced, and such blinks differ considerably in their dynamics
and time course from the ordinary, nonconscious blinking that
occurs thousands of times each day.
Studies using a high-speed camera and long telephoto lens
placed behind a one-way mirror have recorded the normal, non-
conscious blinks of an unknowing subject.1 With a film-recording
rate of up to 500 pictures per second, the resultant images were
subsequently analyzed frame by frame for the details of motion
of each lid, including their instantaneous velocities.
MOTION OF THE UPPER LID
The upper lid is responsible for wiping the anterior surface of
the globe and restoring a clean, ‘new’ tear film with each blink.
From its open, resting position, the upper lid rapidly accelerates
downward until reaching the center of the cornea. It then
decelerates, often slowing to a stop and reversing its motion
before actually contacting the lower lid. Even when such contact
does occur, it is seldom forceful except during strong, voluntary
blinks. Figure 26.1 shows typical time and velocity profiles of
the upper lid for four consecutive nonconscious blinks in four
subjects. The point of zero velocity is the instant of reversal of
motion between the closing and opening phase of the blink.
Note that the opening phase consumes about twice the time of
the closing phase and is particularly slow during the last few
millimeters of lid opening. The reversal of lid motion is rapid,
occurring in less than 2 ms; in voluntary blinking, the lid
remains stationary for much longer, a consistent feature of such
blinks. The maximum downward excursion of the upper lid is,
of course, limited to the width of the palpebral fissure at the
open, resting position of the lids. Many, if not most, blinks are
less than complete, with the amount of lid excursion less than
the maximum possible for a given individual. For the examples
shown, the maximum excursion of the upper lid ranges from
5 to 13 mm and peak velocities from 80 to 300 mm/s.
As indicated by the lid velocity profiles shown in Figure 29.1,
blink velocities vary considerably among individuals and even
between consecutive blinks in the same person. Nevertheless,
by averaging the information for many nonconscious blinks,
the data for a standard, nonforced blink have been obtained
(Table 26.1).
This blinking action of the upper lid efficiently spreads tear
fluid from the marginal menisci over the entire anterior surface
of the globe. This easily can be demonstrated by instilling a small
drop of fluorescein solution from a micropipette into the tear
meniscus along the inferior lid margin. A single blink uniformly
distributes the fluorescein over the cornea. Thus, the instillation
of a small quantity of a miscible tear substitute into the inferior
tear meniscus can be reasonably expected to mix with the natural
tear fluid in the meniscus and be spread over the entire anterior
surface of the globe by the next few blinks of the lids.
MOTION OF THE LOWER LID
The lower lid undergoes little vertical movement, its major
motion being a horizontal translation directed toward the medial
canthus during the closing of the upper lid. This motion
reverses its direction in synchrony with the beginning of the
opening phase of the upper lid. Total translation of the lower lid
is proportional to the extent of movement of the upper lid,
usually in the range of 2–4 mm. 281
 PHARMACOLOGY AND TOXICOLOGY

FIGURE 26.1. Plots of blink motion dynamics

in four subjects. The upper curve of each pair
represents the time course of the upper lid
displacement during its closing and opening
phases. The lower curve in each case is the
time course of the instantaneous velocity of the
upper lid, which is zero at the point where the
lid reverses direction. Note the variation
between individuals. The subject for plot D had
a narrow palpebral fissure; consequently the lid
excursions and velocities were less than
normal.
From Doane MG: Interaction of eyelids and tears in
corneal wetting and the dynamics of the normal
human eyeblink. Am J Ophthalmol 1980; 89:507–516.
SECTION 4

meshwork of small molecules and membrane-bound mucins,

TABLE 26.1. Dynamics of Upper Eyelid Motion During a Blink*
known as the glycocalyx, is generated by healthy ocular
Factor Value epithelial cells and serves to anchor the tear film to the eye.
Above this lies a large phase accounting for well over 90% of the
Duration of closing phase 82.1 ± 2.1 ms
tear volume in which mucins are suspended in an aqueous
Duration of opening phase 175.8 ± 11.0 ms solution. Current models suggest that although mucins arae
Total blink duration 257.9 ± 11.3 ms
scattered throughout the tear film, they are more concentrated
closer to the ocular surface. The outermost layer of the tear film
Maximum closing velocity 18.7 ± 1.7 cm/s is composed of lipids. While the mucins serve to attach the tear
Maximum opening velocity 9.7 ± 0.7 cm/s volume to the ocular surface, and also have lubricating
properties, the lipid layer both protects against external insult
*Each value given is an average of 40 blinks, ± standard error of the mean, in 10
different subjects. and helps delay tear evaporation.
From Doane MG: Interaction of eyelids and tears in corneal wetting and the The primary and accessory lacrimal glands secrete the major
dynamics of the normal human eyeblink. Am J Ophthalmol 1980; 89:507–516. portion of the tear fluid volume – the accessory glands of Krause
Published with permission from the American Journal of Ophthalmology.
Copyright by the Ophthalmic Publishing Company. and Wolfring aid in natural tear production, while it is thought
that the large, primary lacrimal gland is in fact responsible for
reflex tearing. The contributions of the goblet cells in the
conjunctiva (mucin glycoproteins) and the meibomian glands in
TEAR MIXING, TURNOVER, AND DRAINAGE the lids (lipids) are no less important than the lacrimal glands
in maintaining a functional tear layer. Thus, in addition to the
tear film resurfacing action of the blinking lids, the blink also
TEAR VOLUME AND MIXING serves the important function of combining and mixing these
The tear film is a quasilayered structure with numerous tear fluid components into a quasistable mixture. Included in
282 components secreted from many spatially separated sources. A this mixture would be any added tear substitute, suggesting the

importance of the compatibility of the additive and the natural
tear fluid.
The total tear volume on the anterior segment of the eye is
usually between 6 and 7 mL,2 and there is a certain amount of
preferential compartmentalization for this fluid. When available
fluid is deficient, the fornices ‘fill’ first and can contain about
5.9 mL of fluid.2a Blinking carries fluid upward over the corneal
surface, establishing the precorneal tear film, requiring another
1 mL of fluid. The fornices cannot hold more fluid than their
enclosed, defined space allows, nor can the tear film substan-
tially increase its fluid volume. Once these ‘compartments’ are
filled, any excess fluid goes to fill the marginal tear menisci,
which can hold 2–3 mL of tear fluid. Freshly spread tear film is
also drawn out of the meniscus, but in the opposite direction.
Fluid much greater than this amount raises the level of the
inferior meniscus above that of the punctal opening and, as
described in following paragraphs, it is soon drained away via
the canaliculi into the lacrimal sac. Thus, it is of little benefit
to overfill the menisci with an instilled fluid, because any excess
fluid that does not actually overflow onto the lids is quickly
drained away by the blink-driven drainage system.
Within their sustainable volume range, the marginal menisci
act as a variable reservoir. Often, the relative amount of tear
volume in an eye can be somewhat quantitatively assessed by
noting the height of the marginal tear menisci.3 The height of
the inferior tear meniscus often is reduced in patients with
keratoconjunctivitis sicca, although individual variations in lid
apposition, tightness, and thickness can also affect meniscus
height.
For diagnostic purposes, Schirmer strips may be placed in the
eyes for a short period of time to estimate tear volume. Non-
anesthetized Schirmer tests can stimulate reflex, rather than
natural, tear production, which makes for inaccurate tear
volume values. For more exact readings, a fluorophotometer
may be used. The fluorophotometer first measures autofluore-
scence in the tear film. This reading is then subtracted from a
fluorescence value measured following the instillation of a
microdrop of fluorescein in a patient’s eye. The result generated
is an approximale tear volume value, and the procedure can be
repeated to yield a series of results.4
Figure 26.2 is a schematic representation of the above-
mentioned compartmentalization of the tear volume.
MECHANISM OF TEAR FLUID DRAINAGE
The single punctal opening in each of the lid margins is located
at the apex of the lacrimal papillae, in the medial canthal region
of the lids. Each punctal opening leads to a single tubular
conduit, or canaliculus, which makes a right-angle bend
~2 mm from the edge of the lid and then parallels the lid margin
for most of its length. The superior and inferior canaliculi usually
join into a common pathway just before entering the lacrimal tear
sac just posterior and superior to the center of its lateral wall.
There is evidence for a one-way restriction, or valve, in this
common canaliculus, allowing fluid to flow from the canaliculus
into the lacrimal sac but restraining flow in the reverse direction.
A duct, the nasolacrimal canal, descends from the inferior portion
of the sac, opening into the nasal meatus.
The passage of tear fluid through the punctal openings, into
the canaliculi, and onward into the lacrimal sac, is driven by the
squeezing actions and muscular contractions associated with
the blink action of the lids. This process involves a definitive,
rapid sequence of events.5 As the blinking action of the lids
commences, the upper lid begins its downward sweep over the
anterior portion of the globe, with the lower lid starting its
movement medially, carrying with it the fluid in the marginal
meniscus. The lacrimal papillae containing the punctal openings
Tear Film and Blink Dynamics
tend to extend and elevate themselves from the leading edge of
the lids during the blink. Being located near the medial juncture
of the lid structure, this region of the superior and inferior lid
margins meet, often forcefully, by the time overall lid closure is
only one-third to one-half complete. From this point to the
completion of the lid motion associated with the blink, the
punctal openings are largely occluded.
The primary effect of the second half of lid closure is to
squeeze the elastic walls of the canaliculi, forcing any tear fluid
within them onward into the lacrimal sac. Fluorescein experi-
ments indicate minimal regurgitation of fluid out of the punctal
openings, with the firm apposition of the lid margins mini-
mizing this retrograde flow. Detailed high-speed, close-up
photography shows that the region of the lid margins con-
taining the punctal openings remains in tight contact until the
lids are near the end of their opening phase. Then, the region of
the lid margins containing the punctal openings suddenly pops
apart when the force of the separating lids finally overcomes the
suction force holding them together.5 This suction is generated
by the elastic walls of the canaliculi (and to some extent the
lacrimal sac) trying to expand to contain their normal volume
once the pressure of the closing lids is released.
Once the puncta are separated, a rapid, pulsatile flow of tear
fluid is drawn into the puncta from the marginal menisci owing
to the suction force generated within the canaliculi. When tear
volume is normal, this flow typically lasts 1 or 2 s, as long as the
height of the fluid in the meniscus reservoir is sufficient to
maintain contact with the punctal openings. Any instillation of
CHAPTER 26
a tear substitute that temporarily increases the volume of fluid
to a level above the punctal opening prolongs this exit flow, and
the excess volume is quickly removed from the meniscus. Once
the meniscus height falls below the slightly elevated position
of the punctal openings, further drainage stops.6 Because of
differences in blink strength, degree of completion, and fluid
volume in the marginal menisci, not all blinks result in equally
FIGURE 26.2. Schematic representation of tear-fluid
compartmentalization and outflow. Nearly all the effective tear volume
is secreted by the main and accessory lacrimal glands, with an added
contribution from the conjunctival goblet cells (mucin) and the
meibomian glands in the lids (lipid). The tear fluid is first used to fill the
volume between the globe and lids (superior and inferior fornices) and
the tear film over the exposed globe. Any excess fluid then goes into
the reservoir of the marginal menisci, from which drainage via the
punctal openings occurs. Smaller amounts of fluid are lost by
evaporation and absorption by the conjunctiva. 283
 PHARMACOLOGY AND TOXICOLOGY

efficient flow patterns. Because the puncta occlusion by the
opposing lid margin occurs even with half blinks, with
associated squeezing of the canaliculi, some tear fluid is often
drawn into them even after incomplete blinks, although the
amount of fluid drainage is reduced. Figure 26.3 is a schematic
representation of this cycle.
From the time of initial instillation, any applied fluid is
decreasing in its overall tear fluid concentration as time goes by.
The time of contact between the ocular surface and the applied
fluid is directly limited by the rate of drainage from the marginal
menisci; in addition to drainage, the concentration of thera-
peutic agents that does remain is continuously diminished by
newly secreted tear fluid. Thus, any means of increasing the
retention time of instilled solutions at therapeutic levels is of
crucial interest. Clinically, the rate of drainage, or ‘tear flow’,
can be estimated (similarly to tear volume) using a fluoro-
photometer. By recording the amount of fluorescent dye present
in the tear film at various time points after instillation, the
fluorophotometer can determine the rate at which the dye is
being washed out due to tear turnover.4
The stimulation of a faster blink rate (such as by the admin-
istration of a solution that stings or is otherwise uncomfortable)
is undesirable, because this causes a more rapid drainage of tear
fluid from the marginal menisci. An increase in the viscosity of
a tear substitute may result in an effectively prolonged time of
SECTION 4
action. Recall that drainage from the inferior meniscus occurs
from its highest, uppermost portion, which is drawn into the
elevated punctal openings. If a viscous tear substitute is
carefully instilled into the inferior cul-de-sac, it often acts as a
longer-lasting depot of fluid that is not readily drained and is
slowly mixed into the tear film by subsequent blinks. In fact, in
monitoring the concentration of applied agents by interfero-
metry, a few strong blinks often elicit a sudden increase in the
amount of viscous agent in the tear film by forcing the ‘depot’
out of the inferior cul-de-sac many minutes after initial instilla-
tion of the artificial tear solution. However, applied agents that
are too viscous are detrimental to retention time, because they
blur vision, elicit foreign body sensations that stimulate
blinking, and are not well liked by users because of stickiness
and the tendency to collect in the eyelashes.
WETTING AND DRYING OF THE CORNEAL
SURFACE
NATURE OF THE WETTING PROCESS
We can define wetting as the spreading of a fluid over a solid
surface, a complex process from a molecular, surface-chemical
viewpoint. The degree of spreading depends on the relative
forces of cohesion between the like molecules of the fluid and

FIGURE 26.3. Mechanism of lacrimal drainage. Clockwise from the top: (1) At the start of the blink, the lacrimal drainage passages already
contain tear fluid that has entered following the previous blink. (2) As the upper lid descends, the papillae containing the punctal openings
elevate from the medial lid margin. By the time the upper lid has descended halfway, the papillae meet the opposing lid margin, occluding the
puncta and resisting fluid regurgitation. (3) The remaining portion of the lid closure acts to squeeze the canaliculi and sac through the action of
the orbicularis oculi, forcing out contained fluid that has not been absorbed by the mucosa of the sac and nasolacrimal duct. (4) At complete lid
closure, the system is compressed and devoid of fluid. (5) During the start of the opening phase of the blink, the puncta are still occluded and
valving action at the distal end of the canaliculi (and perhaps in the nasolacrimal duct) acts to prevent reentry of fluid or air. As lids open,
compressive action ends and the elastic walls of the canaliculi attempt to expand to their normal shape. This elastic force causes a partial
vacuum, or suction, to form within the canaliculi and sac. (6) The suction force holding the punctal region of the lid margins together is released
when lid separation is sufficient, at about two-thirds of the fully open position. The punctal openings are now accessible for fluid entry from the
marginal tear menisci, and tear fluid is drawn into the canaliculi during the first few seconds following the blink.
From Doane MG: Blinking and the mechanics of the lacrimal drainage system. Ophthalmology 1981; 88:844.
284

the forces of adhesion between the unlike molecules of the solid
surface and those of the fluid. Thus, when a fluid rests on a
solid surface, the relative strength of these two forces deter-
mines the degree of fluid spreading. The stronger the relative
cohesive forces attracting the fluid molecules together, the less
the fluid increases its surface area to spread out on the solid
surface. Thus, in order to spread and wet a surface, the
fluid–solid adhesion forces must be greater than (or at least
comparable to) the fluid–fluid cohesive forces.
However, wettability is more complex than this simple
explanation; it also depends on the degree of polarity and type
of charge of the molecular groups exposed on the surface of the
solid. For instance, exposed polar groups tend to have an attrac-
tion for the polar molecules of water. Materials with nonpolar
surfaces (Teflon, oils) have a low attraction for polar groups
such as those in water and thus are inherently hydrophobic, or
water-repelling. Surface-active agents, or surfactants, can greatly
increase the wettability of a surface by acting as a bridge
between polar and nonpolar molecules. Typically, such agents
have molecules with some exposed moieties that are hydro-
phobic (such as alkyl groups) and others on the same molecule
that are hydrophilic (such as carboxyl groups). Mucin glyco-
proteins are thought to act as a wetting agent in tear fluid.
Virtually all artificial tear preparations contain one or more
chemical surfactants that enhance their wetting of the
cornea.
Corneal epithelial cells secrete glycocalyx, which has a
similar chemical composition and characteristics of other
mucins; therefore, the surface is intrinsically wettable. In
addition, the surface of the cornea is covered by an adsorbed
layer of mucin, perhaps 1 mm thick, allowing the tear fluid to
spread easily over this surface.
BREAKUP OF THE PRECORNEAL TEAR
FILM
The surface of the cornea is rewet with a fresh layer of fluid,
forming the precorneal tear film, by each blink of the eyelids.
This periodic action is necessary owing to the deterioration of
this thin fluid layer between blinks. Immediately after a new
tear film is formed, it undergoes a progressive overall thinning
owing to evaporation and, more importantly, begins to develop
localized areas that thin even more rapidly than the tear layer
as a whole. It is these localized regions, usually small in area,
that result in the first micelles, or ‘dry spots’, observed after a
blink. These spots appear as dark, nonfluorescent areas when
fluorescein is in the tear fluid, because where there is no fluid,
there is no fluorescein and hence no fluorescence. The time
from the completion of a blink to the first appearance of these
dark spots is the tear film breakup time (TFBUT). TFBUTs vary
from blink to blink and person to person – an individual average
TFBUT under 10 s has traditionally been considered indicative
of dry eye syndrome. However, the innovative diagnostic com-
bination of a microdrop of sodium fluorescein (≤5 mL), a yellow
Wratten filter, and a digital video capture system with an
onscreen timer, has yielded more precise TFBUT values. These
measurements have indicated that 5 s is a more appropriate cut-
off point for association of TFBUT with dry eye symptoms.7 Of
course, persistently short blink intervals can somewhat
moderate the symptomatic effects of shortened TFBUTs.
A new diagnostic calculation, called the ocular protection
index (OPI), quantifies the relationship between blink frequency,
TFBUT, and ocular surface protection. The concept is simple:
an interblink interval (IBI) greater than the average TFBUT
value in a given eye indicates that there is a period of time
(namely, the difference between IBI and TFBUT) during which
Tear Film and Blink Dynamics
the ocular epithelium is wholly or in part unprotected by the
tear film. An IBI less than the average TFBUT suggests the eye
is blinking frequently enough to replenish the tear film before it
breaks to expose the ocular surface. To quantify this relation in
a single value, one can simply divide IBI by TFBUT by IBI – any
value greater than or equal to 1 indicates a generally protected
ocular surface, while any value less than 1 suggests at the very
least occasional exposure of the ocular epithelium.8
Examination of TFBUT video images has not only yielded
visual proof of the dynamic nature of tear film breakup, but has
shown that the tear film may break up in any of a number of
unique patterns. The tear film breakup patterns (TFBUP)
identified so far include: spotting, amorphous blob, linear,
fractured, and wispy. Each pattern is generally reproducible by
patient, by eye. It is evident that certain patterns are
considerably more prevalent in certain dry eye populations,
leading to the belief that the pattern present upon breakup is at
least partially indicative of the advancement or nature of the
syndrome. TFBUPs appear to be modifiable by alteration of tear
film composition – for instance, by treatment with a tear
substitute, meibomian gland expression, or stimulation of
reflex tearing. Ultimately, tear film breakup patterns show that
there is more to the dispersion of the tear film than how quickly
it occurs, and these patterns could prove to be a useful visual
diagnostic in the future.9 The causes of tear film breakup are
not, however, as easily explicable as the methods of quantifying
and recording it.
Although evaporative effects progressively thin the tear film
CHAPTER 26
and promote eventual drying and breakup, theoretical calcula-
tions indicate that the times required to thin the tear layer to
dryness should be much longer than those actually observed
for measured TFBUTs.10 Also, long before overall drying of the
anterior surface of the eye occurs, the small, localized areas of
drying discussed earlier are seen. Clearly, evaporation is not the
sole (or even primary) cause of tear film breakup.
The supposed non-wettability of the corneal surface is
thought to be an artefact;10 rinsing the surface with
acetylcysteine returns the surface to its wettable state.11,12 The
presence of glycocalyx makes the surface wettable; however, it
has been shown that newly exposed epithelial cell surfaces,
revealed when overlying cells desquamate, have not yet
developed or fully-expressed their glycocalyx.13 The surface will
hence be relatively non-wettable. Theoretically, even a single
non-wettable cell can initiate a dry spot;14 it may be that the
pattern of dry spots may indicate where surface cells have
recently desquamated. Ultimately, the mechanisms of tear film
breakup are not clearly understood.
It is surmised that over time (i.e., within seconds after a
blink), the mucin layer on the epithelium becomes contami-
nated by nonpolar components of the tear film, primarily lipid
from the superficial layer. This oily layer is, of course, only ~5
or 6 mm above the surface of the mucin layer under the best of
conditions. Microscopic flow patterns, either of thermal origin
or due to the turbulence of the blink action, can bring this float-
ing lipid into contact with the mucin on the corneal surface.
Although a small amount of lipid contamination can be masked
by the mucin molecules, sufficiently large areas eventually
become contaminated whereby the mucin can no longer act as
an effective surfactant. Then, nonwetting areas develop, with
spontaneous thinning of the tear layer immediately above them,
with eventual rupture of the tear film. When these localized
nonwetting areas resist the formation of a new, clean mucin
layer during blinking, persistent tear film breakup over the same
area follows within a few seconds of each blink. A series of
strong, forced blinks often reestablish tear film continuity over
some of these areas by covering them with mucin.
285
 PHARMACOLOGY AND TOXICOLOGY

Disabling the drainage mechanism (through punctal plugs or

surgery) or continuously instilling tear substitutes can tempor-
arily boost tear volume. Ideally, the application of a tear sub-
stitute should aid in wetting the corneal surface and prolong
the TFBUT of the tear film for the entire time between instilla-
tions. Although the initial instillation of a drop of solution
increases tear volume and usually tear film thickness as well,
this effect is largely transitory, and the tear volume quickly
reverts to its prior value as the applied solution is drained away.
Thus, it is unrealistic to expect retention of applied fluid
volume per se to provide long-term benefit unless the solution
is frequently applied to the eye. Various compounds are in
development that approach tear film deficiency from different,
more therapeutic, angles – some seek to upregulate mucin
production so that the tear adheres longer to the ocular surface,
some have antievaporative qualities, and some, like cyclo-
sporine A, are antiinflammatory agents. In the future, neuro-
transmitters and hormonal treatments may also be explored,
assuming the causes of many forms of dry eye to lie in the
neuronal feedback loop. Eventually, it should be possible to
permanently regulate tear volume and related factors such as
blink frequency, through treatment.
SECTION 4
Key Features
Blink Dynamics
• 12–15 blinks per minute is considered normal
• Typically nonconscious, though can be performed consciously
• Upper lid undergoes considerable vertical movement
(5–13 mm) at varying velocities (80–300 mm/s) and spreads
tear film over eye surface
• Lower lid barely moves at all vertically, but does undergo fairly
substantial horizontal motion (2–4 mm)
Tear Mixing, Turnover, Drainage
• The tear film is anchored to the eye by the glycocalyx and
consists of mucins suspended in aqueous at varying
concentrations, covered by an outer lipid layer
• Assessment of the height of the marginal tear menisci and the
geometry of the punctal opening can help approximate tear
volume
• Tear volume can be measured using Schirmer strips or
fluorophotometry
• Fluid drains from the eye through the puncta, proceeds
through the canaliculi, and ends in the lacrimal sac
• Tear flow or drainage is stimulated by blinking and can be
measured using fluorophotometry
Wetting and Drying of the Corneal Surface
• The tear film spreads across the ocular surface postblink, and
its outer lipid layer reduces surface tension
• Tear film breakup is marked by the appearance of dry spots on
the ocular surface

• In healthy eyes, a blink should replenish the tear film prior to

tear film breakup
• Measurements of the rapidity and pattern of tear film breakup
can be used as diagnostic tests for dry eye

REFERENCES
1. Doane MG: Interaction of eyelids and tears
in corneal wetting and the dynamics of the
normal human eyeblink. Am J Ophthalmol
1980; 89:507–516.
2. Mishima S, Gasset A, Klyce SD, Baum JL:
Determination of tear volume and tear flow.
Invest Ophthalmol 1966; 5:264–275.
2a. Yokoi N, Bron AJ, Tiffany JM, et al:
Relationship between tear volume and tear
meniscus curvature. Arch Ophthalmol
2004; 122:1265–1269.
3. Scherz W, Doane MG, Dohlman CH: Tear
volume in normal eyes and
keratoconjunctivitis sicca. Graefes Arch
Klin Exp Ophthalmol 1974; 192:141–150.
4. Göbbels M, Goebels G, Breitbach R,
Spitznas M: Tear secretion in dry eyes as
assessed by objective fluorophotometry.
Ger J Ophthalmol 1992; 1:350–353.
5. Doane MG: Blinking and the mechanics of
the lacrimal drainage system.
Ophthalmology 1981; 88:844–850.
6. Doane MG: Blinking and tear drainage. In:
Bosniak SB, ed. Advances in plastic and
reconstructive surgery. The lacrimal
system. New York: Pergamon; 1984:39–52.
7. Abelson MB, Ousler GW, et al: Alternative
reference values for tear film break-up in
normal and dry eye populations. In:
Sullivan D, et al, eds. Lacrimal gland, tear
film, and dry eye syndromes 3. Kluwer
Academic/Plenum; 2002.
8. Ousler GW, Emory TB, Welch D, Abelson MB:
Factors that influence the inter-blink
interval (IBI) as measured by the ocular
protection index (OPI). (Poster presentation)
The Association of Research in Vision and
Ophthalmology (ARVO); 2002.
9. Ousler GW, Lemp MA, Schindelar MR,
et al: Tear film break up patterns (TFBUP –
a novel method of evaluating tear film
stability (abstract). The Ocular Surface
2005; 3 suppl: S100.
10. Cope C, Dilly PN, Kaura R, Tiffany JM.
Wettability of the corneal surface: A
reappraisal. Curr Eye Res. 1986; 5:777–85.
11. Tiffany JM. Measurement of wettability of
the corneal epithelium. I. Particle
attachment method. Acta Ophthalmol
(Copenh). 1990; 68:175–81.
12. Tiffany JM. Measurement of wettability of
the corneal epithelim. II. Contact angle
method. Acta Ophthalmol (Copehn). 1990;
68:182–7.
13. Gipson IK, Inatomi T. Mucin genes
expressed by the ocular surface epithelium.
Progr Retinal Eye Res 1997; 16:81–98.
14. Sharma A, Coles WH. Physico-chemical
factors in tear film breakup. IOVS ARVO
abstract 1990; 31:552.

286
 CHAPTER
27 Tear Substitutes
Mark B. Abelson, George Ousler, and Russell Anderson
Estimates of the prevalence of dry eye in the general population
range from 11% to 45%,1–3 and this prevalence only appears to
be increasing as factors such as prolonged computer use and
contact lens wear become more and more widespread. Although
treatment options such as punctal plugs, steroids, or prescription
therapy are available to patients, these methods tend to only be
efficacious on a patient-to-patient basis, and the majority of suf-
ferers still primarily manage their dry eye with over-the-counter
tear substitutes. Estimates suggest that at least 7–10 million
Americans use artificial tears.4
Key Features
• Most patients manage dry eye with tear substitutes, which are
over-the-counter eyedrops intended to temporarily help relieve
the clinical signs and symptoms associated with dry eye.
• Demulcents are lubricating compounds contained in artificial
tears that help soothe the ocular surface, while viscosity
agents make for thicker drops that can augment the tear film
and may require less frequent use.
• Although some modern tear substitutes are preservative-free,
many still contain ophthalmic preservatives, which are useful
for their antimicrobial action, but can be potentially irritating to
dry-eye patients with sensitive ocular surfaces.
• In formulating an artificial tear, the other formulary aspects to
consider are electrolytes (these can make for a healthier tear
film), osmolarity/osmolality (a tear should be slightly
hypoosmotic), and pH (drops should be somewhat alkaline).
• Topical cyclosporine A is currently the only approved
prescription therapy for dry eye, but various classes of agents,
including secretagogs, antievaporatives, antiinflammatories,
mucomimetics, and even neuronal feedback loop regulators
are in development as potential dry-eye treatments.
A tear substitute is generally used to supplement a tear film
that is inferior in either quality or quantity in a patient with some
dysfunction of the ocular surface or tear secretory system. As
described in other chapters, this system includes the main and
accessory lacrimal glands, the meibomian glands, the glands of
Zeis and Moll, and the goblet cells. Identification of the
patient’s specific dysfunction, whether it is an aqueous, lipid, or
mucous layer deficiency, helps to determine optimal therapy.5
Whether dry eye is primary or secondary to lid conditions, such
as blepharitis or ocular rosacea, must be determined in order to
initiate the proper concomitant therapy, such as lid hygiene or
oral tetracycline.6,7 Disorders of the ocular surface such as ocular
cicatricial pemphigoid or Stevens–Johnson syndrome must be
identified and treated as described in other chapters.
Tear substitutes may come in various forms – from somewhat
viscous liquids that are dispensed from bottles to thick ointments
squeezed from tubes – and their components are restricted
within the confines of the US Food and Drug Administration
(FDA) monograph on over-the-counter products. This compen-
dium lists all acceptable ingredients, both active and inactive, as
well as acceptable concentration ranges allowed by the FDA in
ophthalmic over-the-counter formulations. Ingredients that have
been historically and traditionally used in ophthalmic products
are included in this list based on the safety profile established
through their numerous years of use. The various ingredients
allowed by the FDA are classified as demulcents, emulsifiers,
surfactants, viscosity agents, and preservatives.
Generally, tear substitutes are hypotonic or isotonic buffered
solutions containing demulcents, viscosity agents, electrolytes
and other components. Historically, all formulations were pre-
served, multidose preparations. However, unit-dose, preservative-
free systems are now common. The type and concentration of
demulcent or viscosity agent, preservative system, and electrolyte
composition are the primary variables in ophthalmic lubricant
formulations. Various diagnostic measures may be used to help
determine the efficacy of an artificial tear, from tear film breakup
time (TFBUT) to corneal and conjunctival vital dye staining or
symptom questionnaires.8,9 Novel diagnostic tools such as ocular
protection index (OPI) and tear film breakup patterns (TFBUP)
will most likely be used to evaluate future tear substitutes.10 In
addition to improving signs and symptoms of dry eye, a tear
substitute should ideally have a long duration of action to mini-
mize the necessary frequency of instillation11 – this is especially
important in the case of formulations containing potentially
irritating preservatives. The primary objectives of the physician
caring for patients with dry eye are to improve subjective com-
fort and to minimize ocular surface desiccation and cell death.
Symptoms can often be reduced but rarely are eliminated.
DEMULCENTS AND VISCOSITY AGENTS
Demulcents are compounds intended to protect mucous mem-
branes and soothe epithelial surfaces. Simultaneously, their
mucilaginous consistency can provide lubricity for the ocular
surface, which can help minimize the abrasive action of the
upper lid on already desiccated epithelial cells. The FDA recog-
nizes six categories of ophthalmic demulcents, with each category
containing one or more compounds: cellulose derivatives, dextran
70, gelatin, liquid polyols, polyvinyl alcohol, and povidone
(Table 27.1).12 The demulcents covered by the monograph are
allowed in ‘over-the-counter’ preparations if they fall within
certain concentration ranges. An ophthalmic preparation may
contain up to three demulcents of any type, and in some cases,
as with dextran 70, combination with another demulcent is
required. Up to three demulcents may also be combined with
either a single ophthalmic vasoconstrictor or a vasoconstrictor/ 287
 PHARMACOLOGY AND TOXICOLOGY

TABLE 27–1 Marketed Tear Substitutes*

Concentration
Demulcent(s) (When Available) Trade Name Preservative
a
Carboxymethylcellulose 0.5% Refresh Tears SOC
a
Refresh Plus None
1.0% Celluvisca None
a
1.0% Refresh Liquigel SOC
b
0.25% TheraTears None
w/Glycerin 0.5% Optivea SOC
c
Glycerin 0.3% Moisture Eyes BAK
c
1.0% Computer Eye Drops BAK, EDTA
w/ Polysorbate 80 1.0% (both) Refresh Enduraa None
d
Hydroxypropyl cellulose 5 mg/insert Lacrisert (biodegradable insert) None
e
Hydroxypropyl methylcellulose 0.3% GenTeal Sodium perborate
GenTeal PFe† None
e
Genteal Gel Drops Sodium perborate
e
0.2% GenTeal Mild Sodium perborate
w/ Dextran 70 Bion Tearsf† None
g
Tears Renewed BAK, EDTA
c
0.8%, 0.1% Moisture Eyes Liquid Gel BAK
SECTION 4

0.8%, 0.1% Moisture Eyes Liquid Gel PFc† None

h
w/ Glycerin Clear Eyes CLR Sorbic acid, EDTA
f
w/ Glycerin & Dextran 70 Tears Naturale Forte Polyquaternium-1
Tears Naturale Freef None
i
w/ Glycerin & PEG-400 Visine Tears BAK
i†
Visine Tears PF None
Methylcellulose 1.0% Murocelc Methylparabens,
Propylparabens
Mineral Oil 4.5% Polyhexamethylene biguanide
j
w/ Light Mineral Oil 1.0% Soothe
Polycarbophil, PEG-400, Dextran 70 Aquasitee EDTA
Aquasite Multi-Dosee† EDTA, sorbic acid
g
Polyvinyl Alcohol 1.4% AKWA Tears BAK, EDTA
e
w/ PEG-400, Dextrose 1.0% Hypotears BAK, EDTA
Hypotears PFe† EDTA
k
w/ Povidone 1.4% Murine Tears BAK, EDTA
f
Propylene Glycol, PEG-400 0.3%, 0.4% Systane Polyquaternium
Systane PFf† None
a
Allergan Pharmaceuticals; Advanced Vision Research; Bausch and Lomb Pharmaceuticals; Merck & Co.; Novartis Pharmaceuticals; fAlcon Laboratories; gAKORN
b c d e

Pharmaceuticals; hMedtech; iPfizer, Inc.; jAlimera Biosciences; kRoss Laboratories.

*Concentrations of the listed components are identified when possible. Ethylenediaminetetraacetic acid (EDTA) is listed in the preservative column for some products,
though it is not technically a preservative. ‘BAK’ is benzalkonium chloride, and ‘SOC’ is stabilized oxychloro complex, both preservatives. Most information is available in
Physician’s Desk Reference for Ophthalmology. 34th edn. Oradell, NJ: Medical Economics; 2006.
†
Preservative-free, unit-dose vials.

astringent combination in order to provide an artificial tear with
additional redness- or discomfort-reducing properties.
Cellulose derivatives are the demulcents most commonly con-
tained in modern tear substitutes and are allowed in concen-
trations between 0.2% and 2.5%. Hydroxypropyl methylcellulose
288 (HPMC) and carboxymethylcellulose (CMC) are the two most
notable cellulose derivatives. Drops containing HPMC can be
formulated as oil-in-water emulsions, and the mucoadhesive prop-
erties of the polymer in combination with an oil can help supple-
ment both the mucin and lipid components of the tear film.13
Cellulose derivatives double as viscosity agents in the sense that
increasing their concentration can increase the viscosity of an

artificial tear and prolong its retention time in the tear film.
This is evident in marketed formulations containing HPMC,
which include either 0.2% or 0.3% concentrations depending on
the severity of the dry eye they are intended to manage. CMC
varies more substantially, with different formulations containing
either 0.5% or 1.0% CMC – the latter tear substitute is a thick,
gel-like liquid. However, when artificial tear viscosity is increased
by means of higher demulcent concentrations, the resultant tear
is often prone to causing blurring of the vision and in some cases
will leave residue on the lid margins as it dries.14 At the same
time, the greater retention time yielded by the gel-like con-
sistency is more likely to improve dry-eye signs and symptoms
than a less viscous tear would.
Liquid polyols (polyhydric alcohols) are also demulcents that
are commonly found in modern artificial tears and are allowable
in concentrations of 0.2–1.0%. While polyols typically do not
double as viscosity agents like cellulose derivatives can, they are
often more effective lubricants.15 A comparative study demon-
strated that a tear substitute containing propylene glycol 0.3%
(PG) and polyethylene glycol 0.4% (PEG 400) provided better
lubricity than a tear containing HPMC, which in turn was more
effective at creating lubricity between two moving surfaces than
a product containing CMC.16 Glycerin and polysorbate are addi-
tional liquid polyols that are often used in varying concentra-
tions as combinative agents in oil emulsion systems that target
the lipid layer of the tear film. Both are also found as ‘inactive’
agents in the cyclosporine A formulation that represents the
only FDA-approved prescription dry-eye therapy.
The remaining demulcents covered by the FDA monograph
include gelatin (allowable in 0.01% concentration, but seldom
used), povidone, and dextran 70 (which can only be incorporated
in conjunction with another demulcent) – all three can serve as
viscosity agents as well as lubricants in artificial tears. Polyvinyl
alcohol (PVA) was one of the original demulcents incorporated
into artificial tears and can be included in concentrations from
0.1% to 4.0%. PVA is still used in some drops and can act alone
or in combination with another demulcent such as povidone.
The stability of the tear film depends on the chemical–physical
characteristics among the three layers. Classically, the mucin
layer was thought to act as a wetting agent by lowering the
surface tension of the relatively hydrophobic ocular surface,
rendering the corneal and conjunctival cells ‘wettable’.17
Evidence has shown, however, that the mucin layer is much
thicker than previously thought, and in fact extends into the
aqueous phase.18 Its role may be similar to that of mucin in
the stomach, where a mucin gel protects the epithelium from
a harsh surrounding environment.19 This may explain why
water-containing lubricants are only partially effective in
restoring the health of the ocular surface. The function of the
tear film’s mucin component is more than that of a wetting
agent. The effect of most available lubricants is probably to help
hydrate available mucin and wash away irritating or toxic
substances in the tear film. While some patients with dry eye
have a deficiency in the aqueous layer, a primary or secondary
mucin deficiency may also be present. The demulcents and
viscosity agents added to artificial tears lubricate and can work
to fortify the mucin layer or the thin outer lipid layer, which
prevents tear film evaporation. The addition of a viscosity agent
to increase residence time can play a role in active drug formu-
lations by prolonging ocular surface contact, thereby increasing
the drug’s duration of action and comfort.
While demulcents such as cellulose derivatives are often used
in high concentrations to increase the viscosity of artificial tears,
other compounds, for instance gels and gelling agents, may also
be incorporated into formulations. Carbopol 980, a gel composed
of linked carboxylic acid polymers, is used in some marketed
tear substitutes to create a highly viscous drop. Hydroxypropyl
Tear Substitutes
TABLE 27–2 Dry Eye Ointments*
Primary Components and Concentration
Trade Name (When Available)
AKWA tears White petrolatum, liquid lanolin, mineral oil
Ointmenta
GenTeal PMb 85% White petrolatum, 15% mineral oil
Hypotears
Ointmentb White petrolatum, light mineral oil
Tears Reneweda
Lacrilube S.O.P.c 56.8% White petrolatum, 42.5% mineral
oil, chlorobutanol, lanolin alcohols
Refresh P.M.c 42.5% Mineral oil, 57.3% white
petrolatum, lanolin alcohols
a
AKORN Pharmaceuticals; bNovartis Pharmaceuticals; cAllergan Pharmaceuticals.
*Concentrations of the listed components are identified when possible. Most
information is available in Physician’s Desk Reference for Ophthalmology. 34th edn.
Oradell, NJ: Medical Economics; 2006.
(HP)-Guar is a gelling agent derived from guar gum that is used
in an artificial tear with the demulcents PG and PEG 400 – its
gelling action is apparently triggered by contact with the tear
film.20 In even more viscous formulations – dry-eye ointments
dispensed from tubes – the primary components are often petro-
latum or mineral oil (Table 27.2). These tear substitutes have
CHAPTER 27
very long retention times and often provide significant symp-
tomatic relief for patients, but due to the excessive blurring
their high viscosities induce, they can usually only be instilled
in the evening prior to sleep.
PRESERVATIVES
The advent of preservative-free single-dose tear substitutes was
an important advancement in the management of dry eye. The
antimicrobial properties of preservatives are almost always
accompanied by mild toxicity, and in dry-eye patients, who often
have sensitive or damaged ocular surfaces to begin with and may
use a tear substitute numerous times daily, this can be particu-
larly detrimental. Preservatives that have stronger antimicrobial
action are also often more toxic to the surface of the eye, so
unpreserved formulations that can be immediately discarded
after use are preferable for many patients.
However, there are drawbacks to the use of single unit-dose
tear substitutes: the expense and the inconvenience of carrying
many vials. For these reasons, some patients may attempt to
reuse their unit-dose vials by recapping them or standing them
upright until it is time for the next dose – this greatly increases
the likelihood of contamination. The ideal artificial tear would be
either a preservative-free unit-dose tear substitute that retains anti-
microbial efficacy for a 24-h span after first use, or a preservative-
free multidose formulation that can maintain sterility even with
frequent use. Both possibilities are being explored, while other
formulations seek to incorporate effective preservatives that are
minimally toxic to the ocular epithelium.
FDA-required components of all multidose ophthalmic prep-
arations since 1953, preservatives must pass specified efficacy
tests to gauge their antibacterial and antifungal action prior to
inclusion in an eye-drop. Traditional ophthalmic preservatives
that were found to have harsh effects on ocular surface cells and
cause discomfort in patients include thimerosal, chlorobutanol,
and sorbate.21–24 These are primarily chemical preservatives, or
detergents, and typically exhibit excellent preservative efficacy.
Another chemical preservative, the antiseptic benzalkonium
chloride (BAK), is probably the most common preservative in 289
 PHARMACOLOGY AND TOXICOLOGY
modern ophthalmic formulations. However, BAK is not ideal
for inclusion in formulations intended for frequent dosing by
dry-eye patients due to its moderate toxicity to the ocular surface
– in an in vitro study, human corneal epithelial cells exposed to
BAK underwent cell retraction as normal cytokinesis, cell move-
ment, and mitotic activity were disrupted causing cell degenera-
tion within 2 h.24 When it is present in a tear substitute, BAK is
usually incorporated in a 0.01% concentration, and is tempered
with ethylenediaminetetraacetic acid (EDTA) – even in this low
concentration, it retains a preservative efficacy superior to many
other preservatives commonly found in artificial tears.25
Belonging to the same class of compound as BAK, polyquater-
nium is a polymeric biocide that has not shown the same
propensity to irritate or disrupt ocular surface cells and, as a
result, is incorporated into several currently marketed artificial
tears. Polyquaternium-1, a quaternary ammonium compound,
exhibited no negative effects on cell movement or mitotic activity
in human corneal surface cells when tested in vitro, nor did it
cause any cell death in the cultures.24 Although it is highly
effective against ophthalmic bacteria, some tests suggest poly-
quaternium to have a more limited ability to eradicate fungi in
certain product formulations.26
In addition to chemical preservatives, a second class known
as oxidative preservatives have become common tear substitute
components. The two primary oxidative preservatives used in
dry-eye formulations are stabilized oxychloro complex (SOC)
and sodium perborate. SOC is a mixture of compounds with
sodium chlorite (NaClO2) as the main component. It is light sen-
SECTION 4
sitive, so that artificial tears containing SOC must be dispensed
from opaque bottles. Although it is considered quite mild and not
prone to irritation of the ocular surface, SOC also has exhibited
weak anti-bacterial action in some preservative efficacy trials.25
Sodium perborate is a bleaching agent with antiseptic properties
that is thought to break down upon contact with the tear film.
However, hydrogen peroxide (H2O2), which sodium perborate
breaks down into, has been shown to have toxic effects on the
cornea – in one study it brought about epithelial cell death in
human corneal cell cultures 12–24 h after exposure.24,27 As an
oxidative preservative, sodium perborate is generally considered
milder on the ocular surface than most chemical preservatives,
and is found in current marketed artificial tears.
Any tear substitute that contains a preservative has the poten-
tial to irritate the ocular surface of a dry-eye patient, especially
a patient who has severe epithelial damage to begin with. Harmful
effects may be minimized by less frequent dosing or limiting
drop volume upon instillation, but ultimately a preservative-
free formulation is the best way to avoid the ocular irritation
ophthalmic preservatives sometimes cause.28
OTHER FORMULARY ASPECTS OF TEAR
SUBSTITUTES
ELECTROLYTE COMPOSITION
Electrolytes occur naturally in physiological fluids and they help
regulate metabolic processes in the tear film. Tear substitutes
with electrolyte compositions have been shown to help improve
and restore damaged corneal surfaces.29–33 One of two ions
can be found in most artificial tears containing electrolytes:
bicarbonate and potassium. Bicarbonate can help in the
recovery of damaged corneal epithelial cells and in the
maintenance of ocular surface health.30 Previous studies suggest
that naturally occurring bicarbonate in the human body is
responsible for aiding in the maintenance of the mucin gel that
lines and protects the stomach.19 Similarly, it is believed that
bicarbonate as an ingredient in tear substitutes may help
290 maintain the mucin layer of the tear film.30 This theory was
supported by a rabbit model study in which treatment with an
electrolyte solution yielded an increase in goblet cell density
while simultaneously decreasing conjunctival staining and tear
osmolarity.33 Potassium can also be a useful element in
ophthalmic solutions due to its ability to retain corneal
thickness.34
Some currently marketed products attempt to mimic the elec-
trolyte composition of human tears. One of these formulations
has demonstrated an ability to increase goblet cell density in
LASIK-induced dry eye.35 Because bicarbonate reacts with air to
produce carbon dioxide, it is often necessary for these electrolyte-
balanced drops to be dispensed from single-dose plastic vials
and packaged in foil.
OSMOLARITY/OSMOLALITY
Analyses of dry-eye patients have demonstrated that dry eye is
consistent with increased tear film osmolarity (crystalloid osmo-
larity), most likely owing to increased evaporation in patients
with lipid layer deficiencies.36,37 Hyperosmolarity may be toxic
to the corneal epithelium, compounding already-present surface
damage.38 For this reason, some artificial tears aim to lower the
osmolarity of the tear film via hypoosmotic formulation.
Colloidal osmolality, mainly dependent on macromolecule
content, is another factor that varies in artificial tear formula-
tions. Colloidal osmolarity, or oncotic pressure, is important for
the control of water transport in tissues – it is defined as osmotic
pressure due to the presence of colloids in a solution. Differences
in osmolality affect the net water flow across membranes. This
water flow is eliminated by applying hydrostatic pressure to the
downside of the water flow. The magnitude of osmotic pressure
is determined by respective osmolalities on the two sides of the
membrane. Damaged epithelial cells swell due either to breaks
in the cell membranes or pumping mechanism dysfunctions. If
a fluid with a high colloidal osmolality is added to this damaged
and swollen cell surface, the oncotic pressure exerted causes cell
deturgescence and a return to normal cell physiology. Thus, an
artificial tear formulation with a high colloidal osmolality may
be of value.
VISCOSITY
Viscosity, or fluid thickness, is still considered one of the most
important properties of an artificial tear. Viscosity is typically
measured in centipoise, and may be enhanced in a formulation
through the inclusion of any of the previously mentioned
viscosity agents (i.e., gelling agents, demulcents, etc.). The more
viscous a tear substitute is, the longer it is expected to remain
in the tear film, and the greater benefit to signs and symptoms
of dry eye it is expected to yield. Of course, as a result of its thicker
constitution, a highly viscous artificial tear or ointment is prone
to blurring the vision after instillation, and in some cases may
leave a residue on the lashes or lids as it dries. Artificial tears
seek to achieve a viscosity that maximizes both clinical efficacy
and visual clarity.
ALKALINITY
Research has suggested that patients with ocular surface disease
or tear film deficiency tend to have higher than average tear film
pH values.39,40 Ocular rosacea, in particular, has been associated
with increased tear alkalinity and it is believed that dry-eye
patients have similarly alkaline tears.39 It is difficult to stand-
ardize pH measurement in tears, because multiple factors such
as goblet cell secretions, tear flow rate, conjunctival metabolism
and carbon dioxide escape can influence pH levels.41 Some esti-
mates place the mean pH in healthy eyes ~7.5–7.6.40,42 It has
 Tear Substitutes

also been demonstrated that when non-dry-eye patients hold
their eyes open for 60 s, the tear-film pH can increase to greater
than 9.40 This effect occurs when bicarbonate in the tear-film
alkalizes in an effort to achieve an equilibrium with the partial
pressure of carbon dioxide in the surrounding air.40 A marketed
artificial tear that contains the gelling agent HP-Guar utilizes
this pH adjustment in the tear film to trigger its gelling action.20
Blinking and tear production appear to reverse increasing alkali-
nity in tears and lower pH levels – it is presumed that artificial
tear instillation has a similar effect.
Several clinical studies have looked at the relationship between
the pH value of artificial tears and patient drop preference and
tolerability.43 On the whole, it has been shown that patients
prefer to dose with slightly alkaline isotonic tear substitutes.44
When artificial tears that vary in pH are tested, patient toler-
ability is greater for drops that are more alkaline.43,44
DRY-EYE THERAPIES AND
INVESTIGATIONAL COMPOUNDS
Tear substitutes, though capable of managing dry eye, do not
generally treat it to a degree constituent of therapy. Currently
only one active compound – cyclosporine A, a partial immuno-
modulator believed to limit the proliferation of T lymphocytes
– is approved and indicated for treatment of dry-eye syndrome
(Table 27.3).45 In the US cyclosporine has only shown clinical
efficacy in patients with aqueous deficient dry eye, although lab
tests have suggested it may increase goblet cell density.46,47 In
dium, ecabet sodium, gefarnate, rebamipide, and 15(S)-HETE,
are being studied in the hope they may upregulate production of
one or more of the tear film layers (primarily the mucin
layer).49–52 Antievaporative agents such as sodium hyaluronate
that are intended to increase tear dwell time by boosting lipid
layer production are also in development. The exploratory com-
pounds known as mucomimetic agents incorporate synthetic
mucins, ideally to enhance both the mucin and aqueous layers
and promote adhesion of tears to the ocular surface. Antiinflam-
matory compounds – both steroids and nonsteroidal anti-
inflammatory drugs (NSAIDs) – are being tested to see if they
can improve signs and symptoms of dry eye.53 Some clinicians
currently prescribe corticosteroid drops off-label on a ‘pulse’
regimen for dry eye, and it is believed that this treatment may
prove quite effective against the signs and symptoms of dry eye.
With any ocular steroid treatment, however, there is always
concern about the possibility of elevated intraocular pressure
(IOP) levels.
Oral supplements, particularly Omega 3 fatty acids, have been
studied for their effects on the lacrimal system and are often
marketed or recommended by physicians for dry-eye manage-
ment or prevention purposes.54,55 In the foreseeable future, dry-eye
treatment may attempt to go directly to the source of the condi-
tion by utilizing neuronal feedback loop regulators to counteract
the neurological changes that are believed to catalyze the onset
of dry eye.56 Hormonal treatments (e.g., androgen) are being
considered and tested as potential therapies due to hormonal
deficiencies associated with some dry-eye etiologies.57,58 Neuro-

its current marketed eyedrop formulation, however, cyclosporine A
is reported to cause discomfort in 17% of patients upon
instillation.46 Some tear substitutes have found further poten-
tial use as concomitant treatment in patients using topical
cyclosporine – clinical trials suggest that this dosing combination
can yield improvements in patient symptoms and clinical signs.48
Many compounds are currently under investigation as poten-
tial dry-eye therapies. Secretagogs, such as diquafosol tetraso-
CHAPTER 27
transmitters could soon be investigated for their potential effects
on dry-eye syndrome as well.
Ultimately, even if dry-eye therapy does move quickly forward,
tear substitutes will not disappear. They are useful for as-needed
dosing, and could prove effective as concomitant medications
with treatments. Also, tear substitutes are becoming increasingly
efficacious at individually managing the signs and symptoms of
dry-eye syndrome.

TABLE 27–3 Prescription Therapy for Dry Eye

Active Components Concentration Trade Name Preservative

a
Cyclosporine 0.05% Restasis* None
a
Allergan Pharmaceuticals.
* Preservative-free, unit-dose vials. All tabulated information is available in Physician’s Desk Reference for
Ophthalmology. 34th edn. Oradell, NJ: Medical Economics, 2006.

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47. Kunert KS, Tisdale AS, Gipson IK: Goblet
cell numbers and epithelial proliferation in
the conjunctiva of patients with dry eye
syndrome treated with cyclosporine.
Arch Ophthalmol 2002; 120:330–337.
48. Sall KN, Cohen SM, Christensen MT,
Stein JM: An evaluation of the efficacy of a
cyclosporine-based dry eye therapy when
used with marketed artificial tears as
supportive therapy in dry eye. Eye Contact
Lens 2006; 32:21–26.
49. Gamache DA, Wei ZY, Weimer LK:
Spellman JM, Yanni JM: Preservation of
corneal integrity by the mucin secretagogue
15(S)-HETE in a rabbit model of
desiccation-induced dry eye. Adv Exp Med
Biol 2002; 506(Pt A):335–340.
50. Hamano T: Dry eye treatment with eye
drops that stimulate mucin production.
Adv Exp Med Biol 1998; 438:965–968.
51. Urashima H, Okamoto T, Takeji Y, et al:
Rebamipide increases the amount of
mucin-like substances on the conjunctiva
and cornea in the N-acetylcysteine-treated
in vivo model. Cornea 2004; 23:613–619.
52. Fujihara T, Murakami T, Fujita H, et al:
Improvement of corneal barrier function by
the P2Y(2) agonist INS365 in a rat dry eye
model. Invest Ophthalmol Vis Sci 2001;
42:96–100.
53. Abelson MB, Sloan J: Nonsteroidal anti-
inflammatory drugs: current ophthalmic
therapy. J Fla Med Assoc 1994;
81:261–263.
54. Miljanovic B, Trivedi KA, Dana MR, et al:
Relation between dietary n-3 and n-6 fatty
acids and clinically diagnosed dry eye
syndrome in women. Am J Clin Nutr 2005;
82:887–893.
55. Brown NA, Bron AJ, Harding JJ, Dewar
HM: Nutrition supplements and the eye.
Eye 1998; 12(Pt 1):127–133.
56. O’Brien PD, Collum LM: Dry eye: diagnosis
and current treatment strategies. Curr
Allergy Asthma Rep 2004; 4:314–319.
57. Sullivan DA: Androgen deficiency and dry
eye syndromes. Arch Soc Esp Oftalmol
2004; 79:49–50.
58. Sullivan DA, Edwards JA: Androgen
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J Steroid Biochem Mol Biol 1997;
60:237–245.
 CHAPTER
28 Viscoelastics
Jack V. Greiner
Viscoelastic substances have viscous and elastic properties. In
ophthalmology, viscoelastics are used in artificial tears and sur-
gical formulations. Viscoelastic artificial tears and contact lens
rewetting solutions are used to treat the ocular surface and relieve
ocular discomfort. Viscoelastics are also used in anterior sur-
gical procedures like cataract extraction, intraocular lens (IOL)
implantation and exchange, keratoplasty, anterior chamber
reconstruction, and in some vitreoretinal procedures.
PHYSICAL AND CHEMICAL PROPERTIES
OF VISCOELASTIC MATERIALS
The ideal viscosurgical material would be noninflammatory, non-
pyogenic, nontoxic, nonantigenic, and highly viscous. It should
be able to pass through small channels such as fine cannulas,
30-gauge needles, or pores of the trabecular meshwork. Elastic
qualities should enable viscoelastic substances to rebound after
mechanical stress or compression. A number of natural and syn-
thetic viscoelastic polymers are used in viscosurgery (Table 28.1).
Healon was the first viscoelastic sodium hyaluronate solution
to be marketed for ophthalmic use. Sodium hyaluronate’s com-
bined viscous, elastic, and pseudoplastic properties make it well
suited for anterior segment surgical applications. Healon is derived
from rooster combs and is available as a 1% sterile solution
sealed in 0.5–1 mL glass syringes (10 mg/mL). Blue-tinted Healon
is also available to facilitate intraocular visualization of the
polymer.1 Provisc is comparable to Healon in terms of sodium
hyaluronate concentration, viscosity, and cohesive properties.
Among Healon formulations, Healon5 has the highest viscosity
and elasticity when exposed to shearing and is most likely to
improve maintenance of anterior chamber depth.2 Healon5
exhibits a dynamic frequency dependence in the presence of
turbulence and phacoemulsification power (continuous high
shear rates). Healon5 has been reported to fragment dispersively
and form a cavity with an outer retentive shell during phaco-
emulsification.2 The cohesive and dispersive properties of
Healon5 are better than Healon and Healon GV for all stages of
phacoemulsification.2
Like Healon, Amvisc is a sodium hyaluronate solution puri-
fied from rooster combs. Unlike Healon, Amvisc is formulated
to a consistent viscosity with a concentration varying between
1% and 1.4% hyaluronate (Healon is prepared to a specific con-
centration of 1% with a variable viscosity). Amvisc Plus has a
higher concentration of sodium hyaluronate (1.6%), and is 30%
more viscous than Amvisc. Healon GV has a 1.4% sodium
hyaluronate concentration and is considerably more viscous
than other ophthalmic viscoelastic preparations.
AMO Vitrax contains 3% sodium hyaluronate, the highest
concentration currently available. Viscoat combines 3% sodium
hyaluronate with 4% chondroitin sulfate. Chondroitin sulfate is
structurally similar to HA. Chondroitin sulfate has a double
negatively charged sulfate group per repeating disaccharide sub-
unit. HA has one negative charged sulfate group per subunit.
Chondroitin sulfate (CS) is not a pseudoplastic fluid; it main-
tains a consistent viscosity at various shear rates. One random-
ized clinical comparison of Healon GV and Viscoat found both
formulations effectively protected the corneal endothelium during
endocapsular phacoemulsification and IOL implantation.3 The
median thicknesses of Amvisc Plus, Healon GV, and Viscoat
remaining adherent to the endothelium after phacoemulsification,
however, were found to be different.4 Viscoat provided the endo-
thelium with the greatest protection, according to the study.4
Different viscoelastic materials are suitable for different types
of surgical procedures (see Table 28.1). Cohesive (or dispersive)
viscoelastics tend to adhere to ocular surfaces, protecting them
without excessive leakage during irrigation. Low cohesive proper-
ties are generally advantageous during iris plane and anterior
chamber phacoemulsification, particularly when endothelial pro-
tection is critical, for example, in Fuchs’ endothelial dystrophy.
Dispersive viscoelastics are difficult to remove. Cohesive visco-
elastics are more easily aspirated from the eye. More cohesive
viscoelastics are desirable for anterior chamber maintenance,
when tissue manipulation and easy removal are the principal
goals. With high positive vitreous pressure, cohesive visco-
elastics have the ability to create and maintain a deep anterior
chamber. Cohesive viscoelastics are effective during capsulor-
rhexis and IOL implantation, particularly when very fine fold-
able lenses are used.
DuoVisc is a viscoelastic ‘system’, containing Viscoat and
Provisc in separate syringes, which allows the surgeon to choose
the appropriate viscoelastic material. Viscoat’s tissue protective
properties are preferable in the initial stages of an anterior segment
procedure such as extracapsular cataract extraction by phacoemul-
sification. Provisc’s cohesive properties would be more appro-
priate for later phases of the procedure, such as expansion of the
capsular opening, maintaining space, and IOL implantation.
Hydroxypropyl methylcellulose (2% solution) also has been used
successfully as an adjunct to anterior segment surgery. Hydroxy-
propyl and methyl groups make this linear polymer of glucose
more hydrophilic than its parent molecule, cellulose. Occucoat
and Cellugel are available in the same concentration but different
viscosities (see Table 28.1). Both are less viscous than Healon.
INDICATION FOR USE
The United States Food and Drug Administration (FDA) has
classified visocelastics as devices (not drugs). Viscoelastics are
indispensable in certain procedures in which the maintenance
of anatomic spaces and traumatic tissue manipulation are
required. They can also be used to lubricate and protect the eye. 293
 SECTION 4

294
TABLE 28.1 Comparison of Viscoelastics

Composition Molecular Weight Viscosity Osmolarity Storage

Product Company (in Saline) (Daltons) (cP) Cohesion (mOsM) pH Conditions (°C)

Amvisc Plus Bausch & Lomb 1.6% HA 1.6 million 55 000 Medium 340 5.5–7.0 2–8
Amvisc Bausch & Lomb 1.2% HA >2.0 million 40 000 High 320 5.5–7.0 2–8
Occucoat Bausch & Lomb 2% HPMC >0.08 million 4000 None 285 7.2 Room temperature
PHARMACOLOGY AND TOXICOLOGY

Viscoat Alcon 3.0% HA NaHA>500 000

4.0% CDS CDS 22 500 50 000 Low 325 7.2 2–8
Provisc Alcon 1.0% HA 2.5 million 30 000– High 310 7.2 2–8
40 000
Duovisc Alcon Small (0.35 mL Viscoat/0.4 mL
Provisc)
Large (0.5 mL Viscoat/0.55 mL
Provisc)
DisCoVisc Alcon 1.7% HA 1.7 million 40 000– Medium 298±32 6.8–7.6 2–8
4.0 CDS 110 000
Cellugel Alcon 2.0% HPMC 0.3 million 20 000 None 315 7.2 Room temperature
Healon AMO 1.0% HA 4.0 million 200 000 High 302 7.0–7.5 2–8
Healon GV AMO 1.4% HA 5.0 million 2 000 000 High 302 7.0–7.5 2–8
Healon5 AMO 2.3% HA 4.0 million 300 000 High 320 6.8–7.6 2–8
CoEase AMO 1.2% HA >2.0 million 40 000 High 320 6.8–7.6 2–8
Vitrax AMO 3.0% HA 0.5 million 40 000 Low 310 7.0–7.5 Room temperature
STAARVisc II STAAR Surgical 1.2% HA >2.0 million 40 000 High 320 6.8–7.6 2–8
Shellgel Cytosol 1.2% HA >2.0 million 40 000 High 320 6.8–7.6 2–8
-1
Viscosity measured at 1 second at 25°C.

In ophthalmology, viscoelastics are most commonly used in
artificial tears, and rewetting solutions. In ocular surgery, visco-
elastics are most commonly used during cataract extraction.
Comparative studies have demonstrated that all viscoelastics
(Table 28.1) effectively maintain the intraocular space and control
posterior pressure while intraocular tissues are manipulated.
CATARACT EXTRACTION
When injected into the cleavage plane between the lens nucleus
and cortex, viscoelastics can greatly facilitate phacoemulsification
of the nucleus during cataract extraction.5
Such ‘viscodissection’ is especially useful when the cataract has
a soft nucleus, negotiating the phacoemulsification tip beneath
the nucleus is difficult, and zonular tears or posterior lens cap-
sular ruptures could occur.6 Viscoelastic materials can also
maintain hydration of the ocular surface for extended periods
during surgery. Nuclear viscoexpression has been recommended
after capsulorrhexis during extracapsular cataract extraction.7–11
The superviscous properties of Healon5 appear to lead to a
higher completion rate of continuous curvilinear capsulorrhexis
in pediatric cataract surgery.12 Differences in osmolarity among
viscoelastic substances (Table 28.1), may explain the differences
in corneal thickness following cataract surgery. Viscoelastic sub-
stances with osmolalities of 305 mOsmol/kg or slightly higher
may be preferable, especially in patients with compromised
corneal endothelial cells.13
VISCOANESTHETICS
Mixtures of viscoelastics and anesthetics such as hydroxypropyl
methylcellulose 2.25% and licocaine 1%14 or sodium hyaluronate
1.5% and lidocaine 1%15 may minimize patients’ pain and dis-
comfort during cataract operations.
RECOVERY OF SUBLUXATED LENS
Sodium hyaluronate has been successfully used for severely sub-
luxated lens removal.16 Injections of SH can elevate the lens,
prevent total luxation, and simplify lensectomy. Viscoelastic dis-
section has been used for relocation of off-axis IOL implants.17
ENDOTHELIUM
Viscoelastics are able to protect the corneal endothelium from
mechanical trauma in anterior chamber surgery especially during
IOL insertion. Metallic instruments can cause cataracts with even
a slight touch to the crystalline lens. Viscoelastics can minimize
such operative complications.
Glasser and colleagues1 compared Healon, Amvisc, and Viscoat
and found that all three viscoelastics provided complete corneal
endothelium protection during contact with an IOL in vitro.
However, a more recent study by Glasser et al18 discovered that
Viscoat was better than Healon at preventing endothelial cell
loss in vivo during phacoemulsification with IOL implantation.
The authors hypothesize that chondroitin sulfate in Viscoat
makes the viscoelastic more adherent to the corneal endothelium,
and therefore, more protective. Viscoat also effectively protects
the endothelium from air-bubble damage.19 Physical trauma to
the endothelium can be prevented by coating the IOL with a
viscoelastic polymer before implantation.
PUPILS
Eyes receiving hydroxypropyl methylcellulose may develop non-
creative semidilated pupils more readily than eyes receiving
sodium hyaluronate, according to one study (Healonid).20 How-
Viscoelastics
ever, a later study reported no statistical difference in pupil size
or reactivity after the use of Occucoat or Healonid in the course
of cataract surgery.21
INTRAOCULAR PRESSURE
IOP may increase postoperatively following the use of visco-
elastics.22 This transient rise in IOP characteristically occurs
6–24 h after surgery and usually resolves spontaneously within
72 h.23 Berson et al24 have suggested that viscosurgery-associated
IOP elevations may be due to mechanical obstruction of aqueous
outflow by the viscoagent. They recommend thoroughly irriga-
ting and aspirating the eye with a balanced salt solution to
remove the viscoagent. In some instances, it may be necessary
to treat the elevated IOP with antiglaucoma medications.
PROTECTIVE EFFECT ON THE CORNEAL
SURFACE
In corneal surgery, viscoelastics are primarily used to protect
corneal endothelial cells. However, viscoagents can also be applied
to the corneal surface during anterior segment procedures to
prevent the trauma and desiccation of the corneal epithelium.
Corneal surfaces coated with viscoagents prior to cataract extrac-
tion do not need to be repeatedly rehydrated with a balanced salt
solution during surgery. The use of a topical viscoagent during
corneal surgery significantly improved corneal epithelial integrity
1 week after keratoplasty,25 according to one study.
CHAPTER 28
REATTACHMENT OF DESCEMET’S
MEMBRANE
One complication of sodium hyaluronate injection, and IOL or
surgical instrument insertion through the corneoscleral or
corneal wound is Descemet membrane detachment.26–28
Sodium hyaluronate29,30 can be used to move Descemet’s mem-
brane back to its normal anatomic position, and avoid further
detachment.
GLAUCOMA FILTRATION SURGERY
Viscoelastic materials can be used in glaucoma filtration pro-
cedures. Viscoelastics have been shown to prevent the collapse
of the anterior chamber and stabilize early postoperative pres-
sure.31–33 One study found glaucoma filtering procedures with
Healon resulted in permanent blebs, more open clefts, less
scarring, less peripheral anterior synechia formation, and sig-
nificantly lower long-term IOP.34 Viscoelastics can also be used
to dilate Schlemm’s canal in viscocanalostomy.35
VITREOUS INCARCERATION
Sodium hyaluronate has been used to treat vitreous incarceration
in patients with corneal decomposition.36,37 Filling the anterior
chamber with Healon may reduce postoperative corneal compli-
cations during neodymium: yttrium–aluminum-garnet treatment
for vitreolysis.
INTRAOCULAR HEMORRHAGE
Viscoagents can be used to control intraocular hemorrhage. Vis-
coelastic materials trap clotted blood in the anterior chamber,
however, so viscoelastics should be used cautiously if blood is
present. Ten percent sodium hyaluronate can be used to manage
suprachoroid hemorrhages postoperatively.38,39 Sodium
hyaluronate allows for good visualization of instruments in the
eye and avoids image minification and distortion from the 295
 PHARMACOLOGY AND TOXICOLOGY

air–fluid interface. Although balanced salt solution can be used,
sodium hyaluronate viscoelastic is less likely to egress through
rents in the posterior lens capsule or between zonular fibers and
therefore provides a more effective and durable expansion of
intraocular volume.38 Viscoelastics can also prolong the main-
tenance of the IOP after filtering surgery.40 Sustaining the IOP
would help facilitate drainage of suprachoroid hemorrhage while
avoiding choroid effusion and hemorrhage incurred by ocular
hypotonia. Using a generous amount of Healon and flattening
the retinochoroid elevations of a suprachoroid hemorrhage pro-
motes expression of blood from the suprachoroid spaces.41
RETINAL DETACHMENT SURGERY
Viscoelastics can be used for retinal detachment repair. For
example, suprachoroid implantation of a viscoelastic substance
can temporarily induce a choroid elevation for closing, retinal
tears.42–46 Sodium hyaluronate has even been used to repair giant
retinal tears.47,48
VITRECTOMY SURGERY
Procoagulate effects of HA after diabetic vitrectomy have been
reported,49 and sodium hyaluronate has been used to perform
delamination at the vitreoretinal juncture in diabetic eye dis-
ease. Such viscodelamination can separate the vitreous cortex
from the fibrovascular epiretinal membranes.50 Viscodelamina-
tion is especially valuable in eyes with combined traction and
SECTION 4
hyaluronate should be applied to the lacrimal sac through the
intact lacrimal canaliculus and probes for bicanaliculonasal
intubation should be inserted. Hyaluronate is thought to coat
and distend the lumen of the lacrimal passage, allowing the
probe tip to find its way to the injured canaliculus.54 Sodium
hyaluronate can help the surgeon find a cut medial canaliculi
when it is injected into the lacrimal sac.55,56
STRABISMUS SURGERY
Sodium hyaluronate has been used in strabismus surgery with
adjustable sutures to minimize tissue drag in the conjunctiva,
Tenon’s capsule, and muscle.56 Healon has been reported to reduce
postoperative muscle adhesions57 and to increase the period of
suture adjustability in operated muscles.58
DRY-EYE TREATMENT
Sodium hyluronate can both subjectively and objectively improve
dry-eye symptoms.59–66 Patients with severe keratoconjunctivitis
sicca respond particularly well to sodium hyluronate.67 Dry-eye
symptoms can also be relieved with topical chondroitin sulfate
solution and viscoelastic artificial tears.66 Viscoelastic contact
lens rewetting solutions are also available.68
CONCLUSIONS
Viscoelastic polymers are valuable surgical adjuncts. They main-

rhegmatogenous retinal detachment. The viscodelamination
technique has a significant risk of retinal breaks, however. The
risk is particularly high when adherent fibrovascular epiretinal
membranes are elevated excessively.50 Healon has been used to
elevate epiretinal membranes from the retina.51
LACRIMAL SURGERY
Sodium hyaluronate, injected into the lacrimal sac, is useful for
identifying the extent of the sac lumen.52,53 Sodium hyaluronate
has been reported to facilitate the passage of lacrimal probes
during lacerated canaliculi repair.54 For such a procedure,
tain anatomic space, manipulate intraocular tissues, and prevent
mechanical trauma to fragile cells such as the corneal endothe-
lium. Viscosurgery may temporarily elevate IOP if the anterior
chamber is not properly irrigated at the end of the procedure.
Comparative data suggest there are no major differences
between the commercially available viscosurgical agents. All
viscoelastics have similar optical clarity, protect tissues, raise
IOP postoperatively and maintain space.28,69,70 When cost is a
concern, methylcellulose preparations should be considered.
Although a number of viscoelastic solutions are available to the
ophthalmic surgeon, no single formulation appears significantly
more efficacious.

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The role of sodium hyaluronate. Can J
Ophthalmol 1984; 19:112.
53. Lerner HA, Boynton JR: Sodium
hyaluronate (Healon) as an adjunct to
lacrimal surgery. Am J Ophthalmol 1985;
99:365.
Viscoelastics
54. Vila-Coro AA, Vila-Coro AA: Hyaluronate
facilitates passage of lacrimal probes for
repair of lacerated canaliculi. Arch
Ophthalmol 1988; 106:579.
55. Seiff SR, Ahn JC: Locating cut medial
canaliculi by direct injection of sodium
hyaluronate into the lacrimal sac.
Ophthalmic Surg 1989; 20:176.
56. Clorefeine GS, Parker WT: Use of Healon in
eye muscle surgery with adjustable sutures.
Ann Ophthalmol 1987; 19:215.
57. Searl SS, Metz HS, Lindahl KJ: The use of
sodium hyaluronate as a biologic sleeve in
strabismus surgery. Ann Ophthalmol 1987;
19:259.
58. Manjoney D, Mathias S, Morris W, et al:
Effect of Healon on adjustable suture
strabismus surgery. Invest Ophthalmol Vis
Sci (ARVO Suppl) 1985; 26:80.
59. Polack FM, McNiece MT: The treatment of
dry eyes with Na hyaluronate (Healon).
Cornea 1982; 1:133.
60. DeLuise VP: Viscodissection as an adjunct
to phacoemulsification. Ophthalmic Surg
1988; 19:682.
61. Stuart JC, Linn JG: Dilute sodium
hyaluronate (Healon) in the treatment of
ocular surface disorders. Ann Ophthalmol
1985; 17:190.
62. Nelson JD, Farris RL: Sodium hyaluronate
and polyvinyl alcohol artificial tar
preparations. A comparison in patients with
CHAPTER 28
keratoconjunctivitis sicca. Arch Ophthalmol
1988; 106:484.
63. Laflamme MY, Swieca R: A comparative
study of two preservative-free tear
substitutes in the management of severe
dry eye. Can J Ophthalmol 1988; 23:174.
64. Bohm E, Rama P, Tallandini L, et al: Low
molecular weight sodium hyaluronate in the
treatment of tear film changes and of dry
eye. Ophthalmologie 1988; 2:353.
65. Orsoni JG, Chiari M, Guazzi A, et al:
Efficacy of hyaluronic acid eye drops in the
treatment of dry eye. Cytologic study using
an optical microscope and computerized
microscope. Ophthalmologie 1988; 2:355.
66. Limberg MB, McCaa C, Kissling GE, et al:
Topical application of hyaluronic acid and
chondroitin sulfate in the treatment of dry
eyes. Am J Ophthalmol 1987; 103:194.
67. Sand BB, Marner K, Norn MS: Sodium
hyaluronate in the treatment of
keratoconjunctivitis sicca. A double
masked clinical trial. Acta Ophthalmol
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68. Physicians Desk Reference
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69. Genstler DE, Keates RH: Amvisc in
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70. McKnight SJ. Giangiacomo J, Adelstein E:
Inflammatory response to viscoelastic
materials. Ophthalmic Surg 1987; 18:804.
297
 CHAPTER
29 Pharmacologic Agents with Osmotic Effects
Gail Torkildsen, Ula V. Jurkunas, and Tolga Kocaturk
Ocular osmotic forces cause prominent signs and symptoms in
many disease states. The mechanism by which water and elec-
trolytes are linked and transferred between epithelial and endo-
thelial cells remains an unsolved problem. Dysfunction of these
layers is thought to figure prominently in many diverse diseases
such as corneal edema, cataract, glaucoma and some vitreous
and retinal detachments and central serous choroidopathy.
Pharmacologic agents exert osmotic effects within the eye and
should be considered in both treatment of disease as well as
avoiding unintended effects.
OSMOSIS, OSMOLARITY, AND TONICITY
Key Features
• Osmotic forces affect the flow of water into or out of tissues,
tear film, aqueous, and vitreous
• Hyperosmotic tear film can induce inflammatory cytokines
• Systemic osmotic agents such as mannitol increase plasma
oncotic pressure and draw water out of the vitreous to lower
intraocular pressure
• Intracameral and intravitreal injections must be carefully
prepared due to the powerful osmotic forces they can exert
• Osmotic forces may be a factor in cataract development
Diffusion is a constant motion of molecules among each other
which results in a solute or solvent moving from a higher con-
centration to a region of lower concentration. If there is a mem-
brane between the two regions and that membrane is permeable
to water only, a net movement of water occurs through the mem-
brane. Water will diffuse from an area of high water concen-
tration into the area of low water concentration. Diffusion of
water across the membrane is called osmosis, and it is driven by
the internal energy of water molecules. The net flow of water can
be prevented by the application of an opposing force, osmotic
pressure. This pressure is directly proportional to the concen-
tration of nondiffusible molecules on the opposite side. As a
result, the direction of water flow is determined by the solute
concentration and not by the molecular weight of the solute.
Facilitated diffusion refers to the interaction of a solute interact-
ing with a carrier protein in a cell membrane to aid the solute’s
passage. Osmolarity (osmoles per liter of water) is a total concen-
tration of solutes in a solution and is independent of whether
the solutes can cross the membrane. Osmolality (osmoles per
kilogram) is a total number of osmotically active particles in a
solution. Tonicity is the effective osmolality or concentration of
the solutes that have the capacity to exert the osmotic force
across the membrane (Fig. 29.1).
A cell membrane, which is a highly complex semipermeable
membrane, has both outer and inner lipid layers and a middle
1M 1M 1M
H2O 1 M Sucrose
Urea H2O Sucrose
Urea
A. Isotonic B. Not Isotonic C. Not Isotonic
Not Iso osmotic Iso osmotic Not Iso osmotic
FIGURE 29.1. Schematic representation of tonicity and osmolarity.
aqueous layer. A lipid-soluble substance passes through the
lipid-soluble layers with greater ease than a water-soluble sub-
stance, whereas the latter transgresses only the middle layer with
comparative ease. Carrier proteins imbedded in a cell membrane
aid the passage of a given substance and facilitate diffusion. The
layers have different permeability to the different substances.
The process of dialysis results when protein is on one side of the
semipermeable membrane. Water moves toward the protein,
and salt flows away from the protein. The final distribution of
salt and protein is described by the Gibbs–Donnan equilibrium
in which the product of cations and anions is the same on both
sides of the membrane and the number of cations on the protein
side equals the sum of anions and proteins on the other side.1
Filtration is the process of forcing fluid across a membrane
exerting pressure. Ultrafiltration results when a hydrostatic force,
such as blood pressure, acts on the solutions that contain protein.
OSMOTIC PHENOMENA IN THE EYE
TEARS
Cellular integrity of air-exposed cells of both the cornea and the
conjunctiva is maintained by the presence of a complex barrier
of isotonic fluid, the tear film. In response to the external stimuli
and the internal requirements of the cornea and conjunctiva the
regulatory mechanisms of the tear film alter its composition,
volume, and structure. The classical view of the tear film as
a three-layered structure, composed of an outer lipid layer, a
middle aqueous layer and an inner mucous layer structure
has been modified. Some authors state that mucous and the
aqueous layers are not distinct, and some suggest that a lipid
layer is a monolayer with polar and nonpolar phases.2 The
regulation of the osmotic flow of fluids between the corneal and
conjunctival epithelial cells and the tear film is mainly achieved
by aqueous phase electrolytes such as Na+, K+, and Cl⫺ that
buffer tear pH and control cell membrane permeability.
The osmolarity of the normal human tear film is 302 ± 6
(SD) mOsm/L, which is higher than serum osmolarity of 299
 PHARMACOLOGY AND TOXICOLOGY
290 mOsm/L.3 Tear osmolarity is the lowest in the morning after
prolonged lid closure, and increases as the day progresses.4,5
The concentrations of chloride and potassium are higher in the
tears than in the serum, and glucose concentration is lower
than tears.2 The importance of tear film osmolarity and the
stable balance of essential electrolytes on the epithelial surface
maintenance have been shown in animal models and in vivo.5–8
Elevated tear film osmolarity of greater than 310 mOsm/L is
often seen in patients with dry-eye syndrome.3,6 The hyperos-
molarity of the tear film indicates an imbalance between the
rate of tear secretion and the rate of evaporation, as seen in
aqueous tear deficiency and evaporative tear loss, respectively.3,6
In aqueous tear deficiency states, lacrimal gland secretion
rate declines and tear osmolarity increases independent of eva-
porative loss.6,7 In keratoconjunctivitis sicca, tear replacement
from the lacrimal gland is decreased, often with striking morpho-
logic changes in both the conjunctival and the corneal epithe-
lium. When the tear osmolarity increases, there is an abnormal
discharge of mucin glycoprotein granules and a decrease in goblet
cell density that contribute to the ocular surface pathology of
dry eyes.8
Hyperosmotic conditions can potentiate cytokine synthesis
by nonimmune resident ocular surface cells, including corneal
and conjunctival epithelial and stromal cells. Expression and
production of matrix metalloproteinases (MMP): MMP-9
(gelatinase), MMP-1 and MMP-13 (collagenases) and MMP-3
(stromelysin) are increased with increasing osmolarity. This
increase in mediated in part by the c-Jun N-terminal kinases (JNK)
SECTION 4
which is a stress activated protein kinase (SAPK).9 Effectors of
cytokine synthesis in dry eye include mitogen-activated kinases
(MAP kinase, p38 kinase), JNK, and I-k kinase (IKK).
Hypersomotic conditions can induce inflammatory processes
which upregulate several gene products. One of these products
is the nuclear transcription factor, NF-kB. In its quiescent state
NF-kB exists as a heterodimer with the protein Ik-Ba. This
masks the nuclear localization signals and DNA binding
domain of the former protein. Under inflammatory conditions
Ik-Ba is phosphorylated, causing a conformational change
which results in its tagging with multiple copies of the ubiquitin
protein. Ubiquinated Ik-Ba is recognized and degraded by the
proteasome, which liberates NF-kB. The free protein is trans-
located to the nucleus, where it binds to the appropriate DNA
sequence and upregulates the production of several inflam-
matory mediators, such as COX-2, iNOS, IL-1, and TNF-a.
CORNEA
The sodium concentration gradient is thought to be the pre-
dominant force acting on the corneal endothelium. This creates
a net osmotic force drawing water out of the stroma via osmosis
while other ions oppose it. In disease states, the ionic gradients
across the endothelium cannot be maintained resulting in corneal
edema and swelling (Fig. 29.2). Corneal edema is characterized
by a marked increase in corneal thickness, and intercellular and
extracellular edema of the basal epithelial cell layer of the
epithelium. In addition, corneal edema is associated with loss
of stromal proteoglycans and hydropic degeneration or cell lysis
of keratocytes.
Water movement within the epithelium is slowed by the pres-
ence of lipid membranes. Zonula occludens or tight junctions
encircle the cells just below the apical surface and constitute an
additional barrier to the passive movement of water, electrolytes
and macromolecules.10 In contrast, water moves rapidly within
the stroma because of the abundance of collagen fibrils, which
are separated by proteoglycans and water. Although endothelial
cells have junctional complexes, they are much more leaky to
300 water than epithelium; the result is relative freedom of water
Lipid & aqueous soluble
Lipid soluble H2O
Blocked by lipid
membranes in
Isotonic Tear Film epithelium
Zonulae Cornea
Epithelium Occludentes
Proteoglycans + H2O
Endothelium Junctional
Complexes
Aqueous Humor
Active Secretion
Na HCO3-
Na+ Ion Pump
Lens
FIGURE 29.2. Osmotic forces in the anterior chamber.
movement.11,12 Thus, only lipid-soluble substances cross freely
the epithelial and endothelial membranes, and water-soluble
substances pass with equal freedom through the stromal layer.
Substances soluble in both lipid and aqueous penetrate the cornea
more easily. Surfacants like benzalkonium chloride (BAK) may
improve the ocular penetration of a drug in a transscleral drug
delivery system without producing toxic reactions by acting on
tight junctions.13
Corneal transparency is directly related to the corneal hydra-
tion. Fluid traverses the endothelium transcellularly in response
to the osmotic gradient created by electrolyte transport and
utilizing the osmotic permeability of aquaporins. Electroosmosis,
whereby a recirculating current causes fluid movement via
paracellular shifts, may be the prominent mechanism of fluid
transport. Trans-endothelial fluid transport can be rapidly
modulated to control stromal hydration in response to small
NaCl osmotic stresses in a way that cushions the shock and
reduces the change in corneal thickness.14
When the endothelial cell density decreases below a critical
level (200–400 cells/mm2), the leak rate of fluid into the stroma
becomes greater than the pump rate of fluid out of the stroma,
producing corneal edema and clouding.15 The blurred vision
from the epithelial edema in the mornings is due to the lack of
tear evaporation under the closed lids. After opening the eyelids,
the evaporation causes transient hypertonicity of the tear film
which extracts the water from the epithelial cells and aids in
clearing the vision.15
OSMOTIC AGENTS
TOPICAL
Pathologic changes in dry eyes produce hyperosmolar tear film
that draws the water from the corneal epithelial cells, reduces
microplicae, disrupts cell membranes and decreases cell vital-
ity.3,8,16 The main aims of dry-eye treatment with topical agents
are tear supplementation and conservation. To counterbalance
the hyperosmotic environment of dry-eye conditions, tear sub-
stitutes have been developed that dilute and decrease the osmo-

larity of the tear film and restore normal tear physiology. Most
tear substitutes are isotonic with natural tear film, and some are
hypotonic.
The reports on the utility of hypotonic versus isotonic tear
substitutes in treatment of dry eyes have been contradictory.
Initial studies by Gilbard et al noted that electrolyte solution with
osmolarity of 175 mOsm/L (TheraTears) effectively decreased
tear osmolarity, increased goblet cell density and improved dry-
eye symptoms.17 Other studies have shown that both isotonic
and hypotonic solutions were equally effective in the relief of
dry-eye symptoms.18,19 The authors postulate that the effect of
hypotonic tear substitutes on the corneal surface is of short
duration of action, and is achieved by isotonic preparations just
as well.19 Even though increased tear osmolarity is present in
dry-eye patients, the focus of tear substitute design should not
be on the tonicity, but rather on tear replacement retention,
mucomimetic action, secondary effects of preservatives,
lubricating properties, and finally actual comfort.
Various polymers are added to the tear substitutes to enhance
tear retention by increasing the viscosity, decreasing surface
tension and enhancing tear film stability. Increasing viscosity
with the addition of polymeric ingredients causes a longer inter-
val of contact with the eye. Sodium hyaluronate, a constituent
of extracellular matrix has been shown to have clear benefit in
promoting corneal epithelial healing and relief of dry-eye symp-
toms.19 A therapeutic soft contact lens, with frequent instilla-
tion of saline or another tear substitute, also prolongs contact of
the tear solution. Ointments are useful when frequent instilla-
tion is not possible.
In contrast to dry-eye treatment, conditions that cause corneal
edema are treated by hyperosmotic agents. They transiently
increase the tonicity of the tear film and enhance water move-
ment from the cornea, especially the epithelial cell layer. Most
frequently used agents in a clinical setting are sodium chloride
2% and 5% solution and ointment (Muro-128) and glycerin
(50–100% preparations). Sodium chloride is most commonly
used in cases of corneal edema due to endothelial dysfunction,
post-LASIK corneal flap edema, and to acute corneal hydrops in
keratoconus.20,21 Sodium chloride drops are particularly bene-
ficial in reducing epithelial edema upon awakening. Hypertonic
sodium chloride ointment at bedtime reduces the amount of
corneal hydration while the eyelids are closed during sleep. Intact
epithelium provides a barrier to solute movement and enhances
the osmotic effect of the hypertonic solutions. Ocular irritation
is a common side effect of hypertonic saline eyedrops.
Glycerin is a fast acting osmotic agent when in contact with
the corneal surface. The effects of glycerin are transient as the
mixture with water decreases the solution’s effective osmolarity.
The main clinical use of glycerin is in corneal edema due to
acute angle glaucoma, or endothethelial dysfunction. In the former,
the application of glycerin aids in gonioscopic examination.
SYSTEMIC AGENTS FOR THE REDUCTION
OF INTRAOCULAR PRESSURE
Acute treatment of ocular hypertension and preparation of the
eye for intraocular surgery are the two prominent therapeutic
indications for systemic delivery of osmotic agents. Osmotic
agents cause rapid reductions in intraocular pressure by increas-
ing blood osmolality which draws fluid from vitreous to blood
thus decreasing vitreous volume and decreasing IOP. In angle
closure glaucoma, the decreased IOP reverses iris ischemia and
improves its responsiveness to pilocarpine and other drugs. If
the blood aqueous barrier is disrupted, osmotic agents can enter
eye and are less effective at decreasing IOP. These medications
may be more effective during inflammation. Osmotic agents
can not be used long term as the osmotic gradient quickly
Pharmacologic Agents with Osmotic Effects
re-equilibrates and can lead to rebound increases in IOP. Osmotic
agents cause a total body diuresis and should not be used in
cardiac and renal patients. Side effects can include headache,
backache, diabetic ketoacidosis, congestive heart failure, and
myocardial infarction due to increased preload on heart. Central
nervous system effects can include confusion and subdural and
subarachnoid hemorrhages.
Osmotic agents include mannitol, glycerin, urea and isosorbide.
IV Mannitol is the most commonly used systemic drug in this
class. Mannitol is not metabolized and the dosage is 1.5–2 g/kg
body weight over 30–45 min. Glycerin can be given orally but is
rapidly metabolized to glucose and should be used cautiously in
diabetics. Dosage is 1–1.5 g/kg body weight. Isosorbide is avail-
able in a 45% oral preparation, and is physiologically similar to
glycerin. It is essentially not metabolized and is excreted by the
kidney. Dosage is 1.5 g/kg body weight.
INTRAOCULAR IRRIGANTS
Irrigating solutions with the corneal endothelium, lens,
trabecular meshwork, vitreous and retina may have important
consequences for cellular survivability and function. An
irrigating solution must maintain both physiologic and anatomic
integrity. An ideal irrigating solution is isoosmotic with intra-
ocular fluids and contains the nutrients necessary for cellular
viability. Currently available intraocular irrigants have osmolarity
of 277–305 mOsm.22 The major ions present in the solutions
are sodium, potassium, magnesium, calcium, and bicarbonate.
CHAPTER 29
Some solutions contain dextrose and reduced glutathione (GSH)
and/or oxidized glutathione (GSSG). Addition of GSH and
GSSG to the irrigating solutions showed a beneficial effect in
preventing corneal swelling by maintaining intracellular levels of
GSH in corneal endothelium.23 GSH is a powerful antioxidant
effective in detoxifying the free radicals released during
intraocular surgery.24 In particular, GSSG an ingredient of BSS
plus (Alcon Laboratories, Fort Worth, TX, USA), was shown to
be beneficial on the maintenance of the barrier function of
corneal endothelium, retinal pigment epithelium, and the
blood–aqueous barrier.25,26
The pH and osmotic tolerance range of the human corneal
endothelium are important considerations when combining
intraocular medications and ophthalmic solutions. The corneal
endothelium has a pH tolerance between 6.8 and 8.2, similar to
the natural aqueous humor bicarbonate buffer system.27 During
phacoemulsification, the osmolality of the anterior chamber can
vary due to medications, viscoelastics, and solutions. Hyperos-
molarity or hypoosmolarity can cause the endothelial cells to
swell, degenerate, become apoptotic, or necrotic. The corneal
endothelial cells have been shown to tolerate a wide range of
osmolalities from 250 to 350 mOsmoles.28 Therefore, both the
pH and osmolality of the intraocular solution are critical in
maintaining the corneal endothelium.29
OSMOTIC FORCES ON THE LENS
Human lens has a requirement for the maintenance of an ela-
borate antioxidant system, failure of which has been associated
with cataract formation. A constant supply of glucose from
aqueous humor serves as a main source of energy for the anaer-
obic glycolysis in the lens.30 In diabetic patients, posterior
subcapsular cataract formation has been associated with pro-
longed irrigation during intraocular surgery. Some surgeons
advocate adding supplemental glucose to the intraocular irrigants
to prevent the cataract formation in the diabetic patients under-
going vitrectomy. The addition of glucose raises the osmolority
from 305 to 320 mOsm, a level consistent with the diabetic
patient’s aqueous humor osmolarity. 301
 PHARMACOLOGY AND TOXICOLOGY

Osmotic stress due to the accumulation of sorbitol in the lens
is most likely the cause of diabetic cataract. Sorbitol accumulates
in the lenses of diabetic animals and the administration of an
inhibitor to aldose reductase (AR), the enzyme that converts
glucose to sorbitol, prevents the formation of diabetic cataracts.
Sorbital along with myo-inositol (MI) and taurine are the major
osmolytes in the lens. For lens epithelial cells, an increase in
extracellular osmotic pressure induces the expression of a Na+-
dependent MI transporter (SMIT), AR, and taurine transporter.
Consequently, intracellular levels of MI, sorbitol, and taurine
are increased to balance the increased osmotic pressure.
Overexpression of SMIT in the lens causes congenital cataract.28
Transporter proteins in the cell wall play a role in how ions
move among cells. One of these is the potassium chloride
cotransporter (KCC) which is involved in the regulation of lens
volume and transparency. Under normal isotonic conditions,
a constitutively active flux of Cl⫺ ions exists in the lens that
regulates fiber cell volume. Under certain conditions, KCC
activity can be increased, not only through dephosphorylation
of the protein, but also by increasing the number of transporters
in the plasma membrane.35
Electrical current flow around the lens may play a role in lens
transparency. A recirculating sodium gradient may drive fluid
into the lens anteriorly and fluid may exit posteriorly. Taking
into account the known presence of membrane channels, trans-
porters, and an aquaporin in lens epithelium, there may exist a
classical epithelial fluid transport mechanism in this layer
which may be of great importance for lens homeostasis.31
SECTION 4
metabolic coupling. From the nonpigmented epithelial cells,
Na+, Cl⫺, and bicarbonate ions are pumped into the clefts
between nonpigmented cells creating an osmotic gradient
which draws water into the clefts. Tight junctions at the apical
side direct fluid into the posterior chamber.33
POSTERIOR POLE
During vitrectomy the irrigating solutions keep the globe
inflated and serve as a vitreous substitute. Studies have shown
that bicarbonate and glucose are especially important in main-
taining normal retinal cell metabolic activity.22 Most additives,
such as antibiotics and epinephrine may decrease the pH of the
solution and cause retinal toxicity.22 The recommendations are
ones of caution when requesting the additions to the intraocular
irrigants, as their efficacy and safety have not been fully
established.
Osmotic forces probably play a role in neuronal degeneration
in the detached retina. Retinal detachment causes a decrease of
the plasma membrane K+ conductance of Müller cells. The
decrease of the K+ currents is associated with a decrease in the
gene and protein expression for the main K+ channel subtype of
Müller cells, Kir4.1. Downregulation of the Kir4.1 protein may
cause an altered current pattern in Müller cells. Impaired spatial
buffering of K+ ions (normally performed by Müller cells by
means of their Kir channels) may contribute to neuronal degen-
eration in the detached retina, by favoring neuronal hyper-
excitation and glutamate toxicity. In the postischemic retina of

the rat, it has been shown that the decrease in K+ currents is

OSMOTIC FORCES IN AQUEOUS
PRODUCTION
Aqueous humor formation depends on hydrostatic pressure and
the oncotic pressure gradient across the ciliary epithelium.
Numerous ion channels and ports have been characterized in
the ciliary epithelium contributing to aqueous formation. Sodium,
choride, and potassium are actively transported from plasma in
the ciliary body stroma into the pigmented ciliary epithelial
cells by a Na+/K+/2Cl⫺ exchanger (symport).32 The pigmented
and nonpigmented epithelial cells are united by electric and
associated with altered osmotic swelling characteristics of Müller
cells, which may contribute to edema development in the retina.
By formation of glial scars and cellular hypertrophy, reactive
Müller glial cells may inhibit regular neuroregeneration in the
detached and reattached retina.34
Investigation of the osmotic phenomenon within the eye
remains an active area of research. Many important ophthalmic
disease states involve imbalances of osmotic forces. Medica-
tions may exert osmotic effects, impacting disease states and
understanding osmotic principles may allow more targeted
therapy.

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enzymes and the eye. Curr Eye Res 2006;
31:1–11.
Pharmacologic Agents with Osmotic Effects
25. Araie M, Shirasawa E, Ohashi T: Intraocular
irrigating solutions and permeability of the
blood-aqueous barrier. Arch Ophthalmol
1990; 8:882–885.
26. Araie M, Kimura M: Intraocular irrigating
solutions and barrier function of retinal
pigment epithelium. Br J Ophthalmol 1997;
81:150–153.
27. Gonnering R, Edelhauser HF, Van Horn DL,
et al: The pH tolerance of the rabbit and
human corneal endothelium. Invest
Ophthalmol Vis Sci 1979; 18:373–390
28. Edelhauser HF, Hanneken AM, Pederson
HJ, Van Horn DL: Osmotic tolerance of
rabbit and human corneal endothelium.
Arch Ophthalmol 1981; 99:1281–1287
29. Edelhauser HF: The Balance between
Corneal Transparency and Edema The
Proctor Lecture. Invest Ophthalmol Vis Sci
2006; 47:1755–1767.
30. Christiansen JM, Kollarits CR, Fukui H,
et al: Intraocular irrigating solutions
and lens clarity. Am J Ophthalmol 1976;
82:594.
31. Jorge Fischbarg, Friedrich PJD, Kunyan
Kuang, et al: Transport of fluid by lens
epithelium. Am J Physiol Cell Physiol 1999;
276:C548–C557.
32. Edelman JL, Sachs G, Adorante JS: Ion
transport asymmetry and functional coupling
in bovine pigmented and nonpigmented
ciliary epithelial cells. Am J Physiol 1994;
266:C1210.
33. Green K, Bountra C, Georgiou C, House R:
An electrophysiological study of rabbit
ciliary epithelium. Invest Ophthalmol Vis Sci
1985; 26:371.
34. Iandiev I, Uckermann O, Pannicke T, et al:
Glial cell reactivity in a porcine model of
retinal detachment. Invest Ophthalmol Vis
Sci 2006; 47:2161–2171
35. Kaa-Sandra NC, Joerg K, Paul JD: Roles
for KCC transporters in the maintenance of
lens transparency. Invest Ophthalmol Vis
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36. Dohlman CH, Hedbys BO, Mishima S: The
swelling pressure of the corneal stroma.
Invest Ophthalmol 1962; 1:158.
CHAPTER 29
303
 Pharmacologic Treatment of Immune Disorders
CHAPTER
and Specifically of Immune Ocular Inflammatory
30 Disease
C. Stephen Foster

Overview TABLE 30.1. Agents Commonly Used to Treat Autoimmune

Inflammatory Conditions
• Immunomodulatory therapy (IMT) is playing an increasingly
important role in the care of patients with ocular inflammatory Class Type of Agent Nonproprietary Names
disease (OID) Alkylating agents Nitrogen mustards Cyclophosphamide
• Outcomes analysis in evidence-based medicine reviews
supports the notion that IMT represents the standard of care Chlorambucil
for certain specific disorders and for most if not all cases of Antimetabolites Folic acid analogs Methotrexate
steroid-dependent chronic OID
Pyrimidine analogs 5-Fluorouracil
• Ophthalmologists would be well advised to acquaint
themselves with these studies and with the Purine analogs Azathioprine
recommendations of the International Uveitis Study Group Natural products Antibiotics Cyclosporine
and of the American Uveitis Society
Dapsone
Tacrolimus
In its broadest scope, the rubric immune disorders would include
Mitomycin
all disorders in which the immune system is abnormal. A
treatise on the pharmacologic treatment of such immune disor- Antibodies Antilymphocyte serum
ders would necessarily include material devoted to the treatment Anti-T-cell antibody
of immunodeficiency diseases, including acquired immunodefi-
ciency syndrome (AIDS) caused by the human immunodefi- Gamma globulin
ciency virus (HIV), as well as material on immunoregulatory
disorders that result in autoimmunity or an overaggressive
immune response. The author’s charge for this chapter is to two agents are usually chosen to represent each class of chemo-
address the latter group of disorders. Because inflammation is therapeutic agent in the following sections.
the paradigm for the expression of autoimmune disease, a dis- One feature common to many of the immunosuppressive
cussion of all therapies for inflammation might be appropriate agents is their ability to interfere with synthesis of nucleic acid,
here, but the pharmacology and use of the steroidal and non- protein, or both. This interference commonly is assumed to be
steroidal antiinflammatory drugs are dealt with in Chapters 25 the immunosuppressive mechanism, because lymphoid cells
and 27. This chapter therefore limits its discussion to the proper- stimulated by antigen to proliferate and produce lymphokines are
ties and uses of the immunosuppressive chemotherapeutic agents exquisitely sensitive to interference with nucleic acid or protein
in the treatment of immune inflammatory or autoimmune synthesis. Bach2 and others have emphasized, however, that the
diseases. effect of immunosuppressive agents cannot be explained solely
Although the use of immunosuppressive and biologic agents by this simple notion. Considering the extraordinary complexity
to inhibit immune reactions dates back at least half a century,1 of the idiotypic–antiidiotypic immunoregulatory network of
the mechanisms of action of most of the immunosuppressive T-lymphocyte subsets, B-lymphocyte subsets, and antigen-
agents are incompletely understood. Often we do not even presenting cells and macrophage subsets, it is remarkable that the
know whether a particular agent is in fact suppressing immune first physicians to explore the possible use of immunosuppres-
responses or suppressing the inflammatory expression of these sive chemotherapeutic agents in the treatment of autoimmune
responses. By definition, immunosuppressive agents suppress inflammatory disorders discovered dosages that produced enough
the development of at least one type of immune reaction: They differential effect on subsets of helper and cytotoxic cells to cause
modify the specific immune sensitization of lymphoid cells.2 immunosuppression.
Table 30.1 lists chemotherapeutic agents useful in the treat-
ment of neoplastic disease, many of which are also commonly ALKYLATING AGENTS
used to treat autoimmune inflammatory diseases. Usually only
one, or at most two, agents from a given class of these chemo-
therapeutic agents has been used extensively enough as an immu-
CHEMICAL PROPERTIES AND MECHANISM
nosuppressive agent in the treatment of immune disorders to OF ACTION
allow us to make wise choices about using such agents to treat Nitrogen mustards, ethylenimines and methylmelamines, alkyl-
autoimmune inflammatory disease. This is why only one or sulfonates, nitrosoureas, and triazenes all act in similar ways, 305
 PHARMACOLOGY AND TOXICOLOGY
FIGURE 30.1. Chemical structure of cyclophosphamide.
through nucleophilic substitution reactions. Of these agents, only
members of the nitrogen mustard family are commonly used as
immunosuppressive chemotherapeutic agents in the treatment of
autoimmune inflammatory disease; of the nitrogen mustards,
only cyclophosphamide and chlorambucil have been used enough
to warrant discussion here.
Cyclophosphamide (Cytoxan), the most potent of the thera-
peutic alkylating agents, is used extensively throughout the
world to treat a variety of conditions (Fig. 30.1). All alkylating
agents act through nucleophilic substitution reactions, and such
reactions with DNA probably account for their predominant
immunosuppressive activity (Fig. 30.2). Breaks occur in single-
stranded DNA. When these breaks are repaired, phosphodiester
bonds form and result in defective cell function. Cross-linking
reactions occur between DNA strands, between DNA and RNA,
and between these molecules and cell proteins, generally result-
ing in death of the affected cell.
Like most other immunosuppressive agents, cyclophos-
SECTION 4
phamide is not immunosuppressive in its native state. After
oral or intravenous administration, it is activated by the liver
P-450 microsome system. Phosphoamidase, which is present in
especially high concentrations in liver microsomes, catalyzes
the conversion of the drug into its active principles, aldophos-
phamide and 4-hydroxycyclophosphamide. In clinical doses,
alkylating drugs are very cytotoxic for lymphoid cells. The effect
on B and T cells appears to be nearly equal, except that large
doses enhance the effect on B cells. Cyclophosphamide has a
potent effect on antibody responses when given with, or even up
to 4 days after, antigen encounter. It suppresses secondary anti-
body responses in previously primed animals and patients.
Cyclophosphamide effectively inhibits delayed hypersensitivity
reactions and is as effective as azathioprine in liver, cardiac,
bone marrow, skin, and pulmonary allograft rejection reactions.
It is the only immunosuppressive agent that can induce immune
tolerance to particulate antigen. The pharmacokinetics and
kinetics of the development of such tolerance are complex. The
drug must be given 24–48 h after antigen priming. Tolerance is
probably mediated, at least predominantly, by regulatory T
lymphocytes that develop after antigen priming. On the other
hand, at least in the murine experimental model, low-dose cyclo-
phosphamide therapy can eliminate regulatory T lymphocytes
that actively mediate tolerance, resulting in release from toler-
ance and in expression of immunoreactivity in the form of a
delayed hypersensitivity reaction to the relevant antigen. The dose
and timing of administration of cyclophosphamide apparently
are critical to its effect on lymphocyte subsets. This, of course,
makes judgments about clinical use of the drug in new applica-
tions difficult. Cyclophosphamide inhibits monocyte precursor
development but has little effect on fully developed macrophages.
It is spectacularly effective in preventing the development of auto-
immune disease in the NZB/NZW F1 mouse model of systemic
lupus erythematosus. Cyclophosphamide is readily absorbed after
oral administration. The standard initial daily dose is 1–2 mg/kg.
The serum half-life is 7 h, and allopurinol prolongs that half-life.
Chlorambucil (Leukeran) (Fig. 30.3) is also readily absorbed
after oral administration. The standard initial daily dose is
306 0.1–0.2 mg/kg. The half-life in plasma is ~1 h, and the drug is
almost completely metabolized. It is the slowest-acting nitrogen
mustard in clinical use, and its cytotoxic effects on bone marrow,
lymphoid organs, and epithelial tissues are similar to those of
the other nitrogen mustards.
NONOPHTHALMIC USES AND POTENTIAL
SIDE EFFECTS
Cyclophosphamide is used extensively to treat Wegener’s granu-
lomatosis, polyarteritis nodosa, and other forms of systemic
vasculitis. It is still sometimes used to treat human allograft
recipients and often to treat bullous pemphigoid. It is some-
times used when severe rheumatoid arthritis is refractory to
more conventional therapy, and it is a common drug of choice
for nephrotic syndrome in children. It is also still sometimes
employed in the ‘polydrug’ approach to malignancies, including
multiple myeloma; chronic lymphocytic leukemia; lung, breast,
cervical, and ovarian carcinoma; neuroblastoma; retinoblastoma;
and some other neoplasms of childhood.
Potential complications of cyclophosphamide therapy include
severe bone marrow depression with resultant anemia, leuko-
penia, thrombocytopenia, and secondary infection; anorexia,
nausea, vomiting, hemorrhagic colitis, and oral mucosal ulcera-
tion; jaundice; hemorrhagic cystitis; gonadal suppression;
alopecia; and interstitial pulmonary fibrosis. Sterile hemor-
rhagic cystitis occurs in 5–10% of patients; this has been attri-
buted to chemical irritation of the lining of the bladder
produced by reactive metabolites of cyclophosphamide, particu-
larly acrolein. This potentially devastating complication, which
can lead to bladder carcinoma, can usually be avoided with
correct administration (i.e., restricting consumption of cyclo-
phosphamide to the early hours of the day and forcing fluid
intake during the remainder of the day). Acetylcysteine or mesna
(sodium 2-mercaptoethanesulfonate) can prevent or reverse
cyclophosphamide-induced hemorrhagic cystitis. If a patient
taking cyclophosphamide develops dysuria or microscopic hema-
turia, the physician should confirm that the patient is taking
the drug correctly and is adequately hydrated and should per-
form emergency cystoscopy to confirm that the source of the
blood is the lining of the bladder rather than the kidney. If, for
example, a patient being treated for Wegener’s granulomatosis
develops microscopic hematuria, cessation of cyclophosphamide
would be inappropriate if the red blood cells are coming from
Wegener’s inflammatory activity in the kidney rather than from
cyclophosphamide-induced cystitis.
Chlorambucil is still the treatment of choice for chronic lym-
phocytic leukemia and primary (Waldenström’s) macroglobuli-
nemia. It is also sometimes used to treat Hodgkin’s disease and
other lymphomas as well as vasculitis associated with rheuma-
toid arthritis and autoimmune hemolytic anemia with cold
agglutinins.
Potential complications of chlorambucil therapy include bone
marrow suppression, gastrointestinal discomfort, azoospermia,
amenorrhea, pulmonary fibrosis, seizures, dermatitis, and
hepatotoxicity. A marked increase in the incidence of leukemia,
lymphoma, and other neoplasms has been reported among
patients receiving long-term adjuvant chemotherapy for breast
cancer and patients being treated for polycythemia vera.
OPHTHALMIC INDICATIONS
Any patient who requires systemic immunosuppressive chemo-
therapeutic agents for an ocular inflammatory disease (OID)
must be managed by an experienced chemotherapist who is, by
virtue of formal training and experience, an expert in the use of
immunosuppressive drugs and in the recognition and treatment
of drug-induced side effects and potentially serious complications.
 Pharmacologic Treatment of Immune Disorders and Specifically of Immune Ocular Inflammatory Disease

CHAPTER 30

FIGURE 30.2. Diagrammatic representation of the mechanism of action of alkylating agents.

The present author’s experience suggests that, in general, the

FIGURE 30.3. Structural formula of chlorambucil.
chemotherapy experts with whom ophthalmologists can most
consistently and effectively collaborate are oncologists or hema-
tologists. The chemotherapist is completely responsible for the
chemotherapeutic aspects of the patient’s care. He or she per-
sonally sees the patient regularly; monitoring blood counts and
blood chemistry without seeing the patient is inappropriate
management. The ophthalmologist apprises the chemotherapist 307
 PHARMACOLOGY AND TOXICOLOGY
regularly of the status of the ophthalmic inflammatory condition.
If the problem is not sufficiently controlled, it is the chemo-
therapist who decides, for instance, whether or not it is safe and
appropriate to increase the patient’s immunosuppressive medi-
cations, to add a second medication with or without stopping the
initial one, or to supplement medications with systemic steroids.
Foster and associates’ published guidelines suggest initial doses
of various agents and one routine for careful hematologic moni-
toring, avoiding depressing the white count below 3500 cells/mm3
and the neutrophil count below 1500 cells/mm3.3 Foster also
suggests avoiding thrombocytopenia below 75 000 platelets/mm3,
including urinalysis every 2 weeks during the initial treatment
period, and then once a month when the patient is on a steady
maintenance drug program.
Cyclophosphamide is the treatment of choice for any patient
with ocular manifestations of Wegener’s granulomatosis or
polyarteritis nodosa. It is also unquestionably the most effective
treatment for patients with highly destructive forms of inflam-
mation in association with rheumatoid arthritis. Few other drugs
have allowed us to intervene successfully in the progression of
rheumatoid arthritis-associated necrotizing scleritis with asso-
ciated peripheral ulcerative keratitis. Interestingly, Watson and
Hazleman4 find that the necrotizing scleritis and peripheral
ulcerative keratitis in some patients with relapsing polychon-
dritis may be more refractory to therapy than that associated
with Wegener’s granulomatosis, polyarteritis nodosa, or rheuma-
toid arthritis. Although dapsone is commonly effective in the
extraocular manifestations of this disease, the author has rarely
SECTION 4
found it effective in abrogating ocular inflammation in this dis-
order. Cyclophosphamide, with or without oral steroid and non-
steroidal antiinflammatory drug therapy, is often required to
treat necrotizing scleritis associated with relapsing polychondritis.
Either cyclophosphamide or chlorambucil is an appropriate
choice for effective treatment of other OID, including
posterior uveitis or retinal vasculitis manifestations of
Adamantiades–Behçet’s disease. Chlorambucil may be the more
effective of the two, but cyclophosphamide, particularly when
given as intravenous pulse therapy, is highly effective. Baer and
Foster,5 and others6 find both drugs to be superior to cyclosporine
(cyclosporin A, CsA) in the care of patients with posterior seg-
ment manifestations of Adamantiades–Behçet’s disease.
Cicatricial pemphigoid affecting the conjunctiva usually
responds to cyclophosphamide therapy. If the patient with cica-
tricial pemphigoid has very active disease that is progressive,
cyclophosphamide is the drug of first choice. Therapy typically
lasts at least 1 year. The relapse rate after discontinuation of
cyclophosphamide is ~20%.7
The use of cyclophosphamide or chlorambucil in the treat-
ment of patients with other OID is slightly more problematic.
There is little question that each can be effective in the care of
youngsters with juvenile idiopathic arthritis (JIA)-associated
iridocyclitis that does not respond to steroids and other conven-
tional treatments, and that in this role these drugs can be sight-
saving. This is a complex area, however, given the age of the
patients and the potential risks for delayed malignancy or sterility
associated with the treatment. The relative risks and benefits
must be explored individually with patient and parents alike.
The author hopes that longitudinal comparative trials in this
patient group will help clarify the issue of relative risks and
benefits of systemic immunosuppressive chemotherapeutic treat-
ment early in the course of chronic iritis associated with JIA.
Other forms of uveitis that do not respond to conventional
treatment or are associated with intolerable steroid-induced side
effects may also respond to cyclophosphamide or chlorambucil
therapy. The guidelines for such an approach vary from clinic to
clinic around the world, but ample precedents exist for this
308 alternative in patients with slowly blinding uveitis.8–12 Whether
the patient has pars planitis or uveitis associated with Reiter’s
syndrome, with ankylosing spondylitis, with inflammatory bowel
disease, or even with ‘idiopathic’ uveitis, the author employs a
stepladder approach to the treatment of that patient’s ocular
inflammation, always using steroids first, and aggressively, via
all potential routes of administration (topical, periocular injec-
tion, intraocular, systemic) and in the largest doses tolerated. It
is typical to obtain informed consent and dispense printed
handouts that describe the potential risks of topical, periocular,
and systemic steroids. If, in spite of this approach, the patient’s
disease is chronic or relapses each time steroids are tapered or
discontinued, the author adds oral nonsteroidal antiinflam-
matory drugs to the treatment plan (with the patient’s consent).
If this combination does not achieve the goal of total quiescence
of all inflammation off all steroids, or if treatment-induced side
effects appear that are unacceptable to patient or doctor, the
patient is offered the alternative of immunomodulatory therapy
(IMT) with a systemic immunosuppressive chemotherapeutic
drug. The choice of that drug depends on the individual patient,
the particular disease, the patient’s age, and the patient’s sex.
Some of the entities the author has treated successfully with
systemic immunosuppressive chemotherapeutic agents, includ-
ing cyclophosphamide and chlorambucil, are as follows: sym-
pathetic ophthalmia; Vogt-Koyanagi-Harada syndrome; birdshot
retinochoroidopathy; multifocal choroiditis with panuveitis;
retinal vasculitis associated with systemic lupus erythematosus;
multifocal choroiditis associated with progressive systemic scle-
rosis; retinal vasculitis associated with sarcoidosis; pars planitis
associated with multiple sclerosis; severe uveitis associated with
ankylosing spondylitis, with Reiter’s syndrome, or with inflam-
matory bowel disease; idiopathic uveitis; and bilateral Mooren’s
ulcer,13 cicatricial pemphigoid; scleritis associated with relapsing
polychondritis with polyarteritis, with Wegener granulomatosis
and with rheumatoid arthritis. One series reported recently was
comprised of 28 patients with uveitis, 10 of them with JIA-
associated uveitis who had failed lesser immunomodulatory
strategies. Sixty-eight percent of the patients were able to
discontinue corticosteroid therapy with uveitis relapse, and 50%
had induction of drug-free durable remission.14
PURINE ANALOGS
CHEMICAL PROPERTIES AND MECHANISM
OF ACTION
Thiopurines, such as mercaptopurine and azathioprine (Imuran)
(Fig. 30.4), interfere with purine metabolism and, so, with
synthesis of DNA, RNA, and protein. Purine analogs interfere
with the synthesis of purine bases. They inhibit purine nucleotide
interconversion reactions and the formation and function of
coenzymes (such as coenzyme A), thereby inhibiting RNA and
DNA synthesis. These agents or their metabolites are incorpo-
rated into DNA and RNA, but that probably is not the locus of
their suppressive effect. These drugs must be converted to active
FIGURE 30.4. Structural formula of azathioprine.
 Pharmacologic Treatment of Immune Disorders and Specifically of Immune Ocular Inflammatory Disease
principles, predominantly in the liver. One such metabolically
active product is thioinosinic acid.
At clinical nontoxic doses of 2–3 mg kg⫺1 day⫺1, azathioprine
has little effect on humoral immunity. Immunoglobulin levels
and specific antibody responses are relatively unaffected. In experi-
mental systems, large doses of thiopurine given within 48 h of
antigen priming can suppress the antibody response and can
induce temporary tolerance to the antigen when given in con-
junction with large doses of the antigen.
Thiopurines appear to exert a relatively selective effect on
T lymphocytes: they prolong renal, skin, lung, and cardiac allo-
grafts; suppress mixed lymphocyte reaction in vitro; depress
recirculating T lymphocytes that are in the process of homing;
suppress development of monocyte precursor cells; inhibit par-
ticipation of K cells (which arise from monocyte precursors) in
antibody-dependent cytotoxicity reactions; and inhibit delayed
type hypersensitivity reactions. On the other hand, they do not
affect the onset or progression of the lupus-like autoimmune
disease in NZB/NZW F1 mice, and their immunosuppression
of renal transplant patients, for example, is partial because such
patients consistently show lymphocyte responsiveness in vitro
(proliferation, lymphokine production, cytotoxicity, cytotoxic
antibody) to donor antigen.
Mycophenolate mofetil (Cellcept), converted to mycophenolic
acid, inhibits inosine monophosphate dehydrogenase, which is
critical to de novo purine synthesis. It is administered orally at
1–3 g day –1.
NONOPHTHALMIC USES AND POTENTIAL
SIDE EFFECTS
Purine analogs, most notably azathioprine, are used extensively
in human heart, kidney, and lung allograft recipients. They have
also been used to treat blistering dermatoses (pemphigus vulgaris
and bullous pemphigoid), rheumatoid arthritis, and regional
ileitis (Crohn’s disease).
The author has suggested an initial dose of 2–3 mg kg⫺1 day⫺1;
dose adjustments are based on clinical response and drug
tolerance. Allopurinol inhibits xanthine oxidase and so inhibits
the conversion of azathioprine to its inactive metabolites; the
dose must be reduced accordingly.
Potential drug-induced complications of azathioprine therapy
include hepatotoxicity, severe bone marrow depression with
resultant anemia, leukopenia, thrombocytopenia, secondary infec-
tion, anorexia, nausea, vomiting, gastrointestinal distress,
diarrhea, rash, fever, and arthralgia. The most notable potential
adverse effect of mycophenolate mofetil is secondary infection.
OPHTHALMIC INDICATIONS
Azathioprine can be effective in patients with ocular inflamma-
tory manifestations of Adamantiades–Behçet’s syndrome.15 The
present author, however, has not found it to be the most effec-
tive drug for this purpose. Still, it can be effective and should be
included in every doctor’s therapeutic armamentarium for this
potentially devastating, frequently blinding disease. Andrasch and
co-workers9 rigorously studied azathioprine in the treatment of
uveitis of various causes. It was judged effective in 12 patients
and ineffective in 10, either because of drug-induced side effects
or because of inadequate response to treatment. Moore16 stopped
the inflammation associated with sympathetic ophthalmia, and
Hemady and associates17 have noted azathioprine’s effective-
ness in patients with JIA-associated uveitis that does not
respond to conventional steroid therapy. It also can be effective
in the treatment of cicatricial pemphigoid18 and in the care of
relapsing polychondritis-associated scleritis.19 The author has
also used it as a steroid-sparing drug for patients with multi-
focal choroiditis with panuveitis, sympathetic ophthalmia,
Vogt–Koyanagi–Harada syndrome, sarcoidosis, pars planitis, and
Reiter’s syndrome-associated iridocyclitis.
Mycophenolate mofetil has been shown to be effective in the
care of patients with ocular cicatricial pemphigoid,20,21 scleritis,22
uveitis,23,24 and orbital pseudotumor.25 Control of inflammation
with mycophenolate mofetil as monotherapy occurred in 65% of
a series studied by the author, with 18% of the patients requiring
discontinuation of the drug because of adverse events.23
FOLIC ACID ANALOGS
CHEMICAL PROPERTIES AND MECHANISM
OF ACTION
Methotrexate (Fig. 30.5), a folic acid analog also known as
amethopterin, binds to folic reductase, thus blocking the con-
version of dihydrofolic acid to tetrahydrofolic acid. This inter-
feres with thymidine synthesis and, so, with DNA synthesis
and cell division. Methotrexate has little effect on resting cells
but pronounced effects on rapidly proliferating cells. It affects
both B and T lymphocytes and can inhibit humoral and cellular
responses when administered during antigenic encounter. The
drug is excreted unchanged in the urine. Folinic acid can reverse
the metabolic block produced by methotrexate, thus rescuing
viable cells.
Methotrexate is absorbed after oral administration, but the
drug can also be given by intramuscular or intravenous routes.
CHAPTER 30
It is excreted unchanged in the urine within 48 h. Renal com-
promise delays excretion and causes undesirable side effects.
Consumption of sulfa drugs, salicylates, phenytoin, chloram-
phenicol, or tetracycline also increases the risk of methotrexate-
induced complications through displacement of methotrexate
from plasma proteins. The drug does not require metabolic
conversion to active principles. The concurrent use of drugs that
affect the kidney, such as nonsteroidal antiinflammatory agents,
can delay drug excretion and lead to severe myelosuppression.
Leucovorin ‘rescue’ may help reverse some methotrexate-
induced toxic effects.
5-Fluorouracil (5-FU) (Fig. 30.6) mimics uracil after intra-
cellular conversion to nucleotide and subsequent incorporation
into both DNA and RNA. The drug is especially toxic to rapidly
dividing cells.
NONOPHTHALMIC USES AND POTENTIAL
SIDE EFFECTS
Methotrexate is used to treat certain types of cancer, acute lym-
phoblastic leukemia, psoriasis, rheumatoid arthritis refractory
to conventional therapy, JIA, and sarcoidosis. Potential compli-
cations include severe bone marrow depression with resultant
anemia, leukopenia, and thrombocytopenia; cirrhosis and hepatic
atrophy; ulcerative stomatitis, nausea, vomiting, and diarrhea;
interstitial pneumonitis; malaise, fatigue, and secondary infec-
tion; rash; cystitis; nephritis; headache, blurred vision, and
drowsiness; and sterility. The hepatic fibrosis and cirrhosis
associated with methotrexate therapy are related to dose and
treatment duration, as well as to alcohol consumption. The risk
FIGURE 30.5. Structural formula of methotrexate. 309
 PHARMACOLOGY AND TOXICOLOGY
FIGURE 30.6.
Structural formula of
5-fluorouracil.
of this potentially devastating complication can be minimized
by administering it only once a week, insisting on total absti-
nence from alcohol, avoiding other drugs that may enhance the
effects of methotrexate, and monitoring the liver carefully and
regularly. 5-FU is used intravenously to treat metastatic breast,
SECTION 4
liver, pancreatic, colon, ovarian, prostatic, and bladder cancer.
Topical 5-FU is used to treat basal cell carcinomas.
OPHTHALMIC INDICATIONS
Idiopathic cyclitis,12 sympathetic ophthalmia,26 ocular mani-
festations of rheumatoid arthritis,27 and the uveitis of JIA are
particularly well suited for once-a-week therapy with oral metho-
trexate. Other varieties of OID, including uveitis including that
associated with Reiter’s syndrome, ankylosing spondylitis,
inflammatory bowel disease, or psoriasis, may also respond to
methotrexate. This drug may be sufficient to control scleritis
associated with the collagen diseases such as Reiter’s syndrome
and rheumatoid arthritis; the author has found it effective in
selected persons with progressive cicatricial pemphigoid. The
suggested regimen is 2.5–7.5 mg once a week, with gradual
escalation of the dose, as indicated by the clinical response, to a
maximum of 50 mg/week.
Regrettably, despite abundant published evidence to the con-
trary, most ophthalmologists consider methotrexate ‘dangerous’.
They undoubtedly remember the complications associated with
high-dose or daily methotrexate therapy in the care of patients
with a malignancy or with psoriasis. Liver toxicity and bone
marrow suppression were indeed prevalent in such patients.
Although the potential risk for such problems in patients treated
with a weekly low dose of methotrexate is not zero, the likeli-
hood of such a problem is clearly low, provided the patient is
managed and monitored correctly.28–33 Proper monitoring is
important; this obviously requires the involvement of an addi-
tional specialist and regular laboratory testing in these patients,
but the alternative of slow degeneration in visual function is
considerably more costly in both human and economic terms.
At the time of this writing, the sole ophthalmic application of
5-FU is subconjunctival injection after glaucoma filtering surgery
in an effort to prevent subconjunctival fibrosis and bleb failure.34
The primary toxic effect of subconjunctival 5-FU consists of
superficial punctate keratopathy and persistent corneal epithelial
defect.
310
SIGNAL TRANSDUCTION INHIBITORS
CHEMICAL PROPERTIES AND MECHANISM
OF ACTION
Cyclosporin A (CsA, Sandimmune, Neoral) (Fig. 30.7) is a fungal
metabolite originally isolated from cultures of Tolypocladium
inflatum Gams and Cylindrocarpon lucidum Booth by Sandoz
Laboratories as part of a screening program of fungal products
with antifungal activity. This undecacyclic peptide is also
produced by C. lucidum. Borel35 found that it had potent immu-
nosuppressive properties. Subsequent work in experimental
models showed the drug to be truly immunosuppressive and
capable of suppressing allograft reactions to heterotopic heart
allografts in rats. CsA also prolonged the viability of renal
allografts in dogs, heart allografts in pigs, and kidney allografts
in rabbits.
Tacrolimus (Prograf) is another fungus-derived immunosup-
pressant, isolated from Streptomyces tsukubaensis. It is struc-
turally similar to rapamycin (Fig. 30.8) and is ~100 times more
potent than CsA in preventing allograft rejection in animals.
Rapamycin (sirolimus, Rapamune) is a macrolide isolated from
an actinomycete.
The mechanism of action of CsA’s and tacrolimus’ immuno-
suppressive properties is incompletely understood, but the best
available evidence suggests that these drugs interfere with
receptors on the surface membranes of certain T lymphocytes
(particularly helper T cells) that recognize DR antigens on other
cells, most notably antigen-presenting cells like macrophages. A
17 kDa protein, cyclophilin, which is a cytosolic protein, binds
CsA and concentrates it intracellularly. Tacrolimus is similarly
bound by another family of immunophilins, FKBP or FK-506-
binding protein. These binding proteins are peptidyl–prolyl cis-
trans isomerases; at least 26 have been identified to date. DR
antigens participate in the production of interleukin-2 (IL-2) by
helper T lymphocytes by rendering the IL-2-producing T cells
sensitive to IL-1. CsA and tacrolimus interfere with helper T-cell
response to IL-1 and block IL-2 production or IL-2 release from
helper T cells. It appears that a complex composed of calcineurin
A, CsA, or tacrolimus, and the relevant immunophilin, inhibits
calmodulin binding, with resultant inhibition of a phosphatase
activity and consequent inhibition of transport of cytoplasmic
NF-AT and NFK6 into the nucleus; the result is inhibition of
IL-2 mRNA transcription. CsA and tacrolimus also may inhibit
IL-1 release from antigen-presenting cells such as macrophages.
Both inhibit expression of IL-3, IL-4, IL-5, and interferon-g.
FIGURE 30.7. Structural formula of cyclosporine.
 Pharmacologic Treatment of Immune Disorders and Specifically of Immune Ocular Inflammatory Disease
FIGURE 30.8. Structural formula of FK-506.
Rapamycin, unlike cyclosporine and tacrolimus, acts not
through calcineurin but rather through engagement of FKBP12,
creating complexes that bind the ‘target of rapamycin’ (TOR),
inhibition of which prevents/blocks signal transduction of
cytokine receptors (e.g., IL-2 and IL-4)
CsA and tacrolimus have a fairly selective suppressive effect
on T lymphocytes, which occurs early in the phase of T cell-
subset interactions. The drugs profoundly decrease antibody
production to T cell-dependent antigens, inhibit cytotoxic activity
generated in mixed leukocyte reaction, and prolong the life of
skin, kidney, and heart allografts in experimental animals and
humans. They also may prevent or mitigate graft-versus-host
disease and may prolong the life of other organ transplants, such
as pancreas and cornea.
NONOPHTHALMIC USES AND POTENTIAL
SIDE EFFECTS
CsA is used extensively for prevention of human allograft rejec-
tion and for the treatment of a variety of other diseases, includ-
ing psoriasis. Tacrolimus has been approved by the Food and
Drug Administration for prevention of human liver allograph
rejection. Potential side effects associated with systemic use of
CsA include an apparent increase in the incidence of B-cell
lymphomas, interstitial pneumonitis, and opportunistic infec-
tions, particularly from herpes simplex virus and Candida and
Pneumocystis organisms, as well as renal tubular necrosis with
compromise of kidney function.
OPHTHALMIC INDICATIONS
CsA may be particularly useful in the treatment of various
forms of posterior uveitis, especially when both retina and
choroid are involved in the inflammatory process and especially
if used as part of a multidrug IMT receipe. Thus, sympathetic
ophthalmia, Vogt–Koyanagi–Harada syndrome, multifocal
choroiditis with panuveitis, and posterior uveitis associated with
Adamantiades–Behçet’s syndrome may lend themselves to effec-
tive treatment with CsA. The author has been disappointed, how-
ever, with the effectiveness of CsA monotherapy compared with
cytotoxic immunosuppressive drugs in treating posterior uveitis
associated with Adamantiades–Behçet’s syndrome when the dose
of cyclosporine is in the acceptable range (5–7 mg kg⫺1 day⫺1)
from the standpoint of risk for kidney damage. Early enthusi-
astic reports of the effectiveness of CsA in the therapy of
Adamantiades–Behçet’s syndrome were based on dosing sched-
ules of 10 mg kg⫺1 day⫺1.29 Unfortunately, it was subsequently dis-
covered that all patients who consumed this dose of CsA long
enough to achieve the desired therapeutic effect in Behçet’s disease
developed renal damage from the drug. In the author’s expe-
rience, the lower, less toxic dose of 5–7 mg kg⫺1 day⫺1, is distinctly
inferior to azathioprine, chlorambucil, and cyclophosphamide
in the care of patients with ocular Adamantiades–Behçet’s disease.
Others report similar disappointment.30 In contrast, it is highly
effective in the care of patients with birdshot retinochoroido-
pathy, even at low doses.36 However, durable drug-free remissions
are much more likely to result from combination CsA-
mycophenolate mofetil IMT. CsA can be of enormous benefit in
the care of patients with severe eczema, especially those with
significant atopic keratoconjunctivitis. Topical CsA was investi-
gated for the treatment of corneal graft rejection and the results
were disappointing. It is, however, effective for keratoconjunc-
tivitis sicca. Two other antibiotics with immunosuppressive
properties that have ophthalmic indications are dapsone and
mitomycin C.
DAPSONE
Dapsone (4,4„-diaminodiphenylsulfone, Fig. 30.9) is a sulfone
used for the antibiotic treatment of leprosy. In addition to its
antibacterial activity, it is a myeloperoxidase inhibitor and stabi-
lizes lysosomal membranes. Its antiinflammatory and immuno-
suppressive effects are most dramatic in dermatitis herpetiformis
CHAPTER 30
and cicatricial pemphigoid. It is in the latter disease that oph-
thalmologists find it most useful. The author found that, pro-
vided the cicatrizing conjunctivitis of cicatricial pemphigoid is not
highly inflamed or rapidly progressive, dapsone halts progres-
sion of fibrosis in 70% of cases.18 And although dapsone may
help patients with relapsing polychondritis, Hoang-Xuan and
co-workers found that treating the scleritis of this disease with
dapsone was disappointing.19
Dapsone may produce profound hemolysis in patients deficient
in glucose-6-phosphate dehydrogenase, so any patient consid-
ered for dapsone therapy must first be evaluated for glucose-6-
phosphate dehydrogenase level. The author begins therapy with
25 mg twice daily; monitor the hemogram, reticulocyte count,
and methemoglobin level biweekly; and increase to as much as
150 mg/day if needed and if tolerated. Additional potential toxic
effects of dapsone include nausea, vomiting, hepatitis, periph-
eral neuropathy, blurred vision, psychosis, and a nephrotic-like
syndrome.
MITOMYCIN C
Isolated from Streptococcus calspitosus in 1958, mitomycin
(Fig. 30.10) reacts with DNA in ways similar to alkylating
agents. It cross-links DNA and inhibits its synthesis. It is a
highly effective antimitotic agent. It is used intravenously to
treat carcinoma of the stomach and colon and sometimes as
adjunctive therapy for cancer of the pancreas, breast, bladder, or
lung. The major systemic side effect is myelosuppression.
FIGURE 30.9. Structural formula of dapsone.
311
 PHARMACOLOGY AND TOXICOLOGY
FIGURE 30.10. Structural formula of mitomycin C.
The ocular indications for mitomycin C are recurrent ptery-
gium and glaucoma filtering surgery. Kunitoma and Mori37 and
later Choon and Fong38 reported favorably on the efficacy of
mitomycin C eye drops in preventing pterygium recurrence
after resection of pterygium that had recurred many times. Singh
and Foster confirmed these observations,39 and also studied giving
smaller doses of the drug than had been previously employed in
an effort to avoid toxicity, and they compared the efficacy of
topical mitomycin C with that of conjunctival transplantation for
treatment of recurrent pterygium.40 It is clear that topical mito-
SECTION 4
mycin C is effective in this role. It is clearly simpler and cheaper
than either conjunctival transplantation or b-irradiation. The
smallest effective dose and shortest duration of therapy are not
yet clear, however. Foster currently uses a single application of
0.02% at the end of surgery.
The efficacy of mitomycin C as an adjunctive component to
glaucoma filtering surgery is now well established, although, as
in pterygium surgery, in glaucoma surgery the ‘best’ concentra-
tion of the drug and best technique and duration of application
of the drug are not yet defined. The author applies it to the
scleral bed of the guarded trabeculectomy site, 0.4 mg/mL in
saturated cellulose sponges, with conjunctiva draped over the
sponges for 4 min, and then vigorously irrigate the area with
45 mL of balanced salt solution after removal of the sponges.
Potential complications of topical mitomycin C ocular ther-
apy appear to be limited to instances of abuse and negligence, to
drug dosage error, and to use of the drug in patients with ocular
surface disorders, such as sicca syndrome and ocular rosacea. The
author is aware of four cases of scleral or corneal ulceration after
such abuse. Applications were continued for 3–6 weeks after
surgery rather than the prescribed 1 week.
BIOLOGIC RESPONSE MODIFIERS
CHEMICAL PROPERTIES AND MECHANISM
OF ACTION
Heterologous antisera to leukocytes relevant to immune reactions
have been used experimentally for immunosuppression since
1956 and clinically in humans since the late 1970s. The most
extensively studied and widely used agent is antiserum prepared
against human lymphocytes. Various antilymphocyte serum (ALS)
preparations have been used; the most potent usually are obtained
after immunization of horses with human thymus or thoracic
duct cells. The greatest immunosuppressive activity usually
appears in the immunoglobulin G (IgG) fraction of the immu-
nized horse 2–4 weeks after immunization begins.
The effects of such antiserums after intravenous administra-
312 tion include leukopenia (highly immunosuppressive preparations
of ALS sharply reduce the number of T lymphocytes); depletion
of thymus-dependent areas in spleen and other lymphoid tissue;
inhibition of delayed hypersensitivity reactions; prolonged
viability of skin, renal, cardiac, liver, and lung allografts; and
suppression of primary and secondary antibody responses if the
antisera are given before antigen priming. Toxic effects of ALS
include anaphylaxis and possible tumorigenesis.
Monoclonal antibodies directed against T lymphocytes (anti-
OKT3 antibodies) have primarily the same effect as ALS, but their
effect is more limited, being aimed only at T lymphocytes rather
than all lymphocytes. Treatment with intravenous OKT3 anti-
bodies (Orthoclone) can reverse renal allograft rejection reactions.
Complications of anti-OKT antibody therapy include increased
risk of malignancy, fever, malaise, severe nausea, and vomiting.
Pooled human immunoglobulin (gammamune) is used not
only for passive immunization to modify hepatitis A, prevent or
modify measles, and provide replacement therapy for patients with
agammaglobulinemia, but also, in its immunomodulatory role,
to treat idiopathic thrombocytopenic purpura, and an expanding
array of other autoimmune diseases. It must be administered
intravenously or intramuscularly and must be given repeatedly
to achieve an immunomodulatory effect. Adverse reactions include
malaise, nausea, vomiting, fever, chills, headache, arthralgia,
and abdominal pain.
NONOPHTHALMIC USES AND POTENTIAL
SIDE EFFECTS
ALS has been used in humans predominantly for organ trans-
plantation, in conjunction with corticosteroid and cytotoxic
drug therapy (usually azathioprine). As mentioned earlier, anti-
OKT antibodies have been used exclusively in humans for
attempted reversal of kidney transplant allograft rejection.
Human immunoglobulin has been used principally as replace-
ment therapy for patients who are hypogammaglobulinemic or
agammaglobulinemic and in treating hepatitis A infections,
herpes zoster infections, and measles infections. Human immu-
noglobulin has also been used as an immunomodulatory agent
for idiopathic thrombocytopenic purpura and in the experi-
mental treatment of systemic lupus erythematosus and severe
atopic dermatitis. Its toxic effects include malaise, fever, chills,
headache, nausea, vomiting, shortness of breath, and back or hip
pain. Patients with prior allergic responses to immunoglobulin
may experience true anaphylactic reactions.
OPHTHALMIC INDICATIONS
To the present author’s knowledge, anti-OKT3 antibody therapy
has been used only once for an ophthalmic indication. The
author treated a woman with bilateral keratoconus whose body
was rejecting her fourth human leukocyte antigen-matched
corneal graft, in the right eye, in spite of aggressive topical,
regional injection, oral and intravenous pulse steroids, and
topical and systemic CsA therapy with seven days’ intravenous
OKT3 monoclonal antibody therapy. Her graft was saved, but
this expensive in-hospital effort was an exercise in heroics that
the author suspects will find little use in ophthalmology. Intra-
venous gamma globulin therapy has been used extensively in
the care of patients with severe eczema, and the author has used
this treatment modality in several patients whose severe atopic
keratoconjunctivitis did not respond adequately to strict envi-
ronmental controls and systemic antihistamine therapy. The drug
must be given each week, and the author prefers the intra-
venous route over the intramuscular one.
We have used IV–Ig to great effect in our care of patients with
ocular cicatricial pemphigoid which was inadequately respon-
sive to more conventional immunomodulatory agents.14
 Pharmacologic Treatment of Immune Disorders and Specifically of Immune Ocular Inflammatory Disease

Daclizumab (Zenapax) is a humanized monoclonal antibody
directed against the alpha chain of the CD-25 glycoprotein,
which is expressed on the surface of activated T lymphocytes. It
is approved and marketed for the treatment of solid allograft
rejections. We41 and others42 have shown that it can be remark-
ably safe and effective in the care of patients with otherwise
treatment-resistant ocular inflammation, particularly uveitis,
but also scleritis, atopic disease and cicatricial pemphigoid. The
author employs it at a dose of 1 mg kg –1, intravenous, every
2 weeks initially, infused over ~1 h.
TNF-a Inhibitors
Infliximab (Remicade) is a mouse–human monoclonal antibody
which neutralizes TNF-a. It is remarkably effective for the
arthritis associated with rheumatoid arthritis, for the dermatitis
associated with psoriasis, and for the colitis associated with
Crohn’s disease and with ulcerative colitis. Multiple authors
have reported small series, unmasked and uncontrolled, attest-
ing to its efficacy in treating various forms of uveitis.43–45 The
drug must be administered intravenously (5–10 mg kg –1 every
2–4 weeks) and it has been associated with development of
malignancies in some instances46,47 and with increased suscep-
tibility to infection and to reactivation of latent tuberculosis.
The present author’s experience suggests that, while treatment
failures are not rare, sufficient evidence for efficacy in sufficient
numbers of cases exists to encourage performance of a placebo-
controlled trial. (Sobrin L, Kim E, Christen WG, Papadaki T,
Letko E, Foster CS. Infliximab for the Treatment of Refractory
Ocular Inflammatory Disease, under review, Archives of Opthal-
mology). The same may be said for adalimumab (Humira) but
not for etanercept (Enbrel).48
Summary
• IMT is the standard of care for many patients with OID
• Ophthalmologists should partner with an ocular immunologist
or with a chemotherapist in order to provide their patients who
have OID with such standard of care
• The appropriate goal is durable remission of the OID: no
inflammation OFF all steroids

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16. Moore D: Sympathetic ophthalmia
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17. Hemady R, Baer JC, Foster CS:
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18. Foster CS: Cicatricial pemphigoid. Trans
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19. Hoang-Xuan T, Foster CS, Rice BA: Scleritis
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Augenheilkd 2001; 218:222–228.
21. Choudhary A, Harding SP, Bucknall RC,
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22. Siepmann K, Huber M, Stubiger N, et al:
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23. Baltatzis S, Tufail F, Yu EN, et al:
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24. Thorne JE, Jabs DA, Qazi FA, et al:
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27. Foster CS, Forstot SL, Wilson LA: Mortality
rate in rheumatoid arthritis patients
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peripheral ulcerative keratitis: Effects of
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Ophthalmology 1984; 91:1253.
28. Graham LD, Myones BL, Rivas-Chacon RF:
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29. Giannini EH, Brewer EJ, Kuzmina N, et al:
Methotrexate in resistant juvenile
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30. Tagwell P, Bennett K, Bell M, et al:
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31. Lehman TJA: Aggressive therapy for
childhood rheumatic diseases. Arthritis
Rheum 1993; 36:71.
32. Wallace CA, Sherry DD: Preliminary report
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33. Rose CD, Singsen BH, Eichenfield AH:
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34. Fluorocil Filtering Study Group: Fluorocil
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35. Borel JF: Comparative study of in vitro and
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36. Vitale AT, Rodriguez A, Foster CS: Low-
dose cyclosporin therapy in the treatment
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38. Choon LK, Fong CY: The pterygium and
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39. Singh G, Wilson MR, Foster CS: Mitomycin
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40. Singh G, Wilson MR, Foster CS: Long-term
follow-up study of mitomycin eye drops as
adjunct treatment for pterygium and its
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transplantation. Cornea 1990; 9:331.
41. Papaliodis GN, Chu D, Foster CS:
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with daclizumab. Ophthalmology 2003;
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et al. Initial evaluation of subcutaneous
daclizumab treatments for noninfectious
uveitis: a multicenter noncomparative
interventional case series. Ophthalmology
2005; 112:764–770.
43. Kahn P, Weiss M, Imundo LF, Levy DM:
Favorable response to high-dose infliximab
for refractory childhood uveitis.
Ophthalmology 2006; 113:864.
44. Rajaraman RT, Kimura Y, Li S, et al:
Retrospective case review of pediatric
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Ophthalmology 2006; 113:308–314.
45. Suhler EB, Smith JR, Wertheim MS, et al: A
prospective trial of infliximab therapy for
refractory uveitis: preliminary safety and
efficacy outcomes. Arch Ophthalmol 2005;
123:903–912.
46. Bongartz T, Sutton AJ, Sweeting MJ, et al:
Anti-TNF antibody therapy in rheumatoid
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meta-analysis of rare harmful effects in
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47. Bucher C, Degen L, Dirnhofer S, et al:
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48. Foster CS, Tufail F, Waheed NK, et al:
Efficacy of etanercept in preventing relapse
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Ophthalmol 2003; 121:437–440.
 CHAPTER
31 Angiogenic Factors and Inhibitors
Michael J. Tolentino, Anthony P. Adamis, and Joan W. Miller
INTRODUCTION
New blood vessel formation can occur either through angio-
genesis or vasculogenesis.1 Vasculogenesis is the formation of
new vessels from the differentiation of angioblasts that sub-
sequently form primitive blood vessels. Formation of new
blood vessels from preexisting microvasculature is called
angiogenesis. Angiogenesis can occur both physiologically and
pathologically. Physiologic angiogenesis occurs mainly in
females during menstruation, ovulation, and the development
of the placenta. Pathologic angiogenesis, on the other hand, can
occur in both sexes. In the fully developed adult, ocular angio-
genesis in most cases is pathologic and is a major component of
several blinding conditions. These conditions include age-
related macular degeneration (AMD), diabetic retinopathy
(DR), neovascular glaucoma, corneal neovascularization,
retinopathy of prematurity, and intraocular tumors and
represent some of the most common causes of blindness in the
United States. Understanding the cascade of events that results
in angiogenesis can hopefully elucidate ways to inhibit this
blinding process. In this chapter we discuss the steps involved
in new vessel formation, research techniques to study angio-
genesis, angiogenic factors involved in ocular neovascular-
ization, and newly discovered angiogenesis inhibitors.
STEPS IN ANGIOGENESIS
There are two types of angiogenesis: sprouting and non-
sprouting (intussusception).2 The events leading to sprouting
angiogenesis begin with dissolution of vessel basement mem-
brane and interstitial matrix. Angiogenesis occurs in response
to angiogenic factors that stimulate the migration and prolifer-
ation of vascular endothelial cells. Canalization is followed by
the formation of branches and loops of confluent sprouts that
eventually support blood flow. New vessels can then begin the
process of maturation and differentiation by the recruitment of
pericytes and the deposition of basement membrane signaling
the end of the neovascular cascade (Fig. 31.1). Nonsprouting
angiogenesis involves the proliferation of endothelial cells that
form a lumen within a preexisting vessel. Interstitial tissue
columns in the lumen of preexisting vessels grow, stabilize, and
partition the vessel lumen, resulting in new blood vessel
formation. Nonsprouting angiogenesis has been described more
in the embryonic lung and in tumor models; however, sprouting
and nonsprouting angiogenesis can occur concurrently.2
Intervention at each step of angiogenesis can be used to
inhibit or stimulate new vessel formation. A balance between
endogenous stimulators and inhibitors leads to the maintenance
of mature vessels and the control of physiologic neovascular-
ization. An imbalance results in pathologic neovascularization.
4. Differentiation 3. Proliferation 2. Migration
Angiogenic
stimuli
1. Dissolution of basement
membrane
and interstitial matrix
FIGURE 31.1. The cascade of angiogenesis begins with an
angiogenic stimulus that leads to the dissolution of basement
membrane and extracellular matrix. This allows the endothelial cell to
migrate and proliferate. After proliferation, the endothelial cell can
differentiate and recruit smooth muscle cells and pericytes, thus
signaling the end of neovascularization.
In ocular neovascularization, overexpression of a stimulator of
angiogenesis has been postulated since the late 1940s.3 It was
apparent then that hypoxia and ischemia result in a release of a
‘factor X’ that results in the formation of new blood vessel
growth.4,5 It is the identification of this factor X and the hope of
inhibiting its effect that have spurred interest in angiogenesis
research in ophthalmology.
ANGIOGENESIS RESEARCH
METHODOLOGY
The process of new blood vessel growth can be studied by
several in vitro and in vivo bioassays. Bioassays are required to
define the angiogenic properties of stimulators and inhibitors of
angiogenesis. In vitro endothelial cell chemotaxis, proliferation,
and lumen formation can be used to define angiogenic or
angiostatic activity. In vivo, there are many bioassays of angio-
genesis. The chick chorioallantoic membrane (CAM) assay is one
of the first in vivo assays used. The corneal neovascularization
micropocket model is probably the most widely used. Others
include chemical- or cautery-induced corneal neovascularization
models; the oxygen-cycling model of retinopathy of prematurity;
and retinal vein occlusion and laser-induced subretinal 315
 PHARMACOLOGY AND TOXICOLOGY
neovascularization models. A murine transgenic model of
retinal vascular endothelial growth factor (VEGF) upregulation
has been developed. Although these models do not fully mimic
true ocular disease, they can be used to test the in vivo effects
of angiogenic factors and inhibitors in the different vessel beds
of the eye.
IN VITRO ASSAYS
Capillary endothelial cell cultures were an important step to
study the angiogenic activity of various factors.6 This technique
allows the angiogenic process to be dissected into several steps.
With endothelial cell cultures, angiogenesis does not have to be
measured as an all-or-nothing event; three separate steps are
measurable: proliferation, motility, and capillary tube
formation.
Endothelial cell proliferation is measured by determining cell
counts, thymidine uptake, and other markers of cellular
proliferation and can be used to determine the endothelial cell
mitogenic activity of various compounds. In the presence of a
known angiogenic compound, cellular proliferation can be used
to screen for angiostatic compounds. Endothelial cell migration
can also be measured using the Boyden chamber assay.7 This
measures the chemotactic activity of various factors.
Capillary tube formation is measured in several ways. In
most cases it requires the growth of endothelial cells into a
three-dimensional collagen matrix to form tube-like structures
and lumens.8 A fragment of human placental blood vessel
SECTION 4
embedded in a fibrin gel can give rise to a complex network of
microvessels during a period of 7–21 days in culture.9 Similar
tube-formation models have been used to assay angiogenic
factors and to screen for angiogenic inhibitors.10–12 Fibrinolytic
activity of cell types may also be predictive of the successful
formation of capillary-like structures.13 The mechanism
underlying capillary formation in these in vitro assays is
dependent on the matrix the cells are grown on. Plating human
umbilical vein endothelial cells on Matrigel results in a
posttranslational-dependent capillary-like formation whereas
plating them on fibrin involves gene transcription and trans-
lation.14 These findings may be helpful in further dissecting the
angiogenic process.
IN VIVO MODELS
Chick CAM Assay
The CAM assay most commonly involves removing a fertilized
chicken egg from its shell and growing it in a culture dish.15
Potential angiogenic substances can be placed on the CAM to
assay their ability to induce angiogenesis. To quantify
angiogenesis, a collagen gel impregnated with an angiogenic
factor is situated between nylon mesh and placed on the CAM
surface. By counting the squares containing new vessels, one
can quantify angiogenesis.16,17 This assay has been used to
identify angiogenic factors and to test angiogenic inhibitors.18
Corneal Neovascularization Models
One of the most widely used angiogenesis assays involves the
implantation of an angiogenic stimulant into a corneal micro-
pocket, which induces vessel growth from the limbus toward
the stimulant (Fig. 31.2). Various models have been described
in mice, rats, and rabbits using endotoxin, basic fibroblast
growth factor, VEGF, and other angiogenic compounds
contained within sustained-release polymers.19–23 The rabbit
models offer the advantage of size, but the mouse models offer
the capability for genetic manipulation. A corneal micropocket
model in knockout or transgenic mice can be a useful assay to
316
FIGURE 31.2. Corneal neovascularization induced in a mouse cornea
by a Hydron pellet impregnated with basic fibroblast growth factor.
determine if a targeted endogenous factor can inhibit or accen-
tuate neovascularization. For these assays to be effective, the
bottom of the pocket has to be within a critical distance from
the limbus. Chemical cautery, epithelial scraping, and xenograft
corneal transplants have been used to develop injury-induced
models of corneal neovascularization.24,25
Branch Retinal Vein Occlusion Models
Retinal vein occlusion models in rabbits, pigs, cats, and
monkeys have been developed using diathermy and photo-
coagulation.26–29 Various degrees of retinal and iris neovas-
cularization have developed in these models. In a pig model,
photodynamic, laser-induced, branch vein occlusion develops
preretinal and optic nerve head neovascularization.30 A minia-
ture pig model of laser-induced branch retinal vein occlusion
develops only preretinal neovascularization.29
In monkeys, branch vein occlusions produce intraretinal
without preretinal neovascularization.31 When two temporal
retinal veins were occluded, iris neovascularization and disk
neovascularization developed in four of six monkeys. Occluding
three retinal veins and performing vitrectomy-lensectomy
resulted in 100% of monkeys developing iris neovas-
cularization, and two of 12 monkeys developed neovascular
glaucoma.32 The use of dye yellow laser produced iris
neovascularization in 70–95% of monkeys without the need
for vitrectomy–lensectomy (Fig. 31.3).33 A grading system
using standardized fluorescein iris angiograms and masked
readers provides semiquantitative analysis, allowing this
monkey model to be used in the evaluation of angiogenic
inhibitors.27,34,35
A laser-induced venous thrombosis rat model of preretinal
neovascularization has been described.28 With an argon
blue-green laser, 70% of the eyes developed retinal neovascular-
ization and traction retinal detachment. Retinal neovascular-
ization included optic disk neovascularization and
neovascularization elsewhere.
Retinopathy of Prematurity Models
Although constant high oxygen exposure was originally thought
to be the cause of retinopathy of prematurity (ROP), oxygen
fluctuations are a more likely cause. In retinopathy of
prematurity animal models, the developing retinal vasculature
is exposed to different cycles of relative hyperoxia and hypoxia.
The hyperoxia produces vasoconstriction of the immature

a b
FIGURE 31.3. Iris neovascularization and laser-induced branch retinal vein occlusions. (a) Laser-photocoagulated retinal veins in a monkey
retina. (b) Subsequent iris neovascularization. (c) Leakage of fluorescein into the anterior chamber, demonstrating florid iris neovascularization.
retinal vessels whereas hypoxia produces vasoproliferation
characteristic of retinopathy of prematurity.
Several species have been used, including rat, cat, mouse, and
dog.36–41 The models use alteration from high to low oxygen
levels in newborn animals to produce preretinal neovasculariza-
tion. In a rat model, alternating the oxygen levels from 40% to
80% for several days followed by room air produced histologi-
cally confirmed preretinal neovascularization in two-thirds of
the animals.37 In a newborn mouse model, 100% of the animals
developed histologically determined preretinal neovascularization
when placed in 75% oxygen for 5 days followed by room air.40
Laser-Induced Choroidal Neovascularization
A monkey model of choroidal neovascularization (CNV) was
first developed using laser-induced retinal vein occlusion and
disruption of Bruch’s membrane. The model was inconsistent,
and 30% of the monkeys developed retinal neovascularization,
with 33% developing vitreous hemorrhage.42 Argon laser burns
to the macular area without retinal vein occlusion produced a
higher percentage of monkeys with CNV (Fig. 31.4).43 Unlike
AMD, this model is injury induced, but the development of
CNV bears many similarities to that of AMD. The model
produces a membrane that leaks fluorescein into the subretinal
space.44 VEGF and, aVb3 integrin which have been implicated
in CNV, are also expressed in this model.45,46
Transgenic VEGF-Dependent Mouse Model
A transgenic mouse overexpressing VEGF in the retina has been
created. To produce a VEGF-induced transgenic model of retinal
and subretinal neovascularization, a bovine rhodopsin promoter
was linked to VEGF complementary DNA. This transgenic
a b
FIGURE 31.4. Laser-induced CNV. (a) Day 1 after laser treatment. (b) Four weeks after laser treatment, demonstrating subretinal
neovascularization. (c) Angiographically demonstrated CNV 4 weeks after laser treatment.
Angiogenic Factors and Inhibitors
c
mouse produced upregulation of VEGF in the photoreceptors
and very limited systemic expression of the transgene. Three
transgenic founders were described, and one resulted in
intraretinal neovascularization that grew into the subretinal
space.47 Although this pattern of retinal neovascularization is
not seen in disease, this model can be a useful means of
studying VEGF overexpression and its inhibitors in the eye.
Diabetes Models
Many models of diabetes have been developed using mice, rats,
CHAPTER 31
monkeys, and dogs.48–52 Both bred rats49 and streptozotocin-
treated rats51 have produced consistent models of diabetes.
Galactose-fed dogs can produce retinopathy similar to that seen
in diabetes.53 The Koletsky spontaneous hypertensive, non-
insulin-dependent rat was observed to have microangiopathic
retinopathy with progressive retinal capillary dropout, and
elevated vascular tortuosity with fluorescein leakage.52 The
Koletsky rat52 and galactose-fed dogs53 are the only two models
of diabetes that develop proliferative retinopathy.
ANGIOGENIC FACTORS IN OCULAR
NEOVASCULAR DISEASE
The discovery of specific factors that are operative in
angiogenesis has facilitated the accelerated pace of angiogenesis
research. Many angiogenic factors have been discovered to date
(Table 31.1), but the contributions of the majority to ocular
neovascular diseases have not been established. The remaining
discussion will focus on four factors for which evidence
supports this role: VEGF, angiopoietins (Ang), ephrins, and
platelet-derived growth factor-B (PDGF-B).
c
317
 PHARMACOLOGY AND TOXICOLOGY

TABLE 31.1. Pro-angiogenic and Anti-angiogenic Factors

VASCULAR ENDOTHELIAL GROWTH
FACTOR
Pro-Angiogenic Factors
Angiogenin
Key Features
Angiopoietin-1
• VEGF is the master regulator of physiological and pathological
Del-1
angiogenesis
Fibroblast growth factors: acidic (aFGF) and basic (bFGF) • Ocular neovascularization is an inflammatory process
Follistatin promoted by elevated levels of VEGF
Granulocyte colony-stimulating factor (G-CSF) • VEGF also promotes the inflammation-mediated vascular
Hepatocyte growth factor (HGF)/scatter factor (SF) damage characteristic of DR and DME
Interleukin-8 (IL-8) • The VEGF165 isoform is especially potent in mediating these
Leptin inflammation-related processes
• VEGF-targeted therapies (pegaptanib and ranibizumab) are
Midkine
approved for treating AMD; pegaptanib has also shown
Pigment epithelium derived growth factor efficacy in treating DME
Placental growth factor
Platelet-derived endothelial cell growth factor (PD-ECGF)
Platelet-derived growth factor-BB (PDGF-BB) VEGF AS THE KEY REGULATOR OF
Pleiotrophin (PTN) ANGIOGENESIS
Progranulin
Among the numerous factors that contribute to the control of
Proliferin
angiogenesis, only VEGF has proved essential for this process in
Transforming growth factor-alpha (TGF-a) the clinic.54 VEGF (also known as VEGF-A) was isolated on two
Transforming growth factor-beta (TGF-b) separate occasions in the 1980s, first as a tumor-derived factor
Tumor necrosis factor-alpha (TNF-a) that increased vascular permeability55 and subsequently as a
Vascular endothelial growth factor (VEGF) mitogen which showed high specificity for endothelial cells;
molecular cloning revealed that these substances were
Anti-Angiogenic Factors
SECTION 4

identical.56,57 Genetic knockout of only a single VEGF allele

Angioarrestin leads to embryonic lethality, demonstrating a critical contri-
Angiostatin (plasminogen fragment) bution for VEGF in embryonic vasculogenesis.58,59 Interestingly,
Antiangiogenic antithrombin III modest overexpression of VEGF (varying from 75% to an
Cartilage-derived inhibitor (CDI) approximate doubling depending on the tissue being examined)
CD59 complement fragment
also proved lethal to the embryo. Thus VEGF levels must be
closely regulated for development to proceed normally.60
Endostatin (collagen XVIII fragment)
VEGF acts through binding to two receptor tyrosine kinases,
Fibronectin fragment VEGFR-1 and VEGFR-2, which respond in typical fashion to
Gro-b ligand binding by activation of signal transduction cascades.61
Heparinases VEGFR-2 is principally responsible for mediating the effects of
Heparin hexasaccharide fragment VEGF on angiogenesis and vascular permeability.62 VEGFR-1
Human chorionic gonadotropin (hCG) has been implicated in mediating monocyte chemotaxis to
Interferon a/b/g VEGF,63,64 a process that may contribute to pathologic angio-
Interferon inducible protein (IP-10)
genesis,65–67 induction of matrix metalloproteinase-9,68 and
release of hepatic paracrine factors.69 Its functions may also
Interleukin-12
include negative regulation of VEGF by sequestering it, thereby
Kringle 5 (plasminogen fragment) making it less available to VEGFR-2.61
Metalloproteinase inhibitors (TIMPs) VEGF is a member of the VEGF-PDGF family (reviewed by
2-Methoxyestradiol Robinson and Stringer70 and by Ferrara61). The VEGF gene is
Pigment epithelium derived growth factor composed of eight exons and seven introns, with alternative
Placental ribonuclease inhibitor splicing resulting in six principal isoforms, containing 121, 145,
Plasminogen activator inhibitor 165, 183, 189, and 206 amino acids. VEGF165, the isoform that
Platelet factor-4 (PF4)
has been most intensively studied, is a heparin-binding, homo-
dimeric, 45 kDa glycoprotein; a significant fraction of VEGF165
Prolactin 16 kDa fragment
is bound to the cell surface and to the extracellular matrix.71
Proliferin-related protein (PRP) Both VEGF189 and VEGF208 are basic, demonstrate strong binding
Retinoids to heparin, and are largely sequestered in the extracellular
Tetrahydrocortisol-S matrix while VEGF121 is acidic, does not bind to heparin, and is
Thrombospondin-1 (TSP-1) freely diffusible.61
(TGF-b) Recently, it has been reported that alternative 3’ splicing of the
Vasculostatin VEGF gene leads to an alternate family of VEGF isoforms, vary-
ing only in the last six amino acids at the carboxyl terminus. These
Vasostatin (calreticulin fragment)
isoforms can bind to VEGFR-2 but cannot activate it.72,73 This
Reproduced, with minor adaptations, with permission from the Angiogenesis isoform family, termed VEGFxxxb, appears to constitute a group
Foundation. From: List of known angiogenic growth factors. In: Understanding
angiogenesis. Available: of physiological inhibitors of angiogenesis and may contribute to
http://www.angio.org/understanding/content_understanding.html; accessed 9 regulation of angiogenesis since downregulation of these iso-
October 9, 2006. forms has been reported in several cancers72,73 as well as in DR.74
318

A variety of different molecular pathways are involved in
VEGF-mediated vasculogenesis and angiogenesis. First, as men-
tioned, VEGF acts as a potent mitogen, with endothelial cells
being the primary targets, although mitogenic effects have been
found in other cell types, including pancreatic duct cells,75
Schwann cells,76 and the retinal pigment epithelium.77 VEGF
can mobilize endothelial cell precursors from the bone marrow
during vasculogenesis78 as well as in pathologic conditions such
as tumor angiogenesis and CNV.79,80 It also promotes the sur-
vival of retinal endothelial cells through the inhibition of apop-
tosis81 and induces them to express and secrete plasminogen
activator82 and matrix metalloproteinases.68,83 These actions
facilitate the growth of blood vessels through the surrounding
stroma and may contribute to an amplification of local VEGF
concentrations, since plasmin can release VEGF from the matrix,71
and matrix metalloproteinases can cleave matrix-bound VEGF
to release active amino-terminal fragments.84 In addition,
VEGF upregulates endothelial nitric oxide synthase, increasing
the production and release of nitric oxide; nitric oxide not
only stimulates angiogenesis but can induce greater synthesis
of VEGF.85–87
Two properties of VEGF that are particularly relevant in
the context of ocular neovascular disease are its actions on
vascular permeability and its regulation by hypoxia. First,
hypoxia is a key positive regulator of VEGF mRNA expres-
sion,88–91 which is mediated through upregulation of hypoxia-
activated transcription factor-161 and may be important in
promoting ocular neovascularization in such conditions as
retinopathy of prematurity and DR. Secondly, VEGF is the most
potent known enhancer of vascular permeability, some 50 000
times more effective than histamine,92 which contributes
significantly to the macular edema and the attending vision loss
in such conditions as AMD and diabetic macular edema
(DME). Both indirect and direct effects contribute to VEGF-
mediated vascular permeability. Its direct effects include the
induction of fenestrations in the plasma membrane of
endothelial cells93 and the dissolution of tight junctions
between cells.94 Indirect mechanisms involve the VEGF-
mediated upregulation of endothelial cell expression of
adhesion molecules such as intercellular adhesion molecule-1
(ICAM-1), which promotes the adhesion of leukocytes that act
to damage the endothelium.95,96
Finally, it should be noted that while the main body of VEGF
research has been premised on its potential as a target in
promoting pathologic angiogenesis, it is becoming increasingly
clear that VEGF is a pleuripotent growth factor, acting in a
variety of contexts, some related to its role in promoting angio-
genesis, and others quite independent of it. Recent work has
demonstrated that VEGF is required for trophic maintenance
of capillaries,97 and regression of the normal vasculature has
been observed in response to nonselective VEGF inhibition.98
In addition, VEGF is known to be important for processes such
as bone growth,99,100 female reproductive cycling,99,101 wound
healing,102,103 vasorelaxation,104 kidney development and
function,105,106 skeletal muscle regeneration107 and protection
of hepatic cells against hepatotoxins.69 Surprisingly, VEGF
has been found to play a key role in neural survival and
may offer a therapeutic strategy against diseases such as
amyotrophic lateral sclerosis.108 This neuroprotective action
may be important in maintaining the health of retinal neurons,
since VEGF has been shown to promote their survival in
conditions of ischemia.109 Finally, conditional gene knockout
experiments have established that VEGF is essential for
development of the choroicapillaris in mice110 while its
secretion by the retinal pigment epithelium provides trophic
support for this tissue.111
Angiogenic Factors and Inhibitors
VEGF IN OCULAR NEOVASCULAR DISEASES
A major research effort has established a causative role for
VEGF in pathologic ocular neovascularization. Clinical studies
have correlated elevations of VEGF in a variety of ocular
diseases while studies in preclinical model systems have helped
to elucidate the cellular and molecular mechanisms con-
tributing to VEGF-mediated pathogenesis of these conditions.
This review will focus primarily on two major areas of in-
vestigation, namely, the role of VEGF in promoting CNV as
well as the importance of VEGF in the etiology of DR and its
associated condition DME. Clinical studies have also demon-
strated elevated levels of VEGF in iridal neovascularization,112
retinal vein occlusion,112 neovascular glaucoma,113 and
retinopathy of prematurity.114
Elevation of Vitreous Levels of VEGF in Ocular
Neovascular Diseases
VEGF is produced by many cell types in the retina,90,115,116 and
a series of studies has confirmed that VEGF is elevated in the
ocular fluid in the majority of patients suffering from ocular
neovascularization but only rarely in those where neovascular-
ization was absent.112,117 Studies of eyes removed at autopsy
demonstrated elevated levels of VEGF in both the retinal pig-
ment epithelium and in choroidal blood vessels of maculae with
AMD when compared to control maculae.118 Several groups
have reported that VEGF was overexpressed in retinal pigment
epithelial cells of surgically excised CNV membranes.119,120
CHAPTER 31
These early studies included patients with DR, with the
proliferative form being associated with higher ocular levels of
VEGF than the nonproliferative form.112,117 There have since
been additional studies confirming these initial reports,121–123
although it was recently reported that VEGF levels were higher
in eyes with nonproliferative DR as compared to the pro-
liferative form.124 In patients with DR, elevations in VEGF also
have been found in association with increased levels of other
growth factors, including interleukin-6,122 stromal-derived
factor-1,123 angiopoietin 2,125 and erythropoietin.126 In DME,
similar correlations have been established between vitreous
levels of VEGF and angiotensin II,127 interleukin-6,128 stromal-
derived factor-1,123 and ICAM-1.129 In some cases, these corre-
lations may reflect the interdependence of VEGF and other
cellular constituents; for example, VEGF induces the expression
of ICAM-1,95,130 while VEGF expression is itself upregulated by
angiotensin II131,132 and stromal-derived factor-1.133 In-
terestingly, in several studies elevated VEGF levels in diabetic
eyes were found to be accompanied by reduced levels of pigment
epithelium derived-factor,124,134,135 which has been reported
to downregulate VEGF expression.136 In contrast to VEGF,
expression of pigment epithelium-derived factor is down-
regulated by hypoxia and upregulated by hyperoxia.137 Finally, a
recent study has provided evidence that expression of the
VEGFxxxb isoform family may be relevant to the etiology of
DR.74 VEGFxxxb constituted 64 ± 7% of the total vitreous VEGF
in 18 control patients compared to only 12.5 ± 3.6% in 13
diabetic patients (p < 0.001), suggesting that development of
DR is accompanied by a switch in splicing from predominantly
nonangiogenic VEGF isoforms to the angiogenic isoforms.74
Preclinical Models Demonstrate the Importance of
VEGF in Ocular Neovascularization
Both AMD and DR are diseases with a complex patho-
physiology resulting from changes over time in the phy-
siochemical structures of the eye and ultimately resulting in
neovascularization. In AMD, the aberrant blood vessels
originate in the choroid while in DR they proliferate from
319
 PHARMACOLOGY AND TOXICOLOGY
retinal blood vessels.138,139 In neither case is there an animal
model that adequately replicates the clinical course of these
diseases, but important insights have nonetheless been gleaned
from experimental systems in which ocular neovascularization
can be induced. In addition to neovascularization, an important
component of the pathology of DR involves damage to the
existing retinal vasculature resulting in excess permeability and
leakage. Both the neovascularization and the vascular damage
appear to be mediated by inflammatory processes in which
VEGF plays a key role, with the VEGF165 isoform behaving as
an especially potent inflammatory cytokine.
Numerous experimental studies using a variety of approaches
have established that elevating VEGF in the eye results in
ocular neovascularization, while inactivating VEGF inhibits its
development. In an early study involving experimental iris
neovascularization induced by laser occlusion of retinal veins
in monkeys, VEGF levels increased in direct proportion to the
degree of induced neovascularization.27 Direct injection of
VEGF into monkey eyes resulted in iris, intraretinal, and
preretinal neovascularization.140–142 The induced blood vessels
were aberrant,142 showing evidence of endothelial cell hyper-
plasia and the excessive tortuosity and leakiness that are
characteristic of CNV.143 Similar neovascular responses have
been induced by overexpression of VEGF from transfected
recombinant DNA in rodents144,145 and in transgenic mice
engineered to overexpress VEGF in the retina.146,147
Several strategies have been employed to demonstrate that
inactivation of VEGF results in inhibition of ocular neovas-
SECTION 4
cularization whether in the iris,35,148 retina,65,149–151 choroid,152
or cornea.25 Agents employed have included VEGF receptor
fusion proteins149 or transfected DNA constructs expressing the
same,150 antibodies to VEGF,25,35 an antibody fragment binding
to VEGF,152 an anti-VEGF165 aptamer,65 and an antisense oli-
gonucleotide against the VEGF coding sequence.148 In an inter-
esting recent finding, studies in a murine model of retinopathy
of prematurity determined that intravitreous injection of
VEGF165b, one of the family of inhibitory VEGF isoforms,
resulted in a significant reduction of the pathologic neovas-
cularization that is normally induced after exposure to an
elevated oxygen environment.153
Finally, it was reported that intravitreous injection of
VEGF164, usually considered to be exclusively proangiogenic,
can be inhibitory to the development of CNV caused by laser
injury in mice.154 In these studies, the effect of VEGF was
proangiogenic when the injection was performed prior to injury
and inhibitory when the injection followed the wounding. The
inhibitory effect involved a complex interaction between VEGF,
VEGFR-1, and VEGFR-2 and was modulated by the activity of
SPARC (secreted protein, acidic, rich in cysteine).154 It remains
to be established whether these findings are specific to the laser
wounding model of CNV or whether they also have relevance to
neovascularization in the clinical setting.
The Role of Inflammation in the Pathogenesis of
AMD and DR
One major theme that has emerged from these studies is the
inflammatory nature of both AMD155–157 and DR.158 Supporting
evidence comes from studies demonstrating that macrophages,
important mediators of inflammation, are present in surgically
excised CNV membranes120,159 and that induction of
experimental CNV was suppressed in the absence of
macrophages.65,66,160 In this context, VEGF165 has been found to
act as a potent inflammatory cytokine.65
Other evidence that inflammation contributes to ocular
neovascular diseases derives from studies showing that certain
haplotypes of factor H, a regulatory component of the com-
320 plement cascade, are associated with an increased risk of
developing AMD157 and laser-induced experimental CNV is
dependent on factor C3, another component of the complement
system;161 this dependence may reflect the importance of C3 in
upregulating VEGF expression in this model.162 AMD also has
been associated with elevated systemic levels of C-reactive pro-
tein, a marker of inflammation,163 as well as ocular Chlamydia
infection.164 Finally, some patients suffering from AMD165 and
DME166 have experienced regression of their lesions when
treated with intravenous infliximab, an antibody against tumor
necrosis factor-a, a major inflammatory cytokine.
The pathophysiology of DR is associated with the
accumulation of polyols and advanced glycation end products,
oxidative damage, and activation of protein kinase C.139,167 This
leads to alterations in the retinal vasculature characterized
by the death of pericytes, thickening of the basement mem-
brane, and adhesion of leukocytes to the endothelium that
contribute to blockages and capillary dropout resulting in local
hypoxia.139,168 In turn, hypoxia is believed to contribute to local
upregulation of VEGF.169 In addition, reactive oxygen inter-
mediates,170 advanced glycation end products,171 and insulin-
like growth factor172 are believed to directly stimulate the
expression of VEGF.
There is now a substantial body of evidence linking eleva-
tions in ocular VEGF levels with damage to the existing retinal
vasculature. This process appears to be mechanistically related
to the pruning of the retinal vasculature in normal
development, a process whereby local adhesion of leukocytes
induces endothelial cell apoptosis.173 Much of our information
has come from a rodent model of diabetes, which is induced by
intraperitoneal injection of streptozotocin, with a key
mechanism being the VEGF-mediated upregulation of ICAM-1.
In common with clinical findings in patients with DR,174 the
onset of diabetes in the rodent model is accompanied by
increased expression of ICAM-1 together with increased retinal
leukostasis; capillary blockage by the leukocytes then leads to
local nonperfusion and leakage, phenomena that can be
prevented by the administration of an antibody directed against
ICAM-1.175 This treatment also reduces the leukostasis-related
injury and death of endothelial cells.96
In the diabetic model, retinal VEGF levels are increased by
3.2-fold after 1 week; this increase is accompanied by increased
vascular permeability and breakdown of the blood–retinal
barrier.176 These effects, as well as the increases in ICAM-1
and retinal leukocyte adhesion, can be significantly reduced by
the inactivation of VEGF through the administration of a
soluble VEGFR–Fc fusion protein.176,177 Reductions in
leukostasis, endothelial cell injury, and the number of acellular
capillaries have been seen in transgenic mice that lack either
ICAM-1 or its ligand on leukocytes, CD18.178 Taken together,
these experiments support a mechanism in which the increased
expression of VEGF in turn leads to increased ICAM-1
synthesis by the endothelial cells followed by increased
leukocyte adhesion and the resultant vascular injury.
The final step in the inflammatory damage is believed to
involve Fas/Fas ligand-mediated apoptosis. During the deve-
lopment of streptozotocin-induced diabetes, FasL expression
was found to be upregulated in neutrophils while Fas expression
was upregulated in the retinal vasculature.179 Systemic
administration of an anti-FasL antibody significantly inhibited
endothelial cell apoptosis as well as the breakdown of the
blood–retinal barrier.179
VEGF165 as Key Mediator in Pathologic Ocular
Neovascularization
Detailed studies of neovascularization in rodent models have
provided new insights into the molecular and cellular events
underlying the response to retinal ischemia and have implicated

one VEGF isoform, VEGF165, as being especially important in
mediating pathologic neovascularization through its
acceleration of inflammatory processes. Ishida et al65 used a rat
retinopathy of prematurity model to compare pathologic retinal
neovascularization to the physiologic neovascularization that
normally occurs in postnatal rats. Compared to physiologic
neovascularization, retinal VEGF expression was dramatically
enhanced during pathologic neovascularization; moreover the
ratio of VEGF164/VEGF120, which was 2.2 ± 1.1 in physiologic
neovascularization, increased to 25.3 ± 8.7 in the pathologic
form (VEGF164 and VEGF120 are the respective rodent versions
of the human VEGF165 and VEGF121 isoforms).65
Studies also revealed that VEGF164 was approximately twice
as potent as VEGF120 in promoting monocyte chemotaxis.130
This finding is notable in that pathologic neovascularization
has been shown to be accompanied by an influx of adherent
leukocytes and was inhibited by inactivation of monocyte
lineage cells;65 these cells may contribute to pathologic neo-
vascularization by secreting VEGF,180,181 thus promoting local
amplification of inflammation. Other experiments involving
laser-induced CNV models demonstrated that the development
of CNV was inhibited when macrophages were depleted with
clodronate liposomes66,160 or in knockout mice lacking
chemokine receptor CCR2, the receptor for monocyte chemo-
attractant protein-1.182 Other evidence for the importance of
leukocytes in experimentally induced CNV comes from studies
demonstrating a reduction in the severity of CNV in mice with
targeted inactivation of either ICAM-1 or the leukocyte
adhesion molecule CD18.67
Another key finding is that intravitreous injection of
pegaptanib, which binds to VEGF164 but not to VEGF121,
inhibited leukocyte adhesion and pathologic ocular neo-
vascularization while leaving physiologic neovascularization
unaffected. In contrast, injection of a VEGFR-Fc fusion protein,
which binds to all isoforms of VEGF, inhibited both physiologic
and pathologic ocular neovascularization (Fig. 31.5).65 It is of
particular interest that VEGF120/188 mice, lacking VEGF164
entirely, develop normal retinal vasculature.65
Studies in the diabetic rat model provided further important
evidence of the specific inflammatory nature of VEGF165 and of
the potential therapeutic value that could result from its
Pathologic neovascularization
1.0
Control
0.8 VEGF164-selective blockade
Nonselective VEGF blockade
P<.01
Area (mm2)
0.6
0.4
0.2
a 0.0
FIGURE 31.5. The role of VEGF164 in pathologic and physiologic retinal neovascularization. (a) Both nonselective blockade with a VEGFR–Fc
fusion protein and blockade of VEGF164 with a pegylated anti-VEGF aptamer significantly inhibited pathologic retinal neovascularization; (b) The
VEGFR–Fc fusion significantly inhibited physiologic retinal neovascularization, but it was not impaired by blocking VEGF164.
Adapted from Ishida S, Usui T, Yamashiro K, et al: VEGF164-mediated inflammation is required for pathological, but not physiological, ischemia-induced retinal
neovascularization. J Exp Med 2003; 198:483–489.
Angiogenic Factors and Inhibitors
inactivation. Injection of VEGF164 into the eyes of nondiabetic
rats was approximately twice as potent as the administration
of VEGF120 in inducing upregulation of ICAM-1 and leukocyte
adhesion, as well as in promoting blood–retinal barrier
breakdown.183 In parallel experiments with diabetic rats, the
injection of pegaptanib, which specifically targets VEGF165/164,
significantly inhibited leukostasis and blood–retinal barrier
breakdown both in early and in late diabetes.183 Taken together
with the finding that inactivation of VEGF165/164 is especially
potent in mediating ischemia-related neovascularization,65
these findings provided support for subsequent trials
investigating pegaptanib for the treatment of AMD and DME.
VEGF INHIBITION IN THE TREATMENT OF
OCULAR NEOVASCULAR DISEASES
The strategy of targeting VEGF for the treatment of ocular
neovascular diseases is based on the premise that inactivation
of a major regulator of angiogenesis should offer therapeutic
benefits for patients with such conditions. The strategy has
proved successful, yielding two therapies, pegaptanib184,185 and
ranibizumab186,187 both of which are administered by intra-
vitreous injection for the treatment of neovascular AMD.
Pegaptanib has shown excellent long-term safety.188 In a first for
an AMD therapy, ranibizumab was shown to improve the mean
visual acuity of patients. In contrast to the laser-ablative
approaches, both pegaptanib and ranibizumab are indicated for
all angiographic subtypes of AMD, effectively obviating the
CHAPTER 31
need for angiographic classification of patients prior to
determining their suitability for treatment.
In addition, in a phase 2 trial involving 172 patients with
DME,189 those receiving intravitreous pegaptanib had better
mean visual acuity than those receiving sham injections as well
as a greater likelihood of reduced central thickness and a lesser
need for photocoagulation therapy; furthermore, many of those
patients who had retinal neovascularization experienced
regression of neovascularization in response to pegaptanib
treatment.190 Similar results were also obtained in a recent
phase 2 trial involving 98 patients testing pegaptanib as a
treatment for macular edema secondary to central retinal vein
occlusion.191
Physiologic Revascularization
30
P<.01
Control
VEGF164-selective blockade
20 Nonselective VEGF blockade
Area (mm2)
10
b 0
321
 PHARMACOLOGY AND TOXICOLOGY
CONCLUSIONS
VEGF is a key mediator of angiogenesis and contributes to
ocular neovascular disease through its effects as an endothelial
cell mitogen, vascular permeability factor, and inducer of
inflammation. Clinical trials demonstrating the efficacy of
VEGF blockade in diseases such as AMD and DME confirm the
important role of VEGF in ocular neovascular diseases and
validate the strategy of attacking the pathogenesis of ocular
neovascular diseases with molecularly targeted agents. Future
therapeutic approaches combining anti-VEGF therapies with
agents targeting other molecular components involved in
angiogenesis may provide even better efficacy.
PLATELET-DERIVED GROWTH FACTOR-B
Key Features
• PDGF-B is a growth factor structurally related to VEGF
• The contributions of PDGF-B to angiogenesis are mediated
largely through its effects on mural cells such as pericytes and
vascular smooth muscle cells
• Mural cells are recruited to the endothelium primarily in
response to endothelial cell-secreted PDGF-B, which activates
PDBF-b receptors on the mural cells and stimulates their
migration and proliferation
• Vasculature that is stably covered with mural cells is largely
resistant to the effects of VEGF withdrawal, making it less
SECTION 4
susceptible to therapeutic intervention with VEGF blocking
agents
• Blocking both VEGF and PDGF may provide improved efficacy
in the treatment of established ocular neovascular lesions
INTRODUCTION
Members of the PDGF family, so named because the first PDGF
was isolated from platelets,192 are dimeric proteins composed
of four different polypeptide chains, PDGF-A-D (reviewed
by Fredriksson et al193). Most PDGFs are homodimeric (i.e.,
PDGF-AA and PDGF-BB), although heterodimers can form
between A and B chains, forming PDGF-AB. All PDGF chains
are structurally related to VEGF and share a highly conserved
growth factor domain. PDGF is produced by a wide range of
different cell types, including fibroblasts, vascular smooth
muscle cells, endothelial cells, retinal pigment epithelial cells,
and macrophages (reviewed by Helden and Westermark194).
Although most of these cell types make both A and B chains,
their expression is differentially regulated.195
The receptors for PDGF are two related tyrosine kinases:
PDGF receptor (PDGFR)-a and PDGFR-b; due to their dimeric
nature, PDGFs can interact with two PDGF receptors simul-
taneously, promoting PDGF receptor dimerization and
autophosphorylation.196 PDGF-A chains can bind to only
PDGFR-a, whereas PDGF-B chains can interact with both
PDGFR-a and PDGFR-b; therefore, homodimeric PDGF-BB can
induce dimerizeration of three combinations, PDGF-aa,
PDGF-ab, and PDGF-bb.194,197 PDGF-b has been detected in
many cells that play a role in angiogenesis, including vascular
smooth muscle cells, capillary endothelial cells, pericytes,
retinal pigment epithelial cells, myeloid hematopoietic cells,
and macrophages; the expression of PDGFR-a appears to be
more restricted though it is notable that platelets express only
PDGFR-a.194
PDGFs have wide-ranging functions, including roles in
embryonic vascular and central nervous system development,
322 wound healing, atherosclerosis, and kidney fibrosis.194 PDGF-B
(used hereafter to describe the homodimeric form PDGF-BB)
has been found to be important in angiogenesis, particularly in
respect to its effects on the recruitment of vascular smooth
muscle cells and pericytes to areas of neovascularization.198
This section will focus on the role of PDGF-B as an angiogenic
factor, with emphasis on its contribution to pathologic ocular
neovascularization.
PDGF-B IN ANGIOGENESIS
In normal embryos, PDGF-B is expressed by vascular endo-
thelial cells and megakaryocytes while expression in neurons
and macrophages occurs postnatally.199 Knockout mice deficient
for PDGF-B die perinatally, with abnormal renal glomerular
development, hemorrhages, thrombocytopenia, and anemia.200
PDGFR-b expression in embryonic mice occurs primarily in
pericytes, with larger arteries being surrounded by several layers
of PDGFR-b-positive mesenchymal cells.201 The phenotype for
PDGFR-b knockout mice is very similar to that of PDGF-B
mice,202 suggesting that PDGF-B mediates its effects largely
through its interactions with PDGFR-b.
Early studies evaluating the potential angiogenic effects of
PDGF-B reported that cultured rat microvessel endothelial cells
expressed both PDGF receptor a and b chains; PDGF-B was
mitogenic for these cells while PDGF-A was not; PDGF-AB was
mitogenic, but the effects were not as great as with PDGF-B.203
Other studies reported that PDGF-B stimulated proliferation of
human microvascular endothelial cells204 and was involved in
formation of capillary-like tubes in cultured bovine aortic
endothelial cells.205
Subsequent work has clarified the role of PDGF in angio-
genesis to be particularly important in recruitment of mural
cells expressing PDGFR-b to developing vasculature. Mural cells
include pericytes and vascular smooth muscle cells. Pericytes
are solitary cells associated with small vessels such as arterioles,
capillaries, and venules and share their basement membrane
with the endothelium while vascular smooth muscle cells form
concentric layers around larger vessels such as veins and
arteries.198 Characterization of PDGF-B knockout mice
identified a specific defect in which pericyte loss resulted in
capillary microaneurysms due to instability of the capillary
walls.201 It was further demonstrated that embryos lacking
PDGF-B or PDGFR-b expression had reduced proliferation of
mural cell progenitors, which normally proliferate at sites of
endothelial PDGF-B expression.198 These findings are
consistent with a model in which PDGF-B that is released by
the endothelium drives vascular smooth muscle recruitment
and migration, resulting in abnormal vasculature when PDGF-
B is not present (Fig. 31.6).198
The localization of PDGF in the pericellular space may be
important in mediating its effects. In studies involving an
endothelial cell line that overexpresses PDGF-B, the majority of
newly synthesized PDGF-B was found to be associated with the
cellular matrix through an interaction with heparin sulfate
proteoglycans and was released in response to a-thrombin.206
The carboxyterminal heparin-binding motif, which is highly
analogous to a similar motif found in certain isoforms of VEGF,
was found to be important in maintaining normal growth and
fertility in knockout mice.207 Mice lacking this domain had a
reduction in pericyte density associated with partial
dissociation of pericytes from the vasculature, consistent with a
model in which PDGF-B retention is required for the formation
of depots or gradients that confine pericyte migration to the
abluminal surfaces of microvessels.207
There is also evidence that VEGF and PDGF-B may work
together in promoting angiogenesis. For example, PDGF has
been found to induce VEGF expression in endothelial cells208

PDGF-B driven
Wild type
vSMC proliferation
and migration
PDGF-B
PCGF-B
PDGF-B
vSMC induction
PDGF-B
Reduced vSMC
proliferation
and migration
PDGF-B or
PDGFA-b
knock-out
FIGURE 31.6. The role of PDGF-B in the development of vessel
walls. Undifferentiated mesenchymal cells (gray) surrounding the
newly formed endothelial tube (yellow) are induced to become
vascular smooth muscle cells (vSMC) and to assemble into a vascular
wall (red). During vessel growth and sprouting, PDGF is released by
the endothelium to drive vSMC proliferation and migration. In mice
lacking PDGF-B or PDGFR-b, there is reduced vSMC proliferation and
migration, which results in vSMC hypoplasia of larger vessels and
pericyte deficiency in capillaries.
Adapted from Hellstrom M, Kalen M, Lindahl P, et al: Role of PDGF-B and
PDGFR-beta in recruitment of vascular smooth muscle cells and pericytes during
embryonic blood vessel formation in the mouse. Development 1999;
126:3047–3055.
and to enhance angiogenesis in gliomas by stimulating VEGF
expression and pericyte recruitment.209 In an assay in which
angiogenesis-promoting gel plugs were implanted subcu-
taneously into mice, VEGF-A and fibroblast growth factor-2
synergistically promoted neovascularization by enhancing
PDGF-B signaling; the mechanism was believed to involve
upregulation of endothelial cell PDGF-B expression by VEGF
and upregulation of PDGFR-b expression by fibroblast growth
factor-2.210
Pericyte recruitment to developing vasculature has been
found to lag behind the formation of the endothelial cell plexus,
providing a ‘plasticity window’ during which endothelial cells
are highly sensitive to VEGF withdrawal.211,212 Intraocular in-
jection of PDGF-B caused detachment of pericytes from newly
formed retinal vessels and abnormal vascular modeling in a rat
retinopathy of prematurity model, presumably by competing
with endogenous signaling; VEGF accelerated pericyte coverage
of developing vasculature.211 Once the pericyte coating was
complete, the vasculature was stabilized and resistant to VEGF
withdrawal.212
PDGF-B IN OCULAR NEOVASCULAR DISEASE
PDGF has been demonstrated to be an autocrine growth
factor213 and a chemotactic factor214 for retinal pigment epi-
thelial cells. These effects appear to be predominantly mediated
through PDGFR-b as shown by studies demonstrating that
rabbit retinal endothelial cells migrated in response to either
PDGF-B or PDGF-AB, but not to PDGF-A.215 Similarly, PDGF-
B, but not PDGF-A, stimulated migration of rabbit corneal
fibroblasts and epithelial cells.216 However, the contribution by
PDGFR-a, which also is activated by PDGF-B, is not clear.
Studies showed that truncated PDGFR-a lacking the intra-
cellular domain was able to block the effects of wild-type
Angiogenic Factors and Inhibitors
PDGFR-a when coexpressed in rabbit conjunctival fibroblasts
and that it was able to reduce the experimental proliferative
retinopathy when these cells were injected into the vitreous of
rabbits.217 In a subsequent study, retinal detachment resulting
from intravitreous injection of rabbit conjunctival fibroblasts
was completely inhibited by coinjecting a retrovirus that
expressed the truncated PDGFR-a.218 Yet, the relative
contribution of PDGFR-a to ocular neovascularization is not
well established.
A variety of models have been used in an attempt to define a
role for PDGF-B in ocular neovascular diseases. To gain a better
understanding of the role of PDGF-B in DR in mice, which
would otherwise be impossible due to the embryonic lethality of
PDGF-B knockouts, mice were engineered with selective
inactivation of PDGF-B in endothelial cells.219 These mice had
vascular aberrations of retinal capillary formation corre-
sponding to pericyte deficiency resulting in areas of proliferative
retinopathy when pericyte density was less than 50% of
normal.219 Similar findings were reported with the use of a
kinase inhibitor that blocks PDGFR signaling.220 In another
model, transgenic mice engineered for photoreceptor-specific
expression of PDGF-B had traction retinal detachment
characterized by proliferation of astrocytes, pericytes, and
endothelial cells.221 These effects were largely blocked by
administration at postnatal day seven of a single intravitreous
injection of an aptamer that binds PDGF-B, confirming the role
of PDGF-B in the pathogenesis of these lesions.222 In a model of
corneal neovascularization, mice treated systemically with a
CHAPTER 31
PDGF inhibitor had a loss of pericytes and reduced vessel
density in the cornea; in this model the inhibitor appeared to be
effective in reducing pericyte coverage in existing vessels as well
as in growing vessels.223
Recent studies involving three different murine models have
further elucidated the respective contributions of the VEGF and
PDGF-B signaling pathways in ocular neovascular disease.224
PDGF-B signaling was inhibited by systemic administration of
an anti-PGDFR-b antibody and VEGF-A signaling was inhibited
by systemic administration of the anti-VEGF aptamer
pegaptanib. One model evaluated the effects of VEGF and
PDGFR-b inhibition on the physiologic development of retinal
vasculature in neonates. The anti-PDGF-b antibody, but not
pegaptanib alone, significantly inhibited retinal blood vessel
growth at postnatal day three; further reductions occurred when
both agents were administered simultaneously. However, in a
CNV model, the anti-PDGFR-b antibody had little effect on
developing or established CNV, while significant reduction
occurred with pegaptanib; addition of the anti-PDGFR-b
antibody provided greater reduction than achieved with
pegaptanib alone.224
A corneal neovascularization model was used to investigate
the effects of the PDGFR-b blockade on regression of
established vasculature. In this model, neovascularization
occurs primarily in the first 7 days postinjury and vessels do not
naturally regress even through 28 days, making it suitable for
assessing the effects of pharmacologic intervention on vessel
regression. When mice were treated with anti-PDGFR-b anti-
body between 10 and 20 days postinjury, mural cells appeared
to detach from corneal neovessels (Fig. 31.7a). When mice were
treated daily immediately after corneal injury, there was
significant reduction in the area of neovascularization with
pegaptanib, but not a significant reduction with the anti-
PDGFR-b antibody; when both agents were combined, there
was a significantly greater reduction than with pegaptanib alone
(Fig. 31.7b).
It is interesting that inhibition of PDGFR-b signaling in these
models resulted in pericyte depletion of established vessels as
well as in developing vasculature but not in quiescent limbal 323
 PHARMACOLOGY AND TOXICOLOGY

PBS control Anti-PDGFR- antibody

PBS Anti-VEGF Anti-PDGFR- Anti-VEGF aptamer +

control aptamer antibody anti-PDGFR- antibody
SECTION 4

FIGURE 31.7. The effects of PDGF-B blockade on mural cells and vascular growth in a corneal neovascularization model. (a) Mice were injected
with anti-PDGFR-b antibody or phosphate-buffered saline (PBS) every day starting at 10 days postinjury and sacrificed at 20 days postinjury.224
Neovessels from mice treated with the anti-PDGF-b antibody had reduced mural cell coverage when compared with PBS-treated mice. Scale
bar = 20 µm. (b) Immediately after corneal injury, mice were treated daily with one of the following: PBS, a pegylated anti-VEGF aptamer, an anti-
PDGFR-b or a combination of the anti-VEGF aptamer and the anti-PDGFR-b antibody. Neovessels are delineated in green. Scale bar = 100 µm.
Quantitative analysis demonstrated that the anti-VEGF aptamer significantly reduced neovascularization when compared with PBS or the anti-
PEGFR-b antibody (p < 0.01), while the combination significantly reduced neovascularization when compared with the aptamer alone (p < 0.05).
Adapted from Jo N, Mailhos C, Ju M, et al: Inhibition of platelet-derived growth factor B signaling enhances the efficacy of anti-vascular endothelial growth factor
therapy in multiple models of ocular neovascularization. Am J Pathol 2006; 168:2036–2053.

vessels.224 These findings are consistent with the notion of a ANGIOPOIETINS

‘plasticity window’ for both PDGFR-b signaling inhibition and
VEGF dependency and provide support for a combination
therapeutic approach using inhibitors of both VEGF and
PDGF-B for treatment of ocular neovascular disease.
CONCLUSIONS
PDGF-B has been shown to provide an important contribution
to angiogenesis, mediated primarily through its effects on mural
cells such as pericytes and vascular smooth muscle cells. The
recruitment of mural cells to the endothelium occurs primarily
in response to endothelial cell-secreted PDGF-B, which
activates PDBF-b receptors on the mural cells and stimulates
their migration and proliferation. Mature vasculature, charac-
terized by a stable coverage of mural cells, is largely resistant to
the effects of VEGF withdrawal and may be less susceptible to
therapeutic intervention. Combined therapies in which both
VEGF and PDGF are blocked may provide improved regression
324 of established ocular neovascular lesions.
Key Features
• Angiopoietins-1–4 (Ang1–4) form a family of growth factors
involved in angiogenesis; only Ang1 and Ang2 currently are
known to have roles in ocular neovascular disease
• Ang1 and Ang2 are ligands for the receptor tyrosine kinase
Tie2, which is expressed primarily on endothelial cells
• Ang1 is produced by cultured vascular smooth muscle cells
and is believed to be involved in maintaining the integrity of the
quiescent endothelium through its interaction with Tie2;
experimental elevation of Ang1 within the context of ocular
neovascularization is inhibitory to the development of
pathological angiogenesis
• Ang2 is produced by endothelial cells and serves as a naturally
occurring antagonist to Ang1/Tie2 angiogenesis; in ocular
neovascularization, when VEGF concentrations are high
elevation of Ang2 promotes neovascularization and when
VEGF concentrations are low elevation of Ang2 is inhibitory
 Angiogenic Factors and Inhibitors

INTRODUCTION
The angiopoietins (Ang1–Ang4) form a family of growth factors.
Ang1 was first identified as a secreted glycoprotein capable of
both binding and inducing the phosphorylation of Tie2, a
receptor tyrosine kinase,225–229 whereas Ang2 is an antagonist
of Ang1 and Tie2 (reviewed by Eklund and Olsen230 and
Maisonpierre et al228). Ang3 and Ang4 represent murine and
human counterparts, respectively, of what appears to be the
same gene product despite their highly divergent structure.229
To date, little is know about the biology of Ang3 and Ang4.
The Ang receptor Tie2, expressed primarily on endothelial
cells,225–229 is part of a family that includes Tie1. Tie2 knockout
mice die by embryonic day 10.5 due to defective formation of
microvessels; in adult mice, Tie2 is expressed both during
angiogenesis and also in quiescent vasculature in many
tissues.231 Tie1 is essential for structural integrity of the
vascular endothelium and hence survival as its gene knockout
in mice leads to death shortly after birth.232 The function of
Tie1 has been much less studied than that of Tie2, whose
actions are the focus of the remaining discussion.
The relative contributions of Ang1 and Ang2 to angiogenesis
and ocular neovascularization are complex. The preclinical and
clinical studies that will be summarized in this section will
show that although Ang1 is essential for the development of the
normal vasculature, experimental elevation of Ang1 in the
context of ocular neovascularization is inhibitory to the deve-
lopment of pathologic angiogenesis. Studies involving Ang2, in FIGURE 31.8. Ang–Tie functions in the regulation of quiescent and

contrast, have shown that depending on the local concentration
of VEGF the experimental elevation of Ang2 may either
promote or inhibit ocular neovascularization; when VEGF con-
centrations are high elevation of Ang2 promotes neovas-
cularization and when VEGF concentrations are low elevation
of Ang2 is inhibitory.
ANGIOPOEITIN-1
Role in Angiogenesis
Knockout mice lacking expression of Ang1 suffer embryonic
lethality characterized by failure to remodel the primary
capillary plexus, a phenotype similar to that seen in Tie2
knockout mice.233 Ang1 has been found to be expressed in close
proximity to developing embryonic vasculature226,234 and is
secreted by cultured vascular smooth muscle cells but not by
endothelial cells.235 These findings are consistent with studies
demonstrating that granular deposits of Ang1 are found in the
extracellular matrix of an Ang1-expressing cultured carcinoma
cell line.236 Adherence of endothelial cells to these cultures
stimulated the release of the granules from the matrix and
resulted in phosphorylation of Tie2 on the endothelial cells.236
In the adult, it is believed that constitutive expression of
Ang1 induces continuous activation of Tie2 and contributes to
maintaining the integrity of the quiescent endothelium,237,238 as
depicted in Figure 31.8.239 Ang1 protects endothelial cells
against apoptosis240 and has been found by some investigators
to induce proliferation of cultured endothelial cells241 while
others have reported contrasting results.242 Treatment with
Ang1 has also been shown to promote proliferation of
endothelial cells in vivo, leading to vessel diameter enlargement
in the venous circulation,243 an effect that is restricted to a
defined and brief postnatal period. In contrast, in adult mice,
the use of collagen oligomeric matrix protein, Ang1, a fusion
protein that is an especially potent activator of Tie2, led to
vessel enlargement in many tissues.244 Overexpression of Ang1
in tumors is associated with tumor vessel maturation and
reduced permeability, together with lowered tumor
growth.245,246
CHAPTER 31
activated vasculature. The quiescent, resting endothelium (upper) has
an antithrombotic and antiadhesive luminal cell surface. Ang1 (shown
as multimeric (white)), is secreted by periendothelial cells at a
constitutive low level. By acting on the endothelium to maintain low
level Tie2 phosphorylation, Ang1contributes to maintaining the
vascular endothelium in the resting state. Ang2 (dimeric (gray)) is
stored in endothelial cell WPB of the quiescent vasculature.
Endothelial cell activation (lower) involves the release of the
endothelial cell WPBs, and concomitant liberation of a variety of
stored factors, including Ang2. The resultant Ang1/Ang2 ratio is now
biased more in favor of Ang2, leading to endothelial destabilization,
and making the endothelial cell layer more responsive to other stimuli,
including proinflammatory cytokines.
Adapted from Ptaff D, Fiedler U, Augustin HG: Emerging roles of the
Angiopoietin-Tie and the ephrin-Eph systems as regulators of cell trafficking.
J Leukoc Biol 2006.
Like VEGF, Ang1 possesses a range of functions important in
angiogenesis, including the ability to promote endothelial cell
chemotaxis242 and to stimulate secretion of plasmin and matrix
metalloproteinases247 as well as to depress secretion of tissue
inhibitors of metalloproteinases.247 These functions are
believed to underlie the vascular remodeling effects of Ang1 in
vivo, as well as the induction of tubule formation in model
systems.238
In some instances, Ang1 has been found to modify or
contribute to the effects of VEGF. For example, Ang1 inhibits
vascular leakage induced by VEGF243,248 and has been found to
antagonize the proinflammatory effect of VEGF through
inhibition of VEGF-mediated upregulation of ICAM-1 and vas-
cular cell adhesion molecule-1249 and to inhibit the induction of
tissue factor expression by VEGF and tumor necrosis
factor-a.250 Ang1 also has been found to induce dose-dependent
capillary sprouting in endothelial cell monolayers; suboptimal
concentrations of Ang1 and VEGF acted synergistically in this
assay.251 Other work using a human fibroblast/endothelial cell
coculture assay showed that Ang1-induced sprouting could be
inhibited by blocking VEGF signaling.252 325
 PHARMACOLOGY AND TOXICOLOGY
In turn, VEGF has been found to impact on the expression of
Ang1. In cultured human retinal pigment epithelium cells,
VEGF upregulated Ang1 mRNA expression in a dose-dependent
manner.253 Similar findings were reported for cultured bovine
retinal pericytes in which both VEGF treatment and hypoxia
significantly increased Ang1 mRNA expression; contrasting
findings were reported for bovine aortic endothelial cells in
which Ang2, but not Ang1, expression was upregulated by these
two stimuli.254
Role in Pathologic Ocular Neovascularization
The effects of Ang1 on endothelial homeostasis have led
investigators to evaluate the role of Ang1 in ocular neovascular
diseases. In studies using a micropocket assay to promote
corneal neovascularization, Ang1 did not affect neovas-
cularization on its own but increased perfusion of the micro-
vasculature when administered in conjunction with VEGF; the
effect was blocked with the addition of excess soluble Tie2.255 In
another model, systemically administered soluble Tie2 also
reduced neovascularization in laser-induced CNV and
ischemia-induced retinopathy in mice, supporting a role for
Ang/Tie2 signaling in pathologic ocular neovascular diseases.256
Evidence that Ang1 plays a protective role against pathologic
ocular neovascularization was provided in studies using rodent
models of DR. Intravitreous injection of Ang1 in early diabetes
normalized expression of VEGF and ICAM-1, reducing
leukocyte adhesion to the retinal vasculature and damage to
endothelial cells and blood–retinal barrier breakdown.257
SECTION 4
Systemically administered Ang1 produced similar effects in
animals with established diabetes.257
Further evidence of the potential utility of Ang1 in ocular
neovascular disease comes from studies with transgenic mice in
which Ang1 expression could be specifically induced in the
retina.258 In this model, elevated retinal levels of Ang1 sup-
pressed the development of CNV following laser wounding and
inhibited the development of retinal neovascularization
following ischemic retinopathy. Moreover, induced elevation of
Ang1 also inhibited the blood–retinal barrier breakdown
following intravitreous injection of VEGF.258 Subsequent studies
in transgenic mice in which retinal expression of both Ang1 and
VEGF could be induced demonstrated that simultaneous
induction of both factors suppressed VEGF-induced CNV and
prevented retinal detachment.259
ANGIOPOIETIN-2
Role in Angiogenesis
Ang2 was first identified through its homology to Ang1.228
Studies in Ang2 knockout mice have demonstrated that Ang2 is
not required for embryonic vascular development, but it is
essential for subsequent angiogenic remodeling and proper
lymphatic vessel development; most Ang2-deficient mice die
within a few weeks of birth.260 However, transgenic over-
expression of Ang2 disrupted embryonic mouse blood vessel
formation resulting in an embryonic lethal phenotype
resembling the loss of either Ang1 or Tie2.228 Genetic rescue
with Ang1 was able to correct the lymphatic but not the
angiogenesis, suggesting that Ang2 serves as an agonist for Tie2
in establishing the lymphatic vasculature but is antagonistic for
Tie2 in angiogenesis.260 Other studies showed that Ang2 bound
Tie2 on endothelial cells with comparable binding affinity
to that of Ang1 but did not induce Tie2 phosphorylation.261
Together these findings support the hypothesis that Ang2 serves
as a naturally occurring antagonist to Ang1/Tie2 angio-
genesis.228 This model is not supported in all contexts, however.
In cultured endothelial cells, Ang2 was able to activate Tie2
326
following long exposures262 and to induce tube formation.262,263
Like Ang1, Ang2 can stimulate matrix metalloproteinase
expression by cultured retinal endothelial cells.264
Endothelial cells are a primary source of Ang2 pro-
duction260,265 where it is stored in Weibel–Palade bodies (WPB)
from which it can be released by a variety of stimuli,239 shown
in Figure 31.8. Expression of Ang2 is upregulated by hypoxia
and VEGF.254,266,267 In contrast to the quiescent maintenance
function exerted by Ang1, expression of Ang2 occurs
prominently at sites of vascular remodeling, where it serves to
destabilize the endothelial layer.230 This was demonstrated in
studies showing that cultured endothelial cells rapidly detach
following exposure to Ang2 and that these effects can be rescued
by Ang1, soluble Tie-2, or VEGF.268 Similarly, local upregulation
of Ang2 in tumors was associated with vascular regression in
the absence of VEGF whereas angiogenesis occurred when
VEGF was present.269 Thus, dependent on the local availability
of molecules such as VEGF, the destabilizing action of Ang2 can
either enhance or decrease local blood vessel formation.
Role in Ocular Neovascular Disease
While both Ang1 and Ang2 have been found in association with
VEGF in proliferative membranes from patient eyes with ocular
neovascular diseases,253,270 Ang2 was particularly localized in
highly vascularized areas of the membranes.270 In patient eyes
with proliferative DR, vitreous levels of Ang2 were significantly
higher than in nondiabetic patients.125 In contrast, while high
levels of Ang2 were detected in eyes with nonproliferative DR
with macular edema during pars plana vitrectomy, Ang2 was
undetectable in eyes with proliferative disease.271 The reason for
these discrepant findings is unclear but may be related to the
possibility that those undergoing vitrectomy were more likely to
have well-established lesions.
The effects of Ang2 in the eye have been found to be de-
pendent on VEGF in some instances. For example, in a micro-
pocket assay of corneal neovascularization, Ang2 administered
in conjunction with VEGF led to longer vessels together with
enhanced sprouting.255 Furthermore, fusion peptides that
inhibited the interaction of Ang2 and Tie2 prevented VEGF-
stimulated corneal neovascularization.272 An aptamer specific
for Ang2 also inhibited fibroblast growth factor-induced neo-
vascularization in a similar model.273
More detailed studies of the relationship of Ang2 expression
have provided evidence for complex interactions with VEGF in
ocular neovascularization, with implications for possible thera-
peutic approaches.274,275 In transgenic mice with inducible
expression of Ang2 and VEGF, induction of Ang2 expression in
the first 2 weeks after birth led to an increased density of retinal
capillaries that had normalized by postnatal day 18, suggesting
that Ang2 expression does not affect mature retinal vessels.274
In mice in which ischemia was induced by transient exposure
to high oxygen between postnatal days 7 and 12, induced
expression of Ang2 had divergent impacts depending on the
time of onset. Between postnatal days 12 and 17, when VEGF
levels were high, induction of Ang2 dramatically increased
retinal neovascularization; if this induced expression was
delayed until P20, when retinal ischemia was less intense and
VEGF levels were concomitantly lower, regression of the
neovascularization was intensified. Finally, in mice with sus-
tained, low-level VEGF expression in photoreceptors, laser-
induced CNV was suppressed by elevated expression of Ang2.
The investigators concluded that while more mature vessels are
not affected by Ang2 expression, its elevation may lead nascent
vessels either to proliferate, or to regress, depending on the ratio
of VEGF and Ang2 concentrations.274 From these findings it
was proposed that elevation of Ang2, in conjunction with

inactivation of VEGF, could be a useful therapeutic approach for
treating ocular neovascularization.275
CONCLUSIONS
The complex roles of Ang1 and Ang2 and their receptor Tie2
have only recently been appreciated in ocular neovascular
diseases such as AMD and DR. Evidence from preclinical and
clinical studies suggests that Ang1 is largely inhibitory to the
development of neovascularization while Ang2 may promote or
inhibit it depending on the local concentration of VEGF. There
is still much to be learned about the mechanisms for these
effects and how they may best be applied to the treatment of
pathologic ocular neovascularization.
EPHRINS
Key Features
• Ephrins are ligands for the Eph class of receptors; both are
membrane-bound proteins
• Ephrins are divided into two subclasses, ephrinA and ephrinB,
as are the receptors EphA and EphB; there are multiple
members in each of these subclasses
• Ephrin/Eph interactions have a wide range of functions in
morphogenesis and development of neural networks,
embryonic vascular development and postnatal angiogenesis;
these interactions are also are a primary determinant of
venous/arterial identity
• Recent evidence supports a role for eprhin/Eph interactions in
pathological forms of ocular neovascularization, suggesting that
modulating these interactions may offer therapeutic options
INTRODUCTION
Among the proteins that play an active role in angiogenesis,
VEGFs stimulate endothelial cell proliferation and migration,
Angs mediate blood vessel plasticity and maturation, and
ephrins are involved in vessel patterning.276 Ephrins are ligands
for the Eph class of receptors, whose name originally derived
from the identification and molecular cloning of a novel kinase
receptor gene from an erythropoietin-producing human hepa-
tocellular carcinoma line.277 Ephrins are divided into two
subclasses, ephrinA and ephrinB, as are the receptors EphA and
EphB; there are multiple members in each of these subclasses
(reviewed by Zhang and Hughes278). In general, interactions
between A and B subclasses are limited, while multiple ligands
are capable of binding to multiple receptors within a subclass.278
At least one interaction between an EphB receptor and an
ephrinA ligand has been reported, however.279
Ephrins are membrane-bound proteins; ephrinAs are tethered
to the cellular membrane and lack any cytoplasmic portion
while ephrinBs are transmembrane proteins that possess a
cytoplasmic signaling domain (Fig. 31.9) (reviewed by Doudelet
and Pasquale280). Thus, ephrinB/EphB interactions may produce
both forward or reverse signaling, depending on the direction
(reviewed by Davy and Soriano281).
Ephs and ephrins have a wide range of functions in mor-
phogenesis, in the development of neural networks, and in
embryonic and postnatal angiogenesis (reviewed by Palmer and
Klein282) and also are involved in controlling trafficking of
circulating cells within the vascular system (reviewed by Pfaff et
al239). In this section, the involvement of Eph/ephrin inter-
actions in promotion of angiogenesis, particularly pathologic
forms of ocular neovascularization, will be discussed.
Angiogenic Factors and Inhibitors
PDZ
P
Ephrin-A Ephrin-B
P
SH2 P
P
EphA EphB
P
PDZ
Ligand binding
CHAPTER 31
Kinase
Cysteine-rich
SAM
Fibronectin type III
FIGURE 31.9. The structure of ephrins and Eph receptors. Both
ephrins and Eph receptors are membrane-bound proteins. Whereas
ephrinAs are tethered to the cellular membrane, ephrinBs have
transmembrane and cytoplasmic signaling domains. Binding of
ephrins to Eph receptors leads to receptor clustering and subsequent
autophosphorylation of multiple tyrosine residues, providing docking
sites for src-homology domain-containing downstream effectors. The
carboxyl terminus of both Eph receptors contains a sterile alpha motif
(SAM) and a PDZ domain (shown here for EphA, but these also apply
to EphB), which promote receptor clustering after ligand binding.
Adapted from Dodelet VC, Pasquale EB: Eph receptors and ephrin ligands:
embryogenesis to tumorigenesis. Oncogene 2000; 19:5614–5619.
EPHRINA
Role in Angiogenesis
Although ephrinA1 is widely expressed in embryonic vas-
culature,283 a clear role for ephrinA/EphA interactions in
embryonic angiogenesis has not been established. In contrast,
ephrinA/EphA interactions have been found to play an active
role in regulating postnatal angiogenesis, as shown by in vitro
and in vivo studies. Pulmonary microvascular endothelial cells
from adult EphA2-deficient mice had normal proliferation and
survival but failed to migrate and form capillary-like structures
in response to ephrinA1 stimulation; assembly into structures
in these cells was restored by overexpressing EphA2.284 EphA2
receptor phosphorylation was shown to be critical for migration
of these cells. Furthermore, EphA2-deficient mice had impaired
angiogenesis in response to implanted ephrinA1-impreganted
sponges, demonstrating that EphA2 is a regulator of
angiogenesis in adult endothelial cells.284
327
 PHARMACOLOGY AND TOXICOLOGY
EphrinA/EphA Interactions Modulate the
Angiogenic Effects of VEGF
EphrinA/EphA interactions are involved in modulating the
induction of angiogenesis by VEGF. Stimulation of EphA
receptor signaling in bovine retinal endothelial cells by
ephrinA1-Fc inhibited VEGF-induced phosphorylation of
VEGFR-2 and subsequent endothelial cell migration and tubule
formation.285 In other work, a soluble EphA2-Fc fusion protein
(which reportedly blocks the interaction of EphA2 with its
ligands) was found to inhibit VEGF-dependent human
endothelial cell migration, sprouting, and survival in vitro but
did not affect endothelial cell proliferation.286 VEGF was found
to induce expression of ephrinA1 on endothelial cells; since the
levels of ephrinA1 expression directly correlated with the level
of phosphorylation of EphA2, it is likely that EphA2 activation
is involved in these effects.286 Hypoxia also was reported to
upregulate the expression of EphA2 and ephrinA1 in a murine
dermal model.287
In subsequent work using bovine retinal endothelial cells,
soluble EphA2-Fc inhibited both ephrinA1- and VEGF-induced
migration and tubule formation.288 VEGF-induced endothelial
cell tubule formation was impaired using cells from EphA2-
deficient mice, providing evidence that EphA2 stimulation is
necessary for maximal induction of neovascularization by
VEGF.288 The mechanism by which soluble EphA2-Fc regulates
VEGF-induced angiogenesis is not clear; an indirect effect on
VEGF signaling is unlikely in that no effects on expression or
phosphorylation of VEGFR-2 in retinal endothelial cells were
SECTION 4
found nor does EphA2-Fc affect VEGF-induced endothelial cell
proliferation. An alternate possibility is that VEGF and Eph
may regulate two distinct pathways.288
EphA/ephrinA in Ocular Neovascularization
EphA/ephrinA interactions have been demonstrated to be in-
volved in pathologic ocular neovascularization as demonstrated
in rodent models. EphA2-Fc-inhibited VEGF-induced angio-
genesis (but not that induced by basic fibroblast growth factor)
in a model in which pellets impregnated with ephrinA2, VEGF,
or basic fibroblast growth factor were implanted into mouse
corneas to provoke neovascularization.286 Intravitreal admini-
stration of EphA2-Fc also reduced the severity of pathologic
neovascularization in a rat model of retinopathy of prematurity
while having no effect on normal retinal vascular development.288
Finally, other rodent studies demonstrated that intravitreal
injection of ephrinA1-Fc inhibited both VEGF-induced
neovascularization and vascular permeability.285
EPHRINB
Essential for Development of Embryonic
Vasculature
The importance of ephrinB/EphB interactions in embryonic
vascular development was demonstrated by the early embryonic
lethality of knockout mice lacking ephrinB2 or EphB4.289,290
Since these two knockouts yield identical lethal phenotypes, it
suggests that EphB4 is the major receptor for ephrinB2 in
cardiovascular development.290 The EphB4/ephrinB2 pair is
believed to be a primary determinant of venous/arterial identity
in that EphB4 is preferentially expressed on veins290 and
ephrinB2 on arteries.289 Targeted disruption of ephrinB2
prevents proper remodeling of veins into branched structures
and disrupts the remodeling of arteries, providing evidence that
reciprocal interactions between arterial and venous endothelial
cells may be required for angiogenesis.289 In addition to EphB4,
ephrinB1 and EphB3 were found to be expressed on embryonic
veins while arteries expressed ephrinB1 in addition to ephrinB2;
328 aortic arches expressed ephrinB1, ephrinB2, and EphB3.291 The
relative contribution of these alternate receptors to embryonic
angiogenesis is not well characterized.
Reverse Signaling is Required for Angiogenesis
Mice that express ephrinB2 lacking the cytoplasmic domain
also demonstrated early embryo lethality due to vascular defects
although the cytoplasmic domain was not found to be required
for activation of EphB4, suggesting that reverse signaling
through ephrinB2 is required for proper embryonic vascular
development.292 An alternate possibility is that ephrinB2 ligand
clustering, which is dependent on the cytoplasmic domain, may
be required for its full activation as has been demonstrated for
ephrinB1.293 EphrinB ligands have a conserved carboxyterminal
sequence that serves as a binding site for PDZ domains,294
named for the first three proteins found to contain this motif
(PSD95, DLG, and ZO-1) and which are important in
clustering and anchoring transmembrane proteins.295
EphB/ephrinB Interactions in Endothelial Cell
Migration and Capillary Formation
The effects of the EphB4/ephrinB2 pair on endothelial cells have
been the most fully characterized of the B subclass, with
forward signaling generally resulting in decreased proliferation
and migration of EphB4-expressing cells and reverse signaling
promoting increased proliferation and migration of ephrinB2-
expressing cells.296 Consistent with this generalization, coating
adhesive culture dishes with ephrinB2-Fc, which stimulates
EphB4, completely blocked adhesion of human umbilical vein
endothelial cells to culture dishes.297 In addition, soluble
ephrinB2-Fc inhibited cell migration, VEGF-driven chemotaxis,
capillary-like network formation, and sprouting angiogenesis.
In turn, soluble EphB4-Fc, which would be expected to stimu-
late ephrinB2 signaling, was proadhesive and stimulated endo-
thelial cell migration and angiogenesis.297 Co-mingling between
endothelial cells expressing either EphB4 or ephrinB2 showed
that forward signaling through EphB4 restricts intermingling of
cells; this separation of EphB4 expressing cells from ephrinB2
cells may be important in segregating arteries from veins.297
The stimulating effects of ephrinB2-Fc on endothelial cell
migration were demonstrated in both human umbilical vein
endothelial cells298 and in cloned human mesenteric micro-
vascular endothelial cells.299 However, contrasting findings were
reported for proliferation, which was not stimulated in human
umbilical vein endothelial cells298 but was stimulated
moderately in microvascular endothelial cells.299 EphB4 and
ephrinB2 were shown to be upregulated by hypoxia in a murine
dermal model, which may contribute to amplification of their
angiogenic effects under conditions of ischemia.287
The contributions of other members of the EphB class to
angiogenesis are not well studied. EphB1-Fc has been shown to
stimulate ephinB1 phosphorylation on human microvascular
endothelial cells and to promote their migration and integrin-
mediated attachment.300 Also, multimeric ephrinB1-Fc pro-
moted attachment and tubule formation in human renal
microvascular endothelial cells and resulted in activation of
EphB1 receptors.293 Thus, in these systems, EphB1/ephrinB1
interactions appear similar to those of EphB2/ephrinB2.
EphB/ephrin B Interactions in Ocular
Neovascularization
Both ephrinB2-Fc298 and ephrinB1-Fc300 have been demon-
strated to promote corneal angiogenesis following implantation
of slow-release pellets into corneal micropockets. In other
experiments using a similar model but involving transgenic
mice that are heterozygous for ephrinB2 and the reporter gene
b-galactosidase, the administration of either ephrinB2 or VEGF
stimulated corneal neovascularization.301 Furthermore, EphB4

mRNA expression was significantly upregulated in the corneal
tissues after 3 days. Although the total length of the neovas-
culature produced by these agents was similar, the extent of the
arterial vessels induced by ephrinB2 was significantly less than
that induced by VEGF. These findings suggest that ephrinB2
induces venous rather than arterial angiogenesis and is consis-
tent with a mechanism of forward signaling through EphB4.301
EphB4/ephrinB2 interactions also have been investigated in
retinal models of ocular neovascularization. In a rat model,
soluble EphB4 reduced the extent of laser-induced CNV and
fluorescein leakage.302 These results suggest that the mechanism
for these effects may involve blocking of ephrinB2 activation by
soluble EphB4 or reverse inhibition of EphB4 signaling and
downstream pathways.302 In a mouse model of oxygen-induced
retinopathy, intravitreal injection of ephrinB2-Fc or EphB4-Fc
decreased formation of pathologic retinal vascular tufts and of
neovascular nuclei anterior to the inner limiting membrane;
superficial and deep vascular beds were not affected.296
Finally, studies in which proliferative membranes were
isolated from the eyes of patients with proliferative DR or
retinopathy of prematurity demonstrated that ephrinB2, EphB2,
and EphB3 were expressed on fibroproliferative membranes but
not EphB4.303 These molecules were detected on both vascular
endothelial cells and stromal mesenchymal cells. The detection
of ephrinB2 on these membranes may indicate a difference
between embryonic and pathologic angiogenesis, in that its
expression is restricted to arteries during embryonic
angiogenesis. The finding that EphB4 was not detected in these
tissues suggests that the lack of expression of EphB4 may be a
contributing factor to the disorganization of neovasculature in
proliferative membranes.303
CONCLUSIONS
Ephrin/Eph interactions are critical for embryonic vascular
development and contribute to postnatal angiogenesis as well.
They have been found to be important in modulating
angiogenic responses to VEGF through both forward and reverse
signaling. Recent evidence documents that ephrin/Eph
interactions also are involved in pathologic forms of ocular
neovascularization in adults, suggesting that modulating these
interactions may offer therapeutic options. Since ephrins/Ephs
are involved in a myriad of other functions, including retinal
axon development304 and the trafficking of immune cells,239 the
consequences of such interventions cannot easily be predicted.
NOTCH
Key Features
• The Notch signaling pathway is essential for cell fate
determination and pattern formation in a wide variety of tissues
• In mammals, the Notch 1 Delta-like ligand 4 (Dll4) is especially
important for arterial patterning in the embryo, while its
expression in the adult is elevated in endothelial cells during
physiological and pathological angiogenesis
• Preclinical findings suggest that interference with Dll4/Notch
signaling may prove to be a useful strategy for inhibiting
pathological angiogenesis
Angiogenic Factors and Inhibitors
INTRODUCTION
Notch is a 300 kDa transmembrane receptor305,306 for which a
mutated phenotype involving notches on margins of the wing
was first described in Drosophila in 1917.307 Notch has since
been found in all animal phyla examined, and is expressed in a
wide variety of tissues where it acts to determine cell fates and
to regulate pattern formation (reviewed by Lai et al308). Ligand
activation of Notch leads to its being cleaved in two sequential
proteolytic steps, releasing an intracellular domain which
translocates to the nucleus and leads to activation of Notch-
targeted genes.308 There are four Notch genes in mammals
(Notch 1 through 4) and groups of ligands for Notch have been
identified in different animal phyla. Following the Drosophila
nomenclature, for which the Delta gene is a major ligand,
mammalian versions include the Delta-like ligand (DLL) series
as well as Jagged1 and Jagged2.308 In keeping with its wide range
of tissue expression, the Notch signaling pathway is important
in numerous processes including liver and kidney development,
somatogenesis, cardiovascular development, neurogenesis and
T-cell differentiation;308,309 in addition, aberrant Notch signal-
ing has been implicated in development of various cancers.310
NOTCH/DELTA SIGNALING IN ANGIOGENESIS
Studies of Notch signaling in mammalian vasculature develop-
ment have focused on the ligand Delta-like 4 (Dll4). Like VEGF,
gene knockout of only one Dll4 gene leads to embryonic
CHAPTER 31
lethality311,312 although expression of the heterozygous lethality
is dependent on genetic background.313 During embryogenesis,
expression of Dll4 is restricted to the arterial endothelium314
where it plays an essential role in arterial patterning311–313 while
expression in the adult is seen in many adult tissues with a high
proportion of endothelial cell types.314 Expression is elevated in
capillaries during the physiological angiogenesis that
accompanies ovarian cycling;314 in addition, Dll4 is expressed
in the endothelium of cancerous tumors.314 Like VEGF, its
expression in cultured endothelial cells is upregulated by
hypoxia.314
Research into the mechanism of Dll4-Notch signaling in
vascular development is still in its early stages, but already has
revealed interactions with other key regulators of angiogenesis;
for example, endothelial cell expression of Dll4 in vitro is
upregulated by VEGF.315,316 Several lines of evidence suggest
that interference with Notch signaling may be a useful
antiangiogenic strategy. Overexpression of an inhibitor of
Notch signaling can partially inhibit vascular development in
collagen gels,315 while overexpression of Dll4 in endothelial
cells can lead to reduced expression of VEGFR-2, as well as to
reduced migratory and proliferative responses to VEGF.317 In
addition, inhibition of venous endothelial cell migration and
differentiation can be effected either by pharmacological
inhibitors or by excess soluble Dll4;318 this latter finding may
reflect the need for cell–cell contact in Notch-mediated
signaling. While the relationship of these model systems to the
processes that mediate angiogenesis in physiological contexts
remains to be established, these studies suggest that several
possible strategies are potentially available for inhibiting
Dll4-Notch signaling as a means of preventing pathological
angiogenesis.
329
 PHARMACOLOGY AND TOXICOLOGY
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 CHAPTER

32 Principles of Toxicology of the Eye

Keith J. Lane, Zhou Chen, and Matthew J. Chapin

Key Features place by regulatory authorities. This chapter will supplement

• Toxicity is a result of harmful byproduct production that occurs
the traditional view of ophthalmic toxicity in several ways. We
during drug metabolism, and consequently toxicity is generally
will identify the cellular processes by which ophthalmic agents
dose dependent. The ease with which the body can convert
exert their toxic affects. We will review common models of
xenobiotic substances (drugs) to hydrophilic byproducts and
ophthalmic toxicology beginning with the traditional Draize
excrete these byproducts is the single largest factor
test originally published in 1944 and followed by a review of
determining tissue toxicity. If lengthy and extensive metabolism
refinements to the model. Regulatory requirements for approval
is required leading to excessive generation of free radicals then
of new topical ophthalmic agents will be reviewed, including
the substance is more likely to buildup in the tissue and cause
descriptions of GLP study designs currently recommended by
toxic effects.
the FDA prior to commencing clinical trials and for new drug
• Ocular irritation and ocular toxicity are independent processes
application (NDA) submission. Finally, we will discuss com-
that are often confused. Irritation involves direct action on pain
mon findings noted during GLP toxicology studies and how
receptors rather than secondary toxic activity related to
these findings are interpreted.
xenobiotic metabolism.
• The Draize test is still considered a standard evaluation for MOLECULAR MECHANISMS OF
assessing ophthalmic irritation potential, however the original OPHTHALMIC TOXICITY
Draize design has been improved to better qualify the potential
toxicity of long-term repeated dosing with topical ophthalmic
At the molecular level, toxicity is a byproduct of a series of
drugs. Improvements to the original Draize test include
reactions by which the body converts hydrophobic, xenobiotic
changes to the scales, better standardization of dosing and
substances into hydrophilic compounds that can be easily
timepoints, improved imaging technology to better assess
excreted. In response to exposure to foreign substances, a series
tissue damage, and the addition of systemic and organ health
of metabolic reactions occur at a cellular level including oxi-
assessments to better account for any systemic effects
dation, hydroxylation, and reduction followed by conjugation
following repeated dosing.
reactions including glucuronidation, sulfation, and acetylation.1
• Regulatory requirements for approval of an ophthalmic agent
These metabolic responses are mediated by a variety of different
vary depending on intended route of administration, intended
enzymes, including those associated with the cytochrome
dosing regimen, and historical knowledge of drug side effect
P-450 system. Free radicals, including nitric oxide (NO), and
profile. The FDA requires a series of genotoxicity studies,
hydrogen peroxide are generated as byproducts during this
acute in vivo toxicity studies in multiple species (featuring dosing
metabolism and tissue damage that occurs with exposure to
via the intended route of administration and via systemic
these volatile molecules is what immediately translates into a
administration), chronic dosing in vivo toxicity studies, and
toxic clinical affect.2
reproductive toxicity studies to establish a margin of safety
The cytochrome P-450 enzymes are critical in the metab-
prior to approving a drug for human use. Close communication
olism of a number of foreign substances and are involved early
with the FDA is recommended to clarify appropriate toxicology
in the process of xenobiotic metabolism. Most P-450 enzymes
study designs and to ensure that a toxicology program fully
have been identified in the endoplasmic reticulum of the cell
supports the intended clinical use of a drug.
and are typically membrane bound. The P-450 system is most
famous for its involvement in steroid biosynthesis, however its
role in detoxification is also well known, with a specific role in
xenobiotic metabolism in the ocular tissues well established.3
INTRODUCTION – PRINCIPLES OF Cytochrome P-450 enzyme systems have been detected in the
TOXICOLOGY OF THE EYE ciliary epithelium, conjunctiva, retina, and the corneal
epithelium.4,5
Historically, ophthalmic toxicology has been presented as a list With xenobiotic metabolism, the P-450 system is activated
of classes of agents and their record of ophthalmic toxicities. immediately upon exposure to the drug, or other foreign sub-
Unfortunately, the traditional approach to qualifying ophthal- stance. P-450 enzymes then catalyze a variety of reactions
mic toxicology is deficient, failing to provide the clinician with including oxidation, reduction, and hydrolation. These enzymes
an encompassing view of the underlying science of toxic reac- are known to activate oxygen and are classified as monoxy-
tions in the eye, the research methods by which toxicity is genases.6 During these early reduction reactions, the P-450
categorized, and the requirements for toxicology testing put in enzyme typically donates an oxygen molecule to the substrate
337
 PHARMACOLOGY AND TOXICOLOGY
to which it binds with the second oxygen molecule reduced as
water. Oxidation, reduction, and hydrolation reactions can
result in free radical production, which in turn contribute
to local toxicity, inflammation, and in some instances
neovascularization.7
Xenobiotic substances may be further metabolized via glu-
curonidation. Glucuronidation involves the interaction of the
xenobiotic with UDP-glucuronate and the enzyme UDP-
glucuronyltransferase and typically transforms xenobiotics into
hydrophilic products which can more easily be removed from
circulation. Glucuronidation is used by the body to recycle
certain molecules such as heme, which is produced during the
metabolism of hemoglobin. Glucuronidation is responsible for
the conversion of heme product bilirubin into bilirubin
diglucuronide, a water-soluble compound that can be easily
removed from the body.
Sulfation is another metabolic method by which the body
eliminates xenobiotic substances. A sulfate molecule is donated
to a substrate by 3-phosphoadenosine-5-phosphosulfate in the
presence of sulfotransferase. Phenols, alcohols, and aromatic
amines are frequently metabolized via sulfation. Free radicals
can be generated during sulfation reactions.
Acetylation provides still another mechanism by which
xenobiotics are further metabolized and prepared for excretion.
N-Acetylation of arylamine is a common route of metabolism
for many drugs in which an acetyl group is transferred to
the arylamine substrate in the presence of arylamine
N-acetyltransferase.8
SECTION 4
Biooxidation reactions occur frequently during xenobiotic
metabolism and the byproducts of biooxidation (free radicals,
super oxides, and hydrogen peroxide) can cause toxicity via a
number of different mechanisms. If free radicals are present at
sufficiently high levels, than lipid peroxidation may occur. Lipid
peroxidation occurs when free radicals incorporate themselves
into polyunsaturated fatty acids, which will eventually cause
the break down of biological membranes.9 Breakdown of the cell
membrane will cause cell death and spilling of cellular contents,
which in turn exacerbates inflammation, causes influx of
inflammatory cells and may lead to further tissue damage and
swelling. Both the lens and the tissue of the retina, because they
are exposed to high levels of light, are especially susceptible to
biooxidation reactions. In the retina, biooxidation and lipid
peroxidation have been related to AMD while in the lens
biooxidation has been shown to contribute to the development
of cataracts.
The ease with which the body can convert xenobiotic sub-
stances (drugs) to hydrophilic byproducts and excrete these
byproducts is the single largest factor determining tissue toxi-
city. If lengthy and extensive metabolism is required leading to
excessive generation of free radicals then the substance is more
likely to buildup in the tissue and cause toxic effects. On the
other hand, if a drug easily metabolized and excreted without
significant generation of free radicals, super oxides, hydrogen
peroxides, and otherwise volatile molecules, it can be assumed
that the agent will have a less toxic profile.
IRRITATION VERSUS TOXICITY:
UNDERSTANDING THE DIFFERENCES
Recognizing the difference between toxicity and irritation is
critical to understanding toxic responses in the eye. Toxicity
generally occurs in a dose-dependent fashion and is reprodu-
cible. There is, in general, a consistent relationship between the
level of toxicity exhibited and the amount of an agent given.
Because toxicity is dependent on metabolism and buildup of the
specific compound administered, toxic effects are not always
338 immediate. Irritation is not entirely related to reactive (and tissue-
destructive) oxygen byproducts that occur with compound
metabolism. Instead irritation is dependent on the physical
characteristics of the compound itself (pH, osmolality, concen-
tration, etc.) and is mediated primarily by immediate activation
of sensory neurons in the eye, rapid prostaglandin release, and
minor tissue damage. Irritation in its most severe forms can be
considered trauma, with severe alkali and acid burns causing
immediate tissue necrosis.
Irritation occurs when a chemical substance applied to the
surface of the eye elicits antidromic sensory activation, alter-
ations in membrane permeability, and/or release of arachidonic
acid metabolites, causing immediate pain, discomfort and
inflammation.10 The exact involvement of the various factors
that contribute to irritation differs depending on the extent and
nature of the ocular irritation and the particular irritating agent.
Antidromic sensory activation quickly differentiates irritation
from toxicity, as toxic effects are apparent following drug meta-
bolism rather than preceding it. Here, the irritant binds directly
at temperature and chemical sensitive receptors found on
sensory nerve endings causing immediate sensation of pain or
irritation. A variety of nerve ending receptors may play a part in
the immediate irritation response. Receptors of the transient
receptor potential (TRP) family are associated with ocular irri-
tation.11 The vanilloid receptor is a member of the TRP family
that is activated by capsaicin, hot temperatures and protons
(H+).12 It is therefore likely that the vanilloid receptor is at least
in part responsible for the irritation that occurs following
administration of an acidic eyedrop.
Others receptors involved in ocular irritation include the
menthol receptor, a member of the TRP receptor family activated
by cold stilumi and neurokinin-1 receptor which binds substance
P in the traditional pain response pathway.13 Additionally,
members of the TRP receptor family are bound by bradykinin,
a well-known proinflammatory peptide released following tissue
damage and involved in inducing pain.
RESEARCH METHODS FOR QUALIFYING
TOXIC EFFECTS
Ocular toxicity and irritation testing is required by all manner
of regulatory authorities for approval of ophthalmic products
and nonophthalmic products, including new raw materials,
additives, or any substance which could at any point be exposed
to the eye. Generally speaking, there is not an in vitro test
available that can effectively predict the human response to
ocular exposure to a specific agent. As a result, animal models
remain standard for evaluating ocular toxicity. The Draize test,
an in vivo model which was originally published in 1944, is still
frequently relied upon to qualify ophthalmic toxicity of both
ophthalmic and nonophthalmic agents, despite the fact that the
Draize test was initially designed to test the ocular irritation
potential of nonophthalmic products. Over time it has become
apparent that the Draize test has some deficiencies. The test
was designed to assess acute irritation only and many agents are
known to be toxic only after long-term, repeated exposures.
While certainly of some value to researchers, new research
methods are being employed which seek to improve on the
Draize model. In this section, we will discuss the original
Draize test and the modifications to the Draize test that aid
with identification and qualification of the toxic potential of
ophthalmic drugs.
THE DRAIZE TEST FOR OCULAR TOXICITY
The Draize test, which was developed some 60 years ago, is still
a standard test for assessing acute toxic potential of ophthalmic
agents.14 The primary deficiency of the Draize test is that it was

designed to assess acute irritation potential (which is frequently
related to pH, osmolality, etc.) rather than long-term toxicity
that may occur after repeated dose administration. Ocular tox-
icity does of course involve reactions that occur with extended
exposure and repeated metabolism of the foreign agent. As per
the original Draize test methodology, only alterations to the
anterior portion of the eye, the conjunctiva, cornea, and iris were
formally assessed. Any changes that occur at the retina cannot
be evaluated using original Draize methodology. Nonetheless,
the Draize test still provides a good idea of the irritation or
acute toxicity potential of a compound and can be helpful for
determining how to label chemical products and other agents
for severity of irritation.
The original Draize design involves topical dosing with
100 mL of an agent to the conjunctiva of the New Zealand White
rabbit. Following dosing, a series of clinical endpoints are eval-
uated at predetermined timepoints. These clinical endpoints
include conjunctival congestion, chemosis, discharge, iritis, cor-
neal opacity (size and degree of) and are graded in accordance
with an incremented scaling system. These scales are combined
to produce a cumulative Draize score which provides an indi-
cation of how irritating or toxic a compound will be to the
human eye. Historically, six rabbits have been used per test
compound when conducting the Draize test.
To a certain extent, the Draize test is predictable. An extre-
mely acidic or basic agent (below pH 2.5 or above pH 11.5) will
cause ocular irritation. Furthermore, dermal tests can be
performed on guinea pigs, rats, mice, and rabbits that can often
determine whether or not ocular dosing will cause irritation. In
many instances, it can be determined whether or not an agent
will cause ocular irritation prior to putting an agent in the
rabbit’s eye. If the Draize test alone is not predictive of long-
term toxicity and is not necessary for identification of acute
irritation, than it may be of questionable utility. Our experience
has been that the Draize test alone is a decent indicator of acute
irritation potential and we typically use the Draize test over
other procedures that involve application via a dermal route;
however we will not test an ophthalmic agent that falls outside
of preestablished comfort matrixes (pH ranges, etc.).
Because it is an acute design, the Draize test also fails to take
into account the possible sensitizing effect of an ophthalmic
agent. A minimum of two doses must be given to allow for
buildup of IgE in the system (sensitization in response to
immunoavailability of dose 1) and subsequent hypersensitivity
response (cross-linking of IgE in response to dose 2).
Other criticisms of the Draize test include the fact that
graders do read responses differently, despite the fact that the
Draize test is based on a standard scale. Additionally, the Draize
test does not mandate specific assessment timepoints and dif-
ferent investigators may score irritation at different timepoints,
leading to inconsistencies in scoring. Finally and perhaps most
importantly, the Draize test does not adequately define a
method for instilling drug. Some investigators may pipette an
agent directly onto the surface of the eye while different
investigators administer the test agent into the conjunctival
sac. These different methods of administration can cause a net
difference in the amount of dose given, as drug may fall out of
the eye more readily with one method than the other, and can
cause a disparity in the amount of drug that comes into contact
with certain ocular tissues. These variations can make it
difficult to compare Draize test results conducted by different
investigators.
IMPROVEMENTS TO THE DRAIZE TEST
While the Draize test is an acceptable means for identifying the
acute irritation potential of a compound that comes into con-
Principles of Toxicology of the Eye
tact with the ocular surface, repeated exposure and assessment
of long-term toxicity mandates different and improved method-
ology. The Draize test is an old test, and since its development
a variety of novel techniques have been developed that can
assess the toxic potential of agents in a variety of different
ocular tissues beyond the anterior region of the eye. Confocal
microscopy, pachymetry, specular microscopy, fluorophotometry,
fluorescein staining, tonometry, and histological assessments
provide a more sensitive means for assessing alterations to the
ocular tissues that may occur with toxicity. Furthermore, the
collection and histological evaluation of all ocular tissues
provides an encompassing picture of the way an agent impacts
the eye, rather than a narrow view of the conjunctiva, cornea,
and anterior chamber.
The McDonald–Shadduck scoring system for ocular lesions15
improved upon the limited Draize scale and is widely used as an
alternative test for scoring clinical signs of ocular toxicity. The
McDonald–Shadduck scoring system provides standardized
scales for assessment of conjunctival congestion, conjunctival
swelling, conjunctival discharge, aqueous flare, iris involve-
ment, corneal opacity (area and severity), pannus, and fluor-
escein staining, expanding on the limited Draize parameters.
That is not to say that the McDonald–Shadduck scale is suffi-
cient; however, when combined with dilated fundus exams to
assess the retina and optic nerve, tonometry to assess IOP,
and histology on all ocular tissues following sacrifice, the
McDonald–Shadduck scoring system provides an acceptable
methodology for evaluating toxic effects related to repeat dosing.
CHAPTER 32
The importance of new technologic developments in imaging
to supplement the standard gross clinical endpoints described
using the McDonald–Shadduck scoring system cannot be
overemphasized. The use of pachymetry can also be employed
to measure subtle changes in corneal thickness that might not
be readily visible under slit-lamp exam.16 Confocal microscopy
can be used to assess pathophysiological events as they occur
in vivo.17 For this reason, the confocal technique holds sig-
nificant promise for use in understanding toxic responses.
Additionally, only a small dose needs to be given to observe
molecular activity at the surface of the eye using the confocal
technique, which is useful as dosing with small quantities of
drug limits potential for irritation. Specular microscopy can be
used to assess changes in endothelial cell permeability following
drug treatment at the ocular surface when combined with
fluorescein administration.18 Changes in the structure of the
corneal epithelium leading to increased penetration of agents
could result in increased toxicity that occurs with increased
exposure. Electroretinography (ERG) is quickly becoming an
important technique for assessing toxicity in the retina. Using
a corneal contact lens electrode to measure the response of the
retina to light stimuli, toxic effects on photoreceptor function
can be identified. A specific pigmented rat model has been
developed, however dog, cat, and monkey models are also
frequently employed.19–21
The Kligman maximization test, first published in 1969, was
developed to account for the sensitization potential of an agent.
While this is not ocular toxicity design, it can be used to supple-
ment an appropriate ocular toxicity study and provide informa-
tion on whether an ophthalmic agent may act as a sensitizer.
The design calls for dermal application but can be used to
screen ophthalmic compounds.22 The Kligman test features
repeated dermal application of the test agent followed by
evaluation of the skin to determine whether or not a hyper-
sensitivity reaction has occurred. Known sensitizers are used as
controls. Since it was originally published, the test has been
altered slightly.23
It is important also to remember that repeated ocular dosing
may have systemic toxic effects, usually less than oral or 339
 PHARMACOLOGY AND TOXICOLOGY
intravenous administration, as drugs delivered to the eye enter
the bloodstream and are often metabolized by the liver. As a
result, a thorough evaluation of toxicity of an ophthalmic agent
should take into account any potential systemic effects by
observing clinical signs, body weight changes, food consump-
tion changes, behavioral changes, hematology, coagulation
studies, urinalysis, clinical chemistry, toxicokinetic assess-
ments, and changes in organ weights/appearance following
necropsy. Any findings should be followed by full histology on
the impacted tissues.
Other basic improvements made to the Draize test include a
decrease in the dose size given and a decrease in the number of
animals used per concentration. The original Draize method-
ology called for a dose size of 100 mL; however, the average
eyedrop size is ~30–40 mL. One hundred mL is far too high a
volume to deliver effectively to the eye, the eye cannot hold
such a high volume of fluid and such a volume is not clinically
relevant. (Remember, the human eye holds ~20–30 mL.) Most
toxicity studies employing topical dosing have decreased drop
size to clinically relevant volumes (30–40 mL). Furthermore, the
number of animals used per concentration of test agent was
defined as nine under original Draize methodology. Under the
Code of Federal Regulations set forth in 1979, this figure has been
reduced to six animals, and further reductions are possible.
DRUG APPROVAL PROCESS:
REQUIREMENTS FOR APPROVAL OF A
TOPICAL OPHTHALMIC AGENT
SECTION 4
The requirements put in place by regulatory authorities for
approval of topical ophthalmic agents are designed to ensure
that the agent is safe, nonirritating, and nontoxic, with an ample
margin of error based on the amount of drug administered. This
simple paradigm can serve as the overall strategy for designing
dose selection and dosing regimen for ‘good laboratory practice’
(GLP) ocular toxicity studies which will be submitted to regu-
latory authorities as a component of an investigational new
drug application (IND) or NDA. In general doses selected and
dosing regimen should approximate or exceed clinical expecta-
tions (preferably exceed). For studies submitted as components
of the IND, the duration of GLP ocular toxicity studies should
exceed or at least equal the duration of proposed clinical studies
to again ensure an appropriate safety margin.
The extent of toxicology testing required for a new ophthal-
mic drug depends on several factors; the amount of toxicology
data already available for that particular drug/class of agents, the
intended dosing regimen/indication, and the route of adminis-
tration. For a new molecular entity, toxicity studies using a
systemic route (e.g., oral, subcutaneous, or intravenous
depending on bioavailability of the compound) should also be
performed to assess potential toxicity with systemic absorption.
For agents that are administered daily for extended periods (e.g.,
ocular hypotensives), toxicology requirements will be greater
than for agents that are administered as needed or irregularly
(antibiotics) or regularly for a specific but relatively short period
of time (e.g., NSAIDs).
This section will describe those standard studies that
regulatory authorities require for approval of topical ophthalmic
agents. While additional studies may be required for certain
classes of agents or chronic indications, or for ophthalmic
agents administered via a nontopical route, the following stu-
dies represent the core toxicology studies that in our experience
should be completed for development and approval of all ocular
ophthalmic products. We will discuss toxicology requirements
of the Food and Drug Administration (FDA) for both IND and
NDA submissions, based on experience working with the FDA
340 as consultants on 26 NDA approvals.
TOXICOLOGY REQUIREMENTS FOR IND
SUBMISSION
Included in the IND submission should be a standard battery of
genetic toxicity tests (Ames reverse mutation assay, in vitro test
with cytogenetic evaluation of chromosomal damage with
mammalian cells or an in vitro mouse lymphoma TK assay, and
an in vivo test for chromosomal damage using rodent hema-
topoietic cells), a non-GLP melanin binding assay, and GLP
ocular toxicity studies in two different species for a duration
that will exceed that of the initial proof of concept clinical trial.
The genetic toxicity studies are standard designs that assay
genotoxic potential of the compound itself and do not use the
ophthalmic solution, rather the active pharmaceutical ingre-
dient (API). The melanin binding study is desired to gain a
better understanding of the pharmacokinetic profile of the test
agent, and is also necessary to determine whether or not pig-
mented animals are needed in follow-up GLP toxicology studies
as an agent which binds melanin will have a different phar-
macokinetic in an albino species. The GLP ocular toxicity
studies involve dosing via the proposed clinical route of admi-
nistration (ocular topical, subconjunctival, intravitreal, etc.).
Clinical signs, gross necropsy and histopathology are all moni-
tored to ensure that the test agent does not cause a toxic effect.
GENETOX STUDIES
The Ames reverse mutation assay is a standard design used to
assess the ability of the test substance to induce reverse
mutations in the histidine and tryptophan genes of Salmonella
typhimurium and Escherichia coli respectively. Histidine and
tryptophan are required for the growth of Salmonella
typhimurium and Escherichia coli respectively. Strains of S.
typhimurium and E. coli with a mutation in these genes are
grown in the presence of exogenous histidine or tryptophan.
These organisms cannot grow without addition of these
proteins unless they are able to reverse the mutation in these
specific genes and produce the protein endogenously.
A range finding assay is performed as an initial step to deter-
mine the concentration at which the test article is cytotoxic.
The test bacterial strains are then exposed to a series of stan-
dard concentrations of the test agent below the established level
of toxicity to determine the ability of the agent to reverse
mutations in the aforementioned genes, with and without a
metabolic activator (Aroclor-induced rat liver S9). Colonies are
plated immediately after exposure and revertant colonies (any
colonies that are able to survive and grow once plated) are
counted. A confirmatory assay is performed with a pre-
incubation period to verify results of the initial assay. Positive
and negative controls are employed to ensure a valid and sensitive
test system.
The chromosomal aberration assay is also a standard design
which tests the ability of the test agent to induce chromosomal
aberrations in Chinese hampster ovary (CHO) cells in the
presence and absence of a metabolic activating system. The
initial step in the chromosomal aberration study is a range-
finding assay which identifies the concentration of active drug
that is cytotoxic. From this concentration, a standard series of
dilutions are performed to create concentrations which will be
used for the actually assay. Drug concentrations are incubated
with CHO cells for a period of 3 h with or without a metabolic
activator (Aroclor-induced rat liver S9). Following this incu-
bation period, cells are assayed for chromosomal structural
aberrations or polyploidy to determine whether or not the test
substance alters these parameters. Typically, a confirmatory
assay with increased duration of exposure (up to 21 h) is also
employed without metabolic activation. Positive controls

mitomycin C and cyclophosphamide are employed to verify
sensitivity of the test system.
The in vitro mouse lymphoma assay is a third genetox assay
required under ICH S2B. While there are a number of cell lines
that can be used, the L5178Y TK+/–3.7.2C mouse lymphoma
cell is standard. This assay identifies mutations that are
associated specifically with cancerous changes and other human
genetic illnesses.24 The in vitro mouse lymphoma assay is not
always requested by FDA prior to IND submission for an
ophthalmic compound. The Ames and chromosomal aberration
assays are almost always performed, however, the sponsor may
choose either mouse lymphoma TK assay or chromosomal
aberration assay as the second in vitro genotoxicity study.
The test agent is administered at several different dose levels
and compared to positive (mitomycin C) and negative controls.
Mitomycin C is a known mutagen that induces polychromatic
erythrocytes.
It is possible that in vivo metabolism of a particular agent
could have mutagenic effects not identifiable using the in vitro
Ames and chomosomal aberration designs and therefore the
mouse micronucleus test is require by the FDA as assurance
that the compound is not mutagenic. This in vivo genetic
toxicity study should be completed prior to Phase 2 initiation.
This of course depends on the nature of the compound and
dosing regimen.
NON-GLP MELANIN BINDING
Melanin is the pigment protein found in the iris, skin, and a
variety of other tissues of the body. The pharmacokinetic profile
of a topical ophthalmic agent is greatly influenced by whether or
not that particular agent binds to melanin. If the agent does
bind melanin, then the iris can act as a sink (holding drug until
it is broken down and metabolized by the body), or it can have
a sustained-release effect, binding melanin for a brief period of
time before being released, sometimes adding to duration of
effect. In either case, in vivo GLP toxicology studies must be
performed in pigmented animals if the active agent does in fact
bind melanin. As an example, the New Zealand White rabbit,
which is the standard albino rabbit used in toxicity testing,
should be substituted out in favor of the Dutch–Belted pig-
mented rabbit (or another pigmented species) if the test agent is
found to bind melanin. Failure to do so could produce false-
positive toxic effects that would not normally occur clinically (if
the iris acts as a sink and facilitates drug removal), or false
negatives (if melanin binding causes a sustained release and
prolonged effect.) A non-GLP melanin-binding study is a quick
and easy in vitro study that can be performed using synthetic
melanin or bovine melanin. The test agent is incubated for a
specific time period with synthetic melanin and is then filtered
and assayed for determination of melanin binding. Differences in
weight of free compound versus compound bound with melanin
are used to determine whether or not melanin binding occurs.
GLP OCULAR TOXICITY STUDY IN
RABBITS
The rabbit has been the preferred standard species for GLP
toxicology studies to support development of ophthalmic
products. While there are certainly differences between rabbit
and human eyes, the size of the rabbit eye and availability
(compared with primates) make them an ideal choice. The
GLP ocular toxicity study in rabbits is usually conducted with
concentrations higher than that which is anticipated as the
clinical dose, with elevated dosing frequency, and for a duration
that exceeds that of the intended proof of concept clinical trial.
As mentioned previously, this study should be performed in a
Principles of Toxicology of the Eye
pigmented species if a positive result is achieved during the
melanin binding study. Both male and female animals are
enrolled to determine if any toxic effects are gender specific.
The GLP ocular toxicology study involves repeated dosing via
the intended clinical route. If the agent being screened is a
topical ophthalmic agent then animals should be dosed top-
ically. If the agent is instead intended for intravitreal or subcon-
junctival administration, then dose should be administered by
intravitreal or subconjunctival route, respectively. Dosing
regimen generally elevates beyond intended clinical regimen to
provide a margin of safety. For example, a topical agent that is
intended for qd dosing may be administered as bid or tid while
an intravitreal agent intended for once a month dosing may be
administered twice a month.
Standard ophthalmic and systemic endpoints are evaluated
during the GLP ocular toxicity study. These parameters include
assessments of body weight and body weight gain, ophthalmic
observations (including fundoscopy, tonometry, slit-lamp bio-
microscopy with fluorescein staining, corneal opacity, iris find-
ings, conjunctival redness, chemosis, and discharge), clinical
observations including appearance of fur, skin, eye and mucus
membranes, behavioral changes, necropsy findings including
weights of major organs, and histology on all ocular tissues.
Ophthalmic observations are typically performed using the
MacDonnald–Shadduct scoring system for ocular lesions.
Organs weighed during necropsy typically include the following
(liver, kidneys, adrenals, testes, prostate, ovaries, uterus, thymus,
lungs, spleen, brain, pituitary gland, heart, thyroid, submandi-
CHAPTER 32
bular gland, stomach, intestines, and pancreas). Any premature
deaths that occur during the toxicity study may need to be
evaluated more thoroughly with full organ histopathology,
depending on the nature of the death. Findings are considered
in terms of observed toxic effects and are distinguished by
animal sex and treatment arm. Dose ranging is typically required
as an element of GLP ocular toxicity studies. Up to three
concentrations are typically run against a vehicle control. Initial
ocular toxicology studies should also include (either as part of
the study or as a separate study) preliminary pharmacokinetic
assessments for systemic absorption.
GLP OCULAR TOXICITY STUDY IN DOGS,
MONKEYS, OR OTHER SPECIES
The GLP ocular toxicity study in beagle dogs or monkeys
utilizes an identical design to the GLP study in rabbits and
fulfills the FDA’s requirement for a second species prior to
initiating clinical testing. As with the rabbit study, dosing regi-
men and dose given generally exceed that which is anticipated
clinically to ensure an appropriate safety margin. Dose ranging
should be included, with three concentrations being a standard
dose-ranging approach. Parameters evaluated should include
assessments of body weight and body weight gain, ophthalmic
observations (including fundoscopy, tonometry, slit-lamp bio-
microscopy with fluorescein staining, corneal opacity, iris find-
ings, conjunctival redness, chemosis, and discharge), clinical
observations including appearance of fur, skin, eye and mucus
membranes, behavioral changes, clinical pathology, necropsy
findings including weights of major organs, and histology on all
ocular tissues.
REQUESTS FOR WAIVERS ON PRE-IND
TOXICOLOGY REQUIREMENTS
Historically, ophthalmic drug products have often been devel-
oped as second-line products following systemic development
for similar indications. Topical antiinflammatory agents were
developed first for systemic inflammation, many angiogenic 341
 PHARMACOLOGY AND TOXICOLOGY
blockers developed for AMD were developed first as anticancer
therapies, and many antihistamines developed for allergic
conjunctivitis were developed first as systemic agents for
treatment of rhinitis. Given sufficient existing toxicology data,
the FDA may grant a waiver on systemic toxicity studies and
one of the required GLP ocular toxicity studies. Typically waivers
are requested for ethical considerations. The Pre-IND meeting
is an important time to review the proposed toxicology plan and
ensure that FDA has the opportunity to comment on the pro-
posed toxicology studies. While systemic studies may be waived
or shortened, ocular toxicology with the final ophthalmic
formulation is still needed.
TOXICOLOGY STUDIES REQUIRED FOR NDA
SUBMISSION
With the IND submission, FDA will require toxicology data to
support the safety of the test agent for the duration of the
intended proof of concept clinical studies. As a component of
the NDA, the FDA will require toxicology studies that support
the long-term safety of the ophthalmic drug product. At the
time of NDA submission, several clinical studies will be com-
pleted, however the duration of dosing for these studies may
vary considerably depending on indication and design. To be
included in the NDA submission are chronic ocular toxicity
studies (with toxicokinetic assessments), reproductive toxicity
studies (segments I, II, and III), absorption, distribution, meta-
bolism and excretion (ADME) studies to identify primary route
SECTION 4
of excretion and distribution of drug in the ocular tissues
following dosing, acute or repeat systemic toxicity as needed, a
mouse micronucleus test to assess in vivo mutagenic potential,
and if applicable, carcinogenicity studies.
The proposed indication, route of administration, and exis-
ting database of studies performed with a particular agent will
greatly influence the toxicology testing required by the FDA for
NDA submission. The following are standard studies recom-
mended by the FDA for approval of a topical ophthalmic agent.
CHRONIC OCULAR TOXICITY STUDIES
The chronic ocular toxicity study is often performed in parallel
with the phase-3 development program; however, for a drug
with a chronic indication, the study should be performed ahead
of the pivotal clinical studies. Typically, the FDA requires a
minimum of two species for a period of 6 months of repeat
ocular dosing for NDA approval of a topical ophthalmic agent.
If ocular and systemic toxicity following ocular dosing does not
appear to be a concern, or other approved ophthalmic drugs in
the same class demonstrated very low systemic and ocular
toxicity potential, chronic ocular toxicity studies in one most
appropriate species may be acceptable.
The chronic ocular toxicity study is usually designed
similarly to the GLP ocular toxicity studies performed prior to
IND submission. Route of administration should be consistent
with the intended clinical route of administration and some
dose ranging should be included. Frequently a recovery group is
employed to determine whether or not any toxicity noted at the
final sacrifice resolves following discontinuation of dosing. This
recovery group may be continued for 4 weeks to several months
after discontinuation of dosing. Doses given and dosing regimen
often approximate or exceed the intended clinical dose and
dosing regimen, with appropriate vehicle controls.
As described above, endpoints evaluated during the chronic
ocular toxicity study generally include assessments of body
weight and body weight gain, ophthalmic observations
(including fundoscopy, tonometry, slit-lamp biomicroscopy with
342 fluorescein staining, corneal opacity, iris findings, conjunctival
redness, chemosis, and discharge), clinical observations including
appearance of fur, skin, eye and mucus membranes, behavioral
changes, necropsy findings including weights of major organs,
and histology on all ocular tissues. Organs weighed during
necropsy typically include the following: liver, kidneys, adrenals,
testes, prostate, ovaries, uterus, thymus, lungs, spleen, brain,
pituitary gland, heart, thyroid, and pancreas. An added element
of the chronic toxicity study includes the evaluation of
pharmacokinetic parameters, hematology, coagulation, urinalysis,
and clinical chemistry. Pharmacokinetic parameters, such as
AUC, Cmax, and Tmax are calculated at specific timepoints during
the study to ensure that there is no buildup of drug following
repeated dosing. As with earlier studies, histology is performed
on all ocular tissues with full histopathology on any animals
that die prematurely. Pharmacokinetic assessments should be
completed after initial dosing and following repeated dosing
(e.g., at least following 1-month dosing).
SEGMENT I, II, AND III REPRODUCTIVE
TOXICITY
The effects of the ophthalmic drugs on all aspects of the
reproductive/developmental process (fertility and early embryonic
development, embryo–fetal development, and prenatal and
postnatal development) should be assessed to ensure that the
test agent does not adversely impact viability of offspring, cause
birth defects or impact fertility. If very low systemic exposures
occur and no systemic effects occur in repeated-dose studies,
it might be acceptable to perform only embryo–fetal develop-
ment studies. If the drug is intended for use in women with no
child-bearing potential, reproductive toxicity studies in female
animals can be waived. These are standard study designs that
many appropriate contract laboratories are experienced in
performing.
ADME STUDY
An ADME study should be performed for submission as a
component of the NDA. The purpose of the ADME study is to
provide the FDA with information on how drug is metabolized,
distribution following dosing, and route of excretion.
Typically the ADME study is performed in rabbits or another
appropriate species. The study drug can be radiolabeled and a
known quantity of radioactivity is administered via the inten-
ded clinical route. Animals are sacrificed at postdose timepoints
and ocular tissues are harvested for assessment of radioactivity.
Ocular tissues assessed include aqueous humor, upper and
lower eyelids, conjunctiva, cornea, iris/ciliary body, lens, optic
nerve, retina, choroid, sclera, and vitreous. Select organs may be
taken and assessed for levels of radioactivity. Plasma is taken at
each timepoint and assessed for levels of radioactivity. Urine
and feces are collected at specific increments following dosing
for determination of route of excretion.
The typical profile for a topical ophthalmic agent is to find
the bulk of radioactivity is located in the cornea, bulbar and
palpebral conjunctiva (eyelids), and aqueous humor at early
assessment timepoints. The majority of the dose given is
excreted within 24 h of dose administration. With topical
ophthalmic products, it is often difficult to get a full mass
balance, as a portion of the dose may be lost during instillation,
even if particular care is taken during instillation. Percent
recovery may vary considerably. For example, per their approval
documents, Optivar (Azelastine HCl 0.05%) percent recovery of
radioactivity was 84% while with Elestat (Epinastine HCl
0.05%), percentage recover was 97% (Summary basis of approval
for optivar (NDA#021127) and Elestat (NDA#021565). The
purpose of the ADME study is not to necessarily raise any

red flags on toxicity per se, but instead to provide a better
description of drug activity, or to help correlate with known
toxic effects.
ACUTE SYSTEMIC DOSE TOXICITY STUDY
The acute systemic dose toxicity study in the rat is performed
to determine the maximum tolerated dose (MTD) and the no
observable adverse effect level (NOAEL). This study features IV
administration using escalating doses to identify the level at
which adverse drug-related effects can be seen. For an oph-
thalmic agent, this is relevant as there should be a considerable
safety margin between NOAEL identified in the acute systemic
dose study and the anticipated systemic availability of the drug
when given via topical ocular dose. This study should employ
higher concentrations than are intended for clinical admin-
istration as some level of toxicity is desired. Again, several
difference doses should be tested.
In this study, a single intravenous infusion is performed.
Blood is collected prior to termination for hematology, clotting
profile, and clinical chemistry evaluations. Animals are eval-
uated for behavioral changes/clinical observations for a period of
14 days following dosing. Necropsies are performed on all
animals. Acute systemic toxicity studies can also be performed
with other routes (oral, subcutaneous, etc.).
CARCINOGENICITY STUDY
In many cases carcinogenicity studies are necessary to support
an NDA filing for ophthalmic products. The ICH Guidance
S1A states that carcinogenicity studies may be waived for drugs
given by the ocular route unless there is cause for concern or
unless there is significant systemic exposure. Causes for con-
cern include a previous demonstration of carcinogenic potential
in the product class, pharmacologic activity, a structure–activity
relationship suggesting carcinogenic risk, positive genotoxicity
results, evidence of preneoplastic lesions in multiple dose
toxicity studies, and/or long-term tissue retention of parent
compound or metabolites resulting in local tissue reactions or
other pathophysiological responses. A request for a waiver
should be submitted to the FDA if the sponsor considers the
drug eligible.
INTERPRETATION OF FINDINGS
In the event that abnormal findings are noted during toxicology
studies, it is the obligation of the researcher to determine the
nature of the findings, evaluate the severity, and determine the
clinical implications of the identified abnormalities. In some
instances, toxic findings are immediate and obvious, causing
animal death and/or overt sickness and immediately halting the
development program. More frequently, however, toxicity does
not manifest in animal death or morbidity. In most instances,
signs of toxicity are subtle and may include differences in organ
weights or organ appearance at necropsy, presentation of some
abnormal ophthalmic signs (such as injection, chemosis,
staining, or discharge), mild alterations in clinical signs, animal
appearance or behavior, changes in clotting, hematology or
clinical chemistry, or abnormal hisptopathological findings.
Subtle findings may present with little or no additional
indication that the animal’s long-term health is in jeopardy. An
animal may act and appear healthy, gaining weight at the same
rate as controls and presenting with no obvious changes, only to
have abnormalities noted at necropsy. It is the obligation of the
researcher to determine the implications of findings during
toxicology studies, and clinical relevance that subtle findings
hold.
Principles of Toxicology of the Eye
In general, findings during GLP ophthalmic toxicology
studies should be interpreted based on any dose-related asso-
ciations and whether or not findings fall within accepted norms
when working with animals. A brief outline of interpretation
of findings (necropsy, ocular signs, and histopathogy) is
included.
NECROPSY
Changes in organ weights/organ appearances at gross necropsy
are among the more common findings associated with topical
ocular toxicity studies. It is important to note that statistically
significant differences within treatment arms do not necessarily
indicate toxicity. The toxicology lab should have a database of
organ weight ranges that are considered normal and it is pos-
sible for organ weight to fall within these accepted norms and
still be statistically different from controls.
The toxicology reviewer will evaluate the extent of findings
and look for an indication of a dose-related response. If a
change in mean organ weight was apparent between the low
dose group and the placebo control however the high-dose group
was not statistically different, then it is less likely that a toxic
effect is occurring. If, however, there is a dose-related effect,
then that can be an indication of toxicity. If the lowest dose
exhibits abnormalities, then the FDA may request additional
toxicology work.
OCULAR SIGNS
CHAPTER 32
In the interpretation of positive findings in ocular toxicity
studies, the incidence, severity, and reversibility should be
evaluated, and toxicological significance and clinical relevance
should be considered. For example, minimal conjunctival red-
ness is common in untreated animals and is considered as
normal and not toxicologically significant.
In ocular toxicity studies, the drug is usually administered to
one eye, the other eye is used as an untreated control. Positive
findings in the drug-treated eyes should be compared with those
in untreated eyes and in vehicle-treated eyes to figure out if the
findings are drug-related, vehicle-related, or spontaneous
findings. In studies with intravitreal injections, injection pro-
cedure-induced inflammation is not unexpected. If the
inflammation in control and drug-treated groups was similar
regarding severity, incidence, and reversibility, it is not toxico-
logically significant.
HISTOPATHOLOGY
Histopathology changes provide an indication of toxicity at the
cellular level that might not be easily identifiable during clinical
exams. The histopathology evaluation can successfully identify
a wide range of conditions, including bacterial or fungal
infection, cancer, or chronic inflammation. Histopathology
performed on dead or morbid animals can provide a diagnosis
of the animal’s condition and cause of death and is therefore an
important tool in the interpretation of toxic findings. Logically,
an increased incidence of bacterial infection may indicate an
immunosuppressive effect of a test agent. Identification of
transformed cells may indicate mutagenic potential.
Histopathological findings should be interpreted in a similar
fashion as other toxic findings. It is important to consider any
dose-related response and it is also important to consider the
historical incidence of infection or disease as it relates to
histopathological findings. A seemingly disproportionate rate of
infection in a particular treatment group may not be toxi-
cologically significant if incidence within the study population
falls within accepted norms. 343
 PHARMACOLOGY AND TOXICOLOGY

SUMMARY body weight gain, necropsy parameters, clinical pathology,

A toxic response occurs as the byproduct of drug metabolism
and generally presents in a dose-related fashion over time. As a
drug is broken down via a variety of different xenobiotic meta-
bolic processes, oxygen radicals are produced. These and other
toxic byproducts cause the tissue damage that manifests as
clinical signs of toxicity. In contrast, irritation occurs via direct
binding of sensory neuron receptors in an acute fashion and
is generally not related to metabolism of the drug. Specific
qualities of the drug product, such as pH, contribute to its
irritation potential, rather than the ease by which it is
metabolized.
The Draize test was originally published in 1944 as a means
of evaluating the single-dose, acute irritation potential of non-
ophthalmic agents. Unfortunately, the test was frequently used
to assess the toxicity profile of ophthalmic drug products. The
original Draize methodology was not designed for this applica-
tion, nor was it adequate to accurately evaluate the sometimes
subtle effects of long-term, repeated ophthalmic administration.
Improved methodologies have been developed for qualifying the
long-term toxicity profile of ophthalmic drug products. Current
methodologies evaluate not only ophthalmic clinical signs
following repeated dosing but histopathology, body weight and
hematology, behavioral changes, and clinical appearance.
The FDA requires that agents are safe, nonirritating, and
nontoxic, with an ample margin of error based on the amount
of drug administered. In general, doses selected and dosing
regimen should approximate or exceed clinical expectations
(preferably exceed). Studies submitted as components of the
IND include GLP ocular toxicity studies in at least two species,
genetox studies, melanin binding studies, and acute irritation
studies. The duration of GLP ocular toxicity studies should
exceed or at least equal the duration of proposed clinical studies
to again ensure an appropriate safety margin. Studies submitted
as components of the NDA include GLP chronic ocular toxicity
studies, reproductive toxicity studies, ADME, carcinogenicity
studies, and systemic toxicity studies.
The requirements for toxicology testing referenced above are
based on our experience as a clinical regulatory group and do not
represent officially regulatory guidance. We believe it is important
for pharmaceutical developers to contact the FDA early in the
development process and remain in close contact with the FDA
through the preclinical and clinical development phases. The
FDA will assist with confirming the appropriateness of the toxi-
cology plan and can help with reviewing and interpreting results
of toxicology studies and implications for drug development.

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344
 CHAPTER
33 Toxicology of Ophthalmic Agents by Class
Millicent L. Palmer, Robert A. Hyndiuk, Mark S. Hughes, Ann Sullivan Baker, Kristine
Erickson, Alison Schroeder, James McLaughlin, Keith Lane, Sarkis H. Soukiasian,
Michael B. Raizman, and Cynthia Mattox
In this chapter, we will outline common toxic and irritative
effects associated with frequently prescribed classes of
ophthalmic drugs. Focus will be on identifying general trends
associated with specific drug classes rather than listing side
affects recorded for individual ophthalmic drugs. Covered under
this chapter are antiinflammatory agents, antiallergics, ocular
hypotensives, antiinfectives, and antiangiogenesis agents
prescribed for age-related macular degeneration (AMD).
Key Features
• Corticosteroids, well known for their highly effective
antiinflammatory profile, are associated with a variety of side
effects including ocular hypertension, cataract formation, and
delayed wound healing and increased susceptibility to
infection.
• Nonsteroidal antiinflammatory drugs in general have a less
adverse side affect profile compared to steroids. These agents
do impact platelet function and therefore carry a risk of
increased ocular bleeding. In addition, NSAIDs have been
shown to inhibit corneoscleral wound healing and create some
susceptibility to infection.
• Topical antiinfective are associated with a wide range of side
effects, including superficial irritation, chemosis, conjunctival
necrosis, epithelial toxicity, and macular infarction (following
intravitreal administration).
• Ocular hypotensive agents produce a wide range of side
effects, given that different classes of hypotensive agents act
via a variety of different mechanism. Beta-blockers are well
known for systemic side effects including bradycardia and
respiratory distress. Adrenergic agonists are known to cause
mydriasis and are associated with a high rate of drug-induced
allergy. Prostaglandins, generally considered the optimum
treatment for management of ocular hypertension, may cause
photophobia, conjunctival injection, pain, breakdown of the
blood aqueous barrier evidenced by anterior chamber cell and
flare, and changes in pigmentation and eyelash growth.
• Antiangiogenesis drugs for use in treating AMD act primarily by
blocking the function of VEGF. While these agents are relatively
new to the market, systemic availability of an administered
anti-VEGF agent could in theory impact the natural function of
VEGF in the body. In addition to its role in angiogenesis, VEGF
has both vasodilative and neuroprotective effects.
TOXICOLOGY OF CORTICOSTEROIDS AND
OTHER ANTIINFLAMMATORY AGENTS
The use of antiinflammatory agents is common in ophthalmic
practice because inflammation, a nonspecific response to tissue
injury,1,2 is frequently encountered. In this chapter, we review
adverse effects caused by local (topical and periocular)
administration of corticosteroids (CSs), the most commonly
used antiinflammatory agents. We also review the adverse
ocular and systemic effects of other agents used to treat ocular
inflammations, including nonsteroidal antiinflammatory drugs
(Nonsteroidal Antiinflammatory Drugs [NSAIDs]), antihis-
tamines and decongestants, mast cell-stabilizing agents, and
immunosuppressive drugs. This section focuses primarily on
conditions of the anterior segment.
SIDE AFFECTS OF CORTICOSTEROIDS
Systemic Side Effects due to Local Corticosteroid
Administration
Systemic side effects due to topical administration are unusual
even with long-term therapy,3 however, measurable and phy-
siologically significant systemic effects associated with frequent
topical use of concentrated preparations of potent CSs have
been reported. Burch and Migeon4 described a 19–72% re-
duction of urinary excretion of 17-hydroxy CSs after bilateral
administration of 0.01% dexamethasone every 2 h for 4 days
(daily systemic dose 0.75 mg); the cortisol production rate
decreased by more than 50% during the experimental period.
Prednisone, 10 mg, or its equivalent daily for 4 weeks may
suppress normal growth in the pediatric population.3 The
administration of a single drop of 0.1% dexamethasone sodium
phosphate four times daily in each eye yields a systemic dose of
0.25 mg.3 Lowering of plasma cortisol levels after 6 weeks of
such a regimen has been observed; the hypothalamic–pituitary
axis functions normally as measured by metyrapone testing.5
Prolonged orbital injections of CSs may also have systemic side
effects, such as adrenal suppression.6
Ocular Hypertension and Glaucoma
One of the most well-known side affects associated with topical
corticosteroid use is an elevation in intraocular pressure (ocular
hypertension). Ocular hypertension and glaucoma have been
well documented after both topical and systemic CS
administration.7,8 In 1950, McLean7 suggested that topical
steroid therapy might increase intraocular pressure (IOP)7;
the first case of ‘cortisone glaucoma’ was reported by Francois
in 1954.8
The dose–response relationship is particularly important in
understanding this undesirable side effect.9–11 Steroid-induced
ocular hypertension can reach clinically significant levels in
~36% of normal subjects on a short-term steroid regimen.10 A
differential susceptibility among individuals expressed as a
skewed distribution has been observed.9 A more pronounced
effect of increased IOP or disturbed aqueous fluid dynamics
has been noted in individuals with suspected glaucoma,12–18 345
 PHARMACOLOGY AND TOXICOLOGY
in those with myopia,19 in older patients, in patients with
glaucoma,12–15,17,18,20–21 in relatives of glaucoma patients,17,22,23–26
in patients with Krukenberg’s spindle,27 and in diabetic
patients.28
A time–response relationship of CS-induced ocular
hypertensive response has important implications. A clinically
significant rise in IOP typically, but not always, requires greater
than 1–2 weeks of topical therapy.10 With systemic steroid
administration, the ocular hypertensive response may require
longer treatment.29 A dose-dependent response after systemic
CS therapy has also been observed.30 A smaller concentration of
drug reaches ocular sites by the systemic route and may explain
the difference in the IOP response time between topical and
systemic routes of administration.10 The magnitude of the
ocular hypertensive response after systemic administration has
been noted to be similar to the response following topical
therapy.29
The mechanism of the steroid ocular hypertensive response
appears to involve an initial increase in aqueous inflow, as
suggested by Linner,31 with a secondary effect on the facility of
outflow.32 Several biochemical mechanisms have been proposed
to explain the decrease in outflow facility based on CS effects on
cells of the trabecular meshwork. These may include inhibition
of prostaglandin mediators33 and alteration of glycosamino-
glycan production or metabolism.32,34,35–37
The CS-induced ocular hypertensive response is usually
reversible, especially with short-duration therapy (weeks)10;
however, irreversible steroid-induced glaucoma has been clearly
SECTION 4
documented, especially in patients with myopia.34,19,38–41
Discontinuation of CSs may result in normalization of the IOP,
but visual field abnormalities and optic nerve damage may be
permanent.34,38,39 Many patients respond to medical anti-
glaucomatous therapy. When medical treatment fails and the
continued use of CSs is required to control ocular inflammatory
disease, argon laser trabeculoplasty and glaucoma filtering pro-
cedures may be required.39
Corticosteroid-Induced Cataract Formation
It is generally accepted that CSs are cataractogenic, commonly
producing posterior subcapsular cataracts (PSCs). The asso-
ciation of PSCs with systemic CS use was first reported by Black
and associates in 1960.42 PSCs developed in patients receiving
moderate or high doses of CSs for greater than 1 year’s
duration. Further work by Oglesby and co-workers,43 Giles and
colleagues,44 and Crews45 indicated that patients receiving
doses of less than 10 mg/day of prednisone or its equivalent or
patients receiving CS therapy for less than 1 year were unlikely
to develop PSCs. However, cataracts have been observed after
even short-term CS therapy,46 and some authors now argue
against the concept of a ‘safe’ noncataractogenic dose.47 Both
systemic and topical administration of CS may induce PSC
formation; those caused by systemic use are bilateral.42–50
Although steroid-induced PSCs occurrence is dose and duration
dependent, the precise relationship of lens changes to the total
dose, the intensity of the dose, and the duration of therapy is
not fully understood.47 Some studies suggest that individual
susceptibility and perhaps even genetic determinants may be
important.46,47,51 Children46,50 and diabetic patients52,53 appear
to be more susceptible.
The pathophysiology of steroid-induced cataracts is similar
to the mechanism of cataract formation proposed for galactose
in that steroids increase the influx of cations,54 resulting in an
increase in the cellular water content, producing cellular
intumescence and disparity of the refractive index from that of
the surrounding medium.39 Glucocorticoids also bind to
specific amino acid groups of the lens cell fibers, leading to a
346 conformational change and exposure of buried sulfhydryl
groups.39 These moieties (i.e., the sulfhydryl groups) form
disulfide bonds and create protein aggregation and a change in
the refractive index.39
Delayed Wound Healing and Effects on Corneal
Reepithelialization
The effect of CSs on corneal wound healing has been the focus
of several investigations.55–68 Corneal wound integrity has been
evaluated by determining the tensile strength,58,59,61,63,64,69
histologic appearance55,58,60 and uptake of tritiated thymidine by
keratocytes.62 Although the results of these studies are some-
what inconsistent, a dose–response effect of CSs on corneal
wound healing has been demonstrated.63 Impaired corneal
wound healing has been less pronounced when steroids were
withheld until after the tenth postoperative day, after which
topical CS treatment did not significantly interfere with the
tensile strength of the healing wound.69
Topical and systemic cortisone derivatives have a depressant
effect on many phases of the healing process. Alterations in
fibroblast proliferation, vascularization, and deposition of extra-
cellular matrix have been observed.56 CSs primarily affect
stromal healing to a greater extent than epithelial healing.
Effects of CSs on corneal epithelial healing have been observed
and may be related to the extent of epithelial injury. Topical CSs
do not impair epithelialization after partial corneal denuda-
tion,66,67 but impairment is observed after complete denudation
in a rabbit model.70
Investigative studies have demonstrated that the enzyme
collagenase is produced in Pseudomonas and herpes simplex
corneal ulcers, alkali burns, and ulcerations associated with
collagen vascular diseases and Stevens–Johnson syndrome.65
CSs may induce rapid destruction or corneal ‘melting’ and even
perforation in these conditions, possibly by enhancing
collagenase activity.3,65
Corticosteroids and Infectious Keratitis
Because CSs alter the host immunologic responses to infection,
their use in the presence of an active infectious process is often
contraindicated.3,67 In addition, chronic use of CSs may alter
normal and pathogenic flora of the lids and conjunctiva.71 The
incidence of corneal thinning and perforation in severe
infectious keratitis may be increased owing to the potential
enhancement of collagenolytic enzymes or decreased collagen
synthesis and wound healing.3,72 In certain cases, judicious use
of CSs may be appropriate to limit the structural damage related
to the inflammatory process. In general, the use of CSs should
be avoided until the infectious process has been controlled by
specific antimicrobial therapy. In the following sections,
important issues regarding the use of CSs are briefly reviewed
by the category of infectious agents: viral (herpes simplex),
bacterial, and fungal.
Herpes Simplex Virus
Topical CS therapy is contraindicated in the presence of active
viral replication associated with herpes simples virus (HSV)
epithelial keratitis.73 The deleterious effects of CSs in
management of HSV infection have been clearly
documented.74–76 Local CSs, however, do not appear to
reactivate latent HSV keratitis or stimulate an episode of
dendritic or stromal keratitis.77 CS therapy plays a role in
controlling the immunologically mediated inflammation of
HSV stromal disease. The results of the Herpetic Eye Disease
Study, a multicenter, randomized, double-masked clinical trial,
revealed the efficacy of topical CSs in HSV stromal keratitis.78
The initiation of CS therapy should be avoided if steroids were
never used previously. Clinically, a careful risk-benefit analysis
should be made on an individual basis.

Bacterial Keratitis
The risks associated with CS therapy in the management of
bacterial keratitis are a subject of controversy. Several reports
favor adjunctive CS therapy in bacterial keratitis. Davis and co-
workers79 demonstrated that concurrent treatment with CSs
did not inhibit the effect of antibiotics in Pseudomonas
keratitis. Aronson and Moore80 claimed that CS treatment pro-
moted the resolution of inflammation associated with
infectious keratitis in their series. A favorable visual outcome,
however, was observed only in mild cases of paracentral
keratitis. Leibowitz and Kupferman81 concluded that the
concurrent use of topical CSs with an effective bactericidal
antibiotic regimen did not enhance the replication of Staphy-
lococcus aureus or Pseudomonas aeruginosa if the CS was not
instilled more frequently than the antibiotic.
CSs have been shown to enhance P. aeruginosa replication
within the cornea if there is inadequate antimicrobial
therapy.82,83 Animal studies have indicated that despite 5 days
of treatment with an effective antibiotic, corneas infected with
Pseudomonas were not sterilized.84 Further recurrences of
Pseudomonas keratitis have been reported in eyes treated with
CSs.85 CSs are contraindicated in eyes that have advanced
corneal thinning with the potential for perforation, owing to
possible enhancement of collagenolytic enzymes or inhibition
of collagen synthesis. A controlled prospective clinical study by
Carmichael and co-workers86 evaluated CS therapy with and
without antibiotic therapy in bacterial ulcers and found no
differences in visual outcome. Considering the risks, steroids
should not be used if there is not a significant chance of
preventing visual loss or of recovering lost vision; that is,
control of inflammation alone should not be the deciding factor.
Fungal Keratitis
The use of CSs in the early treatment of fungal keratitis is
generally contraindicated owing to an enhancement of growth
of both yeast and opportunistic fungi.87 A clinical worsening of
fungal keratitis has been demonstrated after treatment with
CSs.88–91 In contrast to the number of available antibiotics,
there are relatively few antifungal agents. In general, these
agents are poorly soluble and have limited ocular penetration;
therefore, ocular bioavailability and the efficacy of antifungal
agents are less than ideal, reaching only fungistatic, as opposed
to fungicidal, corneal levels.92 CSs may negate the effects of
antifungal therapy and suppress host immune responses. Host
immune responses may be particularly critical in controlling
the inflammation of keratomycoses. The use of CSs to reduce
stromal scarring, intraocular inflammation, and corneal
neovascularization continues to be controversial.93 Adjunctive
CS therapy should be considered only in combination with an
effective antifungal agent or agents in the later stages of a
healing fungal keratitis.93
NONSTEROIDAL ANTIINFLAMMATORY DRUGS
NSAIDs have analgesic, antiinflammatory, and antipyretic
properties.94–102 The antiinflammatory activity is related to the
inhibition of the enzyme cyclooxygenase; this enzyme is
responsible for the conversion of arachidonic acid to prosta-
glandins, which are potent inflammatory mediators.94–100 These
agents are widely used in the treatment of musculoskeletal
disorders such as osteoarthritis, rheumatoid arthritis, anky-
losing spondylitis, and acute gout.94 Topical NSAIDs have been
approved for pain, postoperative inflammation, inhibition of
miosis during intraocular surgery, and for photophobia.
Oral NSAIDs have been useful in the management of
patients with uveitis, particularly recurrent anterior uveitis.96
Foster96 has reported that diflunisal (Dolobid) was the safest
Toxicology of Ophthalmic Agents by Class
and most effective; naproxen (Naprosyn) and indomethacin
(Indocin-SR) were of intermediate efficacy; and piroxicam
(Feldene), sulindac (Clinoril), and ibuprofen (Motrin) have been
the least effective. Long-term maintenance therapy on oral
NSAIDs may help to control inflammation caused by anterior
uveitis without steroids and thereby reduce the steroid
requirement.96 In addition, oral NSAIDs may play a role in the
management of cystoid macular edema (CME) associated with
cataract surgery, posterior uveitis and secondary retinal
vasculitis; NSAID therapy has not been of benefit in the
management of primary retinal vasculitis.96
These agents share several important systemic side effects.94
The most common is the induction of gastric or intestinal
ulceration. In some cases, anemia from gastrointestinal blood
loss may occur. Gastrointestinal side effects are explained on
the basis of two mechanisms. First, local irritation by orally
administered agents allows back-diffusion of acid into the
gastric mucosa, resulting in tissue damage. Inhibition of the
biosynthesis of gastric prostaglandins that inhibit gastric acid
secretion and induce gastric secretion of cytoprotective mucus
in the intestine is the proposed mechanism.94
Additional untoward effects of these agents that are related to
inhibition of the synthesis of endogenous prostaglandins
include altered platelet function, impairment of renal function,
and prolongation of gestation or spontaneous labor.94 NSAIDs
prevent the formation by platelets of thromboxane A2, a potent
platelet-aggregating agent. This results in an increased bleeding
time.94 These agents have a known effect on renal
CHAPTER 33
hemodynamics and fluid and electrolyte balance. In normal
patients, little effect of NSAIDs is seen because the production
of vasodilatory prostaglandins plays a minor role in the
presence of normal sodium balance.94,103 NSAIDs, however,
promote a decrease in renal blood flow and glomerular filtration
in patients with congestive heart failure, chronic renal disease,
hepatic cirrhosis with ascites, or hypovolemia of any cause.94
Salt and water retention may also be induced secondary to
the reduction of prostaglandin-induced inhibition of both
reabsorption of chloride and function of antidiuretic hormone.94
Edema may result in some patients. Hyperkalemia is also
promoted by the use of NSAIDs.94,103 Another renal side effect
is acute interstitial nephritis, with nephrotic-range proteinuria
in 73% of cases.103 Renal failure may be severe enough to
require temporary dialysis in 32% of patients.103 Propionic acid
derivatives have been most often associated with acute
interstitial nephritis.103
NSAIDs bind firmly to plasma proteins and therefore may
displace certain other drugs from binding sites.94 Thus, with
concurrent use of drugs such as warfarin, sulfonylurea
hypoglycemic agents, or methotrexate, an adjustment in the
dosage of these drugs may be required.94 This is particularly
important in patients receiving the anticoagulant warfarin, in
view of the effect of NSAIDs on platelet function.
Use of these aspirin-like agents is contraindicated in patients
with hypersensitivity to NSAIDs and in those with the
syndrome of nasal polyps, angioedema, and bronchospastic
response to aspirin.96 The use of NSAIDs in children should be
restricted to those agents extensively tested in the pediatric
population, namely aspirin, naproxen, and tolmetin.94 Owing to
the association of Reye’s syndrome with aspirin treatment of
children with febrile viral illness, NSAIDs should be strictly
avoided in this clinical setting.94
NSAIDs have the potential to produce photosensitivity rea-
ctions and are a frequent cause of cutaneous reactions. Cutaneous
reactions such as vesiculobullous eruptions, serum sickness,
exfoliative erythroderma, erythema multiforme, and toxic epi-
dermal necrolysis are well summarized in a report by Stern and
Bigby.104 Reactions to piroxicam were reported most frequently.104 347
 PHARMACOLOGY AND TOXICOLOGY
Ocular Side Affects of Systemic NSAIDS
Adverse ocular effects of systemic NSAIDs have been reported;
however, in many cases these are isolated reports or data
obtained from retrospective studies in which a cause-and-effect
relationship cannot be clearly established.105 Generally,
NSAIDs are photosensitizers and have the potential for
inducing phototoxicity of the anterior and posterior segments of
the eye.105 Optic neuritis has been associated with this class of
drugs and is presumed to occur as an idiosyncratic response
that is reversible on cessation of therapy.105 In the case of
ibuprofen, a widely used NSAID, there have been enough
occasional cases in which the drug has been rechallenged that
changes in refractive error, diplopia, and diminished color vision
seem to be well documented.105–107 The occurrence of a
reversible toxic amblyopia has also been described.108–111
Patients taking this drug should be advised to stop if a sudden
decrease in vision occurs.
Interpretation of reports of indomethacin-induced retinal and
macular disease is complicated by almost equal numbers of
contradictory studies.105,112,113 Nevertheless, the potential for
ocular toxicity exists. There are data to support the occurrence
of superficial corneal crystalline deposits secondary to
indomethacin that resolve with discontinuation of the
drug.112,114 There have also been several reports of papilledema
associated with pseudotumor cerebri.105,115
Aspirin has been implicated in increasing the incidence of
rebleeding in traumatic hyphema.116 Therefore, this agent as well
as the aspirin-like NSAIDs should be avoided in this condition.
SECTION 4
Adverse Affects of Topical Ophthalmic NSAIDS
The ophthalmic NSAIDs currently available include flur-
biprofen sodium 0.03% (Ocufen), suprofen 1% (Profenal),
diclofenac sodium 0.1% (Voltaren), bromfenac sodium 0.09%
(Xibrom), nepafenac sodium 0.1% (Nevanac), and ketorolac
tromethamine 0.5% (Acular).97 Both flurbiprofen and suprofen
are approved for the prevention of intraoperative
miosis.97,98,116–118 Ketorolac tromethamine 0.5% is indicated for
the treatment of ocular itch due to seasonal allergic
conjunctivitis.119,120 Diclofenac sodium 1%, bromfenac sodium
0.09%, nepafenac 0.1% and ketorolac tromethamine 0.5% are
indicated for the treatment of postoperative inflammation in
patients who have undergone cataract extraction.117 Nepafenac
is also effective in reducing pain following cataract extraction.
Diclofenac sodium 1%, and ketorolac tromethamine 0.5% have
also been effective in the reduction of pain and inflammation
after excimer laser photorefractive keratectomy.121,123
Several double-masked, randomized studies of the effects of
flurbiprofen on postoperative inflammation have been
published.123–126 Topical administration of flurbiprofen has also
been shown to reduce the inflammation of experimental
anterior uveitis.127 These studies indicate that flurbiprofen does
have some potential as an antiinflammatory agent, but
additional well-controlled clinical trials are needed.
In general, flurbiprofen sodium 0.03% is well tolerated. The
most frequent side effect is transient burning and stinging with
instillation.117 Flurbiprofen has been shown to inhibit
corneoscleral wound healing127,128 and exacerbate epithelial
HSV keratitis,129 effects similar to those seen with topical CSs.
A more recent report by Asbell and co-workers,130 however,
demonstrated that flurbiprofen sodium did not enhance HSV
epithelial keratitis. The strain of HSV, however, was not
specified in this report, and the timing of CS intervention after
infection differed. In a review of topical antiinflammatory
agents in an experimental model of microbial keratitis, a
worsening of Pseudomonas keratitis with topical CSs was
confirmed, and a greater worsening was observed with
348 flurbiprofen sodium 0.03%.131 Concomitant therapy with an
effective antibiotic prevented the steroid- and flurbiprofen-
induced worsening of Pseudomonas keratitis. Pneumococcal
keratitis was not worsened by the use of either CSs or
flurbiprofen in the presence of appropriate antimicrobial
therapy.131
There have been reports that flurbiprofen sodium may
promote bleeding of ocular tissues in the setting of ocular
surgery, particularly in the case of concomitant systemic
dipyridamole, an antiplatelet agent, or oral NSAIDs.117,132 The
manufacturers of all of the currently available topical NSAIDs
advise caution with the use of these agents in patients with
bleeding disorders or individuals taking systemic medications
that may prolong the bleeding time.117,133
A double-masked study evaluating the effects of topical
flurbiprofen sodium 0.03% on the IOP revealed that this agent
did not alter the IOP in known CS responders. In this study,
treatment with flurbiprofen did not prevent the steroid-induced
increase in IOP or the decrease in outflow facility.134
Suprofen may cause minor irritation, itching, redness,
allergic reaction, iritis, pain, chemosis, photophobia, and pun-
ctate keratopathy.116 The use of diclofenac may be associated
with minor symptoms of irritation. Concurrent use of
diclofenac and hydrogel contact lenses may cause burning and
redness.116 Ketorolac tromethamine 0.5% ophthalmic solution
may cause mild, transient burning and stinging on
instillation.119
A case of asthma exacerbated by topical ketorolac has been
reported.135 Caution must be exercised in prescribing topical
NSAID eye drops for patients with a history of asthma, nasal
polyps, and allergy to aspirin or NSAIDs.
Bromfenac sodium and nepafenac are newer agents with
recent approvals and consequently there is less clinical safety
data available on these agents. Initial clinical trials for both
agents demonstrated rare occurrences of a variety of side effects.
Both agents site potential for drug-induced allergy and increased
bleeding of ocular tissues due to interference with platelet
aggregation.
ANTIHISTAMINES AND DECONGESTANTS
Side affects of topical antihistamines are generally mild. Anti-
histamines may cause allergic responses and local irritation.71
H1 blockers also have local anesthetic properties; however, the
concentrations required for this effect are much greater than
those used therapeutically to antagonize the histamine
response.136
Adverse systemic reactions to topical decongestants (alpha-1
adrenergics) are uncommon, but headache,137–139 dizziness,138
nervousness,140 hypotension,141,142 hypertension,137,138,143-145
and cardiac dysrhythmias have been reported. The most
commonly reported ocular side effect is stinging on instillation.
Blurred vision,139,145 mydriasis,146–150 epithelial erosions,151,152
punctal stenosis,153,154 corneal pigment deposition,255 iris
pigment release,148,156 iritis,157 change in IOP137,146,148,149,158,159
and acute angle closure have also been described.139,149,150
A case series report identified acute and chronic conjunc-
tivitis due to over-the-counter ophthalmic decongestants.161
Three clinical patterns in order of decreasing frequency were
observed and include: (1) a pharmacologically induced rebound
conjunctival hyperemia, (2) a toxic follicular conjunctivitis, and
(3) an allergic, eczematoid blepharoconjunctivitis. The authors
note that the longer the duration of eye drops use before
presentation, the longer the recovery period required.
Systemic Antihistamines
Oral antihistamines have a drying effect on the eye that may
worsen or induce keratoconjunctivitis sicca and cause contact

lens intolerance.160,162 Chlorpheniramine maleate, a commonly
prescribed oral antihistamine, which is also available over the
counter, has been shown to decrease tear production signi-
ficantly, as measured by standard Schirmer testing, in normal
patients.163
The most common adverse effect of systemic administration
of antihistamines is drowsiness.161,136,164 This may be
hazardous to those patients who must drive or operate
machinery. These agents also enhance the action of narcotics
and sedatives.136 Astemizole, terfenadine, loratadine, and
fexofenadine have fewer sedative and anticholinergic side
effects. 165,136 Gastrointestinal side effects of oral antihistamines
such as nausea, emesis, anorexia, epigastric distress, and altered
bowel habits may be reduced by ingestion of the medication
with meals.161 Less common central nervous system effects
may include lassitude, dizziness, tinnitus, incoordination,
blurred vision, diplopia, euphoria, nervousness, tremors, and
insomnia.161 The anticholinergic action of these drugs may
induce mydriasis, triggering acute angle-closure glaucoma as
well as a reduction in accommodation by effects on ciliary
muscles.105,161 Rare side effects of visual hallucinations,
temporary blindness, and an absence of pupillary light reflexes
have been induced by overdosage.161
Serious adverse cardiovascular events, including death, cardiac
arrest, torsades de pointes, and other ventricular arrhythmias
have been reported with concomitant use of terfenadine with
erythromycin and related macrolide antibiotics, ketoconazole,
or itraconazole, and significant hepatic dysfunction. Use of
terfenadine is therefore contraindicated in these situations.
Ocular Mast Cell-Stabilizing Agents
Commonly reported side effects of disodium cromoglycate are
transient burning and stinging on instillation.117,166,167
Hyperemia and bulbar conjunctival chemosis have been
reported in 35% of patients.167 Less common adverse effects
include watery and itchy eyes, sties, puffiness, and dryness
around the eyes.168 EDTA, a solution stabilizer in DSCG, has
been implicated as the cause of conjunctival injection in
some cases.168
Clinical trials have indicated that treatment-related ocular
adverse effects of lodoxamide are mild, nonserious, and
transient. Reported adverse effects include minor discomfort,
itching, and pain.169–171 Headache171 and nausea169 are
nonocular side effects that have been reported in rare instances.
Both disodium cromoglycate and lodoxamide may decrease
the steroid requirement, thus reducing the potential adverse
effects from long-term corticosteroid therapy.166,168,169–173 Acute
exacerbations and severe forms of ocular allergy may require
topical steroids, however. In these cases a pulse steroid regimen
with aggressive but brief corticosteroid treatment with rapid
tapering and maintenance therapy with these mast cell-
stabilizing agents may have a therapeutic advantage while
minimizing risks of steroid therapy. The therapeutic effect of
mast cell-stabilizing agents is not as immediate as that of
corticosteroids, taking usually several weeks of regular use for a
desired therapeutic response. Patients should be advised of this
when using these drugs, or compliance may be a problem.
Cyclosporine
Topical Restasis (cyclosporine A) is the only prescription
therapy available for treatment of dry eye. There are few side
affects associated with topical cyclosporine application however
systemic CsA therapy is associated with a number of side
effects. Nephrotoxicity has been observed following systemic
dosing, manifested by decreased creatinine clearance, elevated
serum creatinine levels, and a disproportionate increase in
blood urea nitrogen with preserved urine output and sodium
Toxicology of Ophthalmic Agents by Class
reabsorption.174 It is important to note that the serum
creatinine level underestimates the glomerular filtration rate
and therefore should not be the sole marker of renal toxicity.175
The renal toxicity occurs at the level of the arteriole,
glomerulus, and proximal tubule.39,174 CsA-induced alteration
in renal hemodynamics has been proposed.101 Systemic
hypertension is another significant side effect, occurring in 25%
of patients following systemic CsA therapy; it tends to be more
frequent in those with impaired renal function.39 The exact
mechanism remains unknown but appears to be dose related.
Hypertension is also more common in patients receiving CsA
and steroids than in those receiving CsA alone.176 Leukopenia
is not seen with systemic CsA; however, a normochromic
normocytic anemia is observed in 25% of patients, and other
causes of anemia should be ruled out.39 An increase in the
erythrocyte sedimentation rate has been noted in 40% of
patients, but this does not correlate with the clinical course of
the underlying disease and should not be used as an index of
disease activity.39 An increased incidence of lymphoma was
once thought to be related to CsA use; however, in a large
clinical series of 5000 transplant recipients, the incidence of
lymphoma was no greater in patients receiving CsA than in
those receiving other immunosuppressive agents.177 Other side
effects of CsA include hirsutism, gingival hyperplasia, central
nervous system toxicity, and an increased incidence of viral
infections.39,94,178,179 Several important drug interactions have
been observed with the administration of CsA.39,176,179,180–187
Ocular side effects reportedly due to systemic use include
CHAPTER 33
decreased vision, eyelid or conjunctival erythema, nonspecific
conjunctivitis, urticaria, visual hallucinations, and conjunctival
and retinal hemorrhages related to drug-induced anemia.105
TOXICITY OF OCULAR ANTIINFECTIVES
Topical ophthalmic antiinfectives are prescribed in response to
diagnosis of an active ocular infection and as a preventative
measure in situations where threat of ocular infection exists
(trauma, injury, etc) Common side effects associated with
topical and systemic antibiotics, antivirals and antifungal
agents will be described, with emphasis on the more frequently
prescribed agents of these classes.
ANTIBIOTICS
The antibiotic section briefly covers the ocular toxicity of a
variety of different antibiotics with emphasis placed on the
ocular toxicity of aminoglycosides, cephalosporins, fluoro-
quinolones, saulfanomide and vancomycin, because they are
more commonly employed. For many antibiotics, the mec-
hanism of toxicity is ill understood.
Aminoglycosides
The aminoglycosides are bactericidal antibiotics that
irreversibly inhibit protein synthesis and ribosome function.
Amikacin, gentamicin, neomycin, and tobramycin are in
common use today. The narrow therapeutic index of the
aminoglycosides has stimulated extensive investigation into the
mechanism of aminoglycoside toxicity.
Extensive studies of the mechanism of aminoglycoside
toxicity have been performed owing to the oto- and
nephrotoxicity associated with systemic use of aminoglycoside.
Previous investigators have demonstrated selective accumu-
lation of aminoglycoside within the lysosomes of cultured
fibroblasts.188,189 This may result from the protonation of
aminoglycoside molecules, thus trapping the drug in the low-
pH lysosomal environment. The accumulation of amino-
glycoside produces disturbances in phospholipid catabolism, 349
 PHARMACOLOGY AND TOXICOLOGY
possibly through lowered activity of sphingomyelinase and
phospholipases as demonstrated by Aubert-Tulkens and
associates in cultured rat fibroblasts.188
Libert and colleagues190 evaluated the cellular toxicity of
subconjunctival gentamicin. Electron microscopy revealed an
accumulated substance within the lysosomes that consisted of
granular material with a pleomorphic lamellar structure,
corresponding to the presence of complex lipids. Laurent and
co-workers191 demonstrated that systemic gentamicin induces a
loss of activity of lysosomal sphingomyelinase and
phospholipase A in rats. Furthermore, they found that amikacin
binds more loosely to phospholipid bilayers, induces less
inhibition of phospholipases in vitro, and is taken up less by
tubular cells in vivo. Given amikacin’s lesser nephro- and
retinotoxicity, lysosomal alterations may be an early step in
aminoglycoside-induced toxicity.
Intravitreal injection of aminoglycoside antibiotics is an
established mode of therapy for bacterial endophthalmitis.
D’Amico and co-workers192 have evaluated the comparative
toxicity of intravitreal aminoglycoside antibiotics in a rabbit
model. The observations ranked gentamicin as the most toxic,
then tobramycin, and amikacin as the least toxic: full-thickness
retinal necrosis was induced with 800, 1600, and 3000 mg,
respectively. Toxic lesions secondary to intravitreal amino-
glycoside injection consist of focal areas of lysosomal storage,
with outer retinal necrosis, whereas other areas of the retina
appear normal with the exception of mild accumulation of
complex lysosomal lipids. Owing to the focal areas of toxicity
SECTION 4
and the potential that pigmented eyes may raise the threshold
for toxicity,193 it is particularly difficult to determine the
threshold toxic dose.
After intravitreal aminoglycoside injection, the area of
toxicity is localized to the RPE–photoreceptor outer segment
complex.194,195 The production of lamellar lysosomal inclusions
in the RPE indicates the accumulation of complex lipids. The
aminoglycoside concentrates within the lysosomes and may
interfere with one or more lysosomal enzymes, causing
accumulation of unmetabolized substrates.194 A number of
possible mechanisms have been proposed. Gentamicin-treated
cells exhibit a greater decrease in the activity of sphingo-
myelinase than amikacin.188,189 Alternatively, the cytoplasmic
enzyme phospholipase C correlates well with the nephro-
toxicity. Finally, investigators have proposed a direct effect on
the mitochondria, with disruption of oxidative phosphorylation
with aminoglycoside toxicity. Fleisher and associates196 reported
that intraocular injections of tunicamycin produce photo-
receptor-specific degeneration. The glycosylation of opsin can
be blocked by tunicamycin in vitro in conditions where poly-
peptide synthesis is only slightly decreased. Thus, amino-
glycoside toxicity may be mediated by disturbance in
glycoprotein metabolism. Certainly, some combination of these
mechanisms could be responsible for the pathogenesis of
aminoglycoside toxicity.
Some have proposed that the toxicity of intravitreal
antibiotics can also be effected by the surgical status of
the eye.197 Talamo and co-workers198 showed that posterior cap-
sulectomy and vitrectomy do not change the therapeutic index
(toxic dose:therapeutic dose) for intravitreal aminoglycoside
despite the dramatic reduction in vitreous half-life. Retinal
damage may be related to the peak concentration of the drug to
which the retina is exposed after intravitreal injection. No
additional protection from aminoglycoside toxicity is noted
after vitrectomy.
Macular infarction has been reported after intravitreal
aminoglycoside injection.199,200 Conway and associates201
evaluated the effect of intravitreal gentamicin in the primate
350 retina. Intravitreal gentamicin doses of 1000 and 3000 mg were
employed. The inner retinal layers exhibited considerable
swelling of the nerve fiber and ganglion cell layers; however,
the outer segments and RPE appeared normal by light
microscopy. Electron microscopy of the 3000-µg intravitreal
gentamicin specimens revealed intracellular edema with
massive thickening of the ganglion cell axons. A prominent
inflammatory response was noted on the internal limiting
membrane. Despite these findings there was no evidence of
retinal vasculitis. In areas of the retina that had shown
nonperfusion of the capillary bed, granulocyte plugs were seen
filling the vessels. The authors hypothesize that the inflam-
mation of the inner layers of the retina associated with the toxic
effects of gentamicin may induce granulocytic plugging with
permanent closure of the capillary bed.201,202 Granulocytes
obstruct the lumen by adhering to the endothelium; this
occlusion, combined with oxygen-free radical formation and
lysosomal enzyme activity, may cause ischemic injury.203 The
findings of Conway and co-workers provide strong evidence that
gentamicin toxicity occurs in normal retinal tissue.201 This
effect is in keeping with the known neurotoxicity of
gentamicin.204 Tabatabay and associates205 examined the
immunohistochemical localization of gentamicin in the rabbit
after a single intravitreal injection. Initially, gentamicin was
localized to the ganglion cell layer, inner plexiform and nuclear
layers, and the photoreceptors. By 24 h, gentamicin was
predominantly in the RPE and choriocapillaris. Haines and
associates evaluated the morphologic changes after intravitreal
injection of gentamicin in pig eyes. Three mg of gentamicin was
injected intravitreally to observe the toxicity-related changes
that occurred in the retina. Vacuolization of the nerve fiber layer
and perivascular swelling was seen within 6 h and subsequently
descended deeper into the retina. Vascular endothelial cells,
photoreceptors, and the RPE appeared to be spared from the
toxic effect of gentamicin. By 48 and 72 h after injection,
numerous large and small retinal vessels showed congestion
and leukocyte margination. These changes could not be
prevented by changing the pH to the gentamicin to 7.2. Thus,
the authors conclude that the gentamicin toxicity effect is not a
pH-related phenomenon but that the primary targets for
gentamicin are the neurons and the glia of the inner retina, and
as a result, retinal infarction occurs secondarily owing to
leukocyte plugging.206 In addition, Haines and colleagues
speculate that the predisposition for macular infarction is due
to the dependent position of the macula during surgery as well
as the higher density of ganglion cells in the perimacular
area.206
A survey of retinal specialists from the Retina, Macula, and
Vitreous Societies revealed 101 cases of macular infarction due
to aminoglycoside administration.207 Interestingly, 93 cases
were associated with gentamicin, five with amikacin, and three
with tobramycin. Of the 93 gentamicin cases, 21 used
intravitreal doses of 100-200 mg, a dose considered to be non-
toxic. Twenty-three cases of gentamicin toxicity resulted from
prophylactic subconjunctival injections after cataract
extraction. Although dilution errors cannot be ruled out, this
reference clearly points out that the safe therapeutic window for
ocular use of aminoglycoside is sufficiently narrow to be a
significant clinical problem. The authors advocate reserving
aminoglycoside for known or highly suspicious gram-negative
infections.207 They recommend: (1) abandoning routine use of
subconjunctival aminoglycoside after ocular surgery (using
cefazolin instead), and (2) avoiding intravitreal aminoglycoside
in the prophylaxis of penetrating ocular trauma.208 The
authors207 recommend vancomycin (or clindamycin) and
ceftazidime or imipenem210 for penetrating ocular trauma.
More recently, Campochiaro and Lim reported on the results
of a survey of 13 patients who received 200–400 mg of amikacin

sulfate or 100–200 mg of gentamicin sulfate for prophylaxis
or treatment of endophthalmitis. Low-dose gentamicin or
amikacin can cause macular infarction, even with doses
prepared by hospital pharmacists using typewritten protocol. Of
note, several cases exhibited very discrete macular involvement,
causing the authors to speculate on the role of a localized
increase in concentration in dependent areas of the retina.210
Generally, retinal specialists recommend vancomycin, 1 mg,
and ceftazidime, 2.25 mg, for intravitreal injections. However,
the choice of antibiotic may vary based on the patient’s clinical
circumstances.
Cephalosporins
Cephalosporins are b-lactam antibiotics that interfere with
bacterial cell wall synthesis. The first-generation cephalosporins
include cefazolin, cephalothin, cephapirin, cephradine, cephaloxin,
and cefadroxil. The second-generation cephalosporins include
cefamandole, cefuroxime, cefonicid, cefoxitin, ceforanide, and
cefaclor. The third-generation cephalosporins include cefepime,
cefoperazone, cefotaxime, ceftizoxime, moxalactam, ceftazidime,
and ceftriaxone. Although many cephalosporins have been
evaluated for use in ocular infections, cefazolin, ceftazidime,
and cefuroxime are the most often used cephalosporins. The
intravitreal use of cefazolin has been supplanted mainly by
vancomycin.
Common side effects of these agents include superficial
irritation (conjunctival injection, chemosis, conjunctival
necrosis and lid edema.211 Some photoreceptor toxicity has been
noted in preclinical studies performed with intravitreally
administered ceftazidime.102
Fluoroquinolones
The fluoroquinolones are structurally related to nalidixic acid.
These agents block enzymatic activity of bacterial DNA gyrase
and alter the structure and functioning ability of bacterial DNA.
Currently, ciprofloxacin, norfloxacin, and ofloxacin are
available in the United States, although a number of fluoro-
quinolones are under study, including pefloxacin.212 Topical
0.3% ciprofloxacin is tolerated well. Only mild untoward ocular
events are noted, the most frequent one being a white
crystalline precipitate, commonly located in the superficial
portion of the corneal defect. This precipitate has been iden-
tified as ciprofloxacin. Hobden and colleagues demonstrated the
efficacy of transcorneal iontophoresis of 1% ciprofloxacin for
therapy of aminoglycoside-resistant Pseudomonas keratitis.213
They found no evidence of toxicity with the 1% formulation.
Stamer and co-workers214 evaluated the effect of ciprofloxacin
on rabbit corneal endothelial viability. A concentration of 10
µg/mL of ciprofloxacin had no effect on endothelial cell counts
or viability, whereas 100 mg/mL caused a 2% reduction in viable
endothelial cells. Haller-Yeo and associates215 evaluated
intravitreal ciprofloxacin; they reported no toxicity in cat eyes
with doses of 1, 10, 100, and 1000 mg when evaluated by light
microscopy and electrophysiology. Steven and co-workers216
evaluated the intraocular use of ciprofloxacin in phakic and
aphakic rabbits. Corneal decompensation occurred in aphakic
vitrectomized rabbits with intravitreal doses of 100 µg of
ciprofloxacin, whereas retinal toxicity was noted on electron
microscopy with doses higher than 250 mg. At 1000 µg, electron
microscopy revealed loss of the outer rod segments, followed
by atrophic changes of the inner rod segments as well as the
outer and inner nuclear cell layers. Marchese and colleagues
evaluated the toxicity and pharmacokinetics of ciprofloxacin.
They studied the pigmented rabbit model and injected doses of
100, 200, 500, and 1000 mg. An evaluation was performed by
indirect ophthalmoscopy, electrophysiology study, and
histology. Focal areas of retinitis were observed after injections
Toxicology of Ophthalmic Agents by Class
of both 500 and 1000 mg but not with 250 mg of ciprofloxacin.
In addition, electrophysiology revealed that the amplitude ratios
were significantly reduced after the 1000-mg dose. At the 100-
or 250-mg ciprofloxacin dose, histologic sections were compar-
able between control eyes, and ERG ratios were unchanged from
the baseline level.217
Kawasaki and associates found that 200 mg of ofloxacin did
not cause deterioration of the b-wave, c-wave, or the oscillatory
potential over a 2-month period in the rabbit model.218 Mochizuki
and colleagues studied the effects of ofloxacin on the rabbit ERG
in vivo.219 They also determined that 200 mg of ofloxacin did
not cause an alteration in the ERG in the rabbit model.219
Sulfonamides
The sulfonamides interfere with bacterial utilization of p-
aminobenzoic acid (PABA). These drugs are bacteriostatic, and
the various preparations have different chemical, phar-
macologic, and antibacterial properties. Flach and associates220
demonstrated that topical 25% sulfisoxazole diolamine
ointment and exposure to ultraviolet light resulted in a photo-
toxic reaction. Boettner and co-workers221 found that topical
use of sulfadiazine ointment for 1 year caused formation of
multiple small white concretions in cysts of the palpebral
conjunctiva, identified by spectroscopy as sulfadiazine. Hook
and colleagues141 reported a case of transient myopia induced by
sulfonamides. A-scan measurements and cycloplegic refraction
demonstrated the primary mechanism of sulfonamide-induced
myopia to be lens thickening from ciliary body edema.
CHAPTER 33
Vancomycin
Vancomycin is a bactericidal antibiotic that inhibits bacterial
cell wall synthesis through interference with glycopeptide
polymerization. Pryor and associates222 evaluated topical and
subconjunctival administration of vancomycin in rabbits. They
found no evidence of toxicity with subconjunctival doses of
12.5 or 25 mg, whereas a 5% aqueous solution given every 5 min
for 30 min revealed minimal superficial punctate keratopathy.
Fortified vancomycin in doses of 14–25 mg/mL has been reported
to cause irritation, conjunctival injection, and superficial punc-
tate keratopathy. Although subconjunctival vancomycin injec-
tions have been reported to cause conjunctival necrosis and
sloughing,223 Lindquist and co-workers demonstrated the safety
and efficacy of vancomycin in corneal storage media.224 They
found no evidence of endothelial damage at doses of 150 mg/mL
vancomycin in gentamicin-free Dex-Sol. Kattan and Pflugfelder225
evaluated the corneal toxicity of vancomycin in corneal storage
media and found no evidence of endothelial damage with concen-
trations of 5 mg/mL. Garcia-Ferrer and associates223 evaluated
the antimicrobial efficacy and lack of corneal endothelial
toxicity of Dex-Sol corneal storage medium supplemented
with vancomycin, 10 mg/mL. Choi and Lee226 documented an
8.8% decrease in endothelial cell counts with transcorneal
iontophoresis of vancomycin in rabbit eyes, compared with
5.4% decrease with balanced saline solution.
Intravitreal vancomycin has been evaluated extensively.
Homer and associates evaluated the toxicity, clearance, and
therapeutic effectiveness of intravitreal vancomycin in a rabbit
model of staphylococcal endophthalmitis.155 Concentrations of
vancomycin ranged from 0.25 to 500 mg/0.1 mL. At doses
higher than 5 mg/0.1 mL the vitreous exhibited a whitish
reaction, although ERG abnormalities were associated with
doses higher than 2 mg. Histologic study of doses of 2–5 mg
revealed toxicity localized to the retina with photoreceptor outer
segment degeneration. In contrast, Smith and co-workers228
evaluated the toxicity, clearance, and efficacy of intravitreal
vancomycin in an experimental rabbit model of methicillin-
resistant S. epidermidis endophthalmitis. Doses of 1, 2, and 351
 PHARMACOLOGY AND TOXICOLOGY
5 mg were evaluated by light microscopy. There were no
discernible retinal abnormalities except one eye injected with
5 mg confirmed the absence of extensive toxicity. Smith and
co-workers believe that the histologic change noted by Homer
and co-workers155 might be the result of tissue processing.
Borhani and associates evaluated vancomycin in the vitrectomy
infusion solution. They found that concentrations of 8, 16, and
32 µg/mL of vancomycin in infusion solution caused no
abnormal ERG or histologic changes. However, electro-
physiologic depression and abnormal histologic changes
occurred with concentrations of 100 mg/mL of vancomycin in
the infusion solution.228
Pflugfelder and colleagues229 evaluated the retinal toxicity,
clearance, and interaction of intravitreal vancomycin with
gentamicin in phakic and aphakic vitrectomized rabbits.
Clinically, with doses higher than 2 mg there was immediate
clouding of the vitreous and within 24 h opacification of the
retina. By 2 weeks the retinal opacification had cleared, but the
RPE showed pigment clumping and atrophy. Electrophysiologic
testing revealed no evidence of toxicity up to 2 mg; however,
there was marked reduction in the a- and b-wave amplitudes in
the eye that received a 5-mg dose. Ultrastructural studies of
doses greater than 2 mg revealed a number of pathologic
changes, including: (1) hypertrophy of the RPE with abnormal
clustering of pigment granules in the cytoplasm; (2) loss of
photoreceptor outer segment–RPE interdigitation due to
retraction of apical microvilli of RPE; (3) appearance of lucent
vacuoles in the RPE basal cytoplasm beneath the plasmalemma
SECTION 4
infoldings; (4) gross disorganization of the photoreceptor outer
segments with distention and displacement of the inner seg-
ments past the external limiting membrane; and (5) accumu-
lation of cellular debris in the subretinal space. Pflugfelder and
colleagues229 found that lensectomy and vitrectomy increased
the intraocular clearance of vancomycin but did not alter the
threshold for retinal toxicity. Oum and associates230,231 studied
the effect of combined and repeated injections of intravitreal
vancomycin and aminoglycoside. They found increasing retinal
toxic reaction with repeated injections. The exact biochemical
mechanism of toxicity is unknown.232
ANTIFUNGAL AGENTS
Ocular fungal infections continue to challenge ophthal-
mologists.233 The selection of appropriate antifungal chemo-
therapy is limited by the paucity of effective drugs.234,235 The
only approved ophthalmic antifungal is 5% natamycin;
however, amphotericin B, flucytosine, miconazole, and
ketoconazole have a role in the management of ocular fungal
infections.236,237
Natamycin
Natamycin is a tetraene polyene and is the only antifungal
available in the United States in a topical form. Topical nata-
mycin is well tolerated. Superficial punctate keratopathy has
been reported with prolonged use 238,239 Foster and co-
workers240 demonstrated that natamycin did not retard the
healing of corneal epithelial defects. Ellison and Newmark158
demonstrated conjunctival necrosis after subconjunctival
injection of natamycin.
Intraocular use of natamycin is not well tolerated.241 Anterior
chamber injection of 250 mg of natamycin is tolerated in a rabbit
model; however, with a dose of 500 mg corneal decompensation
and iridocyclitis develop. Ellison and Newmark242 reported the
intravitreal effects of pimaricin: 25 mg was not toxic but was not
therapeutic either; doses higher than 50 mg destroy the retina.
Other formulations of amphotericin B have been evaluated
352 topically. Amphotericin B methyl ester, which is water soluble
and has reduced toxicity, has been evaluated in a 1%
formulation and has been found to be nontoxic and to penetrate
better than amphotericin B.243 Owing to leukoencephalopathy
associated with systemic administration, further work on
amphotericin B methyl ester has not been pursued.
Intravitreal injection of amphotericin B has been studied in
detail. Foster and associates244 reported a case of Volutella
fungal infection after cataract extraction that was treated with
three intravitreal injections of 35-40 mg over 1 month. The eye
was sterilized, although blind with a corneal pannus, updrawn
pupil, and total, funnel-shaped retinal detachment. Green and
co-workers245 reported successful sterilization of a postcataract
fungal infection with 20 mg of intracameral amphotericin B
combined with topical and subconjunctival amphotericin B,
although the final acuity was extremely poor. Axelrod and
associates246 evaluated the toxicity of intravitreal amphotericin
B in a rabbit model. They found that doses of 25–500 mg of
intravitreal amphotericin B resulted in retinal detachment with
a proteinaceous exudate and cloudy vitreous with monocytes in
the vitreous cavity. They proposed that the amphotericin B
alters cell membranes, with resultant transudation of subretinal
fluid. Of note, they found that sodium deoxycholate is not toxic
to the retina, and intravitreal doses of 5–10 mg of amphotericin
B produced no abnormalities by electrophysiologic testing or
light microscopy. In addition, 25 mg of amphotericin B injected
close to the retina resulted in immediate focal retinal necrosis.
Axelrod and Peyman247 demonstrated that, in the setting of
experimental fungal endophthalmitis, 5 mg of intravitreal
amphotericin B was nontoxic (as determined by light
microscopy) in rabbits. Souri and Green248 documented that
intravitreal doses of amphotericin B as small as 1 mg resulted in
focal retinal necrosis in the rabbit when injected adjacent to the
retina.
Different formulations and delivery systems for intravitreal
amphotericin B have been evaluated. Amphotericin B methyl
ester, although it has much less antifungal activity, is a water-
soluble compound with a much wider range of therapeutic
doses. McGetrick and associates185 found that amphotericin B
methyl ester showed no evidence of retinal toxicity by light
microscopy or electrophysiologic studies when intravitreal
doses were 50 mg or less. Doses of 100 mg of amphotericin B
methyl ester resulted in degeneration of the photoreceptor layer;
this was caused by the drug and not by the ascorbic acid used to
solubilize the antifungal agent. Raichand and co-workers249
evaluated the toxicity of amphotericin B methyl ester in
vitrectomy infusion fluid and found that the maximal nontoxic
dose was 75 mg/mL; at 100 mg/mL. Electrophysiologic studies
revealed a decreased response, although no toxic damage was
appreciated by light microscopy. Amphotericin B methyl ester
was found to cause leukoencephalopathy when used sys-
temically and has not, therefore, been a candidate for
intraocular use.
Imidazoles
The imidazoles are a group of synthetic antifungals that are
fungistatic in low concentration and fungicidal in high
concentrations. They possess a broad spectrum of antifungal
activity. They inhibit ergosterol synthesis at low concentrations
and interfere with the mitochondrial oxidative and peroxidase
enzymes.
Ketoconazole
Ketoconazole is a weakly dibasic synthetic imidazole that
inhibits ergosterol synthesis. Foster and co-workers240,250
evaluated the toxicity of 1% ketoconazole with Cremophore EL
as the carrier. They found a slight delay in corneal epithelial
wound healing. Grossman and Lee251,252 evaluated transscleral

and transcorneal iontophoresis of ketoconazole in a rabbit
model. Subconjunctival ketoconazole (50-mg) injections were
compared with iontophoresis and produced no evidence of
toxicity at the doses employed. Intravitreal ketoconazole in
dimethyl sulfoxide (DMSO) was evaluated by Yoshizumi and
Banihashemi.253 The ocular toxicity of experimental intravitreal
DMSO has been evaluated254; a single 0.1–mL injection of
100% DMSO results in transient focal retinal edema and a 50%
decrease in the amplitude of the photopic, flicker fusion,
scotopic, and combined photopic and scotopic response.
Electrophysiologic response returned to normal after 1 month
and retinal edema resolved within a week. Intravitreal
injections of ketoconazole in DMSO at doses of 2240 mg
resulted in retinal edema and necrosis with marked photo-
receptor outer segment loss; electron microscopy of the RPE
revealed degeneration of mitochondria and a decline in the
number of melanin granules.253 Doses of 720 mg of intravitreal
ketoconazole produced toxic vacuolizations of the inner
segments of the photoreceptors detected by electron microscopy.
The study determined that doses up to 540 mg produced no
ocular toxicity, giving a much wider therapeutic window than
miconazole.
Itraconazole
Itraconazole is a triazole derivative with broad-spectrum
antifungal activity in vitro and in animal models. This
antifungal drug is lipophilic and practically insoluble in water.
Schulman and colleagues injected intravitreal itraconazole in
doses ranging from 10 to 100 mg devolved in 100% DMSO into
the eyes of New Zealand rabbits. Ocular toxicity studies
performed 5 weeks after administration showed no substantial
retinal or histologic changes in eyes injected with either 100%
DMSO or 10 mg of itraconazole. Higher doses cause focal areas
of retinal necrosis.255
Fluconazole
Fluconazole is a bis-triazole, potent antifungal with low toxicity
and excellent water solubility; it is currently available in oral
and intravenous forms. Brooks and associates256 found that
topical fluconazole, 100 mg/mL, appeared to be equivalent to,
and potentially less toxic than, amphotericin B in an experi-
mental Candida keratitis model. Schulman and co-workers255
evaluated the toxicity of intravitreal fluconazole in the rabbit.
They found no corneal, lenticular, or retinal changes by light
microscopy and no evidence of depressed electrophysiologic
testing at doses of 100 mg. Fluconazole has excellent ocular
penetration when taken systemically; further work is required
to evaluate the efficacy and toxicity of ocular fluconazole
treatment.
ANTIVIRALS
Great strides have been made in the chemotherapy of ocular
viral diseases since the introduction of idoxuridine in 1962.
Currently, ophthalmic preparations of idoxuridine (IDU),
vidarabine, and trifluridine (TFT) are available. Acyclovir is
available for systemic use and as a dermatologic preparation;
ganciclovir is available for systemic and intraocular use.257
Idoxuridine.
The adverse ocular effects of topical IDU are common and
result from direct toxicity or allergic reactions. Local irritation,
with conjunctival injection, follicular conjunctivitis, allergic
blepharoconjunctivitis,258 and perilimbal filaments have been
reported.259 Lass and associates260 have reported IDU-induced
conjunctival cicatrization. Corneal problems such as superficial
punctate keratitis, delayed corneal wound healing, and corneal
Toxicology of Ophthalmic Agents by Class
edema have been reported.259 Punctal scarring and occlusion
have also been reported, particularly after long-term therapy.
The mechanism for the observed toxicity is believed to be the
activation of IDU in normal cells, particularly rapidly dividing
cells, resulting in disruption of normal DNA synthesis.
Vidarabine
Adenine arabinoside (vidarabine, Ara-A) is a purine analog. Ara-
A is phosphorylated by viral thymidine kinase, then
triphosphorylated. The active form inhibits DNA polymerase
and ribonucleotide reductases, thus blocking viral DNA
synthesis. Ara-A is available in a 3% ophthalmic ointment and
an intravenous suspension (200 mg/mL).257
Similar to IDU, the adverse effects of Ara-A are due to direct
toxicity or to allergic reactions. Local ocular reactions include
conjunctival injection, follicular conjunctivitis, and punctal
scarring. With prolonged treatment, conjunctival cicatrization,
corneal scarring, or permanent punctal occlusion can result.261
Lass has reported that Ara-A has insignificant effects on corneal
epithelial wound healing, although there is significant delay in
stromal wound healing.261 Kaufman and associates262 have
found that subconjunctival injections of Ara-A can be toxic;
daily subconjunctival injection of 5% Ara-A results in
significant conjunctival inflammation; 20% injections result in
the formation of conjunctival granuloma.
Different methods of delivery of Ara-A have been evaluated.
Hill and associates263 found that iontophoresis (0.5 mA in 4
min) of Ara-AMP (vidarabine adenosine-5-phosphate) resulted
CHAPTER 33
in higher corneal and intracameral levels without evidence of
toxicity. Pulido and associates264 evaluated the toxicity of
intravitreal injections and infusions of vicarabine in rabbits.
Intravitreal injections of 80 mg/0.1 mL Ara-A revealed no
abnormalities in electrophysiologic testing or light microscopy.
However, after vitrectomy/lensectomy, disorganization of the
external retina was visible by light microscopy in rabbits that
received infusions of 100 mg/mL Ara-A.
Trifluridine
TFT is a halogenated pyrimidine with three fluorines in place of
the 5-methyl group of thymidylate. It is a potent inhibitor of
thymidine synthesis, which in turn inhibits DNA synthesis. It
is preferentially incorporated into viral DNA, thus producing
defective DNA. TFT is available as a 1% ophthalmic solution.
The adverse ocular effects of topical TFT are due to direct
toxicity or to allergic reaction. Local reactions include con-
junctival injections, superficial punctate keratopathy, fila-
mentary keratitis, and punctal occlusion with prolonged
treatment.259 Udell265 has reported conjunctival cicatrization
after topical TFT, whereas Maudgal and associates266 have
reported corneal epithelial dysplasia after TFT.
Carmine and associates267 reported no evidence of toxicity of
1% TFT in normal rabbit eyes. However, with a standard
corneal epithelial defect and 8 days of eight-times-daily 1% TFT,
they noted pathologic changes in the regenerating epithelium,
which resolved when the TFT was discontinued. They also
noted that stromal wound healing was affected with decreased
tensile strength; this was confirmed by Gassett and Katzin.263
Wellings and associates268 have demonstrated that TFT is more
effective than IDU, associated with fewer failures and less
toxicity, although the toxicity may reflect failure to control the
herpetic keratitis rather than a toxic reaction to IDU. Hyndiuk
and associates269 documented a case of reversible crystalline
epithelial keratitis with 1% TFT, which presented with
superficial punctate keratopathy and gray epithelium with fine
linear retractile crystalline intraepithelial deposits. Maudgal
and associates270 did report on conjunctival ischemia, corneal
epithelial dysplasia, filamentary keratitis, and punctal stenosis 353
 PHARMACOLOGY AND TOXICOLOGY
with the use of 2% TFT (with 1% neomycin) in an experimental
model of herpes simplex keratouveitis. The complications
increased with prolonged use.
Peyman and associates212 have investigated the toxicity of
intravitreal TFT. Pang and associates270 found that intravitreal
injections of 200 mg/0.1 mL and vitrectomy infusion solutions
of 60 mg/mL were not toxic to rabbits. With injections of 500 mg
a mild decrease in ERG functions was noted; however, no
damage was seen by light microscopy. With injection of 1000 mg
(and infusions of 100 mg/mL) there was a moderate depression
in b-wave amplitudes, and photoreceptor clumping and
degeneration were noted by light microscopy. Liu and
associates271 evaluated liposomal delivery of intravitreal TFT.
Injections of 42.9 mg revealed no evidence of toxicity by clinical
examination, ERG, or light microscopy, and vitreal drug levels
remained for 28 days in the range of ID46 for many strains of
herpesvirus and human cytomegalovirus (CMV).
Acyclovir
Acyclovir, a purine analog similar to Ara-A, is activated by
virus-induced thymidine kinase to the monophosphate form
and then to the triphosphate form. It inhibits viral DNA poly-
merase. Acyclovir is available as a 5% dermatologic ointment, a
3% ophthalmic ointment (not available in the United States),
and in oral and intravenous formulations. The adverse ocular
effects of topical acyclovir (not approved for ophthalmic use) are
mild: local irritation with mild superficial punctate keratitis and
follicular conjunctivitis.274 One report of punctal stenosis with
SECTION 4
the topical preparation was reported,261 but it is not known
whether stenosis was due to acyclovir or to herpes zoster
keratouveitis.
Because of the low toxicity of acyclovir, it has been inves-
tigated for intraocular injection. Pulido and associates275
investigated intravitreal injections and infusion solutions of
acyclovir in rabbits. They found that injections of 240 mg/0.1
mL revealed no evidence of abnormalities on histopathologic
examination or electrophysiologic testing. Infusion solutions
containing 400 mg/mL revealed disorganization of the external
layers of the retina after lensectomy or vitrectomy.
Ganciclovir
Ganciclovir is a synthetic nucleoside analog of 2„-deoxy-
guanosine, similar to acyclovir. Virus-specified thymidine
kinase converts ganciclovir to the monophosphate form, which
is then converted to the di- and triphosphate form, which
competitively inhibits virus DNA polymerase, thus preventing
viral replication. Ganciclovir is available for systemic use.
The rising incidence of AIDS and the widespread use of
immunosuppressive drugs have caused an increase in CMV and
herpesvirus retinitis. In particular, ganciclovir is effective in
CMV retinitis; unfortunately, its systemic toxicity (bone
marrow suppression) has limited its use. Much research has
focused on intraocular injections and new delivery systems for
intraocular ganciclovir.
Foscarnet
Phosphonoformate (PFA, foscarnet) is a highly water-soluble
pyrophosphate analog that effectively inhibits in vitro
replication of HSV-1 and HSV-2, varicella zoster, and CMV
through noncompetitive binding to the exchange site of virus
DNA polymerase, thus blocking viral DNA synthesis. Foscarnet
has been used as an alternative to ganciclovir in the systemic
treatment of CMV infections. A number of investigators have
evaluated the effects of intravitreal foscarnet. She and co-
workers found that single doses of intravitreal foscarnet in doses
ranging from 200 to 1000 mg/0.1 mL are nontoxic to the retina
354 in New Zealand albino rabbits.276 Of note, their evaluation
consisted of clinical examinations and light microscopy. Dia-
Llopis and colleagues reported no evidence of toxicity with
intravitreal injection of 1200 mg in an AIDS patient with CMV
retinitis; again toxicity was evaluated by post mortem light
microscopy.277
In order to evaluate the effect of repeated intravitreal injec-
tions of foscarnet, Turrini and co-workers evaluated the retinal
toxicity of two, four, and six intravitreal injections of 3.6 mg
of foscarnet in 16 pigmented rabbits using ophthalmoscopy,
histology, and electrophysiology.278 All rabbits revealed evidence
of yellowish punctate retinopathy in the midperiphery and
posterior pole after the first injection. After four or six
injections, there was a significant decrease in the scotopic ERG,
whereaas after six injections there was a significant decrease in
the mesopic ERG. Of note, light microscopy revealed mild
vacuolization and rarefaction in the photoreceptors and inner
nuclear layers. After six intravitreal injections, focal areas of
photoreceptor layer destruction was observed.278
TOXICOLOGY OF ANTIGLAUCOMA DRUGS
Therapeutic agents designed to treat ocular hypertension reduce
intraocular pressure either by reducing aqueous humor
production or by enhancing aqueous humor outflow (through
the trabecular meshwork or via uveoscleral pathway). These
agents are commonly prescribed as ocular hypertension impacts
a high percentage of the elderly population. In this section,
ocular side effects will be broken down by agent classes.
CHOLINERGIC DRUGS
Much has been learned about the ophthalmic toxicology of
cholinergic drugs from testing in animals and humans, from
observations in accidental poisonings, and in connection with
medical uses of both direct-acting agents and anti-
cholinesterases in the treatment of glaucoma, accommodative
strabismus, and myasthenia gravis. Most commonly known
effects include miosis (constriction of the pupil), induction of
pupillary cysts, enhancement of accommodation (i.e.,
adjustment of the lens of the eye to focus on near objects),
formation of cataracts, and reduction of IOP. A variety of other
effects are less well known or are less well established. Systemic
poisoning, which has occasionally been caused by the use of
anticholinesterase eye drops, is manifested by both muscarinic
and nicotinic symptoms, which can include paralysis of the
respiratory muscles mediated by stimulation of nicotinic
receptors. Although the ocular and systemic side effects of
anticholinesterases are generally more frequent, similar
patterns are noted with direct-acting muscarinic agents such as
pilocarpine. Therefore, direct-acting agonists and anticholi-
nesterases are considered as a group.
ADRENERGIC AGONISTS
Nonselective adrenergic agonists, such as epinephrine,
stimulate both a- and b-adrenergic receptors. They exert their
therapeutic effect on IOP by reducing aqueous outflow
resistance. Although adrenergic agonists have been used in the
treatment of POAG for over 100 years, the site of action of the
outflow resistance-decreasing effect is unknown. The
mechanism of action is through stimulation of the b-adrenergic
receptors.279–283 Adrenergic receptors are found on almost all
ocular tissues, and adrenergic agonists are known to affect a
number of ocular physiologic parameters, including smooth
muscle tone in the iris and ciliary body, aqueous humor
production, and intraorbital and extraorbital vascular tone. The
incidence of cardiovascular stimulation after topical ocular

treatment is a potential major side effect that limits the
therapeutic use of epinephrine. The prodrug formulation
dipivefrin has allowed smaller doses of epinephrine to be
administered, limiting the risk of adverse systemic effects.
In in vitro studies, epinephrine in clinically relevant doses
was toxic to trabecular cells.284 In contrast, examination of the
outflow pathway tissue of normal cynomolgus monkey eyes
after 6 months of topical treatment with epinephrine revealed
no apparent toxicity to the outflow pathway tissues.285
However, the ciliary muscle appeared to be displaced anteriorly,
narrowing the chamber angle. Also, changes in the ciliary
processes consistent with hypersecretion were noted in some
sections and hyposecretion in others.286
Experimentally, epinephrine dramatically reduces blood flow
to the ciliary processes in the cynomolgus monkey287 and the
albino rat288 but not to the ciliary muscle288 as observed by a
functional resin-casting method. Similar results were noted
using radioactive microspheres in the albino rabbit eye, in
which 2% epinephrine administered topically three times a day
over a 5–6-week period resulted in decreased blood flow to the
iris and ciliary processes but not to the posterior uvea or optic
nerve head.289
As reviewed by Grant,290 epinephrine-induced retinal toxicity
was not recognized until the 1960s. A reduction in visual acuity
can occur with the long-term administration of epinephrine.
Generally, the reduction in visual acuity is reversible within
several months.
Much has been written about the incidence of hypertension
and heart palpitations after the administration of topical
epinephrine. An extensive review is presented by Grant.290
Additional caution is necessary when epinephrine is employed
in combination with a local anesthetic, such as occurs in the
course of otolaryngologic procedures. Additionally, if a patient is
taking b-blockers, the possibility of serious complications
resulting from additional epinephrine results.291 What is
sometimes observed is a hypertensive crisis that is immediately
followed by cardiac slowing and possible cardiac arrest. Despite
the potential for adverse cardiovascular effects, the use of
intraocular epinephrine has become standard practice in
cataract surgery, and no untoward effects on the cardiovascular
system have been noted.292–295
ADRENERGIC b-RECEPTOR-BLOCKING
DRUGS
The b-blockers timolol, betaxolol, and levobunolol are widely
used in the treatment of primary open-angle glaucoma. Both
timolol and levobunolol are nonselective b-blockers (e.g., they
bind b1- and b2-receptors with nearly equal affinity). Betaxolol is
somewhat selective for the b1-receptor. More recently, carteolol
has been introduced into the medical treatment of glaucoma.
Carteolol is a nonselective b-blocker that also has some
intrinsic sympathomimetic activity (ISA).
Much has been written about the tendency of b-blockers to
cause cardiovascular and respiratory problems.290,295,296
Theoretically, the selectivity of a b-blocker for b1- or b2-receptors
would make it a better choice for use in patients with asthma
and cardiovascular insufficiency, respectively. However, the
drugs currently in use do not have a selectivity sufficient to
prevent their binding of all b-receptors at therapeutic
concentrations.
In addition to receptor selectivity, several other phar-
macologic parameters determine the profile of side effects
associated with a given b-blocker. b-Blockers with some ISA,
such as carteolol, pindolol, acebutolol, and penbutolol, are less
likely to cause cardiovascular insufficiency, bronchospasm, or
adverse changes in serum lipids.297 The degree of lipid solubility
Toxicology of Ophthalmic Agents by Class
should influence how much drug needs to be given topically to
reach therapeutic levels in the anterior chamber. Also, the
degree of plasma protein binding influences how much free drug
is available to the systemic circulation. b-Blockers also differ in
their activity as membrane-stabilizing (and anesthetic) agents.
All these factors influence the degree of local and systemic
toxicity.
A recent area of investigation has involved the development
of prodrugs of timolol and levobunolol that might allow greater
corneal permeability; therefore, the required topical dose of drug
could be reduced, minimizing possible systemic toxicity.298,299
Timoptic-XE (a formulation of timolol that forms a gel on
contact with the ocular surface) administered once a day was
shown to be equally effective in lowering IOP as the equivalent
concentration of topical timolol administered twice a day. The
safety profile is similar to that of equivalent concentrations of
timolol.300
The ocular administration of b-blockers results in rapid
systemic absorption of the drugs in sufficient quantities to
affect the heart and the lungs.301 Early clinical trials showed
timolol to be without serious systemic side effects. However, as
summarized by Nelson and colleagues,302 many of these early
studies did not include patients with underlying cardiovascular
or respiratory problems. As of 1985, the US Food and Drug
Administration and the National Registry of Drug-Induced
Ocular Side Effects have tabulated a total of 450 case reports of
serious cardiovascular or respiratory complications, 32 of which
resulted in death, after the administration of topical timolol. Of
CHAPTER 33
the 212 patients for which a medical history was provided, 92%
had either cardiovascular or respiratory problems.302 Therefore,
a careful medical history is necessary before prescribing topical
b-blockers for the treatment of glaucoma in order to eliminate
the possibility of exacerbating an underlying condition.
In addition to the contraindications noted later, b-blockers
should not be used in combination with calcium channel
blockers, since sudden death has been reported after the
systemic administration of a b-blocker and verapamil.303,304
b-Blockers cause bronchial constriction as a consequence of
binding to b2-receptors in the bronchi. b-blockers that are
nonselective (such as timolol) may compromise ventilation in
patients with obstructive lung disease, asthma, or bronchospasm.
The National Registry of Drug-Induced Ocular Side Effects has
received over 200 reports of topical timolol-induced respiratory
problems. Sixteen fatal attacks of status asthmaticus have
occurred after the application of topical timolol.305
ADRENERGIC a-RECEPTOR BLOCKING
DRUGS
Clonidine is a relatively selective a2-adrenergic agonist that is
used clinically as an antihypertensive agent. The hypotensive
effect is mediated by the activation of a2-receptors in the central
nervous system.306 Topically, clonidine reduces IOP307–311 and
aqueous humor flow.312 It is thought to act by binding
a2–receptors in the ciliary body that inhibit adenylate cyclase.313
Apraclonidine is a p-amino derivative of clonidine, which is
incapable of penetrating the blood–brain barrier. Therefore, the
use of topical apraclonidine should prevent the systemic
hypotension that can occur with the use of topical clonidine.
Apraclonidine is as effective as clonidine in lowering IOP314,315
and has seen use clinically in preventing the large elevations in
IOP that occur after argon laser iridotomy,316,317 argon laser
trabeculoplasty,317,318 and Nd-YAG posterior capsulotomy.317,319
There are indications of possible usefulness in the treatment of
POAG,320,321 particularly when a patient on maximally tolerated
medical therapy is awaiting surgery. Long-term use of
apraclonidine requires frequent monitoring due to the frequent 355
 PHARMACOLOGY AND TOXICOLOGY
occurrence of tachyphylaxis. Brimonidine, an a2-adrenergic
agonist that is 10-fold more selective than apraclonidine, also
binds to imidazoline receptors. It functions similarly to
apraclonidine, by reducing aqueous inflow and uvealscleral flow.
Brimonidine (0.5%) was developed for post-argon-laser-
iridotomy and (0.2%) for glaucoma treatment. The advantage of
brimonidine over apraclonidine is that there appears to be a
lower incidence of allergic reaction and tachyphylaxis does not
occur.
CARBONIC ANHYDRASE INHIBITORS
Inhibition of carbonic anhydrase in the ciliary processes of the
eye reduces aqueous humor secretion, presumably by slowing
the formation of bicarbonate ions with a subsequent reduction
in sodium and fluid transport. The result is a reduction in
IOP.323
Acetazolamide (Diamox) has been used in the treatment of
glaucoma. It has been administered orally on account of the
inability of the compound or other carbonic anhydrase
inhibitors such as methazolamide, ethoxzolamide, and
dichlorphenamide to cross the cornea.324 Even though
systemically administered carbonic anhydrase inhibitors are
effective in lowering IOP, the constellation of side effects
associated with their use has limited the clinical usefulness of
carbonic anhydrase inhibitors in the treatment of glaucoma.
Recently, as summarized by Podos and Serle,325 three
derivatives of acetazolamide that are permeable to the cornea
SECTION 4
have been introduced. They are effective in reducing the IOP
with systemic drug levels too low to produce systemic side
effects.326 Most literature concerns the effects of MK-927.327–337
There is some evidence that MK-417, the enantiomer of MK-
927, is slightly more effective in lowering the IOP with
multiple-dose administration to patients with glaucoma.336
Finally, early results with a third derivative, MK-507, suggest
that it may be longer lasting than the other two derivatives.337
Dorzolamide has been developed as a long-awaited carbonic
anhydrase inhibitor that can be administered topically rather
than systemically. It inhibits carbonic anhydrase type II,
reduces the IOP by 21.8% (bid) to 26.2% (tid), and is used alone
or as an adjunctive therapy. Although it is administered
topically, the potential for systemic absorption exists.
Therefore, its use is contraindicated with oral carbonic
anhydrase inhibitors.323
PROSTAGLANDINS
Prostaglandins were discovered in the eye in the course of a
search for mediators of ocular inflammation. Prostaglandins D2,
E2, and F2a are synthesized by ocular tissues338 and are actively
transported out of the eye.339 Aside from playing a role in
intraocular inflammation, there is some evidence that
prostaglandins play an endogenous role in normal physiologic
processes.339 Some prostaglandins may actually attenuate an
inflammatory response.340 Prostaglandin F2a causes a dramatic
reduction in IOP in monkey eyes,341–347and in normal348,349 and
glaucomatous365 human eyes, which is apparently mediated by
increased nonconventional outflow.342,344–346,340 Latanoprost, a
prostaglandin F2a analog that has been introduced for the
treatment of glaucoma, is a prodrug and is metabolized by
corneal esterases. Latanoprost reduces the IOP ~27% when
administered once daily in the morning and, interestingly,
~35% when administered once daily in the evening.
Prostaglandin E2 also apparently reduces the IOP in human
eyes.351 However, the potential for an irritative response is
apparently greater with prostaglandin E1 and prostaglandin E2
356 than with prostaglandin F2a.347
The major ocular side effects that result from prostaglandin
use relate to their capacity to influence the blood-aqueous and
blood–retinal barriers. Prostaglandin E2 (0.02%) administration
to human eyes is associated with a transient mild eye ache,
photophobia, and conjunctival vasodilatation without clinical
evidence of ciliary flush or anterior chamber cells and flare.351
Intravenously administered prostaglandin E1 resulted in retinal
vasodilation in normal human adults.352 In a single-dose study,
administration of the trimethalamine salt of prostaglandin F2a
in doses ranging from 62.5 to 250 mg resulted in reddened skin
of the lower lid, ocular irritation, conjunctival hyperemia, and
headache without evidence of pupillary changes or anterior
chamber cells or aqueous flare.348
In another study, chronic administration of the more lipid-
soluble isopropylester of prostaglandin F2a in doses of 1 mg once
daily or 0.5 mg twice daily for 2 weeks resulted in a significant
reduction in IOP in normal human eyes that was associated
with a dose-dependent hyperemia, foreign body sensation, pain,
and photophobia with no evidence of ocular inflammation.349
No studies in animals or humans have noted a systemic side
effect related to the topical application of prostaglandins.
The ocular side effects associated with latanoprost are a
foreign body sensation, punctate epithelial keratopathy,
stinging, conjunctival hyperemia, blurred vision, itching,
burning, and iris pigmentation. In preclinical studies,
latanoprost was found to increase pigmentation in the iris of
monkeys.353 Additionally, in a 6-month study comparing
latanoprost with timolol in open-angle glaucoma and ocular
hypertensive patients, 10% of patients developed increased iris
pigmentation. All these patients had hazel irises.354 Latanoprost
increases the amount of brown pigment in the iris by increasing
the number of melanosomes within melanocytes, rather than
melanocyte proliferation. The increase in brown pigment does
not progress after discontinuation of treatment, but the
resultant color change may be permanent.353,354
TOXICOLOGY OF AGE-RELATED MACULAR
DEGENERATION DRUGS
The progressive deterioration of central vision in exudative
(wet) age-related macular degeneration (AMD) is caused by
choroidal neovascularization (CNV). Although the majority of
AMD cases are nonexudative, most severe vision loss is attri-
butable to wet disease, and consequently pharmaceutical
development efforts have focused on wet AMD.
Currently approved treatments for CNV associated with wet
AMD include verteprofin, a lipophilic molecule; pegaptanib
sodium, an aptamer; and ranibizumab, a monoclonal antibody.
In addition, bevacizumab, a monoclonal antibody related to
ranibizumab, is sometimes used off-label. Pegaptanib, ranibi-
zumab, and bevacizumab inihibit the vascular endothelial
growth factor (VEGF), an angiogenic factor.
ANTIANGIOGENESIS DRUGS
In the healthy eye, the blood–retinal barrier (BRB) isolates the
eye from systemic circulation. However, neovascular disease
can compromise BRB integrity, making it semi-permeable to
intraocular drugs.355 Excess anti-VEGF in nonocular vascular-
ization could down-regulate healthy angiogenesis systemically.
Cardiovascular and cerebrovascular events have been observed
in short-term studies of anti-VEGFs, but the risk of chronic
exposure, which is theoretically more of a concern for systemic
safety, has not yet been quantified.
VEGF has many physiologic roles which could be adversely
affected by a VEGF inhibitor. Its pivotal role in the angiogenic
cascade as a growth and permeability factor is needed for wound

healing. VEGF also has vasodilative and neuroprotective
effects,356 and helps maintain vessels such as the coronary
artery.357 Therapeutic down-regulation of VEGF using wet
AMD drugs could theoretically impact any or all of these
processes. Systemic side effects of intraocular anti-VEGFs have
thus far been rare, but since no data exist on prolonged anti-
VEGF exposure, serious adverse reactions cannot be ruled out.
Our understanding of the adaptation of the angiogenic cascade
in the presence of anti-VEGF agents is incomplete. Because of
these gaps in our knowledge, it is impossible to be sure of the
long-term toxicity of anti-VEGF drugs.
All 3 anti-VEGF drugs used intraocularly to treat AMD —
pegaptanib, ranibizumab, and bevacizumab — are administered
via intravitreal injection. While the procedure delivers
therapeutic levels of medication to the posterior segment of the
eye, it has been associated a number of vision-affecting compli-
cations, including endopthalmitis, retinal detachment, vitreous
floaters, traumatic cataract, and vitreous hemorrhage.358,359
Anti-VEGF drugs are also used to inhibit abnormal
neovascularization associated with tumor growth in cancer
patients. Adverse events associated with intravenous beva-
cizumab used to treat colon cancer include cerebral infarctions,
myocardial infarctions, other arterial thromboembolic events,
hemorrhage and gastrointestinal perforations.360
Although ophthalmic anti-VEGF therapies sucha as rani-
bizumab, are similar to oncological drugs, such as bevacizumab,
their systemic side effects are less severe. The doses needed to
inhibit abnormal ocular neovascularization are far lower than
the doses needed to disrupt angiogenesis in malignant tumors.
Also, intravitreal injection limits systemic exposure, even if the
blood–retinal barrier is breached by disease.
Intravenous verteporfin used in conjunction with a
nonthermal red laser, is also used to treat wet CNV. The
combination treatment, known as photodynamic therapy
(PDT), involves a photosensitive compound which, when
activated by a low-power laser dissociates into volatile oxygen
free radicals. The unstable oxygen compounds injure the
neovascular endothelium so that it secretes procoagulant and
vasoactive factors which occlude abnormal vessels in the
macula. PDT can selectively treat diseased tissue while leaving
healthy tissue intact.
Since PDT is a combination drug and device therapy, it is
difficult to attribute specific adverse reactions to a specific stage
of treatment. That said, the following side effects have been
observed in exudative AMD patients treated with PDT:
blepharitis (1.7%), conjuctivitis (6.7%), dry eye (2.7%), ocular
itching (3.5%), retinal capillary nonperfusion (0.2%), retinal
detachment (1.0%), subretinal hemorrhage (2.2%), and vitreous
hemorrhage (1.7%).361 Because the drug is photosensitive, uncon-
trolled exposure to light could activate the drug, causing it to
occlude normal and abnormal vessels without distinction.362
Some cohort studies recommend high dose vitamin
supplements for nonexudative AMD projected to progress to
exudative AMD.363 Vitamins C, E, beta-carotene, and other
carotenoids365 such as zeaxanthin and lutein, have been
considered for both prophylactic and therapeutic treatment of
AMD.366 The antioxidant (vitamins C, E and beta carotene)
plus zinc formulation used in the Age-Related Eye Disease
Study (AREDS) has been shown to reduce the risk of developing
advanced AMD.381a At daily recommended levels, antioxidants
are safe and essential, but the much higher dosage required to
treat AMD has been associated with side effects. Beta-carotene
has been shown to increase the risk of lung cancer in
smokers,367 and zinc supplementation can lead to systemic
copper deficiency.368 High doses of vitamin E and C have been
associated with an increased risk of heart failure in patients
with vascular disease or diabetes.
Toxicology of Ophthalmic Agents by Class
Pegaptanib, ranibizumab, and bevacizumab all disrupt the
angiogenic cascade at the beginning of the neovascular process
by binding to VEGF, the most important and abundant protein
mediator type. The pharmological differences between the three
treatments explain the drugs’ different efficacies. Pegaptanib
selectively blocks VEGF165, one subtype of the VEGF-A splice
variant class that also includes VEGF121, VEGF189, and
VEGF206.369 Ranibizumab and bevacinzumab bind to all
VEGF-A isoforms, and therefore have broader inhibitive,
antiangiogenic effects.
Ranibizumab
Serious side effects of ranibizumab attributable to intravitreal
injection include endopthalmitis (1.3%), and intraocular
inflammation (1.7%). Adverse events potentially related to the
drugs’ systemic toxicity include myocardial infarction (2.1%)
and cerebral vascular events (0.9%).343,371 It is important to
note, however, that the aforementioned clinical data was
gathered in studies of limited duration, and may not reliably
predict the consequences of long-term management of
exudative AMD with ranibizumab.
Pegaptanib
Adverse events associated with pegaptanib sodium include
traumatic lens injury (0.7%), retinal detachment (0.6%),
vitreous floater (33%), endophthalmitis (1.3%) and retinal
detachment (0.6%).372 No extraocular complications were
observed, but since patients at risk of cardiovascular and
CHAPTER 33
cerebrovascular events were excluded from major clinical
studies, the systemic safety of the drug cannot be assumed.
Bevacizumab
Because bevacizumab has not been FDA-approved for
ophthalmic use, there is no official data regarding its intraocular
toxicity. The small case studies that do exist claim intravitreal
bevacizumab has no systemic toxicity,373–376 but since they were
not random or controlled, their conclusions can be viewed
skeptically. Also, the side effects associated with ranibizumab
are probably comparable, to a greater or lesser extent, to the side
effects of bevacizumab, due to the molecular similarity of the
two compounds.
OCULAR TOXICITY OF SYSTEMIC
MEDICATIONS
The intermittent or chronic administration of certain oral,
transdermal, and parenteral (including intrathecal and
intracarotid) medications may produce a variety of side effects
in one or more areas in the eye and visual system. It is
important to recognize the ocular effects following the systemic
application of drugs. Although adverse effects are frequently
encountered within the first 2 weeks of therapy, they may be
delayed. The ophthalmologist may not initially relate the ocular
side effects to the systemically applied drugs. This is especially
true if there is a long latent period between drug intake and the
pathologic eye changes or if the toxic effects of the drug are
persistent or even progressive after withdrawal of the drug, as
with phenothiazine.
The toxic effect of certain drugs may be cumulative and dose
dependent. For example, the retinopathy associated with
chloroquine therapy may appear years into therapy. Therefore,
the daily dose and duration need to be monitored. The toxic
effect of other drugs may be idiosyncratic and occur after a
single dose, as in Stevens–Johnson syndrome (in which a variety
of ingested drugs such as sulfonamides, barbiturates,
salicylates, phenylbutazone, penicillin, phenytoin, and others
have been implicated) or with ibuprofen-induced optic neuritis. 357
 PHARMACOLOGY AND TOXICOLOGY
Toxicity may depend on the solubility characteristics of the
drug and its ability to gain access through certain barriers such
as the blood–brain barrier or the blood–ocular barrier. The route
of administration becomes critically important, since such
barriers may be bypassed (as in the case of intrathecal
administration of certain chemotherapeutic agents). Massive
concentrations of a drug may be locally delivered in a fashion
that bypasses the hepatic metabolism to limit systemic toxicity,
but with significant local toxic manifestations (as in the case of
intracarotid administration of nitrosoureas, such as
bischloroethylnitrosourea (BCNU), for the treatment of primary
central nervous system tumors).
The route of drug delivery to the eye and its specific
characteristics influence the type of toxicity. Drugs that gain
access to the eye through tears may manifest ocular surface
abnormality in the form of toxic conjunctivitis or epithelial
keratitis (e.g., certain antimetabolites such as methotrexate).
Many systemic agents, including oral antihistamines, cause
ocular drying that can exacerbate keratoconjunctivitis secca and
cause extreme discomfort and ocular surface irritation. Drug
access into the eye via the aqueous humor may produce
lenticular or posterior corneal changes (e.g., the pigmentary
deposits on the anterior lens capsule and posterior cornea seen
with chronic phenothiazine use or the formation of posterior
subcapsular cataracts due to lens epithelial toxicity from
antimetabolites such as busulfan).
A variety of drugs used for a diverse range of medical
conditions may manifest a similar pattern of toxicity if they
SECTION 4
possess similar chemical–physical properties. A whorl-pattern
epithelial keratopathy may be produced by the group of drugs
that possess cationic amphiphilic properties (e.g., amiodarone
used for cardiac arrhythmias, chloroquine used as an
antimalarial and in collagen vascular disease, indomethacin
used as an analgesic and antiinflammatory, and suramin used
as an antiprotozoal and as a reverse transcriptase inhibitor of
human T-cell lymphotrophic virus III. By binding to polar lipids
and thus accumulating in the lysosomes of epithelial cells,
these agents produce a ‘lysosomal disorder’ similar to the
lysosomal enzyme disorder seen in Fabry’s disease and with a
similar clinical pattern. Drugs that have affinity for particular
chemical components often manifest their toxicity in areas
where high concentrations of these components are present.
Since the uvea has the highest melanin content of any tissue in
TABLE 33.1. Ocular Toxicity and Side Effects of Systemic Medications
Drug Class Uses Route
Anesthetic (inhalation)
Methoxyflurane Methyl ether Inhalation Inhalation
anesthetic
Antianginal
Atenolol b-Adrenergic- Antianginal and Oral, IV
(including blocking antihypertensive
labetalol, agent
metoprolol,
nadolol, and
pindolol)
Diltiazem (also Calcium Antianginal Oral,
nifedipine, channel sublingual,
verapamil) blocker IV
358
the body, it is not surprising that drugs with a high affinity for
melanin (e.g., chloroquine and hydroxychloroquine) induce
retinal-uveal toxicity.
Not all ocular changes due to systemic drugs require
discontinuation of the drug, since some may be inconsequential
and ultimately reversible, as with the whorl-like corneal
epithelial deposits seen with the cardiac antiarrhythmic
amiodarone or the ocular hypotensive effects of orally
administered b-blockers used for the therapy of hypertension or
angina pectoris. However, other adverse ocular reactions may be
irreversible, as with ethambutol-associated optic neuropathy.
Thus, it is critical to be aware of the nature of the toxicity and
the prognosis in order to plan the appropriate strategies for
patient monitoring and management.
Side effects caused by one member of a given chemical family
are often, but not always, caused by other members of the same
drug group. Therefore, knowledge of side effects of one drug
should alert one to monitor for side effects when drugs from a
similar family are used. Experimental trials of new agents must
include monitoring for the side effects anticipated by the
chemical family.
The ophthalmologist must be familiar with the appropriate
visual tests and monitoring requirements appropriate for
particular drug regimens. It is critical to
1. Identify the toxic agent and know its chemical family
2. Know if the effects are reversible or irreversible in order to
determine the appropriate plan of action
3. Be aware if a drug toxicity is cumulative or dose dependent
and monitor the specific parameters carefully
4. Be familiar with the appropriate diagnostic tests
Comprehensive reviews of ocular toxicity exist.377–381 Table 33.1
summarizes important side effects with practical information
for recognizing and managing toxicity: The information has
been limited to effects seen in humans and has been divided
into clinically useful categories of anterior segment, posterior
segment, and clinically relevant systemic toxicities.
ACKNOWLEDGMENTS
The preparation of this manuscript was supported in part by grants from
the National Eye Institute (EYO 7321), Research to Prevent Blindness,
and the Massachusetts Lions Eye Research Fund.
Side Effects Dose Comments
Relationship
Crystalline retinopathy Prolonged Calcium oxalate
anesthesia crystals in retinal
pigment epithelium
and retina
Sicca syndrome, Yes Work-up myasthenia
visual hallucinations, if patient exhibits
myasthenic extraocular muscle
neuromuscular- paresis
blocking effect
(may worsened
myasthenia gravis)
Rare; ocular irritation Yes Reversible
with periorbital
edema and blurred
Continued
 Toxicology of Ophthalmic Agents by Class

TABLE 33.1. Ocular Toxicity and Side Effects of Systemic Medications—cont’d

Drug Class Uses Route Side Effects Dose Comments

Relationship

vision, retinal
ischemia, and
transient blindness
Practolol b-Adrenergic- Antianginal and Oral, IV Keratoconjunctivitis Duration related Side effects reversible
blocking antihypertensive sicca, conjunctival in early stages, but
agent cicatrisation, decreased tear
keratitis with production may
opacities, and persist. This drug
myasthenic for general use has
neuromuscular- been withdrawn
blocking effect from the market.
Mechanism of toxicity
may be related to
production of anti-
bodies to practolol
metabolite.
Oculomucocutaneous
findings not seen
with other
b-adrenergic-
blocking agents
Propranolol b-Adrenergic- Antianginal and Oral, IV Sicca syndrome, Yes Reversible
blocking antihypertensive myasthenic neuro-
agent muscular-blocking-
effect, visual
hallucinations, and

CHAPTER 33
?inflammatory
orbital pseudotumor
Antianxiety
Alprazolam Benzodiazepine Antianxiety Oral, IV, IM Decreased No Side effects reversible
(including corneal reflex,
clonazepam, accommodation,
flurazepam, and depth
triazolam) perception,
abnormal
extraocular muscle
movement, allergic
conjunctivitis,
?mydriasis
precipitating
narrow-angle
glaucoma
Antiarrhythmic
Amiodarone Benzofuran Antiarrhythmic Oral, IV Whorl-like (vortex Dosage and Keratopathy due
derivative (ventricular) pattern) epithelial duration to cationic
keratopathy (98%), related amphophilic
resulting in photo- (keratopathy) with properties of drug
phobia (3%), halos minimal corneal that binds to polar
(2%), and blurred deposits with lipids and produces
vision (1%), sicca dosages <200 a lysosomal
syndrome, lens mg/day but in disorder, as in
opacities, skin nearly all patients Fabry’s disease.
pigmentation, with >400 mg/ Onset of
papillopathy and day; unclear for keratopathy as
optic neuropathy, papillopathy early as 6 days but
pseudotumor usually by 6 weeks;
cerebri, usually resolves in
?retinopathy 3 months after
(hypopigmentation) discontinuation but
may have
prolonged effect
owing to long half-
life. Keratopathy not
indication to
discontinue drug,
but papillopathy is a
relative indication

Continued 359
 PHARMACOLOGY AND TOXICOLOGY

TABLE 33.1. Ocular Toxicity and Side Effects of Systemic Medications—cont’d

Drug Class Uses Route Side Effects Dose Comments

Relationship

Digitalis Digitalis Antiarrhythmic and Oral, IV 11–25% side effects Yes Reversible. Toxicity
glycoside for congestive with toxic doses. may be made
heart failure Color vision worse with
abnormalities concomitant
(yellow-blue), visual quinidine therapy
sensations and (ERG may be
hallucinations, helpful). Color
scotomas, retinal testing (yellow-blue)
toxicity with may be helpful in
abnormal ERG adjusting dosage
amplitude
Disopyramide Anticholinergic Antiarrhythmic Oral, IV Blurry vision, Yes Side effects due to
decreased accom- anticholinergic
modation and effects, which are
lacrimation; reversible
mydriasis may
precipitate narrow-
angle glaucoma
Procainamide Procaine Antiarrhythmic Oral, IV Rare; lupus-like No
hydro- syndrome with
chloride scleritis
analog
Anticonvulsant
Phenytoin Hydantoin Anticonvulsant Oral, IV, IM Nystagmus, lens Yes Nystagmus may
opacities, benign persist for months
SECTION 4

intracranial after
hypertension, discontinuation.
ocular teratogenic Fine nystagmus at
effects therapeutic doses;
coarse nystagmus
in toxic states
Antidepressant
Amitriptyline Tricyclic Antidepressant Oral Mydriasis and Yes Reversible
(including anti- cycloplegia (may
desipramine, depressant precipitate narrow-
imipramine, angle glaucoma),
nortriptyline) aggravate kerato-
conjunctivitis sicca
owing to anticholin-
ergic effects,
oculomotor
abnormalities
Carbamazepine Iminostilbene Antidepressant, Oral Blurred vision, Yes; side effects Reversible with
derivative pain associated extraocular muscle with dosages decrease in dosage
with trigeminal abnormalities with >1.2 g
neuralgia diplopia, downbeat
nystagmus,
sluggish pupil and
papilledema with
toxic doses, retinal
pigmentary changes
Doxepin Tricyclic Antidepressant Oral, IV Blurred vision, Yes Reversible
(including anti- (also for mydriasis,
amoxapine, depressant psychoneurotic accommodation
clomipramine) anxiety) disturbances, and
aggravation of
keratoconjunctivitis
sicca due to anti-
cholinergic effects.
Nystagmus and
ophthalmoplegia
with toxic states
Methylphenidate Piperidine Antidepressant and Oral, IV (see Rare; mydriasis. Yes, usually with Illicit IV use of crushed
derivative for hyperkinetic Comments) Talc retinopathy overdose tablets is responsible
syndrome in (see Comments) for talc and
children cornstarch (used as
360 fillers) retinopathy

Continued
 Toxicology of Ophthalmic Agents by Class

TABLE 33.1. Ocular Toxicity and Side Effects of Systemic Medications—cont’d

Drug Class Uses Route Side Effects Dose Comments

Relationship

Phenelzine Monoamine Antidepressant Oral Rare; mydriasis, Yes, usually with MAO inhibitor activity
oxidase miosis, anisocoria, overdose increased with
inhibitor nystagmus, concomitant use of
diplopia, and other MAO
myasthenic inhibitors and
neuromuscular tricyclic
blockade antidepressants
Antihistamine
Brompheniramine Alkylamine See Oral See Pyrilamine. See Pyrilamine Alkylamine has the
(also chlor- Cyproheptadine Facial dyskinesia lowest incidence of
pheniramine, with chronic use ocular side effects
dexbrompheni-
ramine,
dimethindene,
triprolidine)
Cyproheptadine Phenothiazine Antihistamine used Oral Rare. Atropine-like Side effects usually
(Periactin) (also analog in allergic or effects causing disappear even with
azatadine) vasomotor mydriasis and continued use.
rhinitis, allergic decreased May precipitate
conjunctivitis secretions narrow-angle
aggravating glaucoma
keratoconjunctivitis
sicca
Diphenyhydramine Ethanolamine See Oral See Pyrilamine Toxic doses

CHAPTER 33
(Benadryl) Cyproheptadine responsible for
visual hallucinations
and nystagmus
Pyrilamine (also Ethylene- See Oral See Cyproheptadine. Visual hallucinations See Cyproheptadine
tripelennamine) diamine Cyproheptadine With long-term use, with overdose
anisocoria,
decreased
accommodation,
and blurred vision.
Facial dyskinesia
(blepharospasm),
visual hallucinations
Antihyperlipidemic
Niacin (nicotinic Vitamin Antihyperlipidemic Oral Metamorphosia, Yes, >1.5/day Symptoms precede
acid) blurring, central or findings. Amsler
paracentral grid may
scotoma, demonstrate central
maculopathy, visual change.
‘atypical CME’ with Reversible
no accumulation
of fluorescein on
angiogram
Antihypertensive
Clonidine a-Adrenergic Antihypertensive Oral Miosis and mydriasis Yes Reversible; unclear if
agonist (toxic doses), retinal findings
?retinal coincidental or drug
abnormalities related
(depigmentation,
degeneration, tears)
Hydralazine Phthalazine Antihypertensive Oral, IV Nonspecific ocular Transient Reversible
derivative irritation, lupus-like
syndrome with
episcleritis, retinal
vasculitis, and
exophthalmos
Antiinflammatory
Ibuprofen Nonsteroidal Antiinflammatory, Oral Rare. With rechallenge Optic neuritis and Optic neuritis and
(Motrin, Advil) anti- analgesic, refractive error toxic amblyopia toxic amblyopia are
inflammatory antipyretic changes, diplopia, are idiosyncratic reversible with
drug that Osteoarthritis, photophobia, dry visual acuity
inhibits rheumatoid eyes, decrease in returning to normal 361
Continued
 PHARMACOLOGY AND TOXICOLOGY

TABLE 33.1. Ocular Toxicity and Side Effects of Systemic Medications—cont’d

Drug Class Uses Route Side Effects Dose Comments

Relationship

cyclooxy- arthritis, gout, color vision, optic in 1–3 months, but

genase ankylosing neuritis with central color vision not
(propionic spondylitis, scotomas, toxic returning for up to
acid) cystoids amblyopia 8 months. May be
macular edema, irreversible is drug
?ocular is not discontinued
inflammation
Indomethacin See Ibuprofen See Ibuprofen Oral Decreased vision, ? Corneal deposits is
(Indocin) (indole) color vision defects, not a indication to
hypersensitivity discontinue the
reactions, including drug
Stevens-Johnson
syndrome, corneal
deposits, including
whorl-like epithelial
deposits,
papilledema
secondary to orbital
pseudotumor
Ketoprofen See Ibuprofen See Ibuprofen Oral Nonspecific In general,
(Orudis) (propionic conjunctivitis and nonsteroidal
acid) dermatologic antiinflammatories
reactions, are photosensitizers
cholinergic crisis,
and papilledema
secondary to
SECTION 4

orbital pseudotumor
Naproxen See Ibuprofen See Ibuprofen Oral Whorl-like corneal Optic neuritis is This drug is a
(Naprosyn) (propionic opacities, optic idiosyncratic photosensitizer;
acid) neuritis ?role in
maculopathy or
necrotizing
vasculitis
Piroxicam (Felden) See Ibuprofen See Ibuprofen Oral Rare and insignificant Idiosyncratic Most widely
(oxicam and prescribed
enolic acid) nonsteroidal anti-
inflammatory
worldwide
Prednisone Corticosteroids Antiinflammatory Oral Cataracts (PSC), Cataracts usually Exophthalmos may
Adrenocortical ocular hypertension dose related, not completely
insufficiency and glaucoma, increased risk of reverse. Increase,
replacement pseudotumor cerebri pressure elevation then slowly taper
and papilledema with ocular dose in
with withdrawal, hypertension, pseudotumor
exophthalmos with glaucoma, or cerebri. Pressure
long-term use, family history of may rarely remain
decreased tear glaucoma and elevated after
lysozyme, diabetes discontinuation.
?decreased Cataracts may
resistance to rarely progress after
infection, discontinuation;
myasthenic may be reversible
neuromuscular- in children
blocking effect
(extraocular muscle
paresis, ptosis),
delayed wound
healing
Sulindac (Clinoril) See Ibuprofen See Ibuprofen Oral Rare and insignificant Idiosyncratic
(indene)
Antimalarial
Chloroquine (see Quinolone Antimalarial and Oral Whorl-like corneal Yes (cumulative Toxicity greater with
also hydroxy- antirheumatic epithelial deposits, dose); little toxicity chloroquine than
chloroquine) Rheumatoid Hudson-Stahli line, if 3.5 mg/kg/day, with
arthritis, lupus accommodation, <250 mg/day hydroxychloroquine.
erythematosus motility, subcapsular for small patients, Corneal changes
cataracts, central <100 g total reversible. Rental
362
Continued
 Toxicology of Ophthalmic Agents by Class

TABLE 33.1. Ocular Toxicity and Side Effects of Systemic Medications—cont’d

Drug Class Uses Route Side Effects Dose Comments

Relationship

and paracentral < year changes may be

scotoma, irreversible or
photophobia, progressive after
nyctalopia, discontinuation.
photopsia, macular Since early changes
pigmentation, loss are nonspecific
of macular reflex, and patients with
macular edema, toxicity may be
bull’s-eye asymptomatic,
maculopathy, bone routine testing is
spicule formation, required. Every 6
optic disc pallor, months: vision,
vascular attenuation history, Amsler grid,
(end stage), ERG and central visual field
EOG abnormalities with red target,
color testing, ?ERG,
?EOG
Dapsone Sulfone Antimalarial and Oral Rare. Optic atrophy Dose related With massive doses
anti-inflammatory
Hydroxychloroquine Quinolone See Chloroquine See See Chloroquine. See Chloroquine
(see also Chloroquine Safe <6.5 mg/kg/
chloroquine) day or 400 mg/day
for smaller patients
Quinine Alkaloid Antimalarial Oral Toxic amblyopia, Dose related Use on rise, especially
Nocturnal leg sudden vision loss, (massive); in ‘street’ drugs.

CHAPTER 33
cramps, retinal arterial occasionally with Vision loss may be
myotonia constriction, venous low chronic acute or
congenital, congestion, retinal administration progressive with
myokymia, edema, macular usually some return
attempted pigmentary changes, of vision. Prenatal
abortions disc edema, optic maternal ingestion
nerve hypoplasia, may cause optic
myasthenic nerve hypoplasia.
neuromuscular Acute therapy
blockade unclear
(extraocular muscle
paralysis, ptosis)
Antimicrobial
Atovaquone Analog of Antiparasitic and Oral Vortex keratopathy
ubiquinone Pneumocystis
carinii in AIDS
Cefazolin (including Cephalosporin Antibacterial Oral, IV, IM Rare. Allergic reactions, No Side effects reversible
first, second, including Stevens-
and third Johnson syndrome,
generations) ?retinopathy
(cephaloridine)
Chloramphenicol Dichloracetic Antibacterial Oral, IV Rare. Decreased vision, Dose related; total Findings most often in
acid optic neuritis, optic >100 g or children. Most
derivative atrophy, toxic duration > 6 feared side effect is
amblyopia, weeks aplastic anemia,
retinopathy which is
idiosyncratic
Ciprofloxacin Fluoroquinolone Antibacterial Oral, IM, IV Rare. Blurred vision, Dose related; Quinolone group
photophobia, altered ?duration related common to quinine
color vision, toxic and chloroquine
optic neuropathy may be responsible
for optic nerve
toxicity. Optic
neuropathy is
slowly reversible
Clofazimine Phenazine Antibacterial used Oral Eyelid and conjunctival Yes Side effects are
derivative for leprosy pigmentation, reversible
corneal epithelial
changes

Continued
363
 PHARMACOLOGY AND TOXICOLOGY

TABLE 33.1. Ocular Toxicity and Side Effects of Systemic Medications—cont’d

Drug Class Uses Route Side Effects Dose Comments

Relationship

Doxycycline (also Polycyclic Antibacterial Oral Eyelid skin conjunctival Orbital pseudotumor Most side effects are
tetracycline, naphthacene hyperpigmentation, not dose related; reversible. Orbital
minocycline) carboxamide hyperpigmented pigmentation pseudotumor
conjunctival cysts, dose related mostly seen with
blue-gray sclera tetracycline and
pigmentation minocycline ?due
(minocycline), orbital to greater lipid
pseudotumor, solubility. Sclera
extraocular muscle pigmentation
paralysis, (minocycline)
aggravation of frequently
myasthenia gravis associated with
pigmentary
changes elsewhere
Ethambutol Tuberculostatic Oral Color vision Dose related. Optic neuritis
abnormalities, visual Infrequent with symptoms usually
field changes doses ≤15 mg/ noted at 3–6
(scotomas), axial kg/day months. Increased
and paraxial optic toxicity with renal
neuritis disease. With
regular doses,
home visual acuity
and color vision
testing
recommended. With
higher doses,
screen patient at
SECTION 4

2- to 4-week
intervals. Visual-
evoked response
helpful in detecting
subclincal toxic
effects. Visual
recovery variable.
?Treat optic nerve
toxicity with zinc
sulphate or
parenteral
hydroxycobalamin
Gentamicin Aminoglycoside Antibacterial IV, IM, Papilledema secondary No Most side effects are
(including intrathecal to pseudotumor reversible after
tobramycin, cerebri, myasthenic discontinuation
streptomycin) neuromuscular
blockade (paralysis
of extraocular
blockade and
ptosis), blindness
and optic atrophy
with intrathecal
administration
Isoniazid Hydrazide of Antitubercular Oral Rare. Optic and No Side effects usually
isonicotinic retrobulbar neuritis seen in
acid with visual field and malnourished or
color vision chronically ill
abnormalities patients. Many side
effects can be
prevented by daily
administration of
pyridoxine
Nalidixic acid Naphthyridine Antibacterial Oral Visual disturbances, Most not dose Side effects reversible
color vision defects, related if dosage is
papilledema due to decreased or drug
increased discontinued.
intracranial pressure, Increased
lupoid skin changes intracranial pressure
reported in persons
younger than
age 20

364 Continued
 Toxicology of Ophthalmic Agents by Class

TABLE 33.1. Ocular Toxicity and Side Effects of Systemic Medications—cont’d

Drug Class Uses Route Side Effects Dose Comments

Relationship

Penicillin (including Penicillin Antibacterial Oral, IM, IV Rare. Allergic reactions,

semisynthetic including Stevens-
penicillins) Johnson syndrome.
Aggravation of
ocular signs of
myasthenia gravis
(ampicillin), including
paralysis of extra-
ocular muscles,
diplopia, and ptosis,
pseudotumor cerebri
Rifabutin Synthetic Antitubercular and Oral Anterior uveitis, May be dose related Occurs with
rifamycin prophylaxis hypopyon uveitis, with increased concomitant use of
against white-yellow incidence with rifabutin with
Mycobacterium opacities 600 mg/day, less clarithromycin and
avium complex vitreous common with fluconazole.
in AIDS 300 mg/day Immunologically
mediated process
rather than direct
drug toxicity;
resolves with
topical
corticosteroid
therapy frequently
without
discontinuation of

CHAPTER 33
rifabutin
Rifampin Hydrazone Antitubercular and Oral Conjunctival Yes Reversible ocular side
derivative of antibacterial hyperaemia, effects in 5–15% of
rifamycin B conjunctivitis (may patients and more
be exudative), frequently seen with
orange staining of intermittent use
contact lenses
Sulfamethoxazole Sulfonamide Antibacterial, Oral Myopia due to lens No Side effects rare and
(including other ?anti- thickening from reversible
sulfa-containing inflammatory ciliary body edema,
medications allergic reactions,
such as including Stevens-
sulfadiazine and Johnson syndrome,
sulfasalazine) anterior uveitis,
optic neuritis
Suramin Nonmetallic Antiprotozoan Oral Vortex-like epithelial Dose related Side effects usually
polyanion used for keratopathy, ocular depend on
adjuvant therapy irritation, optic nutritional status.
in AIDS patients atrophy Optic atrophy
(inhibitor of secondary to
reverse inflammatory
transcriptase response to dead
of HTLV III) microfilariae.1
Keratopathy due to
lysosomotropic
properties that
inhibit lysosomal
enzymes.
Reversibility of
keratopathy unclear
at present owing to
prolonged half life
Antineoplastic or Immunosuppressive
Busulfan Alkylating Cancer Keratoconjunctivitis Yes, 2–6 mg/day for
agent chemotherapy: sicca, posterior months to years
chronic subcapsular
leukemia, cataract with
polycythemia polychromatic
vera, sheen 10–30%
myelofibrosis

Continued
365
 PHARMACOLOGY AND TOXICOLOGY

TABLE 33.1. Ocular Toxicity and Side Effects of Systemic Medications—cont’d

Drug Class Uses Route Side Effects Dose Comments

Relationship

Chlorambucil Alkylating Cancer Oral Rare but includes Yes

agent chemotherapy: keratitis,
chronic leukemia hemorrhagic
Immunosup- retinopathy, and
pression: oculomotor
vasculitis with disturbances
RA, Behçet’s
disease,
autoimmune
hemolytic
anemia
cis-Platinum Alkylating Cancer IV, Blurred vision (62%), Yes, >600 mg/m2 Blurred and color
(cisplatin) agent chemotherapy: intracarotid impaired color vision abnormalities
testicular cancer, vision (23%), retinal are reversible
breast cancer, toxicity (ERG) (84%),
bladder cancer, macular
lung cancer, pigmentation (46%),
gastrointestinal disc edema,
cancer, retrobulbar neuritis,
lymphoma, cortical blindness.
osteogenic With intracarotid
sarcoma administration,
ipsilateral vision
loss due to retinal
and optic nerve
ischemia (15–60%)
SECTION 4

Cyclophosphamide Alkylating Cancer Oral, IM, IV Blurred vision (17%), Yes

agent chemotherapy: keratoconjunctivitis
lymphoma, sicca (50%), pinpoint
breast cancer pupil due to
Immuno- parasympa-
suppressive: thomimetic effect
rheumatoid
arthritis,
Wegener’s
granulomatosis,
Mooren’s ulcer,
cicatricial
pemphigoid,
Behçet’s
disease, Graves’
disease
ophthalmopathy
Cytosine Pyrimidine Cancer IV Keratoconjunctivitis, Yes Resolution of
arabinoside analog chemotherapy: central punctate symptoms in
acute leukemia, opacities with weeks,
refractory subepithelial granular prednisolone
lymphoma deposits, microcysts, phosphate or 2-
reversible superficial deoxycytidine
punctate keratitis prophylaxis
Intrathecal (38–100%)
Optic neuropathy (may
be potentiated by
cranial irradiation)
Doxorubicin Antimicrobial Cancer Lacrimation (25%), red
(Adriamycin) anthracycline chemotherapy: discoloration
that binds sarcoma, of tears
DNA leukemia,
lymphoma
Fludarabine Purine analog Cancer Decreased vision due Yes
chemotherapy: to optic neuritis or
leukemia cortical blindness,
encephalopathy
5-Fluorouracil Pyrimidine Cancer IV Blurred vision, ocular Most are reversible Massage, topical
analog chemotherapy: pain, photophobia, 6–14 months for corticointubation
breast cancer, lacrimation, cicatricial changes
GI cancer, conjunctivitis,
GU cancer blepharitis, keratitis
366
Continued
 Toxicology of Ophthalmic Agents by Class

TABLE 33.1. Ocular Toxicity and Side Effects of Systemic Medications—cont’d

Drug Class Uses Route Side Effects Dose Comments

Relationship

(25=n38%),
cicatricial ectropion,
punctal and
canalicular stenosis,
blepharospasm,
oculomotor
disturbance,
nystagmus, optic
neuropathy
Actinic keratosis Topical Systemic absorption
may cause similar
corneal and external
disease findings
Methotrexate Folic acid Cancer Oral, IM, IV Periorbital edema, Yes (IV) Resolves off therapy;
analog chemotherapy: photophobia, ocular artificial tears
leukemia, solid pain and burning,
tumors blepharitis,
Immunosup- conjunctivitis, and
pressive: decreased tear
rheumatoid Intrathecal, production (25%),
arthritis, intracarotid optic neuropathy,
psoriasis, uveitis macular edema and
pigment epithelial
changes
Mitomycin C Antimicrobial Cancer Blurred vision
that cross chemotherapy:

CHAPTER 33
links DNA solid tumors
Mitotane Antimicrobial Cancer Neuroretinopathy, disc
DDT chemotherapy: edema, retinal
derivative adrenocortical hemorrhages, retinal
cancer edema, cataracts
(3–16%)
Nitrogen mustard Alkylating Cancer IV, Necrotizing uveitis Most likely
agent chemotherapy: intracarotid and vasculitis
lymphoma, (intracarotid)
brain tumor
Nitrosoureas Alkylating Cancer Oral, IV, Usually benign. Yes (dose and ERG; pressure on eye
(BCNU, CCNU, agent chemotherapy: intracarotid Conjunctival rapidity of during infusion or
methyl CCNU) primary CNS hyperemia and infusion) with Honan’s balloon to
tumor, blurred vision (4%), intracarotid limit toxicity
lymphoma, ?optic neuritis, administration
multiple ipsilateral periorbital
myeloma, colon edema, orbital pain
and gastric and congestion,
cancer conjunctivitis,
chemosis,
neuroretinal toxicity
(70%) (NFL infarcts,
intraretinal
hemorrhages, and
disc edema, with
intracarotid
administration)
Plicamycin Antimicrobial Cancer Periorbital pallor
(mithramycin) Inhibits RNA chemotherapy:
synthesis by testicular cancer,
binding DNA hypercalcemia
Tamoxifen Antihormonal Cancer Oral Whorl like epithelial Yes (120–200 mg/m2 May be irreversible
estrogen chemotherapy: keratopathy, for >1 year; Toxicity unlikely
antagonist breast cancer maculopathy with cumulative dose with standard
superficial white of 90–230 g) doses242 but has
refractile opacities been reported.243,244
associated with Presence of a
cystoid macular few intraretinal
edema, optic disc crystals in absence
edema, posterior of macular edema
subcapsular or vision loss or
cataracts presence of 367
Continued
 PHARMACOLOGY AND TOXICOLOGY

TABLE 33.1. Ocular Toxicity and Side Effects of Systemic Medications—cont’d

Drug Class Uses Route Side Effects Dose Comments

Relationship

posterior
subcapsular
opacities does not
warrant
discontinuation of
drug
Vincristine Vinca alkaloid Cancer IV Cranial nerve palsy Yes Increased toxicity with
Yes chemotherapy: (50%), internuclear hepatic dysfunction.
leukemia, ophthalmoplegia, Resolves in
lymphoma, corneal hypesthesia, 3 months
solid tumors optic neuropathy– ?Irreversible
demyelination, Reversible in
night blindness, 1–14 days
and cortical
blindness
Antipsychotic
Chlorpromazine Phenothiazine Antipsychotic Oral, IM, IV Similar to thioridazine. Pigmentary changes
Pigmentation of may be reversible
skin, conjunctiva,
and cornea,
pigmentary
retinopathy (fine)
Haloperidol Buterophenone Antipsychotic Oral, IM Decrease or paralysis Yes Transient and
derivative of accommodation, reversible side
mydriasis that may effects
SECTION 4

precipitate narrow
angle glaucoma1
and ?cataracts
Lithium carbonate Lithium salt Antipsychotic Oral Ocular irritation and Yes Reversible; toxic drug
photophobia, response related
blurred vision, to blood levels
extraocular muscle (>2 mEq/L);
abnormalities, exophthalmos may
exophthalmos, be seen at normal
papilledema due levels owing to
to pseudotumor effect on thyroid
cerebri
Thioridazine Phenothiazine Antipsychotic Oral, IM, IV Decreased vision, Dose and duration Symptoms improve
paralysis of related. after
accommodation, Rare, <1000 mg/day discontinuation, but
mydriasis due to Recommended dose fundus changes
anticholinergic <300 mg/day; may progress
properties, corneal maximum
pigment deposits 800 mg/day
(epithelium and
Descemet’s
membrane), corneal
edema, lens
surface deposits.
Granularity of
posterior pole,
transient disc and
retinal edema,
nummular retinopa-
thy, paracentral
and ring scotoma,
abnormal ERG and
EOG, myasthenic
neuromuscular
blockade, extra-
ocular muscle
paralysis, diplopia,
ptosis
Antiparkinsonism
Amantadine Tricyclic amine Parkinson’s disease Oral Rare. Transient Dose related Side effects reversible
Antiviral used in decreased vision, with discontinuation
prophylaxis of superficial punctate
368
Continued
 Toxicology of Ophthalmic Agents by Class

TABLE 33.1. Ocular Toxicity and Side Effects of Systemic Medications—cont’d

Drug Class Uses Route Side Effects Dose Comments

Relationship

influenza A keratitis, sudden

vision loss, visual
hallucinations
Benztropine Anticholinergic Parkinson’s disease Oral Decreased Dose related Ocular side effects
(also biperiden, Control of accommodation; more common with
chlorphe- extrapyramidal rarely mydriasis benztropine versus
noxamine) disorders may precipitate biperiden
narrow angle
glaucoma,
hallucinations
Levodopa b Adrenergic- Parkinson’s disease Oral Mydriasis may Dose related Side effects reversible
blocking precipitate narrow
agent angle glaucoma,
miosis, ptosis,
blepharospasm,
visual hallucinations,
oculogyric crisis
Antirheumatic (see also Antiinflammatory and Antineoplastic or Immunosuppressive)
Allopurinol Xanthine Chronic Oral ?Cataract, ?macular Unclear with
oxidase hyperuricemia, edema and cataracts and
inhibitor gout hemorrhage, toxic maculopathy,
epidermal necrolysis not dose related
(Lyell’s syndrome) toxic epidermal
necrolysis

CHAPTER 33
Gold Heavy metal Rheumatoid IM Conjunctival and Yes, >1 g, 1 g/day Cornea and lens
arthritis, lupus corneal deposition, for years for deposits do not
erythematosus occasionally lens lenticular affect visual acuity
deposition, rarely deposition and are not an
ptosis, diplopia, indication for
nystagmus discontinuing
therapy
Deposits reversible
after
discontinuation
Antispasmodic
Dicyclomine Anticholinergic Antispasmodic Oral Rare. Decreased Yes Due to mild
vision, mydriasis antichoinergic
(rarely may activity. Side effects
precipitate narrow reversible and not
angle glaucoma), indication to
decreased discontinue drug
accommodation,
and photophobia
Dermatologic
Canthaxanthine Carotenoid Tanning agent for Oral Metamorphopsia, Yes; total 30–40 g, Increased retinopathy
(non– vitiligo, decreased vision, >50% with ingestion of
provitamin photosensitive yellow, refractile retinopathy; other carotenoids
A) dermatitis inner retinal total >60 g,
deposits >55–100%
surrounding fovea retinopathy
Chrysarobin Keratinolytic Topical Keratoconjunctivitis Yes Symptoms rarely last
for weeks after
discontinuation
Methoxsalen (also Psoralen Vitiliginous lesions Oral, topical ?Cataracts Used in conjunction
trioxsalen) with ultraviolet light
for photochemo-
therapy (PUVA).
Patient requires
adequate UV
blocking goggles
after therapy

Continued

369
 PHARMACOLOGY AND TOXICOLOGY

TABLE 33.1. Ocular Toxicity and Side Effects of Systemic Medications—cont’d

Drug Class Uses Route Side Effects Dose Comments

Relationship

Immunosuppressive (see Antineoplastic or Immunosuppressive)

Industrial
Methanol Alcohol Industry Oral, Decreased vision, Variable, as low as Primary site of injury is
(rubbing, inhalation nystagmus, 1 oz the optic nerve.
wood) mydriasis, disc and Emergency medical
retinal edema, therapy (respiratory
central and support, dialysis,
cecocentral ethanol) is required.
scotoma, optic Vision may improve,
atrophy and usually in 6 days
excavation
Stimulant (Gastrointestinal and Urinary Tracts)
Bethanechol Quaternary Gastrointestinal Oral, Rare. Occular irritation Side effects may
ammonium and urinary sub- with lacrimation, continue long after
parasympa- tract stimulant cutaneous decreased the drug is
thomimetic accommodation, discontinued
and miosis
Miscellaneous
Deferoxamine Chelating Removal of IV, sub- Cataracts, visual loss, Duration related Toxicity may be rapid
mesylate agent excess systemic cutaneous optic neuropathy, in onset and
iron retinal pigmentary irreversible.
degeneration Retinopathy
reported with single
subcutaneous dose
SECTION 4

Pamidronate Biphosphonate Inhibitor of bone IV Mild to severe anterior Anterior uveitis

resorption used uveitis and frequently bilateral
in hypercalcemia nonspecific and may require
of malignancy, conjunctivitis topical therapy
painful bone
metastases, and
Paget’s disease
Abbreviations: AIDS, acquired immunodeficiency syndrome; BCNU, carmustine; CCNU, lomustine; CME, cystoid macular edema; CNS, central nervous system; DDT,
chlorophenothane; EOG, electrooculogram; ERG, electroretinogram; GI, gastrointestinal; GU, genitourinary; HTLV, human T cell lymphotrophic virus; MAO, monoamine
oxidase; NFL, nerve fiber layer; PSC, posterior subcapsular cataract; PUVA, psoralen ultraviolet light application; RA, rheumatoid arthritis; UV, ultraviolet.

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J Ophthalmol 1993; 21:130–131.
359. D’Amico DJ, Bird AC: VEGF inhabition
study in ocular neovascularization-1
(Vision-1); Safety evaluation from the
pivotal macugen (pegatanib sodium)
clinical trials. ARVO Absract 2004; 2363.
360. The United States Food and Drug
Administration. Avastin (bevacizumab).
Drug label approved October 11, 2006.
361. Azab M, Benchaboune M, Blinder KJ, et al:
Verteporfin therapy of subfoveal choroidal
neovascularization in age-related macular
degeneration. Retina 2004; 24:1.
362. Azab M, Benchaboune M, Blinder KJ, et al:
Verteporfin therapy of subfoveal choroidal
neovascularization in age-related macular
degeneration. Retina 2004; 24:1.
363. Age-related Eye Disease Study Research
Group: A randomized, placebo-controlled,
clinical trial of high-dose supplementation
with vitamins C and E, beta carotene, and
zinc for age-related macular degeneration
and vision loss: AREDS report no 8. Arch
Ophthalmol 2001; 119:1417–1436.
364. Hall NF, Gale CR: Prevention of age related
macular degeneration. BMJ 2002; 325.
365. West S, Vitale S, Hallfrisch J: Are
antioxidants or supplements protective for
age-related macular degeneration? Arch
Ophthalmol 1994; 122:99–104.
366. Seddon JM, Ajani UA, Sperduto RD, et al:
Dietary carotenoids, vitamins A, C and E, and
advanced age-related macular degeneration.
JAMA 1994; 272:1413–1420.
367. Omenn GS, Goodman GE, Thornquist MD,
et al: Effects of a combination of beta
carotene and vitamin a on lung cancer and
cardiovascular disease. N Engl J Med
1996; 334:1150–1155.
368. Hoffman HN, Phyliky RL, Fleming CR:
Zinc-induced copper deficiency.
Gastroenterology 1988; 94:508–512.
369. Park JE, Keller GA, Ferrara N: The vascular
endothelial growth factor (VEGF) isoforms:
differential deposition into the subepithelial
extracellular matrix and bioactivity of
extrecellular matrix-bound VEGF. Am Soc
Cell Bio 4:1317–1326.
370. Rosenfield PJ, Brown DM, Heier JS, et al:
Ranibizumab for neovascular age-related
macular degeneration. N Engl J Med 2006;
355:1419–1431.
371. Brown DM Kaiser PK, Michels M, et al:
Ranibizumab versus verteporfin for
neovascular age-related macular
degeneration. N Engl J Med 2006;
355:1432–1444.
372. Gragoudas ES, Adamis AP, Cunningham
ET, et al: Pegaptanib for neovascular age-
related macular degeneration. N Engl J
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373. Manzano RP, Peyman GA, Khan P, Kivilcim
M: Testing intravitreal toxicity of
bevacizumab (Avastin). Retina 2006;
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Electrophysiologic findings after intravitreal
bevacizumab (Avastin) treatment. Retina
2006; 26:270–274.
375. Spaide RF, Klancnik J, Sorenson J, et al:
Intravitreal bevacinzumab for choridal
neovascularization secondary to age-related
macular degeneration. Invest Ophthalmol
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376. Pieramici D, Avery R, Rabena MD, et al:
Bevacizumab (Avastin) in the treatment of
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Springfield, IL: Charles C Thomas; 1986.
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of ocular drug therapy and ocular side
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Little, Brown; 1991.
380. Physicians’ desk reference: Oradell, NJ:
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complications of systemic cancer
chemotherapy. Surv Ophthalmol 1989;
34:209.

CHAPTER 33

377
 SECTION 5 PRINCIPLES OF EPIDEMIOLOGY
Edited by Frederick L. Ferris III and Emily Y. Chew
CHAPTER
34 Epidemiology and Clinical Research
Jie Jin Wang and Tien Y. Wong
CLINICAL RESEARCH AND THE
SCIENTIFIC HYPOTHESIS
Clinical research seeks to answer a scientific question by con-
ducting a study in humans. This question may cover etiology,
pathogenesis or risk factors of a particular disease, its natural
history and prognosis, and possible treatment options.
Table 34.1 shows the principles typically followed in con-
ducting clinical research. Each clinical research project should
have a sound hypothesis that the proposed study will address.
Researchers will need to: (1) logically display all the evidence
supporting the hypothesis (research background information);
(2) ask a research question that is answerable with the proposed
study (study aim); (3) design a feasible project to provide the
highest quality of evidence as possible to answer the research
question (research methods); and (4) finally conduct the study.
Common examples of clinical research questions include the
following: Does smoking (risk factor exposure) increase the risk
of age-related macular degeneration (disease outcome)? Does
the use of systemic steroids (treatment exposure) increase the
risk of multiple sclerosis following optic neuritis (prognostic
outcome)? Will a retinal photography screening program
(intervention exposure) reduce blindness from diabetic retino-
pathy (effectiveness outcome)?
ESTIMATES FOR FREQUENCY OF
DISEASES (RATES)
In epidemiology research, rates are preferred to absolute
numbers. A rate provides the proportion of individuals with a
particular disease or a certain characteristic which facilitates
comparison between groups or studies, while the absolute
number of cases provides very little information because the
size of the group (the denominator) can vary widely. The two
rates commonly used in clinical research and epidemiological
studies are the prevalence rate and the incidence rate.
PREVALENCE RATE
The prevalence rate refers to the frequency with which a disease
or condition is present in the study sample of a specific
population under study at a particular point in time. Prevalence
relates to a condition present at the time of examination or
assessment (at a point in time), regardless of when that
condition developed. Prevalence is calculated as follows:
Prevalence (at a point in time) = n ÷ N
where n is the number of all cases with the condition at the
point in time, and N is the total size of the study sample.
In the Blue Mountains Eye Study, there were 253 participants
who had diabetes and 82 of these participants had signs of
diabetic retinopathy.1 Thus, the prevalence of diabetic
retinopathy is 82 ÷ 253 = 32.4% among diabetics, with one in
three persons presenting diabetes affected with retinopathy.
The prevalence rate is important in assessing the disease
burden in a country or community, indicating the proportion of
people who are blind at a certain point in time in a population.
In the US, a study in 2004 estimated that the prevalence of
blindness, defined according to the WHO definition as best-
corrected visual acuity of <20/400 in the better eye, was 5 per
1000 or 0.5% in persons aged 40 years and older.2 Extrapolating
that to the US population, the authors estimated that more
than 900 000 Americans 40 years and older were blind.
Prevalence rates allow comparison between studies. For
example, the prevalence of blindness reported in that US study
can be compared with the prevalence of blindness of 4.3% in
an Indian population of similar age range, showing that the rate
is eight times higher in India.3 A study in Beijing, China among
4319 persons 40 years and older reported that the prevalence
of myopia was 21.8%.4 This prevalence rate is higher than
similarly aged white persons in Australia (17%),5 but con-
siderably lower than similarly aged Chinese adults in Singapore
(38.7%).6
The prevalence rate of a specific condition can also be
compared between different time points to assess time trends,
i.e., whether the prevalence has increased or decreased after a
period of time. Examples of such time trend comparisons in the
Blue Mountains Eye Study have been reported for diabetic
retinopathy and cataract, which were assessed initially in
1992–94 and then subsequently in 1997–98.7,8
INCIDENCE RATE
In contrast to prevalence, the incidence rate refers to the fre-
quency with which new cases of a disease or condition develops
over a defined period of time. Incidence is also called absolute
risk (as opposed to relative risk (RR), which is described below)
and is calculated as follows:
Incidence (over a defined period) = n ÷ N
where n is the number of new cases that develop over the
period chosen, and N is the total number of study subjects who
were free of the disease at the beginning of the time period.
The incidence rate is the rate a condition develops over a
time period in a particular population. In a study on the inci-
dence of retinopathy among people with diabetes, Klein and
colleagues observed how many new cases of retinopathy devel-
oped over a 10-year period: 75% in participants with type-1
379
 PRINCIPLES OF EPIDEMIOLOGY

TABLE 34.1. Principles of Clinical Research

1. Define overall goal of project
2. Perform comprehensive
literature review
3. Identify specific research
questions
4. Design an appropriate study
5. Select sample size
6. Select study population
7. Identify study site
8. Identify methods to measure
exposure and outcome
9. Determine if masking is needed
10. Write a detailed protocol
11. Standardize study forms and
procedures
12. Examine for possible bias
13. Review information as study
SECTION 5
Define a clear overall goal of the research project. First, is the subject important? Does the project
address etiology, pathogenesis, natural history, treatment, or impact?
Familiarize with all relevant background information. Identify what is known and unknown, what has
been done, and what are the remaining gaps in the literature
Specify a focused question and the underlying hypothesis behind the question. These questions
should be directly answerable by the study
Select an appropriate study design that can answer the specific questions with the highest quality
evidence, taking into account feasibility and resources to do this study
Prior knowledge and pilot studies help determine the expected strength of association and expected
difference in study groups. Decide on a sample size that has adequate power, but is sufficiently cost
effective
Selection of cohort for cohort studies and selection of cases and controls for case control studies, etc.
Where will the study be conducted?
Both exposure and outcome should be measured using objective, standardized methods
Ideally, both study exposures and outcomes should be determined in a masked fashion
Provide clear documentation of study progress. Important reference of all procedures; and basis of
‘Introduction’, ‘Methods’, and ‘Discussion’ sections in paper
Procedures should be tested before study commencement usually in pilot settings. Quality
control procedures should be in place prior to study start and examined at frequent intervals
thereafter
Determine if there are major biases early in the study
Review factors for nonparticipation and response rates. If there is a major loss to follow up,

progress
14. Examine the primary results at
the conclusion of the study
15. Perform appropriate
statistical tests
16. Compare study results with
other studies
17. Consider alternative
explanations
18. Discuss inferences
19. Consider limitations
20. Conclusions and future research
21. Publish the study!
evaluate the patterns that might explain why. Patients lost to follow up are important sources of
selection bias
Results should be reported starting with primary endpoints. Subgroup
(often interesting) results should only be reported after the primary results
The best studies report simple statistics as true associations are usually obvious. Avoid
report-complicated statistics
Consistency between studies increases the likelihood that the observed results are real
Are the observed results due to chance, confounding or bias and are they clinically meaningful
Explain the meaning behind the study results. What do the results show? Do the observations have a
sound biological basis? Speculate on how the study may change clinical practice, but do not stretch
the conclusion beyond what the data show
All studies have limitations. Were all confounders appropriately controlled for? Are there selection
information and other types of biases? Is there a significant loss to follow-up?
Conclude by directly answering the research question. Consider future research. The best studies
usually lead to further questions

diabetes, 70% in participants with type-2 diabetes who were on
insulin treatment, and 50% in participants who were not on
insulin treatment.10
The incidence rate is commonly used to examine the effects
of treatment, typically in the setting of randomized clinical
trials (see section on Study Designs). In this situation, the
investigator compares the incidence of an outcome in a group
who received a particular treatment with the incidence of the
same outcome in the group who did not receive this treatment.
For example, the question whether antioxidant supplemen-
tations can reduce the risk of age-related macular degeneration
(AMD) was tested in the randomized control trial (RCT), the
Age-related Eye Disease Study, which compared the incidence of
advanced AMD in a group of patients taking antioxidant sup-
plements with another group not taking these supplements.11
380 The association of dietary intake and/or supplements of
antioxidants with the incidence of developing advanced AMD
can be evaluated in an observational cohort study.12
Prevalence and Incidence
The prevalence and incidence rates are closely interrelated. If a
condition is irreversible (e.g., AMD or glaucoma), and if people
with it have the same mortality rate as the rest of the
population (an assumption that is not often true), then a
condition with a high incidence will also be highly prevalent.
Many epidemiological studies have provided estimates of both
prevalence and incidence of disease in specific populations.
Prevalence rates are assessed from a cross-sectional analysis of
the baseline data and incidence rate is subsequently determined
with follow-up of the same study sample over a period of time.
For example, Leske and colleagues studied glaucoma in a
population sample of black persons in the Barbados Eye Study.

They initially reported the prevalence of glaucoma at baseline
(all glaucoma cases at the time of the survey),13 and then
subsequently reported the 4-year incidence of glaucoma (new
glaucoma cases that developed between the baseline and follow-
up examination at 4 years) in the same population.14 Although
both prevalence and incidence are estimates of frequency of a
disease, the incidence rate is more valuable in understanding
the etiology of a disease, while the prevalence rate is more
valuable to help policymakers in evaluating the impact of a
disease on demand for health services.
STUDY DESIGNS
Clinical studies fall into one of two large categories: controlled
and uncontrolled (Table 34.2). Anecdotal case reports and case
series are the uncontrolled studies. Controlled studies have at
least one concurrent comparison group, generally a ‘standard
care’ or untreated group. In ophthalmology, as in medicine in
general, there have been many uncontrolled case series studies
reporting ‘new’ and ‘exciting’ findings, many of which have
been shown to be unproven in subsequent controlled studies.
Examples of these include the use of intravitreal steroids as a
primary monotherapy treatment for neovascular AMD15 and
the effectiveness of optic nerve sheath decompression surgery to
improve vision in patients with nonarteritic anterior ischemic
optic neuropathy.16
TABLE 34.2. Study Designs
Controlled Studies
Experimental Randomized clinical trials
Observational Cohort studies
Case-control studies
Cross-sectional studies
Uncontrolled Studies Case reports and case caries
Controlled studies can be further divided into experimental
and observational studies. The preferred experimental study
design in clinical research is the RCT. The major difference
between experimental and observational studies is that in
observational studies, investigators have no control over the
allocation of intervention or exposure factors, while in the RCT,
the intervention or exposure is randomly allocated, making it
likely that both known and unknown confounders will be
similar in the intervention and control groups.
One of the major issues in controlled studies is comparability
between study groups in factors other than those under study.
In clinical research, it is important to define, for example, expo-
sures (risk factors, treatments, interventions) and outcomes
(disease development or progression). The link between
exposure and outcome can be typically expressed by the classic
2 µ 2 table (Table 34.3). This table can be used to understand
estimates of the associations between exposure and outcome
(see further ahead).
RANDOMIZED CONTROLLED TRIALS
An RCT compares an outcome (often incidence or progression
of a disease) among groups with and without particular expo-
sure(s) or intervention(s). The RCT is similar to an observa-
tional cohort study except that the allocation of exposure or
study intervention to participants is not self-selected (as in
observational cohort studies) but by random chance.
Epidemiology and Clinical Research
TABLE 34.3. Classic Two by Two Table in Research
Outcome
Yes No Total
Exposure Yes A C A+C
No B D B+D
Total A+B C+D A+B+C+D
The incidence or risk of outcome in the exposed group is A/(A + C).
The incidence or risk of outcome in non-exposed group is B/(B + D).
The relative risk of outcome in association with exposure is
{A/(A + C)} ÷ {B/(B + D)}.
Randomization is used to assign study participants with equal
probability to the intervention group(s) or the control group.
This makes it likely that patient characteristics (both known
and unknown) will be equally distributed between groups except
for the intervention under investigation, if the sample size is
sufficiently large. Because this random allocation is dictated by
chance only, with no subjective influence on the intervention
allocation from either study subjects or study investigators, the
RCT is classified as an experimental study.
The RCT provides the highest quality of evidence in clinical
research and is the ideal study design to investigate the effec-
tiveness and safety of a new treatment. For studying etiological
CHAPTER 34
causes, harmful exposures or established treatments, however,
it is often unethical and not feasible to use RCT design. For
example, to determine if smoking is associated with risk of
AMD, it is obviously not possible or ethical to randomly ‘assign’
participants to cigarette smoking.
The Diabetes Control and Complications Trial (DCCT) was
a classic RCT that assessed the effect of intensive glycemic
control on the development of diabetic retinopathy and other
vascular complications in patients with type-1 diabetes.17 The
researchers randomly assigned patients to tight glycemic
control versus ‘standard’ glycemic control, and compared the
incidence of retinopathy over a 6.5-year period between the two
groups. The study found that the incidence of retinopathy was
75% lower in the group assigned to tight glycemic control, thus
concluding that glycemic control is important in preventing
diabetic retinopathy. The results of this RCT provide the
cornerstone of diabetes management.
Appropriate conduct of the RCT is paramount to the inter-
pretation of study results. Issues such as noncompliance with
the treatment, substantial or selective loss of follow-up of study
participants, the occurrence of unexpected adverse events and
the application of the study findings from the study population
to appropriate target populations in the community are
important considerations. Even the procedure of randomization
itself is not a guarantee that patient characteristics are evenly
distributed. In some studies, particularly those with small
sample sizes, the distribution of characteristics may not always
be comparable, and it is essential to check this prior to making
comparisons and drawing conclusions from the RCT. In an
RCT studying whether lisinopril, an ACE inhibitor, would
reduce the rate of diabetic retinopathy progression in type-1
diabetes, the European controlled trial of lisinopril in insulin-
dependent diabetes mellitus (EUCLID) study showed that
patients randomly assigned to lisinopril treatment had a lower
risk of diabetic retinopathy progression than controls.18
However, the investigators found that at baseline patients in the
lisinopril group had lower baseline hemoglobin A1C levels than
control patients. Thus, whether the observed lower risk of
retinopathy progression in the treatment group was actually due 381
 PRINCIPLES OF EPIDEMIOLOGY
to the effect of the intervention (lisinopril) or due to the
‘selection’ of patients with good glycemic control in the treat-
ment group is unclear.
PROSPECTIVE COHORT STUDIES
A prospective or cohort study is a study that follows a group of
individuals over time with an aim to determine the rate at
which a disease outcome occurs over the time interval. It is also
used to examine the prognosis (e.g., visual loss) of the disease
over time. Prospective cohort studies provide data on the
incidence of the disease and can answer research questions per-
taining to etiology or risk factors, intervention, and prognosis.
Prospective cohort studies can also be used to determine if the
efficacy observed in RCT translates into ‘real world’ effective-
ness in community-based populations. For both RCTs and
cohort studies, assessment of risks is as important as assess-
ment of efficacy.
In a prospective cohort study one can assess whether the
incidence or risk of a disease (e.g., glaucoma) is related to a
particular factor (e.g., race), a group of individuals is initially
recruited from a target population. These individuals are
classified on the basis of presence or absence of exposure to the
specific risk factor in question (i.e., white and black race), and
all individuals must be initially free of the disease under
investigation (i.e., do not have glaucoma). The study sample is
then followed over time to assess the incidence rates of the
disease, which are then compared (i.e., incidence of glaucoma in
SECTION 5
whites versus blacks). The relative incidence is expressed as the
RR or risk ratios (see section on Measure of Associations).
In ophthalmology, two classic examples of prospective cohort
studies have examined the relationship between cigarette
smoking at baseline and the 5-year incidence of AMD: the
Beaver Dam Eye Study and the Blue Mountains Eye Study.19,20
Both studies showed that cigarette smoking was associated with
a threefold higher risk of AMD, independent of age and other
risk factors. The results of these two studies, and others, have
led to an increased public health awareness of the potential
blinding effects of smoking.
Advantage and Disadvantage of RCT and
Prospective Cohort Study
Traditionally, it has been claimed that observational studies find
stronger treatment effects than RCT. However, this is not
always the case. A review of 136 reports covering 19 diverse
treatments showed that the treatment effects from obser-
vational studies and RCT were similar.21
The major difference between the two study designs,
however, is that the exposure or intervention factors are self-
selected in cohort studies, and therefore cohort studies are
vulnerable to selection bias and other confounding issues
(see section on Bias). Observational studies of hormone replace-
ment therapy (HRT) in middle-aged women provide a classic
example of how confounding can lead to spurious conclusions.
More than 50 observational studies showed apparent benefits of
HRT on a variety of cardiovascular and health outcomes. In
2002, however, a large RCT, the Women’s Health Initiative
study, showed that long-term use of HRT was associated with
an increased risk of invasive breast cancer, heart disease, stroke,
and pulmonary embolism.22 The RCT was terminated before
study completion and discontinuation of HRT was recom-
mended for the 16 000 participating women. Observers
concluded that the difference between data from previous
observation studies and the RCT was due to uncontrolled
confounding.23 In the observational studies, women who were
on HRT were healthier and saw doctors on a more frequent
382
basis than women who did not receive HRT. When HRT users
had better outcomes, it was erroneously credited to a beneficial
effect of HRT. This example illustrates that in cohort studies,
factors that determine whether a person received a particular
exposure or treatment can result in a significant difference in
the study outcomes. Often these factors are unclear, and there
are many unknown reasons why participants may have selected
a specific treatment. These factors can easily confound the
assessment of effectiveness of intervention. However, as
discussed above, while the RCT is the ideal study design for any
new treatments, it is not always feasible or ethical to conduct
an RCT. In these cases, a cohort study provides the next best
level evidence.
CASE-CONTROL STUDY
A case-control study, sometimes referred to as the retrospective
study, differs from prospective cohort studies in one major
aspect. In the cohort study, the exposure factor is defined at the
beginning of the study and the outcome at the conclusion of the
study. In contrast, in a case-control study, the outcome is first
determined at the beginning of the study and the exposure is
‘retrospectively’ assessed. The principles of conducting a case-
control study are as follows. first, the investigator chooses two
patients groups with and without the outcome of interest. The
group in which the individuals have the disease or outcome
(cases) is compared to the group in which they do not have the
disease or outcome (controls) for whether they had been
exposed in the past to the study factors of interest. Instead of
prospectively following the study groups over a period of time as
in cohort studies, exposure data are collected retrospectively
from cases and controls, or examined on study participants after
recruitment in the case-control study. By comparing the
frequency of different exposures, risk factors or characteristics
between the two groups, the investigator can determine if a
specific risk factor occurs more frequently in cases than
controls. If so, it suggests that this factor may be associated
with the disease outcome. The measure of this association in
the case-control study is the odds ratio (OR) (see section on
Measure of Associations).
Acute endophthalmitis is a devastating postoperative compli-
cation after cataract surgery. The risk factors for endophthal-
mitis are unclear. In a case-control study, Wong and Chee
investigated the risk factors associated with endophthalmitis
following cataract surgery.24 In a review they identified 34
patients with acute endophthalmitis presenting within 6 weeks
after cataract surgery (cases), and selected another group of
cataract surgery patients who did not have endophthalmitis
(n = 102). Findings showed that endophthalmitis cases were
more likely to have silicone intraocular lens (five out of 34 cases
or 15.1%) compared with controls (four out of 102 controls or
4%). Thus, patients implanted with silicone lens were about
four times (15.1% vs 4%) more likely to have endophthalmitis
as patients implanted with polymethylmethacrylate lens. This
association is expressed as the OR (see section on Measure of
Associations).
Selection of cases and controls requires clearly defined
diagnostic criteria, ideally including gold standard tests being
used to diagnose the disease. Case definitions should be based
not only on a patient’s clinical history or symptoms but also on
objective evidence from pathological or other diagnostic tests.
Controls should be representative of the referent population
from which cases are selected (i.e., comparable), and should
have the same probability of being selected as cases, if they had
the disease as cases do. Cases and controls can be recruited
from patients in the hospital (hospital-based case-control study)

or from the community (population-based case-control study).
The latter is less likely to be subject to selection bias and thus
will provide better quality of evidence than the former.
An example of a case-control study that examined risk factors
for AMD is the Eye Disease Case-Control Study.25 In this study,
the investigators recruited 421 patients with neovascular AMD
and 615 controls and examined possible risk factors through
interviews, clinical examinations, and laboratory analyses of
blood samples. The study was one of the first to identify that
AMD was associated with cigarette smoking and hyper-
cholesterolemia.
Advantages and Disadvantages of the Case-
Control Study and the Cohort (Prospective) Study
Cohort and case-control studies have relative advantages and
disadvantages (Table 34.4). Compared with clinical trials, these
observational approaches studies are more prone to problems
associated with bias and uncontrolled confounding. Case-
control studies are typically much less expensive and less time
consuming than cohort studies. Case-control studies do not
provide estimates pertaining to incidence rates and absolute
risks, but only ORs for association assessment. There are also
biases with known and unknown bias directions (such as selec-
tion bias and recall bias) inherent with case-control studies.
While they can be a useful means of generating research
questions, case-control studies cannot by themselves provide
sufficient evidence for causal inferences.
TABLE 34.4. Comparison of Cohort and Case-Control Studies
Cohort Studies Case-Control
Studies
Causal inference Can be made Cannot be made
Estimating incidence rates Yes No
Estimating relative risks Yes No
Cost High Low
Time Long Short
Loss to follow-up Potential problem Not an issue
Studying rare diseases Inefficient design Efficient design
Studying multiple outcomes Able to Not able to
Studying multiple risk factors Possible Possible
Nested Case-Control Study
Sometimes a case-control study can be ‘nested’ within a larger,
population-based cross-sectional or cohort study. This type of
‘hybrid’ study incorporates the advantage of a population-based
sampling design and the cost-effectiveness of performing
investigation of specific study factors on cases and randomly
selected or matched controls (instead of the whole study
population). In a case-control study nested in a population-
based cohort study, incident cases are usually defined at follow-
up visits, and study factors collected or assessed at baseline are
retrospectively examined; thus a recall bias on these prior
collected factors can be avoided. An example of this type of case-
control study was conducted in the Beaver Dam Eye Study, in
which investigators examined the relationship between retinal
microvascular signs (exposure) and cardiovascular mortality
(outcome).26 Cases were study participants who had died from
cardiovascular disease since the baseline examination and three
controls per case were selected from the baseline cohort,
Epidemiology and Clinical Research
matched on age and gender. Retinal photographs taken at
baseline were measured for various retinal microvascular signs
and the frequency of these signs were compared in cases and
controls. The study showed that retinal microvascular signs
were more frequent in cases than controls, and concluded that
these signs may be a risk marker for cardiovascular mortality.
The advantage of this design included the fact that retinal
photographs were taken prior to the occurrence of outcome and
that measurement of retinal microvascular signs from baseline
retinal photographs was limited only to cases and controls (total
of ~1200 photographs) and not the entire study population
(~5000 photographs).
CROSS-SECTIONAL STUDY (SURVEY)
A cross-sectional study is usually conducted in a representative
sample of the target population, either within a geographically
defined community or randomly drawn from the entire popu-
lation. In a cross-sectional study, the exposure and disease
outcome are determined at the same time. This study design is
useful for estimating the prevalence of diseases as well as cross-
sectional associations with diseases.
The National Health and Nutrition Examination Survey
(NHANES) in the US is a typical cross-sectional survey.27 The
NHANES used a multistage probability sampling design to
recruit and examine the prevalence of visual impairment in
representative populations aged 12 years and older in the US,
reporting a prevalence of visual impairment (presenting visual
CHAPTER 34
acuity of 20/50 or worse) of 6.4%. This type of study is
important for public health planning purposes.
Like the case-control study, cross-sectional studies allow one
to determine possible associations between exposures or risk
factors and disease outcomes. A major limitation of this study
design, however, is that the temporal relationship between
exposures and disease outcomes is not known, and therefore,
evidence from cross-sectional studies, as with other obser-
vational study approaches, can be used to generate research
questions (hypothesis generation), but not causal inference
(hypothesis testing). For example, in a cross-sectional study of
the association of myopia and cataract, Wong and colleagues
showed that participants who had high myopia were more likely
to have nuclear cataract.28 However, it is not possible to
conclude that the observed association is due to myopia leading
to the development of nuclear cataract, or due to nuclear
cataract itself as a cause of ‘index myopia’.
MEASURE OF ASSOCIATIONS
There are two common measures of associations between
exposure factors and dichotomous outcomes: the RR (including
hazard ratio, used in prospective studies with time-dependent
outcomes) and the OR. For continuous exposure and outcome
factors, correlation and linear regression models are often used
to assess the degree of association. The latter statistics will not
be discussed here.
RELATIVE RISK
The RR provides an estimate of the difference in incidence or
risk associated with an exposure compared with the incidence
or risk associated with the absence of the exposure. The RR
indicates the risk of developing the outcome/disease in the
exposed group (people with a risk factor) relative to the risk in
those who are not exposed (people without the risk factor). RR
is often calculated in cohort studies and the RCT. The RR is
383
 PRINCIPLES OF EPIDEMIOLOGY
defined as the ratio of two absolute risk measurements and
calculated as follows:
RR = Absolute risk among exposed ÷ Absolute risk among
unexposed
The RR can be interpreted as follows:
• RR = 1; the risks are the same for the exposed and
unexposed, and the exposure is therefore not related to the
disease.
• RR > 1; the risk is higher for the exposed than the
unexposed, and the exposure is positively related to the
disease.
• RR < 1; the risk is lower for the exposed than the
unexposed, and the exposure is protective to the disease.
ODDS RATIO
The OR involves comparing ‘odds’, or likelihood, and is defined
as the ratio of the odds of being a case in the exposed group
compared to the odds of being a case in the unexposed group.
The ‘odds’ is not a proportion, but the probability that an event
occurs relative to the probability that the event did not occur.
Thus, odds = p ÷ (1⫺p), where p is probability of having an
event (or being exposed) and 1⫺p is the probability of not
having the event (or not exposed). The OR is usually calculated
when absolute risk or incidence rates cannot be calculated and
is therefore usually used in case-control or cross-sectional
SECTION 5
studies.
OR = Odds of disease in the exposed ÷ Odds of disease in the
unexposed
The OR can be interpreted as follows:
• OR = 1; the odds of having the disease is the same for the
exposed and unexposed, and the exposure is not related to
the probability of having the disease.
• OR > 1; the odds of having the disease is higher for the
exposed than the unexposed, and the exposure is
associated with an increased probability of having the
disease.
• OR < 1; the odds of having the disease is lower for the
exposed than the unexposed, and the exposure is
associated with a reduction in the probability of having the
disease.
BIAS
The validity of a study is the extent to which the observed
association (e.g., smoking and AMD) is attributed to the study
exposure (i.e., smoking) rather than other factors. Bias occurs
when the true associations are distorted due to systematic
(nonrandom) differences during sample selection (selection
bias), assessment of exposure and outcome factors (information
bias, measurement error) or other factors. There are many types
of biases, but only the major types are discussed here.
SELECTION BIAS
Selection bias occurs when the study population differs in some
systematic way that influences the study results and can render
them invalid. Observational studies, particularly case-control
studies, are prone to selection bias. For example, an investigator
may be interested in studying the association of hypertension as
a potential risk factor for AMD in a case-control study. The
investigator may choose as controls a sample of patients seen in
the eye clinic for other conditions, as long as they do not have
384 AMD. It is possible that the rate of diabetes among these eye
clinic controls is higher than the rate of diabetes in the general
population, as persons with diabetes are more likely to see an
eye doctor for retinopathy assessment. If this were the case, the
control group taken from eye clinic patients may have a higher
prevalence of hypertension than a control group selected from
the general population. Thus, even if hypertension was truly a
risk factor for AMD, the investigator may find that the
frequency of hypertension in AMD cases and controls in the
study samples are similar, and falsely conclude that hyper-
tension is not associated with AMD.
To enhance comparability, investigators may select controls
matched for cases on some important characteristics, most
commonly age, gender, and race. In the above example, the
investigator may choose to match for diabetes status between
AMD cases and controls. The closer the match, the more valid
are the comparisons between cases and controls. However, it is
often not feasible to match for more than three characteristics
between cases and controls. Also, if you match on a factor you
can not assess whether it is associated with the disease.
A particular form of selection bias seen in cohort studies is
survival bias. If an investigator were to conduct a cohort study
of the association between Alzheimer’s disease and AMD, but
participants with Alzheimer’s disease and AMD were more
likely to die prior to the follow-up visits, the investigator may
not be able to detect a true association between Alzheimer’s
disease and AMD. In this situation, selection bias, due to dif-
ferential loss to follow up, affected the true association between
Alzheimer’s disease and AMD.
Selection bias may occur even in an RCT if the study parti-
cipants were lost to follow up either substantially or diffe-
rentially after randomization.
INFORMATION BIAS
Information bias occurs during the collection of study exposure
or outcome factors. Interview data are particularly prone to
information bias, particularly if the interviewer or the patient
believes that a particular question on a risk factor is related to
the study outcome. For example, in a case-control study of
smoking and AMD, patients with AMD may be more likely to
‘remember’ and report a past history of smoking than controls,
who may dismiss a short prior history of smoking. This type of
information bias, called recall bias, can lead to either an over- or
underestimation of the true association.
DIAGNOSTIC AND SCREENING TESTS
SENSITIVITY AND SPECIFICITY
There are two estimates that are fundamental to evaluating
diagnostic and screening tests: sensitivity and specificity. The
sensitivity is the probability of a positive test in subjects who
TABLE 34.5. Sensitivity and Specificity of a Test Disease
Total Yes No
Test Yes True False Positive TP + FP
Positive (TP) (FP)
No False True Negative FN + TN
Negative (FN) (TN)
Total TP + FN FP + TN
Sensitivity of test = TP/(TP + FN).
Specificity of test = TN/(FP + TN).
Positive predictive value of test = TP/(TP + FP).
Negative predictive value of test = TN/(FN + TN).

have the disease, while the specificity is the probability of a
negative test in subjects who do not have the disease. Table 34.5
shows how these estimates are calculated. An ideal (usually not
feasible) test is to have both sensitivity and specificity of 100%.
Nonmydriatic retinal photography has been suggested as a
possible means to screen for diabetic retinopathy. However, this
type of photography may miss some patients with diabetic
retinopathy (false negative) and may also falsely identify
patients without retinopathy (false positive). The question is,
what is the sensitivity and specificity if nonmydriatic photo-
graphy as a screening test for diabetic retinopathy, compared
with 7-field, mydriatic retinal photography (the current gold
standard)? The sensitivity tells us how frequently patients with
diabetic retinopathy are identified correctly from nonmydriatic
photography (true positive), and the specificity how frequently
patients without retinopathy have normal photographs (true
negatives).
To test a new diagnostic tool, the investigators usually need a
gold standard tool to compare against but each disease may not
always have a gold standard diagnostic test that is accepted by
the field. Kuo and colleagues, for example, compared a single
field, nonmydriatic retinal photography with a detailed ocular
examination by ophthalmologists, but it is not clear that the
latter can be considered ‘gold standard’ in diagnosis of diabetic
retinopathy.29
An important characteristic of the sensitivity and specificity
analysis is that the results are independent of the prevalence of
the disease. As can be seen in Table 34.5, each statistic is
column specific: sensitivity only involves those with the disease
and specificity those without the disease.
VARIABILITY AND RELIABILITY
When two physicians examine the same patient for the presence
of a disease, they often do not arrive at the same diagnosis. The
variability between the two physicians for the same disease is
called interobserver variability. Additionally, when the same
physician examines the same patient again at another time, he or
she may not arrive at the same diagnosis at the subsequent
examination. The latter is termed intraobserver variability.
Interobserver and intraobserver variability provides an
estimate of the reliability of the measurement or test by dif-
ferent observers and by the same observer over time. It is not a
reflection of the validity or accuracy of the test (which is defined
above by the sensitivity and specificity), as the same observer
may have good reliability but make the same error during
repeated measurement; thus, the intraobserver variability can
be low but not valid or accurate.
To minimize interobserver and intraobserver variability,
objective measures with detailed criteria (including reference
photographs where appropriate) and frequent standardization
across observers and instruments are recommended. Stan-
dardized measures, such as automated blood pressure device, or
computer assisted imaging, will help to reduce the measure-
ment noise introduced by human errors.
STATISTICS
SAMPLE SIZE
Apart from quality of study design and quality of data collected,
an important determination of eventual study success is the
calculation of the required sample size to detect a statistically
significant association or difference between study groups.
There are numerous studies where a true difference existed but
the difference was not statistically significant because the sam-
ple size was insufficient to demonstrate the true assoiation.
Epidemiology and Clinical Research
On the other hand, a study with a very large sample size
could detect even small associations or differences between
study groups that although statistically significant may not be
clinically meaningful (see section on Clinical Versus Statistical
Significance).
STATISTICAL SIGNIFICANCE: p VALUE
VERSUS CONFIDENCE INTERVALS
A test of significance is used to determine whether an observed
association is due to chance. A p value provides the probability
that an association observed in a study (e.g., smoking and
AMD) might have arisen purely by chance. A p value of <0.05
implies that the likelihood that this observation has arisen by
chance alone is less than 5%. In other words, the probability
that this association was seen, when in fact there was no real
association, is less than 5%. A value of p < 0.01 indicates that
the probability that the association was due to chance alone is
less than 1 in 100. A small p value does not prove that the
association is absolutely ‘real’ but only that the probability that
the association was a chance finding is highly unlikely. The p
value is only an accurate assessment if there is no important
bias or confounding affecting the study groups.
The confidence interval (CI) is another test commonly seen
in clinical research and is a summary of precision around a
point estimate. A 95% CI indicates that if the study was
repeated multiple times, the observed associations would lie
within the CI boundaries 95% of the time. The narrower the CI,
CHAPTER 34
the more precise is the observed point estimate. CI is more
informative than a p value. For example, a ‘nonsignificant’ p
value by itself provides no information about the power of the
study to find a difference between groups. However, the breadth
of CI indicates how large a difference is likely to exist between
study groups, whether the results are statistically significant or
nonsignificant.30
Both the p value and CI depend on sample size and the degree
of variability of the data, standard deviation and standard error.
Studies with small sample size and large standard deviation or
standard error provide estimates with wide CTs, and are less
likely to be able to detect a significant difference.
CLINICAL VERSUS STATISTICAL
SIGNIFICANCE
A result can be statistically significant (i.e., p value < 0.05) but
may not be clinically meaningful. A small size of the effect of an
intervention or a weak association between a risk factor and an
outcome can have still a very small p value, if the study sample
size is large enough.
Whether a result is clinically meaningful is usually a matter
of clinical judgment. Investigators should ask this question: Is
the association seen or the difference between study groups
large enough to be clinically important and worth achieving?
When investigators conclude that their study shows a ‘highly
significant’ result, in most instances the investigators mean
the results were statistically significant. Investigators rarely
comment on whether the differences are large enough to affect
clinical practice. For example, a new drug might lower IOP by
2 mmHg more than another drug, but while the improvement
is statistically significant and likely to be real, it may not be
clinically meaningful.
INFERENCES AND CAUSALITY
The goal of most studies is to determine whether an exposure
is associated with an outcome in the ‘real world’. However,
reported associations in studies should be described as they are, 385
 PRINCIPLES OF EPIDEMIOLOGY
TABLE 34.6. Principles of Causality
Principles of Causality
Strength of association
Consistency of association between studies and coherence of
evidence
Specificity of association. Removal of the exposure is associated
with a reduction in risk of outcome
Temporal relationship between exposure and outcome
Dose–response of association
Biologic plausibility of the association
Confirmation in experiments
and no more and no less can usually be inferred than what was
actually observed. Additional studies and information are
usually needed to show causality between the exposure and
outcome. For example, if AMD rates are observed to be higher
in whites than blacks, this does not necessarily imply that
genetic factors play a role in the etiology of AMD.31 Further
SECTION 5
studies showing evidence that whites have some genes that are
different from blacks and that these genes are responsible for
AMD development are needed. If patients with diabetes are
more likely to undergo cataract surgery than persons without
diabetes, it does not necessarily imply that diabetes is a cause of
cataract, until there is cumulated evidence from other studies.
In general observational studies can not test for causality in the
way that clinical trials can. However, there are many examples
where the totality of the evidence leads to a conclusion of
causality despite the lack of clinical trial results. At this point
there is little doubt that cigarette smoking causes lung cancer
even though there are no clinical trial data. There are ample
observational studies using different approaches demonstrating
consistently high ORs for lung cancer among smokers, as well
as biologic plausibility confirmed by animal models.
CAUSALITY
The demonstration of an association between an exposure and
an outcome in a study is only the first step. To assess the
validity of the observed association, one must address whether
the observed association is a true association, or is due to
chance, confounding or bias. After determining the validity of
an association, one can consider whether it is causal.
First, one should determine if the association is likely to be a
chance observation? This question is usually determined by
examining whether the association is statistically significant, as
indicated by the p value or the CI. However, a statistically
significant association at the level of a p value of < 0.05 only
suggests that the probability of the association occurring by
chance is less than 5%. There is still a probability of 1 in 20 that
the observations occurred by chance alone, which is referred
to as a type I error (i.e., a significant finding that is not true).
Thus, statistical significance is not sufficient and other criteria
for causality are needed for correct interpretation of study
findings.
Second, is the observed association between an exposure and
outcome due to an indirect association with a third factor, a
386 confounder? In other words, is the link between the exposure
Examples
Strong association of cigarette smoking and risk of lung cancer34
Smoking and AMD demonstrated in three population-based studies35
Control of hyperglycemia and reduction in risk of diabetic retinopathy17
Hyperglycemia and duration of diabetes link to subsequent
development of diabetic retinopathy36
Increasing dose of inhaled corticosteroid use and increasing risk of
posterior subcapsular cataract risk37
and outcome explained by their common association with a
third factor (confounder)? For example, pterygium has been
observed to be more common in countries nearer the equator.32
Is residence near the equator (exposure) therefore a direct cause
of pterygium (outcome)? More likely, while this association may
be real, the geographic location itself is not a direct cause but a
surrogate for a longer duration of sunlight exposure, which is
associated with the geographic location (countries near the
equator). In this case, sunlight exposure is the confounder for
the association between residence near the equator and
pterygium.
In another study, people with diabetes who were using
insulin were more likely to have diabetic retinopathy than those
not using insulin. Does this then imply that use of insulin is a
direct risk factor for retinopathy? A more probable explanation
is that people who use insulin have more severe diabetes and
poorer glycemic control putting them at a higher risk for
diabetic retinopathy. In this example, poor glycemic control is
the confounder for the observed association between insulin use
(exposure) and diabetic retinopathy (outcome).
Third, is the observed association due to bias that may lead
to spurious inferences? An association can be due to selection
or information biases that the researchers have not taken into
consideration. Some study designs (e.g., RCT) minimize
possible biases and confounding factors better than other study
designs (e.g., case-control studies).
Finally, assuming that the association is not due to chance,
confounding, or bias, and is likely to be real, the question still
remains as to whether the association is due to a causal re-
lationship. Criteria for causal inference were proposed by
Bradford Hill and are shown in Table 34.6.33
GENERALIZATION OF STUDY FINDINGS
Having demonstrated and precisely defined an association that
appears to be real and causal, the final process of assessing a
study is to determine whether the findings are widely applicable
or generalizable. The purpose of research is not confined to a
simple demonstration of an association in the study sample.
Equally important, one must assess whether the study results
can be extrapolated to the community and to clinical practice.
In other words, the aim of clinical research is to translate
research findings to clinical practice, in order to improve health
services and health outcomes. Ideally, a random sample of the
entire relevant population should be studied. In practice, this is
 Epidemiology and Clinical Research

rarely feasible and investigators have to assume that persons

Key Features
with characteristics similar to those enrolled in their study will
• Clinical research answers a scientific question by conducting
respond in similar ways, although this is not always true.
studies in humans. This question may cover etiology,
pathogenesis, risk factors, natural history, and treatment
CONCLUSIONS options for this disease.
• The prevalence of a disease refers to the frequency of existing
There are several challenges for clinical research in
disease in a specific population at a particular point in time.
ophthalmology. Investigators need to move progressively from
• The incidence of a disease refers to the frequency with which
conducting a purely descriptive type of clinical studies
new cases of a disease develops over a defined period of time
(hypothesis development) to controlled trials (hypothesis
(e.g., one month or one year).
testing). Research studies should incorporate objective,
• Clinical studies can be divided into uncontrolled and controlled
quantitative measurements of exposures and outcomes.
studies. Anecdotal case reports and case series are the
The complex etiology and pathogenesis of most chronic eye
uncontrolled studies. Controlled studies can be further divided
diseases poses an additional challenge for clinical researchers. It
into experimental and observational studies.
is likely that identifying new genetic factors and examining
• The experimental study in clinical research is the randomized
gene–environmental interactions will become increasingly
control trial, in which the intervention (e.g., treatment) to study
important in understanding how these chronic diseases
participants is randomly allocated.
develop. Finally, improvement in methods to measure change
• Observational studies include the cohort or prospective study,
and progression of disease outcomes (e.g., progression of
the case-control or retrospective study or the cross-sectional
cataract) and standardizing and uniformly using definitions and
study. In all these studies, there is a control group in which
classification for diseases (e.g., glaucoma) will enhance the
comparisons to disease or risk factors can be made.
quality of clinical research outcomes, leading to further
• As far as possible, clinical research studies should incorporate
understanding of the nature and impact of the major blinding
objective, quantitative measurements of both exposures (risk
ocular diseases.
factor), and outcomes (disease).

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Hyman L: The Barbados Eye Study.
Prevalence of open angle glaucoma. Arch
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14. Leske MC, Connell AM, Wu SY, et al:
Incidence of open-angle glaucoma: the
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119:89–95.
15. Gillies MC, Simpson JM, Luo W, et al:
A randomized clinical trial of a single dose
of intravitreal triamcinolone acetonide for
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degeneration: one-year results. Arch
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16. The Ischemic Optic Neuropathy
Decompression Trial Research Group:
Optic nerve decompression surgery for
nonarteritic anterior ischemic optic
neuropathy (NAION) is not effective and
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Trial Research Group: The effect of
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diabetes mellitus. N Engl J Med 1993;
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 CHAPTER
35 Epidemiology of Age-Related Cataract
Barbara E. K. Klein
Key Features
Risk Factors for Age-Related Cataracts
Diabetes (cortical cataracts)
Smoking (nuclear cataracts)
Sunlight exposure (cortical cataracts)
Controversial Risk Factors for Cataracts
Dietary/nutritional supplements
Alcohol intake
Genetics
PUBLIC HEALTH SIGNIFICANCE
Cataract is thought to be the most common cause of blindness
worldwide1 and is the leading cause of diminished vision in the
United States.2 It is the single most common cause of blindness
in black Americans being associated with 36.8% of this important
loss of function.3 It is estimated that 20.5 million Americans
older than 40 years of age have cataracts in either eye and that
this prevalence will rise to 30.1 million by 2020.4 In developed
countries such as the United States, cataract surgery procedures
are both successful and widely available, but they carry a signifi-
cant cost. It has been estimated that if cataract formation were
delayed by 10 years, the need for cataract extraction surgery
might be reduced by 45%.5
AGE-RELATED CATARACT:
METHODOLOGIC CONSIDERATIONS
Prevalence data help define the magnitude of the disease burden.
While prevalence surveys may not be undertaken primarily for
purposes of developing health policy, they may provide a gross esti-
mate of the potential need for surgical and rehabilitative services.
A major advance in epidemiologic research on cataract has
been the development of photographic documentation and codi-
fied grading schemes to identify the presence and severity of
cataracts.6 Much of the data accumulated are based on clinical
examinations whose validity and reliability are difficult to assess.
The data we report, because of the relative paucity of use of lens
photography and gradings, include studies of several different
national and international populations using both grading of
standardized lens photographs and clinical gradings. We group the
cataract types for global descriptions of prevalence of (any)
cataract because many studies do not describe cataract preva-
lence by type of opacity. This is appropriate when visual func-
tion is the object of the analysis. However, when searching for
potentially etiologic relationships, it is necessary to investigate
specific cataract type.
PREVALENCE STUDIES
The Eye Disease Prevalence Research Group was composed of
investigators of several population-based studies of age-related
eye disease in order to estimate current prevalence of these
conditions in adult Americans and to project these findings to
the year 2020. Data on cataract or cataract surgery were
included for 31448 adults 40 years of age or older, 1897 of
whom contributed to cataract prevalence.4 Data for ~15 000 of
these persons were derived from photographic documentation.
For all studies, roughly comparable definitions were used to
classify cataract presence. The data for estimates for white
Americans came from five studies containing substantial num-
bers of persons with European background, two studies with
substantial numbers of persons of African ancestry, and one
study of Mexican-Americans (only cataract surgery data). The
pooled estimates from these data indicate increased prevalence
with increased age in blacks and whites. Both black and white
women had higher prevalence than their male counterparts.
With regard to cataract surgery, persons of Mexican ancestry
had higher prevalence than blacks or whites in each age group
for both women and men.
Studies in the Punjab (India), Tibet and Saudi Arabia, although
not recent, confirm the importance of age in the prevalence of
cataract in these populations.7–9 However, it is difficult to compare
prevalence between them and to compare those data estimates
from the Eye Disease Prevalence Research Group studies. It is
likely that in these countries the burden of blindness is largely
attributable to cataract, as is true in Andhra Pradesh (India)
(44% of cases of blindness)10 and Nigeria (44% of blindness)11
but may reflect the decreased ability to assess the presence of
other sight-limiting conditions.
TYPES OF AGE-RELATED CATARACTS
There are three common types of age-related cataract: nuclear,
cortical, and posterior subcapsular. There is ample reason to
believe that their causes, environmental and genetic, differ both
qualitatively and quantitatively.12–18 Therefore, identifying the fac-
tors that influence the risk of a cataract severe enough to require
surgery is likely to be quite complicated although very important.
Data by cataract type are available for the Barbados Eyes Study,
the Beaver Dam Eye Study, the Blue Mountains Eye Study, the
Salisbury Eye Evaluation project (SEE), and the Melbourne Visual
Impairment Survey.19 Nuclear cataract occurred in ~0.2–0.4%
of whites 40–49 years of age and in ~44–50% in those who were
75–79 years of age. The data for blacks are limited, but prevalence
appears to be lower at each age than in whites. Cortical cataract
is also relatively common, and it appears to be more frequent in
389
 PRINCIPLES OF EPIDEMIOLOGY
blacks than whites. Posterior subcapsular cataract (PSC) is the
least common, but its prevalence is also related to age. Racial/
ethnic differences are uncertain.
RISK FACTORS FOR CATARACT
FORMATION
DIABETES
The role of diabetes in the development of cataract has been
controversial. In the past, some investigators thought that
cataracts did not occur more often in diabetics but rather were
simply diagnosed more often because of the increased frequency
of visual examinations in this group. Diabetics may also be
more likely to undergo cataract extraction because of the need
to visualize the retina clearly in order to monitor the devel-
opment and progression of retinopathy. Thus, it is important to
evaluate the association of diabetes and cataract in population-
based surveys and not by studies of either persons undergoing
screening or cataract extraction. The Framingham Eye Study
and the National Health and Nutrition Examination Survey
(NHANES) found a three- to fourfold excess risk of cataract
among diabetics less than 65 years of age.18 Because of the rela-
tively strong association indicated by these data (and the biologic
plausibility of the association), it is now generally accepted that
diabetics have a higher risk of cataract, but it is not clear why.
Not all cataracts are uniformly increased in diabetics.20 Cortical
cataracts are the type most often associated with it20 but are the
least likely to lead to cataract extraction.21
SMOKING
Most epidemiologic studies have noted an increased risk of cataract
among smokers. In a cross-sectional study, Klein and associates22
assessed smoking and cataract among diabetics and found a
positive association in those who were diagnosed with diabetes
after age 30. Data from many other studies of various designs
SECTION 5
have found an increase in the risk of nuclear cataract to cigarette
smoking. Flaye and co-workers23 found that nuclear cataracts were
2.5 times as common among current smokers compared with
nonsmokers. Among ex-smokers, cataracts were more common in
those who had smoked heavily, whereas no increase in risk was
noted in past light or moderate smokers. In a case-control study,
current smokers, defined as those who had smoked at least one
cigarette per day for at least 1 year and still smoked, had a 70%
increased risk of nuclear cataract.24 Among 21 316 US male
physicians,25 current smokers of 20 or more cigarettes per day
had a twofold increase in cataract risk relative to nonsmokers.
In case-control studies conducted in India,26 Italy,27 and
Maryland,28 no association between smoking and cataract was
reported. In general, positive results are most consistent for a
relationship between smoking and nuclear cataract. An increase
in risk has not been noted for cortical cataract and, except for the
reports by Bochow et al,28 Christen et al,25 and Hankinson et al,29
most studies have had a limited ability to assess the effect of
smoking on PSC cataract. Although findings have not been
entirely consistent, smoking does appear to be one of the best
confirmed risk factors for (nuclear) cataract.
One hypothesis to explain the risk is that smoking increases
oxidative stress in the lens, however, direct evidence to support
this hypothesis is lacking.
SUNLIGHT EXPOSURE
Sun exposure is known to be damaging to a number of tissues
390 in the eye. Both cortical and PSC cataract have been induced by
UV irradiation in animal studies.30 UV radiation could increase
the risk of cataract through disruption of the membrane–cation
transport system or injury to nucleic acids in the epithelial cells
of the lens.30
Studies have been conducted to assess the association between
sun exposure and cataract formation in persons in geographically
defined areas. The advantages of such studies are that they can
be performed quickly, at low cost, and may generate important
hypotheses concerning exposure–disease relationships. However,
it is not possible to know whether persons developing the
disease of interest were those exposed and, even if exposed, at
what level of exposure. In addition, data are often lacking on
other potentially important variables that might serve to alter
the relationship between exposure and disease.
In a study conducted in Australia by Hollows and Moran,31
the cataract status of 64 307 aborigines and 41 254 nonaborigines
was examined. Cataract prevalence was higher in the higher UV
light zones. Another study conducted among 30 565 lifelong resi-
dents of Nepal32 found that altitude and cataract were inversely
associated in this study; this was attributed to the blockage of
sunlight at higher altitudes by neighboring mountains. Neither
of these studies was able to control for potentially important
variables, such as smoking status and diet.
Data from the NHANES were used to assess the association
between cataract and annual sunlight exposure. The prevalence
of cataract increased with increasing exposure. In another
assessment of sun exposure using these data, UV radiation was
estimated33 at each site using data on latitude, elevation, and
cloud cover. An increase in cataract prevalence was associated
with increase in UV-B exposure. For example, controlling for
age, education, diabetes, race, and urban/rural residence, the
prevalence of cataract for those exposed to UV-B at a level
similar to that in Tucson, Arizona, was 58% higher than for
those exposed to UV-B levels similar to those in Albany,
New York.
A detailed study of sun exposure and cataract was conducted
of 838 Chesapeake Bay watermen in Maryland34 to assess sun
exposure since adolescence, the use of eyeglasses and hats,
medical history, smoking, and diet. By incorporating laboratory
data on the effectiveness of eyeglasses and hat use in blocking sun
exposure of the lens and data from UV monitors, the investi-
gators calculated annual and cumulative sun exposure for each
individual. The risk of cortical cataract was 60% greater (risk ratio
(RR) = 1.60; 95% confidence interval (CI) = 1.01–2.64) with a
doubling of cumulative sun exposure. Bochow and colleagues28
examined 160 persons with PSC and 160 controls, matched by
age, sex, and referral pattern. Sun exposure, both annual and
cumulative, was calculated as it was in the Watermen Study,
and as in that study, the positive association between cataract
and sun exposure was statistically significant.
Recently, West et al modeled the risk of cortical cataract in
the US presumably due to the increase in UV radiation that is
due to stratospheric ozone depletion. Ambient UV exposures were
estimated based on extensive questionnaire data from the SEE
project participants.35 Estimates of exposure for age–gender–race
categories were based on questionnaire responses, and these were
extrapolated to various locations around the country (which are
expected to vary with ozone depletion over the coming years).
Based on these analyses, the authors calculated that there
would be 167 000–830 000 additional cases of cortical cataract
by the year 2050. They posit that were these cases to result in
cataract surgery, the costs could be monumental.
The results of animal and ecologic studies, in conjunction with
the strength of the proposed biologic mechanism, all support an
association between sun exposure and cataract, although the
exact nature and strength of the association remains uncertain.
It is also not certain whether UV exposure is associated with only

cortical cataract or whether PSC is also increased with UV
exposure. Difficulties in quantifying exposure (i.e., collecting data
on time spent outdoors in the sun, the level of UV radiation in
specific locales, and the use of eyeglasses and hats, and in
sorting out possible ethnic differences in susceptibility) make
such studies complex.
DIET/SUPPLEMENTS
The possible effects of dietary or supplemental vitamins on
cataract development have been assessed in some epidemiologic
studies, although results have been inconclusive.
The Age Related Eye Disease Study (AREDS), a randomized
controlled clinical trial designed to determine whether anti-
oxidant vitamins and/or zinc decreased the risk of progression
of age-related macular degeneration (AMD), also evaluated
incidence and progression of age-related cataracts. There was no
evidence of a beneficial effect of the study preparations on any
of the three age-related cataracts.36 However, in subsequent
analyses using the propensity score, which adjusts for factors
associated with the use of multivitamin there was a beneficial
protective effect such as on the development of cataracts,
especially for the nuclear type.36a In the Beaver Dam Eye Study,
past use of multivitamins was protective for severity of
prevalent nuclear sclerosis.37
A combined antioxidant nutrient score has been reported to be
inversely associated with cataract.24,38 Further data on specific
antioxidants are needed before any public health recommenda-
tions can be made.
Some studies focus on individual vitamin supplements,
although ignoring intake from food sources may under-
estimate exposure. Even when high in particular vitamins,
foods contain other nutrients and structural components that
influence absorption and availability of any particular vitamin
or mineral.
Levels of vitamin C are reduced in the cataractous lens39 and
levels are reportedly increased with vitamin C supplementation.40
Leske and colleagues24 assessed dietary intake of vitamin C in a
large study with 945 case-patients and 435 controls and noted
a decreased risk of nuclear cataract among those in the top 20% of
intake. The NHANES study showed no association with cata-
ract prevalence when either vitamin C intake (calculated from a
24-h dietary recall) or usual frequency of fruit consumption was
assessed.41
Vitamin E (tocopherol), a fat-soluble antioxidant, breaks the
chain reaction of lipid peroxidase formation in cell membranes
and may help maintain the integrity of cell membranes in the
lens.42 One case-control study24 revealed a statistically significant
decrease in cortical and mixed cataract among persons in the
highest quintile of vitamin E intake and found an inverse asso-
ciation between plasma vitamin E and nuclear cataract.43 An
inverse association was also reported in the Baltimore
Longitudinal Study of Aging.44
Several studies have assessed the association between either
carotene or retinol and cataract. The Baltimore Longitudinal
Study on Aging reported no substantial association with plasma
ß-carotene and cataract.44 Leske and co-workers24 reported that
total vitamin A was protective for cortical, nuclear, and mixed
cataracts, whereas in the large Italian case-control study,27
retinol intake was not associated with risk of cataract.
Associations with specific foods were not evaluated in any of
these studies. Incidence data from the Beaver Dam Eye Study
cohort gave little evidence of a protective effect of carotenoids in
serum and nuclear cataract.45
Riboflavin is required for the synthesis of flavin adenine
dinucleotide, a cofactor for the antioxidant enzyme glutathione
reductase.46 In several animal species, a deficiency in riboflavin
Epidemiology of Age-Related Cataract
results in cataract formation.47 Leske and associates noted a
40% decrease in the risk of cortical cataract among persons in
the highest quintile of dietary riboflavin intake24 and found an
inverse association of the highest levels of plasma riboflavin with
both nuclear and PSC catarae. A number of other studies26,27,48
have found no association of riboflavin with cataract. In a clini-
cal trial conducted in China among adults with multiple chro-
nic nutrient deficiencies, a supplement combining riboflavin
and niacin resulted in a 41% lower prevalence of nuclear
cataract,49 suggesting that cataract risk is increased at or near
deficiency levels.
LOW-PROTEIN OR AMINO ACID-
DEFICIENT DIETS
Both low-protein diets and specific amino acid deficiencies have
been proposed as risk factors for cataract based on animal models.
Two studies, both conducted in India, have examined low-
protein diets as a risk factor for cataract. In a cross-sectional
study, Chatterjee and colleagues7 found that persons with the
lowest reported intake of beans, lentils, meat, milk, eggs, and
curd had a 1.5- to 2.5-fold increased risk of cataract when
controlled for age, caste, marital status, education, and weight.
In a case-control study, Mohan et al26 collected information on
usual monthly consumption of foods containing protein, thiamine,
riboflavin, vitamin A, ascorbic acid, vitamin E, and calcium.
Unfortunately, it was not possible to discern whether the
nutrient associated with risk of cataract was protein or another
dietary constituent.
ALCOHOL INTAKE
A positive association between alcohol consumption and cata-
ract, particularly PSC, has been reported.50,51
ASPIRIN USE
A possible relation between aspirin use and decreased risk of
CHAPTER 35
cataracts has been reported.52 In another case-control study, the
odds ratio associated with aspirin use was 0.25 (95% CI =
0.10–0.66).53 In contrast, Klein and colleagues22 assessed aspirin
use among 1370 diabetic patients and found no association
with cataract. This was confirmed in a randomized controlled cli-
nical trial of aspirin for patients with diabetes.53a Other studies in
persons not selected by diabetes status have failed to find any
association of aspirin and cataracts.54,55
POSTMENOPAUSAL HORMONE USE
Two recent cross-sectional studies have noted an inverse relation-
ship between current postmenopausal hormone use and cata-
ract. However, the studies were somewhat inconsistent in that
the inverse association was noted for nuclear cataract in one56
and cortical cataract in the other.57
SEVERE DIARRHEA
It has been proposed that the dehydration and uremia
associated with severe diarrhea could increase the levels
of cyanate in the body and that cyanate-associated
carbamylation of lens proteins would result in cataract
formation. To date, few studies have addressed this association.
In a matched case-control study conducted in India, case-
patients were four times as likely as controls to have reported at
least one severe episode of diarrhea; they were 21 times more
likely to have reported two or more bouts of diarrhea (95% CI =
8.9–31.0).58 391
 PRINCIPLES OF EPIDEMIOLOGY

GENETIC FACTORS analyses by Klein et al,62 genetic effects may be obscured or

There have been recent reviews in the literature describing sites
in the genome related to congenital or early-onset cataract.59,60
A study of twins61 indicated greater concordance of nuclear and
cortical in monozygotic compared to dizygotic twins. In the
population-based Beaver Dam Eye Study, Heiba et al and Klein
et al described evidence of familial effects on nuclear15,62 and
cortical cataracts.16 Iyengar et al discovered multiple loci asso-
ciated with cortical cataract in this population. Judging from the
modified by environmental factors.
CONCLUSION
Age-related cataracts are common throughout the world. There
are many factors – environmental, personal, and genetic – that
appear to influence their frequencies. Further research, both exper-
imental and observational, is needed to develop interventions
that will have an impact on their prevalence.

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CHAPTER 35
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 CHAPTER
36 Epidemiology of Primary Open-Angle Glaucoma
Anne L. Coleman, Steven L. Mansberger, and M. Roy Wilson
The ultimate goal in the management of a chronic disease such
as primary open-angle glaucoma (POAG) is to design interven-
tion programs that can prevent, or at least control, the debili-
tating outcomes associated with the disease. The chapters on
glaucoma in this book are devoted to providing a better under-
standing of the pathophysiologic mechanisms of disease, as well
as treatment strategies. This chapter uses epidemiology to
describe the prevalence, incidence, risk factor, treatment, and
screening of POAG.
Epidemiology studies are directed to finding the distribution,
determinants, and frequency of disease in groups of persons in
order to improve our understanding of prevalence, incidence,
pathogenesis, and treatment. Implicit in the definition of ‘epi-
demiology’ is the fact that disease is not randomly distributed
throughout a population; instead, the frequency differs among
subgroups. Knowledge of this uneven distribution, and of the
factors that influence this distribution, may provide valuable
clues as to what factors are important in pathogenesis and
development of glaucoma.
POAG AS A PUBLIC HEALTH PROBLEM
A recent meta-analysis estimated the overall prevalence of
POAG in the United States to be 2.2 million persons based on
2000 US census data.1 Worldwide, 48 million persons2,3 are
thought to have POAG. This high prevalence is likely to increase
in the future because of the aging population. In the year 2000,
people aged 65 and older made up 12.4% of the US population.
By 2040, this group will increase to 20.4% and comprise one
fifth of the population. During the same period, the 3 million
Americans aged over 85 are expected to triple to 9.8 million.4
Overall, this change in population demographics will increase
the prevalence of glaucoma in the United States by 50% to
3.4 million persons by the year 2020.1 A similar demographic
change will occur worldwide and will increase the prevalence of
glaucoma by 100%.2
The real public health impact of POAG in the US is the visual
limitation it causes. Studies estimate that approximately 0.37%
of US adults over the age of 40 years are bilaterally blind, which
corresponds to over 400 000 (119 million µ 0.37%) persons. This
figure increased to almost 5 million globally.5 Glaucoma is the
second leading cause of blindness.3
The societal costs are staggering. The estimated expenditures
for treatment are at least $1.6 billion. The federal government
provides $1.05 billion per year in income support to assist
persons blind from glaucoma (e.g., Social Security disability
income, automatic Medicare and Medicaid eligibility, and
income tax credits).6 Additional costs which are difficult to
estimate, include lost earnings and requirements for care taking
services. A recent clinical trial7 evaluating the quality of life in
persons undergoing initial surgical versus medical treatment for
glaucoma reported that more than 50% of newly diagnosed
glaucoma patients were worried about going blind at the start
of the study. This percentage decreased to ~25% at 5 years
of follow-up. Females and older subjects had more func-
tional disabilities related to activities of daily living during
the study, and subjects randomized to surgery had more local
eye symptom complaints than subjects randomized to medi-
cations. Several investigators have found that even mild visual-
field loss is associated with decreased vision-related quality
of life.8,9
PREVALENCE
Prevalence of disease is one of the cornerstones to epidemiology-
based knowledge and control programs. Many studies report the
prevalence of glaucoma in a variety of ethnic groups and regions;
however, the methods and definitions vary. The prevalence of a
disease would best be estimated: (1) on a well-defined population,
(2) by examining and reporting on all of the defined population
or a specified sample of the defined population, (3) if sampling
is used, sampled subjects should represent the population, with
no subgroup systematically excluded from examination, and
(4) by specifying and consistently applying the case definitions
for glaucoma. Studies based on self-selected or small nonrepre-
sentative segments of the population are particularly susceptible
to bias. A population-based study design is preferable. Yet, even
among studies using this design, methodological shortcomings
are often present, and study results must be compared with
caution.
Major differences exist in the case definitions of glaucoma in
prevalence studies. Studies have used elevated intraocular pres-
sure (IOP), optic nerve pathology, and/or visual-field defects to
define glaucoma. More recent studies have excluded a specific
level of IOP as part of the definition of glaucoma but have
uniformly required the presence of a glaucomatous-appearing
optic disk and/or visual-field changes.
However, visual-field testing creates a myriad of diagnostic
dilemmas when performed as part of a prevalence study, espe-
cially when the majority of participants are novice to perimetry.
For example, a common finding in participants new to peri-
metry is an abnormal or unreliable test result in the presence of
a normal optic disk.10,11 In such cases, should the study proto-
col require repeat visual-field testing or should repeat testing be
performed only in patients with an abnormal optic disk? Some
subjects are unable to perform visual-field testing reliably, but
have an obviously glaucomatous disk. Should these subjects be
classified definitively as having glaucoma? Partly because of
these dilemmas, researchers have developed a definition of glau-
coma to be used for prevalence studies based on cup-to-disk 395
 PRINCIPLES OF EPIDEMIOLOGY
ratio (C/D) and visual-field results.12 This definition includes
three categories for glaucoma: category 1 includes patients with
glaucomatous optic neuropathy and confirmed visual-field loss;
category 2 includes participants with a C/D greater than 97.5
percentile of the population, but who are unable to perform
visual-field testing satisfactorily; and category 3 includes partici-
pants with a history of glaucoma surgery or extremely elevated
IOP, but the examiner is unable to view the optic disk. This
definition may lead to a more uniform definition of the preva-
lence of glaucoma in the future that will allow researchers to
more easily compare studies.
An example of the difficulty in defining glaucoma in preva-
lence studies is highlighted in the different prevalence rates in
the surveys performed in Ferndale, Wales (0.47%),13 Dalby,
Sweden (0.86%),14 and Framingham, Massachusetts (1.6%).15
Why did these populations have an almost fourfold difference in
prevalence? One reason may be the difference in the case
definition for glaucoma. The diagnostic criteria for POAG in the
two European studies were based on abnormal disk cupping and
loosely defined visual-field defects. Thus, these studies may
have excluded subjects with high IOPs and field defects only.
Conversely, the Framingham study relied solely on rigorously
defined visual-field criteria and did not require subjects who
were not suspected of having glaucoma (such as those with
normal IOP), to undergo perimetric examinations. Overall,
these methodological differences would make it difficult to
compare study results.
Over the last 30 years, investigators have performed popu-
lation-based prevalence studies in most regions of the world and
in most ethnic populations, which add to our understanding
of primary open-angle glaucoma. Quigley and Broman5
summarized the worldwide prevalence of glaucoma. They used
generalized estimating equations to account for different sample
sizes and age distributions, which allowed them to collate results
from 34 different studies into separate regions that were expected
to have similar glaucoma prevalence. The eight regions
included: European, Middle East/North Africa, Latin American,
African (south of the Sahara), South East Asia, Indian group,
SECTION 5
China group, and Japan. They included studies with random
population-based sampling, high proportion of examinations
(>50%), high proportion of visual-field testing (>50%), optic
disk evaluation by an ophthalmologist, and definition of glau-
coma based on optic disk and visual-field criteria. The propor-
tion of open-angle glaucoma in each region from highest to
lowest was: African (4.2%), Japan (3.3%), Latin America (3.2%),
Europe (2.0%), India (1.8%), China (1.4%), Middle East (1.3%),
and SE Asia (1.2%). The overall prevalence of POAG worldwide
was estimated to be 2.0%. These results provide valuable
information for forecasting the burden of OAG in these groups.
The authors also estimate the prevalence of angle closure glau-
coma, which is the cause of a high proportion of glaucoma in
several regions,16–18 and is described further in other chapters.
Some prevalence studies describe intra-regional and intra-
ethnic variations in the prevalence of open-angle glaucoma
especially in Asia, Africa, and Latin America. For example, the
Tajimi Eye Study in Japan19,20 found 7:1 ratio of POAG
(including normal tension glaucoma) to primary angle-closure
glaucoma (PACG). However, PACG was more common in
participants of Chinese descent with a ratio of POAG:PACG of
1.6:1 in Chinese living in Singapore16; and a 1:3 ratio in
Mongolia, China.18 Similar discrepancies can be found in the
population-based studies among Africans and Latin Americans.
Data from the West Indies11,21 and from Baltimore22 suggest a
very high POAG prevalence for Africans, but almost a twofold
difference in prevalence between the two studies with a
prevalence of 8% and 4%, respectively. Recent studies suggest
396 similar regional differences in Ghana23 (8.5%) when compared
to Tanzania24 (3.1%) and South Africa25 (2.1%). Finally, the
Proyecto VER population-based study in Arizona26and the Los
Angeles Latino Eye Study (LALES)27 show differences in the
prevalence of POAG of 2.0% and 4.7%, respectively. These
results underscore the fact that regions and ethnic groups may
vary greatly in the prevalence of POAG. While many of these
differences may come from genetic and environmental influences,
some may be the result of different definitions of glaucoma and
varied age stratification within the sampled population.
A population-based prevalence survey among Alaska’s
Northwestern Inuit found a glaucoma prevalence of 0.65%, but
most were of the angle-closure variety.28 This study suffered
from the fact that the diagnosis of open-angle glaucoma was
based on visual-field defects, with the tangent screen in the
presence of either an elevated IOP (>21 mmHg) or a C/D of
more than 0.5. Because a substantial proportion of persons with
glaucoma present with normal IOPs and may have C/D of less
than 0.5, the reported prevalence of POAG (0.06%) was
undoubtedly underestimated. Nonetheless, it is probably safe to
conclude that the prevalence of POAG in this population is very
low. Two other studies have been reported recently in American
Indians. They show a prevalence of glaucoma of 5.6% in
Oklahoma Indians29 and a prevalence of 6.2% in Northwest
American Indians.30 The latter study is noteworthy in that 90%
of the glaucoma were ‘normal-tension’ glaucoma with IOP
<21 mmHg and no cases of angle closure glaucoma were
detected. Overall, these findings in mainland American Indians
were similar to those found in the Japanese and different from
the findings in Alaskan Inuit.
Other interesting results from recent prevalence studies
improve our understanding of POAG. The Tajimi study19
(Japan) showed that 92% of participants with POAG had IOP
<21 mmHg and showed an age-associated decrease in IOP.
Similar age decreases in IOP were found in a study in Ireland.31
Pseudoexfoliation, which is known to be a common cause of
open-angle glaucoma in Northern Europe and Greece, has also
been found to be a significant cause of open-angle glaucoma in
India32 and Africa.25,33 Finally, most prevalence studies27,34,35
show a high prevalence of undiagnosed glaucoma, from 50% to
as high as 93%.36 Overall, these studies underscore the import-
ance of prevalence studies, which has generated considerable
research interest in morphologic, genetic, and environmental
influences into the causes of glaucoma.
INCIDENCE
Prevalence data are valuable, but they do not provide estimates
of disease over time, nor do they permit etiologic inference.
Incidence data, on the other hand, provide this information.
They give a direct measure of the rate at which individuals in a
given population develop disease and the probability of risk of
the disease. Despite their desirability, reliable POAG incidence
data are scarce.
One can divide studies measuring open-angle glaucoma inci-
dence into two types: (1) those that are population-based and
(2) those that target a specific ‘high-risk’ subpopulation. Because
of the relatively low incidence, large cohorts and long follow-up
periods are necessary to obtain a sufficient number of newly
diagnosed glaucoma cases to ensure valid estimates; thus only
a few such studies have been conducted.
Several population-based studies were designed to yield inci-
dence data. A 9-year incidence of 4.4% (95% CI: 3.7–5.2) was
reported for a black West Indian population.21 Although issues
related to study design preclude making a direct comparison, a
glaucoma annual incidence of between 0.19% and 0.24% was
reported in the white population of Dalby, Sweden.37 In the
Melbourne Visual Impairment Project,38 there was a 1.1%

5-year incidence of OAG, while in Olmsted County, Minnesota 39
the annual age-adjusted incidence of OAG was estimated to be
0.0145% in a predominantly Caucasian population. The
incidence estimate from Olmsted County was most likely an
underestimate because it was based on a review of medical
records while the other population-based studies involved the
reexamination of study subjects. The Rotterdam study reported
a 5-year incidence in glaucoma of 0.6% in participants greater
than 55 years old.
Because of this low incidence, various investigators have
attempted to determine incidence rates in selected high-risk
subpopulations. The Collaborative Glaucoma Study,40 which
was a prospective study conducted over a 13-year period to
identify factors that influence the development of glaucomatous
visual-field defects, enrolled relatives of patients with open-
angle glaucoma. This was augmented with a group of persons
with IOPs greater than 20 mmHg. Glaucomatous visual-field
defects developed in 1.7% of the 5886 eyes included in the
analysis, over a maximal time period of 7 years. The annual
incidence rates ranged from 0.25% to 0.54%. Using life table
analyses for different levels of IOP, for the 5-year survival (free
of visual-field defects) rate for eyes with IOPs greater than or
equal to 20 mmHg was 93.3%, whereas it was 98.5% for eyes
with lower IOPs.
Other studies followed cohorts of subjects with higher-than-
normal IOP for variable time periods.42–43 Unfortunately, these
studies do not permit the calculation of incidence rates and
comparison of the results of these studies is difficult because of
different inclusion criteria and diagnostic criteria. Despite these
caveats, the studies had two major findings: (1) visual-field
defects develop infrequently, even in subjects with higher-than-
normal IOP; and (2) the higher the baseline IOP, the greater the
risk of subsequently developing visual-field defects. In the
Ocular Hypertension Treatment Study (OHTS) the 5-year inci-
dence of developing glaucoma was 9.5% in ocular hypertensive
subjects who were randomized to no treatment.44
The lack of good population-based incidence data has
prompted the derivation of incidence estimates from age-specific
prevalence data.45 Such estimates are regarded as gross approxi-
mations, and their use is usually restricted to certain specific
purposes such as the planning of epidemiologic studies.
RISK FACTORS FOR DISEASE
DEVELOPMENT
IOP is the most important known risk factor for glaucoma
development. Evidence clearly indicates that increased IOP can
cause glaucoma. Experimentally induced high IOP in animals
results in typical glaucomatous cupping.46,47Acute angle closure
and many cases of unilateral high IOP glaucoma support a
cause–effect relationship between high IOP and glaucomatous
damage. Even at normal IOP levels, asymmetric IOP has been
noted to correlate with asymmetric cupping and field loss, with
greater damage occurring on the side with higher pressure.48,49
Population surveys provide additional support that there is
an increase in the prevalence of POAG with increasing levels of
IOP.13,14,48 However, many subjects with elevated IOP do not
have glaucoma, and longitudinal studies of subjects with ele-
vated IOP have demonstrated that most patients with ocular
hypertension never develop glaucoma. Thus, elevated IOP is
frequently not sufficient and is, in fact, not a necessary condi-
tion for glaucomatous damage. Population surveys have con-
sistently demonstrated that 30–90% of subjects (depending on
ethnicity) did not have elevated IOP at the time of
diagnosis.14,15,22,31,50 Even if researchers could perform multiple
IOP measurements, they would find a significant proportion of
subjects with glaucoma and statistically normal IOP. The fact
Epidemiology of Primary Open-Angle Glaucoma
that some eyes with high IOP do not develop glaucomatous
damage and some eyes with low IOP suffer definite glauco-
matous damage indicates that other factors may contribute to
the pathogenesis of glaucoma.
Data regarding the possible role of myopia are conflicting.
Although a number of studies have demonstrated an associa-
tion between myopia and POAG,51,52 these studies were clinic-
based studies (rather than population-based), and the potential
for selection bias must be considered, because persons with
refractive errors are more likely to seek eye care and have a
higher probability of being diagnosed with glaucoma. In the
BMES, there was a threefold increased risk of POAG in indi-
viduals with myopia of ⫺3.0 D or greater.53 This increased risk
was independent of IOP and other glaucoma risk factors.
Disks with large C/D also tend to have a larger disk size with
proportionately more neural rim tissue.54 Thus, whether larger
C/D, per se, predispose to glaucomatous damage is unclear. An
enlarged C/D, as well as asymmetric cupping, may be a sign of
early disease. From a practical standpoint, subjects with
‘suspicious’ disks must be observed closely for development of
signs of clinically significant glaucoma.
The transient nature of disk hemorrhages makes it difficult
to assess the importance of this factor for subsequent glauco-
matous damage. Fairly consistent evidence indicates a poorer
prognosis in glaucomatous eyes with disk hemorrhages com-
pared with those without hemorrhages.55–57 Although disk
hemorrhages have been shown to precede retinal nerve fiber
layer defects, glaucomatous changes of the optic nerve head,
and glaucomatous visual-field defects, it is not known how
frequently this occurs.58–61 An extreme view, one that is not well
supported or accepted, proposes that disk hemorrhages precede
all cases of open-angle glaucoma.62 A recent study showed that
the presence of optic disk hemorrhages resulted in mean risk of
visual-field deterioration over 9 years of 80% (hazard ratio
of 5.4) and 89% (hazard ratio of 3.6) in normal tension and
primary open-angle glaucoma patients, respectively.63 Another
study showed optic disk deterioration but no visual-field
changes with a history of optic disk hemorrhage.64 Although
CHAPTER 36
clinicians may consider advancing glaucoma therapy in a glau-
coma patient with a recent disk hemorrhage, 70% of subjects in
the BMES who had disk hemorrhages had no other signs of
glaucoma.65
Recent studies have demonstrated the association of thin
central corneal thickness (CCT) with existing glaucoma, with
progression to glaucoma from ocular hypertension, and pro-
gression of existing glaucoma.66–70 In contrast, one randomized
controlled clinical trial did not show an association of CCT
with progressive glaucoma.57 The explanation for why CCT is a
risk factor for glaucoma is not known. The simple explanation
is that it is a surrogate for the known risk of IOP as the actual
IOP is higher than the measured IOP in eyes with thin
CCT.71–73 However, the OHTS study showed a strong,
independent association of CCT with development of glaucoma
suggesting that a linear or nonlinear correction of IOP by CCT
would not explain the association of CCT with glaucoma. This
indicates that CCT may correlate with other biomechanical
factors associated with glaucoma such as lamina cribrosa com-
pliance and scleral compliance. One histological study showed
no association between CCT and lamina cribosa thickness;
however, artifacts and sectioning methods may have prevented
accurate measurements.74 Further studies are needed to explore
this relationship.
Race, age, and family history are nonocular factors related to
glaucoma risk. As indicated earlier, blacks, Hispanics, and
American Indians have a disproportionately high prevalence of
POAG when compared to Caucasians.22,26,27,30 Although race-
specific incidence data are not yet available for all of these 397
 PRINCIPLES OF EPIDEMIOLOGY
groups. Precisely why certain ethnic groups are more likely to
develop glaucoma is not known. However, some researchers
have found higher IOP, thinner CCT,75 and larger C/D in blacks
when compared to Caucasians, and suggest these factors as
causal. However, data conflict as to whether blacks have higher
IOP than do whites,76,77 and as discussed previously, the
relevance of larger C/D is uncertain.
Nearly every population-based study has demonstrated that
the prevalence of glaucoma increases with advancing
age.11,14,15,21,22,31,50,78–82 The oldest age groups have prevalence
estimates approximately three to eight times higher when com-
pared to persons in their 40s. Further, the Collaborative Glaucoma
Study identified age as the major predictor of glaucoma
incidence.40 As with race, the exact causal mechanisms are
unknown, and underlying susceptibility factors must be inves-
tigated. The higher IOP noted with increased age in most
studies were not found among the Japanese.78 Yet, glaucoma
prevalence increased with age in the Japanese, suggesting that
the optic nerves of the elderly are more susceptible to damage
for reasons other than just higher IOP.
Familial factors are also important in the underlying suscep-
tibility to POAG. Several ocular parameters associated with
POAG, such as IOP and C/D, are known to be influenced by
heredity.59 Relatives of glaucoma patients would thus be expected
to exhibit abnormalities of these parameters more often and to
more likely be diagnosed as having glaucoma, either from selec-
tion bias, or better case finding, or from a true increase in the
prevalence of glaucoma in relatives. Most studies investigating
risk in relatives had selection and recall bias because of incom-
plete ascertainment of relatives, therefore, accurate estimates of
the exact risk of POAG in relatives are lacking. Other chapters
in this book explain the genetic associations of glaucoma.
Systemic factors provide other information about POAG risk.
Diabetes,79 systemic hypertension,51 and various other vascular
abnormalities such as migraines80 have been implicated as risk
factors for glaucoma. Much of the data regarding possible asso-
ciations between these factors and glaucoma are contradictory.
The role of diabetes as a risk factor for POAG is controversial.
SECTION 5
The Blue Mountain Eye Study found an association between
diabetes and glaucoma;81 while the Baltimore Eye Survey did
not detect an association overall but did find an association
among persons in whom glaucoma had been diagnosed prior to
the survey examination.82 Persons with diabetes are more likely
to be in the health care system and thus lead to a bias in having
glaucoma detected. Surprisingly, the Ocular Hypertension
Treatment Study found that the presence of diabetes was pro-
tective toward developing glaucoma. Overall, the relationship of
diabetes to glaucoma development is controversial.
Although the evidence that systemic hypertension is a risk
factor for glaucoma is not strong, the hypothesis that microcir-
culatory effects on the optic disk may lead to increased glau-
coma susceptibility is biologically plausible. The Rotterdam
Study reported an association of systemic hypertension with
high-tension glaucoma but not with normal-tension glaucoma.83
The Blue Mountains Eye Study investigators reported a 1.5 times
increased risk of open-angle glaucoma in subjects with systemic
hypertension, independent of IOP and other glaucoma risk
factors.84 The Baltimore Eye Survey reported modest, positive
but not statistically signficant associations of increased systolic
and diastolic blood pressure with POAG. However, lower
perfusion pressure (blood pressure ⫺ IOP) was strongly asso-
ciated with an increased prevalence of POAG.85 These results
suggest that POAG may be associated with a change in factors
related to ocular blood flow.
Migraine and peripheral vasospasm may be important in the
development of some cases of glaucoma, particularly those in
398 which IOP is in the normal or low range.86 Investigations of
other associations of other systemic factors with POAG have
been scant, and the results have been inconclusive. One
interesting observation has been that among Japanese, IOP has
not been found to increase with age as it does in Western
populations.87 One explanation for this apparent discrepancy is
that IOP is related to body build, and that Japanese typically do not
get obese with age when compared to Americans and Europeans.
Another interesting finding has been the relationship between lean
body mass and increased prevalence of POAG among the
participants of the Barbados Eye Study.88 These results suggest that
anthromorphologic considerations may warrant further study.
It is unclear whether POAG is more frequently associated
with men or with women. A higher prevalence among women
was reported in Dalby14 and Blue Mountains,89 a higher preva-
lence among men in Tierp90 Framingham,15 and Barbados,21
and no difference in St Lucia,11 Wales,13 Baltimore,22 Beaver
Dam,50 Melbourne,91 Los Angeles,27and Arizona.92
A variety of risk factors may be present in an ocular hyper-
tension or glaucoma patient, but each patient encompasses a
unique combination of risk factors that the clinician must take
into account.93 Recently, investigators have developed risk
calculators to estimate the risk of developing glaucoma from
ocular hypertension using data from the OHTS study.94,95 The
OHTS and European Glaucoma Prevention Study (EGPS)
demonstrated that age, corneal thickness, IOP, pattern standard
deviation (PSD), diabetes mellitus status, and vertical C/D were
independent predictive variables for the development of
glaucomatous optic disk or visual-field changes. The OHTS
and EGPS combined their data set to provide more precise
estimates.
Key Features: Reliable Risk Factors for Glaucoma
• Increased IOP is the most important risk factor
• However, elevated IOP is frequently not sufficient and is, in
fact, not a necessary condition for glaucomatous damage
• Increasing age
• Racial differences, more common in African-Americans and
other ethnic groups
• Family history
• Thin CCT
• Controversial risk factors for glaucoma include:
• Myopia
• Presence of disk hemorrhages
• Diabetes
• Hypertension
TREATMENT ISSUES
Thus far, treatments for POAG have focused exclusively on
lowering IOP. Several trials have documented the efficacy of
medications, laser, and surgery in lowering IOP. However, only
recently, have randomized clinical trials demonstrated the value
of these treatments in reducing the occurrence or progression of
visual-field damage.
The Ocular Hypertension Treatment study44,66 and European
Glaucoma Prevention Study96 determined the efficacy of ocular
hypotensive treatment in ocular hypertension patients; the
Collaborative Initial Glaucoma Treatment Study97 and the Early
Manifest Glaucoma Treatment study57 examined treatment of
early or newly diagnosed glaucoma patients; the Collaborative
Normal-Tension Glaucoma Study98 (NTGS) examined patients
with mild to moderate glaucoma and normal IOP; and the
Advanced Glaucoma Intervention Study99 investigated surgery
or laser in patients with moderate to severe glaucoma. These
studies guide clinicians in their treatment of glaucoma patients,

examining the full range of glaucoma from preperimetric
glaucoma to advanced glaucoma.
Ocular hypertension is present in ~8% of adults over the age
of 40 years in the United States.100 The Ocular Hypertension
Treatment Study recently demonstrated that treatment of ocular
hypertension decreases the risk of development of visual-field
loss.44 In contrast, the European Glaucoma Prevention Study
showed no benefit of treatment with dorzolamide eye drops
compared with placebo.96 The latter study had the benefit of
placebo control, but it suffered from high loss to follow-up
(30%), no target IOP for treatment, low clinical applicability
(only dorzolamide treatment) and only one baseline IOP
measurement. These methodological flaws would tend to
decrease effect of treatment. Despite these different results, most
clinicians recommend treating ocular hypertension patients at
high risk for developing glaucoma.
Recently, randomized controlled trials have demonstrated
that patients with definite glaucoma, regardless of disease stage,
should be treated. The Early Manifest Glaucoma Trial and the
Collaborative Initial Glaucoma Treatment Study evaluated the
treatment of newly diagnosed glaucoma patients. The Early
Manifest Glaucoma Trial (EMGT) randomized patients with early
glaucoma either to argon laser trabeculoplasty plus betaxolol
(n = 129) or to monitoring without immediate treatment
(n = 126).101 These were newly diagnosed glaucoma patients
found during a community glaucoma screening. The rate of
progression was 45% in the treated group versus 62% in the
untreated group. The treatment reduced IOP by ~20% and
decreased the risk of worsening glaucoma by 50%.57 The
Collaborative Initial Glaucoma Treatment Study (CIGTS)
enrolled 607 patients with newly diagnosed open-angle
glaucoma and randomized them to treatment with topical
ocular hypotensive medications or trabeculectomy surgery.102
The 5-year outcomes reported in the CIGTS demonstrated that
both medications and surgery resulted in reduced IOP, and both
groups of patients had similar low rates of visual-field
progression.97 Only 11% of patients treated medically versus
14% of patients treated with surgery had significant progression
during follow-up. Medically treated patients were less likely to
develop cataracts, suffer noncataract-related visual acuity loss,
or complain of ocular side effects.
The Collaborative Normal-Tension Glaucoma Study (CNTGS)
evaluated the treatment of moderate to advanced normal ten-
sion glaucoma (<20 mmHg) by randomly assigning 240 patients
to treatment versus no treatment. It required patients to have
documented progression or a specific visual-field defect. The
treatment included medications, laser, or surgery to reduce IOP
by at least 30%.98 The rate of progressive visual-field loss was
slower in the treated group than in a group that did not receive
treatment when the analysis was adjusted for the effect of
cataracts.
Finally the Advanced Glaucoma Intervention Study (AGIS)
evaluated the treatment of uncontrolled primary open-angle
glaucoma. The study randomly assigned 591 persons to a treat-
ment sequence of argon laser trabeculoplasty, trabeculectomy,
and trabeculectomy (ATT sequence): or trabeculectomy, argon
laser trabeculoplasty, and trabeculectomy (TAT sequence). The
main outcome measures of the study were visual acuity and
visual field, although the study also evaluated IOP, complications
of treatment, time to treatment failure, and need for adjunctive
medications. The study reported no difference in visual acuity
and visual-field outcomes by treatment regimen.99 A post hoc
subanalysis found that the ATT sequence was favored for black
patients while the TAT was better for whites for a visual acuity
outcome.103
Overall, these randomized controlled clinical trials demonstrate
that lowering IOP decreases the risk of subsequent visual-field
Epidemiology of Primary Open-Angle Glaucoma
loss, or optic disk deterioration in ocular hypertension and glau-
coma patients. These studies provide important information
guiding the treatment of ocular hypertension and glaucoma.
SCREENING FOR GLAUCOMA
Glaucoma, as one of the leading causes of blindness, may be a
strong candidate disease for screening programs. It is asymp-
tomatic in the early stages and treatment decreases the risk of
visual-field loss. Unfortunately, major impediments to wide-
spread glaucoma screening are a lack of a screening test(s) with
appropriate diagnostic precision and lack of evidence that
screening for glaucoma prevents visual impairment. The fol-
lowing section outlines the deficiencies and strengths of current
screening tests.
Tonometry has been used as a screening test for glaucoma for
more than 40 years. However, we have yet to identify a set of
tonometric criteria that adequately classify persons in terms of
their disease status. Data from a number of studies have
demonstrated the futility of using the widely accepted cutoff of
21 mmHg for screening purposes.105 Moreover, no matter what
IOP level is chosen, the balance of sensitivity and specificity (or
diagnostic precision) is unacceptable.
Optic disk evaluation has other difficulties. Ophthalmoscopy
and optic disk photography are difficult to obtain in many
participants for reasons such as ocular media abnormalities and
difficulties with cooperation such as blinking. The Baltimore
Eye Study photographers had difficultly attaining optic disk
photos in over 20% of participants.100 Photography requires
technical expertise to perform and needs expert opinion to grade
the optic disk photos. Finally, experts disagree when grading
optic nerve photos.105
Standard achromatic automated perimetry (SAP) has good
diagnostic precision for glaucoma106,107 but is nonspecific
because abnormal results can occur from other conditions such
as cataracts and retinal disease. Even normal eyes can have
abnormal results from small pupil size,108–110 uncorrected
refractive error,111,112 fatigue,113 and learning effects.114–118
CHAPTER 36
Abnormalities from uncorrected refractive error are a particular
problem as refractive error are a common source of visual
impairment in the community.119
Overall, the traditional methods of detecting participants at
risk for glaucoma in the community are fraught with difficulties
that reduce their feasibility and diagnostic precision. Investi-
gators are researching new methods of screening. Their goals
are to develop a screening program with high diagnostic preci-
sion and immediate results, as well as being able to be
performed by paraprofessionals such as ophthalmic technicians
and nurses. These include new methods of visual-field testing
and examining the optic disk.
Studies have reported results with smaller, faster visual-field
machines such as frequency doubling technology perimetry,120–126
oculokinetic perimetry127 and laptop computer techniques.128
Despite the fact that these techniques have been available for
several years, they still require validation in population-based
screening settings.
Objective structural testing with optic imaging devices such
as confocal scanning laser ophthalmoscopy (CSLO), scanning
laser polarimetry, and ocular coherence tomography has
promise for screening for glaucoma. They are able to image the
optic disk without dilation and in patients with cataract or
other mild media abnormalities. Studies indicate that they have
similar and reasonable diagnostic precision for early glaucoma
when compared to normal subjects.129 However, the machines
are expensive and somewhat difficult to transport. Further
studies will need to determine the diagnostic precision in an
unselected screening population. 399
 PRINCIPLES OF EPIDEMIOLOGY

One of the most important issues is that the prevalence of SUMMARY

glaucoma in the unselected, general population is relatively low.
Thus, the predictive power of a positive test result will be low.
Only a small proportion of those identified as glaucomatous by
the screening test – even with a highly valid and suitable test –
will actually have the disease; the remainder will nonetheless
undergo costly, unproductive diagnostic work-ups. Focus has
gradually shifted from widespread population-based screening
to case-finding in high-risk individuals to obtain a high yield of
true cases.
Much of our knowledge of glaucoma epidemiology has come
from population-based prevalence studies. These studies have
documented the relatively common occurrence of higher-than-
normal IOP without evidence of glaucomatous damage, glauco-
matous damage with normal IOP, and the influence of age and
race on disease prevalence. Research is now available on glau-
coma incidence, investigating possible risk factors for disease
development, and evaluating factors that influence glaucoma
progression and outcome.

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 CHAPTER

37 Epidemiology of Diabetic Retinopathy

Hanna R. Coleman and Donald S. Fong

INTRODUCTION according to their severity: (1) intraretinal microvascular abnor-

mality, (2) hemorrhages and microaneurysms, and (3) venous
Diabetic retinopathy is the most common microvascular abnormalities. The ETDRS also developed a severity scale that
complication of diabetes and is a leading cause of visual considers the natural history of diabetic retinopathy as well
impairment among Americans.1 Blindness is 25 times more as the orderly progression of risk of severe visual loss.8 It was
common in diabetics.2,3 Approximately 3.4% of the population based on the modified Airlie House classification of diabetic
is estimated to be affected with diabetic retinopathy and this retinopathy but has not proven to be easy or practical.9,10
translates to ~4.1 million persons with diabetic retinopathy A simpler diabetic retinopathy severity scale was developed
with 1 in 12 having vision threatening diabetic retinopathy.4 by the Global Diabetic Retinopathy Group.11 It consists of
Future projections of the rates of development of diabetic five levels with increasing risks of retinopathy and is described
retinopathy are significant increases of public health import- in Table 37.1.
ance as the population will live longer with a high prevalence The ETDRS also showed that clinically significant macular
of developing diabetes in the future. Diabetic retinopathy can edema (CSME) can lead to moderate visual loss if not treated
develop over a long period of time. Knowledge of the demo- by focal photocoagulation. Macular edema is defined as thick-
graphic distribution of this disease and understanding of the ening in the macula as seen by biomicroscopy or fundus photo-
clinical risk factors is crucial in patient management. This graphy. The diabetic macular edema (DME) disease severity
chapter presents descriptive and analytic epidemiologic data for scale separates eyes with apparent DME from those with no
diabetic retinopathy in the US. apparent thickening or lipid in the macula and is summarized
in Table 37.2.
DEFINITION AND DIAGNOSIS These two clinical disease severity scales are intended to be
a practical method of grading severity of diabetic retinopathy
There are two common types of diabetes mellitus: insulin- and DME that will allow observers to recognize and categorize
dependent, known as type-1 and non-insulin-dependent, type-2
diabetes mellitus. The latter is more common, comprising
90–95% of all cases in the US. These two types differ in their TABLE 37.1. International Clinical Diabetic Retinopathy Disease
clinical characteristics, etiologies, and pathophysiologic basis. Severity Scale
A main difference is their propensity to develop diabetic
ketoacidosis in the basal metabolic state. Insulin is required Proposed Disease Severity Level Findings Observable Upon
Dilated Ophthalmoscopy
in type 1 to prevent ketoacidosis, whereas in type 2, keto-
acidosis is unlikely even with poor glycemic control. Typically, No apparent retinopathy No abnormalities
type 1 presents acutely with polyphagia, polydipsia, and poly-
uria. Type 2 is insidious and may be present for years before Mild nonproliferative diabetic Microaneurysms only
retinopathy
being diagnosed clinically.
From the ophthalmic standpoint, patients with type-1 Moderate nonproliferative More than just
diabetes have a higher risk of developing severe proliferative diabetic retinopathy microaneurysms but less
diabetic retinopathy. However, a greater percentage of cases of than severe NPDR
severe proliferative diabetic retinopathy (PDR) are caused by Severe nonproliferative diabetic Any of the following:
type 2 because of the higher prevalence in the general population. retinopathy More than 20 intraretinal
There is evidence that retinopathy begins to develop at least hemorrhages in each of
7 years before the clinical diagnosis of type-2 diabetes is made.5 4 quadrants
Diabetic retinopathy has been traditionally divided into Definite venous beading in
2+ quadrants
nonproliferative and proliferative categories. Diagnostic corre- Prominents IRMA in 1+
lation among various specialists has varied from 50% to 85%.6,7 quadrant
The recognized gold standard for grading the severity of dia- And no signs of
betic retinopathy in clinical trials is the Early Treatment proliferative retinopathy
Diabetic Retinopathy Study (ETDRS) severity scale. The
Proliferative diabetic retinopathy One or more of the following:
ETDRS was a randomized clinical trial of photocoagulation Neovascularization
versus deferral of photocoagulation that allowed the obser- Vitreous/preretinal
vation of the natural history of diabetic retinopathy. It identi- hemorrhage
fied three retinal lesions that are predictive of progression 403
 PRINCIPLES OF EPIDEMIOLOGY
TABLE 37.2. International Clinical Diabetic Macular Edema Disease Severity Scale
Proposed Disease Severity Level
Diabetic macular edema apparently
absent
Diabetic macular edema apparently
present
If Diabetic Macular Edema is Present, it can be Categorized as Follows:
Proposed Disease Severity Level
Diabetic macular edema present
* Hard exudates are a sign of current or previous macular edema. Diabetic macular edema is defined as
retinal thickening and this requires a three-dimensional assessment that is best performed by a dilated
examination using slit-lamp biomicroscopy and/or stereo fundus photography.
levels of retinopathy and the presence of most DME resulting in
more appropriate and consistent referrals to treatment centers.
INCIDENCE AND PREVALENCE
There are no national data on the prevalence or incidence of
diabetic retinopathy in the US. Population studies have been
performed on select populations such as inhabitants of
Rochester, Minnesota; Framingham, Massachusetts; Pittsburgh,
Pennsylvania; Mexican-Americans in San Antonio; blacks in
the Bahamas and Pima Indians.12–18 The largest population-
SECTION 5
based study using fundus photography to document diabetic
retinopathy was the Wisconsin Epidemiologic Study of
Diabetic Retinopathy (WESDR). It identified previously diag-
nosed diabetics in 11 counties in Wisconsin. Because the deter-
mination of insulin dependency can be difficult, the WESDR
investigators divided the diabetics by age of diagnosis into two
cohorts. The first cohort was composed of patients whose
disease was diagnosed prior to the age of 30. The second cohort
was a stratified random sample of patients whose diagnosis was
made after the age of 30. The prevalence of diabetic retinopathy
determined between 1980 and 1982 was 50.1%. The prevalence
of PDR with high-risk characteristics was 2.2%.19,20 Younger-
onset patients with diabetes had the highest prevalence of any
retinopathy, proliferative retinopathy, and macular edema.
The 4-year incidence of developing retinopathy was 40.3%,
whereas the incidence of developing PDR with high-risk
characteristics was 2.4%. The younger-onset group had the
highest incidence rate of progression to PDR, whereas the older-
onset patients with diabetes had the highest incidence of
macular edema.
Although WESDR provides the only population-based data
for calculating rates, there are caveats common to this
epidemiologic study: The population in Wisconsin is relatively
homogeneous ethnically, consisting of few blacks, Hispanics,
and Asians. Since the study was based on previously diagnosed
individuals with diabetes, rates on type 1 may be skewed,
because up to one-half of all persons with type 2 are undi-
agnosed.21 Generalizing the WESDR data to the US population,
404 11 000 new cases of DME, 22 000 new cases of proliferative
Findings Observable Upon Dilated
Ophthalmoscopy
No apparent retinal thickening or hard exudates
in posterior pole
Some apparent retinal thickening or hard exudates
in posterior pole
Findings Observable Upon Dilated
Ophthalmoscopy *
Mild diabetic macular edema
Some retinal thickening or hard exudates in posterior
pole but distant from the center of the macula
Moderate diabetic macular edema
Retinal thickening or hard exudates approaching the
center of the macula but not involving the center
Severe diabetic macular edema
Retinal thickening or hard exudates involving the
center of the macula
DR, and 5000 new cases of blindness are estimated to occur
each year.22
RISK FACTORS
A number of risk factors have been identified for the
development and progression of diabetic retinopathy.
DURATION
The strongest predictor is duration of diabetes. In younger
type-1 group in WESDR, the prevalence of any retinopathy
was 8% at 3 years, 25% at 5 years, 60% at 10 years, and 80% at
15 years. The prevalence of PDR was 0% at 3 years and
increases to 25% at 15 years.23 In the Pittsburgh Epidemiology
of Diabetes Complications Study (PEDCS) for patients with
type-1 diabetes, longer duration of diabetes was also observed
in those with PDR than in those with no retinopathy.24
Among groups comprising mostly type-2 diabetes, retino-
pathy was more frequent earlier after the diagnosis; 23% had
retinopathy at 3 years and 2% had proliferative retinopathy.25
However, after 20 years of duration of disease, smaller
proportion of older-onset individuals had any proliferative
retinopathy than in the younger-onset group.
The incidence of developing retinopathy also increases with
increasing duration. The 4-year incidence of developing
proliferative retinopathy in the WESDR younger-onset group
increased from 0% during the first 5 years to 27.9% during
years 13–14 of diabetes. After 15 years, the incidence of
developing PDR remained stable. In a cohort study of patients
with type-1 diabetes from the Joslin Clinic, the incidence rate
(cases of PDR/1000 person-years) for development of PDR
was 1.5 in patients with less than 10 years of diabetes, rises to
30 during the second decade of diabetes, and remains at this
level for the next 25 years.26
In the WESDR older-onset group, the 4-year incidence of
developing proliferative retinopathy in those with less than
5 years of follow-up was 2%. In the Rochester, Minnesota
study, the 20-year cumulative incidence of PDR was 4% and
2% in obese and nonobese patients with type-2 diabetes,27

respectively. Among Mexican-American patients with type-2
diabetes in the San Antonio study, duration was again
significantly associated with the development of retinopathy.
HYPERGLYCEMIA
The relationship between hyperglycemia and retinopathy has
been strongly documented. In WESDR,28 the level of glycemic
control was measured by the level of hemoglobin Alc.
Among patients with IDDM less than 18 years of age, those
who developed retinopathy had higher levels of glycosylated
hemoglobin than those who did not develop retinopathy
(10.4% vs 12.1%, p < 0.001).29 No association was found in
the older age groups between Hg Alc and the development of
any retinopathy or proliferative diabetic retinopathy. In the
Pittsburgh Prospective Insulin-Dependent Diabetes Cohort
Study, seven of 62 newly diagnosed patients with IDDM
developed retinopathy. Those who developed retinopathy had
a higher level of Hg Alc than those who did not (13.0 vs 11.7%,
p < 0.05).30
Among older-onset patients with diabetes, subject in the
highest quartile of glycosylated hemoglobin are 2.5 times as
likely to have retinopathy as those in the lowest quartile.
This relationship existed even after controlling for duration
of diabetes. In the Rochester, Minnesota study of type-2
diabetes, after controlling for other factors, elevated fasting
blood sugar was associated with an increased risk of developing
diabetic retinopathy and PDR.31 Evidence for the role of
hyperglycemia in the development of retinopathy is present
also in experimental studies in animals; poorly controlled
diabetic animals are more likely to develop retinopathy.32
Several earlier clinical trial studies attempted to demonstrate
the effect of tight glycemic control but were difficult to interpret,
because of small sample sizes, short follow-ups, and failure to
adjust for patients with different levels of retinopathy.33–35 To
address these issues, the Diabetes Control and Complications
Trial (DCCT) was designed and executed in 1441 patients with
type-1 diabetes.36 The DCCT asked whether (1) intensive treat-
ment of glycemia would prevent or delay the progression of early
non-PDR (primary prevention cohort), and whether (2) intensive
glycemic control would prevent the progression of early
retinopathy to more advanced forms of retinopathy (secondary
intervention) cohort.
In the primary prevention cohort, the cumulative incidence
of a three-step increase in retinopathy level sustained over
6 months was quite similar between the two groups, during
the first 36 months. From that point on, there was a persistent
decrease in the intensive group. From 5 years onward, the
cumulative incidence was ~50% less in the intensive group.
During a mean follow-up of 6 years, retinopathy developed
in 23 patients in the intensive group and 91 in the conventional
group. Intensive therapy reduced the mean risk of retinopathy
by 76% (95% confidence interval: 62–85).
In the secondary intervention cohort, the intensive group
had a higher cumulative incidence of sustained progression
during the first year. However, by 36 months, the intensive
group had lower risks of progression. Intensive therapy reduced
the risk of progression by 54% (95% confidence interval: 39–66).
In addition, the risk for proliferative diabetic retinopathy, severe
NPDR, and laser photocoagulation was also reduced.
The protective effect of intensive therapy for retinopathy
was found to be consistent in all subgroups. It was also protec-
tive against neuropathy, microalbuminuria, and albuminuria and
reduced the development of hypercholesterolemia. Cardio-
vascular disease was reduced by 57% in the EDIC study.36a The
incidence of severe hypoglycemia was three times higher in the
intensive group which poses a significant problem. Because of
Epidemiology of Diabetic Retinopathy
the associated dangers of hypoglycemic episodes, the DCCT
recommends that therapy should be individualized for each
patient.
After 6.5 years of follow-up, the DCCT ended, and all
patients were encouraged to maintain strict control of blood
sugar. These patients are followed in the Epidemiology of
Diabetes Interventions and Complications trial (EDIC), which
includes 95% of DCCT subjects, half from each treatment
group. A total of 1294–1335 patients have been examined annu-
ally in the EDIC. Further progression of diabetic retinopathy
during the first 4 years of the EDIC was 66–77% less in the
former intensive treatment group than in the former conven-
tional treatment group.37 The benefit persists even at 7 years.38
This benefit included an effect on severe diabetic retinopathy,
including severe nonproliferative diabetic retinopathy, prolifer-
ative diabetic retinopathy, clinically significant macular edema,
and the need for focal or scatter laser therapy. The decrease in
the mean hemoglobin A1C (HbA1C) from 9% to ~8% did not
drastically reduce the progression of diabetic retinopathy in the
former conventional treatment group, nor did the increase in
HbA1c from ~7% to ~8% drastically accelerate diabetic retino-
pathy in the former intensive treatment group. Thus, it takes
time for improvements in control to negate the long lasting
effects of prior prolonged hyperglycemia, and once the biological
effects of prolonged improved control are manifest, the benefits
are long lasting. Furthermore, the total glycemic exposure of the
patient (i.e., degree and duration) determines the degree of
retinopathy observed at any one time.
In 2005, the DCCT/EDIC study group reported that
intensive treatment reduced the risk of any cardiovascular
disease event by 42% (95% CI: 9–6%; p = 0.02) and the risk
of nonfatal myocardial infarction, stroke, or death from
cardiovascular disease by 57% (95% CI, 12–79%; p = 0.02).39
For the first time, intensive treatment of hyperglylcemia was
proven to be beneficial in reducing the risk of cardiovascular
disease in persons with diabetes.
In 1998, the United Kingdom Prospective Diabetes Study
Group (UKPDS)40 compared the effects of intensive blood-
CHAPTER 37
glucose control with either sulfonylurea or insulin and
conventional treatment on the risk of microvascular and
macrovascular complications in patients with type-2 diabetes
in a randomized controlled trial. It similarly found that inten-
sive control substantially decreases the risk of microvascular
complications for these patients. There was a 25% reduction
in the risk of the ‘any diabetes-related microvascular endpoint’,
including the need for retinal photocoagulation in the intensive
treatment group compared to the conventional treatment group.
After 6 years of follow-up, a smaller proportion of patients in
the intensive treatment group than in the conventional group
had a two-step progression (worsening) in diabetic retinopathy
(p < 0.01). Epidemiologic analysis of the UKPDS data showed
a continuous relationship between the risk of microvascular
complications and glycemia, such that for every percentage
point decrease in hemoglobin A1C (e.g., 9–8%), there was a 35%
reduction in the risk of microvascular complications.
In summary, intensive treatment of type-1 diabetes delays
the onset and slows the progression of diabetic retinopathy,
nephropathy, neuropathy, hypercholesterolemia, and cardio-
vascular disease. While the DCCT also confirmed the early
worsening of retinopathy with intensive glycemic control seen
in the early treatment it showed that tight control still leads
to subsequent protection. The benefits of tight glucose
control were similarly found in patients with type-2 diabetes.
The results of both the DCCT and UKPDS show that while
intensive therapy of glucose reduces the risk of the develop-
ment and progression of diabetic retinopathy, it does not
prevent retinopathy completely. 405
 PRINCIPLES OF EPIDEMIOLOGY
BLOOD PRESSURE
Epidemiologic observations suggest that hypertension increases
the risk of diabetic retinopathy and macular edema. In WESDR,
progression of retinopathy was associated with higher diastolic
blood pressure (BP) at baseline and an increase in diastolic
BP over a 4-year follow-up period.41 Among older-onset
patients, increased diastolic BP was associated with a higher
incidence of macular edema.42
The UKPDS reported the effectiveness of tight BP
control.43,44 It randomized 1148 hypertensive patients with
type-2 diabetes to less tight (<180/105 mmHg) and tight BP
control (<150/85 mmHg) with the use of an angiotensin
converting enzyme (ACE) inhibitor or a b-blocker. Patients in
the tight control group had a 34% reduction in progression
of retinopathy and a 47% reduced risk of deterioration in
visual acuity of three lines compared with the less tight
control group. There were also reductions in deaths related to
diabetes and stroke and no clear adverse reactions to tight BP
control. There was no difference in the efficacy of ACE
inhibitors or b-blockers with regard to progression of DR in
type-2 diabetics, suggesting that BP control and not the type
of medication is most important in those with hypertension.45
The EUCLID study group investigated the effect of lisinopril
in patients with type-1 diabetes who were normotensive
and normo- or microalbuminuric.46 After 2 years, the clinical
trial showed a statistically significant 50% (p = 0.02) reduction
in retinopathy progression by at least one level; 13.2% of
159 patients on lisinopril progressed versus 23.4% of 166
patients on placebo. After adjusting for center and glycemic
control, the protective effect (0.55, p = 0.06) was similar
but not statistically significant. This borderline effect in this
study and the findings from ABCD may be due to the small
benefit of incremental lowering of BP in normotensive
patients.
IMPAIRED RENAL FUNCTION
SECTION 5
Retinopathy and nephropathy are two important microvascular
complications in diabetic patients with hyperglycemia and
hypertension.47 The Microalbuminuria Collaborative Study
Group found that retinopathy was not an independent predic-
tor of albuminuria, but the ETDRS and the WESDR study
found that the presence and severity of DR are still indicators
of the risk of developing proteinuria.48–50 Conversely,
proteinuria is a known predictor of the development of PDR
in type-1 diabetics51 and gross proteinuria is also associated
with a 95% increased risk of developing DME among persons
with type-1 diabetes.52 There is controversy as to whether this
association is due to hyperglycemia, or whether nephropathy
is an independent risk factor for diabetic retinopathy.
Use of angiotensin converting enzyme inhibitors (ACE-I)
slow the progression of nephropathy. Serum pro-renin concen-
trations have recently been directly correlated with the
severity of DR and components of the renin–angiotensin
system (RAS) have been found in the eye. These observations
imply that the use of ACE-I may also protect against the
development and progression of DR.53 However, the association
between the RAS and the development and progression of DR
is not straightforward, and initial results looking at the
influence of ACE-I upon DR in normotensive diabetics have
had equivocal results. A large randomized, double-masked,
placebo-controlled trial examining the efficacy of ACI-I (and
ACE receptor blockers) in both type-1 and -2 diabetics is cur-
rently underway. Patients with refractory retinopathy and
macular edema should have an evaluation of their renal
406 status.
HYPERLIPIDEMIA
Dyslipidemia is a known risk factor for diabetic renal disease,
but the effect of serum lipids on DR and macular edema is
still under investigation.47,54–58 There is observational evidence
that elevated lipids may increase the morbidity of macular
edema, and affect the risk of diabetic retinopathy severity.
Among insulin-using patients in WESDR, the presence of reti-
nal hard exudates was significantly associated with increased
serum cholesterol levels.59 Likewise, patients in ETDRS who
had elevated serum cholesterol or low-density lipoprotein
levels at baseline were more likely to have retinal hard exudates
than those with normal levels.60 Development of retinal hard
exudates was also 50% more likely in those patients with
elevated serum total cholesterol or triglyceride levels. Because
the risk of loss in visual acuity was correlated with the degree
of retinal hard exudates, reducing serum lipid levels in patients
with diabetes and retinopathy may be particularly important.
In addition, severe hard exudates can lead to the development
of subretinal fibrosis, a complication that can lead to perma-
nent loss of vision.61 Whether intensive lipid-lowering therapy
will reduce the severity of retinopathy or the resultant losses
in visual acuity remains to be tested in prospective trials.
VESSEL CALIBER
WESDR showed that for patients with type-1 diabetes, larger
retinal arteriolar caliber is an independent predictor of
incident retinopathy in children and adolescents.62 For type-2
diabetics, variations in retinal vascular caliber are related to a
number of systemic and ocular factors but further evaluation
may provide insight into diabetic changes as well.63
DEMOGRAPHICS
The prevalence of PDR is higher in young males, but there is
no difference in the incidence of progression between the
sexes.18 Among older diabetics, there is no sex differential. In
the WESDR younger-onset group, children 10–12 years old
compared to those less than 10 years old have a 4-year relative
risk of 3.6. This increased risk is believed to be due to the
10- to 12-year-old children passing through puberty during
the 4-year period of follow-up. Prior to puberty, children rarely
develop diabetic retinopathy regardless of the duration of
diabetes. Among type-2 diabetes, younger age at examination
was a strong risk factor for the 4-year progression of diabetic
retinopathy. PDR was not seen in those whose age at exam-
ination was older than 75 years of age.
Pima Indians have the highest prevalence and incidence of
type 2 diabetes. The 20-year cumulative incidence was 14%.26
Mexican-Americans also have a high prevalence of type-2
diabetes and diabetic retinopathy when compared with non-
Hispanic whites. They have three to five times the prevalence
of type-2 diabetes and are more likely to develop any retino-
pathy and severe retinopathy (preproliferative and proliferative
retinopathy).18,48
The epidemiology of diabetic retinopathy in Asian-Americans
is limited. In one study of Japanese-Americans living in Seattle,
the prevalence of diabetic retinopathy was reported to be 11.5%.
Additional epidemiologic studies will be needed.64
There are not many studies of the prevalence of the disease
in African-Americans, but the 9-year diabetic retinopathy inci-
dence in the Barbados Eye study, a population with similar
ancestry, was 39.6% (38.0% for minimum, 9.0% for moderate,
and 2.6% for severe/proliferative DR). Of persons with pre-
existing DR at baseline, 8.2% progressed to proliferative DR.
The CSME incidence was 8.7%. All incidences tended to

increase with diabetes duration.65 A more recent study of
whites, African-Americans, Hispanics, and the Chinese in the
multiethnic study of atherosclerosis (MESA) found race not
to be a significant risk factor in diabetic retinopathy.66
GENETICS
In studies of identical twins, the retinopathy was observed
to have similar onset and severity.67,68 HLA-DR antigens have
been examined in WESDR and at the Joslin Clinic. After con-
trolling for duration of diabetes, diastolic BP, proteinuria, and
history of hypertension, the DR4 allele was associated with
an increased risk of proliferative retinopathy in WESDR. The
Joslin Clinic study showed an increased risk of PDR in DR3 and
DR4 homozygote that was neutralized in the presence of
myopia. DR3 and DR4 heterozygote showed decreased risk.69
Other studies have shown no association between HLA
antigens and diabetic retinopathy.70,71
PREGNANCY
There are few studies on the effect of pregnancy on diabetic
retinopathy.72–74 One review reported that 8% of women with
minimal to no retinopathy had progression during their preg-
nancy; if proliferative retinopathy was present, 25% pro-
gressed.75 In one prospective study, the risk of progression of
retinopathy was 2.3 higher during pregnancy as compared to
controls.76 The role of tight control that is instigated at the
beginning of pregnancy may play an important role in this
accelerated progression of retinopathy during pregnancy.77,78
Progression of retinopathy during pregnancy was proven to be
secondary to both the tight control and to the pregnancy itself.79
ALCOHOL
In a case-control study examining the relationship between
alcohol and retinopathy of IDDM from the Joslin Clinic, the
percentage of subjects consuming alcohol was similar in those
subjects with and without proliferative diabetic retinopathy.33
In a prospective study of 296 diabetic men, the relative risk in
heavy drinkers of developing severe retinopathy was 3.5 (95%
confidence interval, 1.2–8.4).80
SOCIOECONOMIC STATUS
One case-control study reported an association between pro-
liferative retinopathy and working-class occupational status
and lower income in patients with IDDM.81 Among Mexican-
Americans in San Antonio, SES determined by Duncan’s socio-
economic index, education, and income was not associated
with retinopathy status.82 Similarly, in Oklahoma Indians
socioeconomic factors also were not found to be a risk factor
for retinopathy.36
Key Features: Medical Risk Factors for Progression of
Diabetic Retinopathy
• Hyperglycemia. Observational and clinical trials support for
beneficial effects of achieving tight glucose control for
reducing the risk of diabetic retinopathy by 35–70%
• Hypertension. Modest reduction in both systolic and diastolic
BP result in reduction in diabetic retinopathy progression
• Hyperlipidemia. Observational data to suggest that progression
of diabetic retinopathy and the development of macular edema
may result from dyslipidemia. Clinical trials are underway
• Pregnancy may increase the risk of progression of diabetic
retinopathy
Epidemiology of Diabetic Retinopathy
TREATMENT AND FUTURE DIRECTIONS
Because diabetic retinopathy is a significant source of visual
loss among diabetics, the National Eye Institute (NEI) and
other research institutions sponsor several multicenter clinical
trials to determine the optimal management regimen for
patients with diabetic retinopathy.
DIABETIC RETINOPATHY STUDY
The Diabetic Retinopathy Study (DRS) was the first multi-
center randomized controlled clinical trail in ophthalmology.83
The study addressed the question of whether photocoagulation
therapy was beneficial in patients with diabetic retinopathy in
over 1700 patients with severe nonproliferative or proliferative
diabetic retinopathy.
The study showed a reduction, after only 2 years, in the
cumulative event rate (visual acuity <5/200, at two consecutive
4-month follow-up visits) from 16.3% in untreated eyes to
6.4% in treated eyes (z = 5.5). This early benefit persisted
at 5 years; the difference between the treated group compared
to the control group was even greater (z = 11.0). In addition,
argon laser compared to xenon arc photocoagulation was
found to cause fewer side effects. The study further identified
eyes that were at high risk for severe visual loss and for which
photocoagulation was of particular benefit. The features of
these eyes can be summarized as follows: (1) neovascularization
of the disk, severity greater than standard photo 10A; (2) any
neovascularization of the disk if accompanied by vitreous
or preretinal hemorrhage; and (3) vitreous hemorrhage
accompanied by one-half disk area of neovascularization
elsewhere.84
EARLY TREATMENT DIABETIC RETINOPATHY
STUDY
The ETDRS was designed to determine when in the course of
diabetic retinopathy it is most effective to initiate scatter or
CHAPTER 37
pan-retinal photocoagulation, whether photocoagulation is
effective in the treatment of DME, and whether aspirin treat-
ment is effective in altering the course of diabetic retinopathy.
The results from the ETDRS showed that early treatment
compared with deferral of photocoagulation until high-risk
characteristics were observed is associated with a small
reduction in the incidence of severe visual loss. The 5-year
rates of severe visual loss were 2.6% in the early-treatment
group versus 3.7% in the deferral of treatment group. The
relative risk of severe visual loss in eyes randomized to early
photocoagulation compared to eyes assigned to deferral was
0.77 (99% confidence interval, 0.56–1.06). Furthermore,
scatter laser photocoagulation is probably not beneficial for
eyes with mild or moderate nonproliferative diabetic
retinopathy. However, in those with type-2 diabetes, additional
analyses of visual outcome in ETDRS patients with severe
NPDR to non-high-risk PDR suggest that the recommendation
to consider scatter photocoagulation prior to the development
of high-risk PDR is particularly appropriate for patients with
type-2 diabetes. The risk of severe vision loss or vitrectomy was
reduced by 50% in those who were treated early compared
with the deferral until high-risk PDR developed.
Regarding the management of DME, the ETDRS demon-
strated that eyes with CSME should be considered for
treatment. Eyes assigned to immediate focal photocoagulation
were about half as likely to double their visual angle (12% in
those treated versus 24% in those assigned to deferral, z = 2.58)
at 3 years.85 It also showed that aspirin did not affect the
course of retinopathy. 407
 PRINCIPLES OF EPIDEMIOLOGY
DIABETIC RETINOPATHY VITRECTOMY
STUDY
The Diabetic Retinopathy Vitrectomy Study (DRVS) was a
multicenter, randomized clinical trail that addressed the risks
and benefits of performing pars plana vitrectomy in eyes with
severe proliferative diabetic retinopathy. The DRVS was divided
into three studies. The first study, DRVS-Group N, was a
natural history study to examine the course of severe PDR
managed by conventional therapy. It showed that decreases in
visual acuity were more likely during the first year than the
second year of follow-up. The second study, DRVS-Group H,
examined the timing of vitrectomy in eyes with severe vitreous
hemorrhage of less than 6 months’ duration. Four-year
follow-up showed that the proportion of eyes with visual acuity
of 10/20 or better was higher (p <0.05) in the early vitrectomy
group than in the deferral group.86 The benefit of early
vitrectomy to patients with IDDM, but not to patients with
NIDDM, remained after 4 years of follow-up. The third study,
DRVS-Group NR, was a randomized clinical trail comparing
early vitrectomy with conventional management in eyes with
extensive, active neovascular, or fibrovascular proliferations
and useful vision. After 4 years of follow-up, the percentage
of eyes with visual acuity of 10/20 or better was 44% in the
early-vitrectomy group and 28% in the conventional-
management group (p < 0.05). The proportion with very poor
visual outcome was similar in the two groups. The advantage
of early vitrectomy tended to increase with increasing severity
of new vessels. In the group with the least severe new vessels,
no advantage of early vitrectomy was apparent. Thus, the
decision to perform early vitrectomy on eyes with severe PDR
and good vision remains complex.
PKC INHIBITORS
The relation between metabolic abnormalities and diabetic
microvascular complications has long been suspected but the
underlying pathologic mechanisms are not clearly understood.
SECTION 5
Early data suggest that protein kinase C (PKC) is an important
factor. Hyperglycemia increases diacylglycerol (DAG), an acti-
vator of PKC. There are multiple isoforms of PKC but PKC-b2
isoform is preferentially activated in tissues that usually are
damaged in diabetes such as retina and kidney. Ruboxistaurin
(RBX LY333531) is a specific inhibitor of PKC-b87 and has been
found to block vascular complications of diabetes, including
abnormalities in retinal blood flow, neovascularization, and
VEGF-mediated effects on permeability in animal models.88–90
Early phase-1 safety studies with orally administered RBX
showed that it was well tolerated with no significant adverse
effects. It also showed that while it did not prevent progression
of retinopathy to proliferative disease it was associated with
less visual loss especially in patients with clinically significant
macular edema.91,92 A phase-2 study showed that RBX did
TABLE 37.3. Published Reports of Vitrectomy for Diffuse Macular Edema
Study Eyes Vitreous Findings Previous Focal
Photocoagulation (%)
Lewis et al 10 Thickened hyaloid
Van Effenterre et al 22 Thickened hyaloid
Harbour et al 7 Thickened hyaloid
Tachi and Ogino 58 Attached hyaloid
Pendergrast 59 Thickened hyaloid
408
not delay the progression to sight threatening macular
edema.92a
CORTICOSTEROIDS
Corticosteroids, a class of substances with antiinflammatory
properties, have been demonstrated to inhibit the expression of
the VEGF gene and reduce the induction of VEGF by proin-
flammatory mediators in a time and dose-dependent manner.93,94
Multiple case series using 4 mg/0.1 mL of intravitreal triamci-
nolone suggest efficacy in the treatment of macular edema,
however, elevated intraocular pressure and cataract formation
are important side effects and their long-term effect on func-
tional outcome is still unknown.95,96 To investigate the safety
and efficacy in DME, the NIH is sponsoring a clinical trial
investigating the efficacy of intravitreal triamcinolone for
DME through the Diabetic Retinopathy Clinical Research
Network.97
ANTI-VEGF TREATMENTS
A number of anti-VEGF antibodies are under investigation.
(Macugen) Pegaptanib is a 28-base oligonucleotide ligand
(aptamer) that binds VEGF and was approved for treatment
of neovascular AMD.98 It is currently being investigated in a
phase-3 clinical trial on DME. Lucentis (ranibizumab) is an
antibody fragment directed against VEGF. There are plans to
investigate this product in the treatment of DME.99,100 Avastin,
(bevacizumab) the larger compound from which Lucentis is
derived is also being evaluated for its effect on macular edema
as well as neovascular proliferation.101
VITRECTOMY
Various literature reports suggest that vitrectomy surgery may
be helpful in eyes with refractory macular edema.102 In some
eyes, tangential tractional forces from the vitreous may be the
reason for visual loss. The role of the vitreous has been
described in cystoid macular edema secondary to uveitis, reti-
nitis pigmentosa, and aphakia.103 Diabetic eyes with macular
edema may have a lower rate of posterior vitreous separation
(20%) than those without macular edema (55%).104 Tangential
vitreomacular traction may arise from contraction of the
premacular hyaloid membrane and cause increased per-
meability of the retinal vasculature or retinal detachment. In a
recent study using optical coherence tomography to evaluate
the macula in eyes with DME and thickened posterior hyaloid,
a shallow macular traction detachment was observed in eight
of nine eyes.
These observations have led some investigators to recom-
mend vitrectomy for patients with refractory DME
(Table 37.3).105–109 In every series, eyes that improved with
vitrectomy had an intact/attached macular posterior vitreous
Resolution of >2 Lines of Snellen
Edema (%) Acuity Increase (%)
90% 80% 60%
64% 45% 86%
57% 57% 57%
19% 98% 53%
86% 73% 47%
 Epidemiology of Diabetic Retinopathy

hyaloid attachment. However, one cannot conclude that eyes
with diffuse macular edema undergoing vitrectomy had a more
favorable clinical course than eyes that did not undergo
vitrectomy. The natural history of these eyes is unknown
because the study did not provide a comparison group. The
efficacy of vitrectomy surgery will likely require investigation
with a randomized clinical trial.
SUMMARY
Diabetic retinopathy is a major cause of blindness in the
United States. It occurs in both insulin-dependent diabetes
mellitus and noninsulin-dependent diabetes mellitus. Because
NIDDM accounts for 90% of diagnosed cases of diabetes, most
cases of PDR are due to NIDDM even though the retinopathy
is more severe in IDDM.
Many risk factors for diabetic retinopathy have been studied.
The most important risk factors are duration of diabetes and
hyperglycemia. Further studies are necessary to improve these
patients’ overall health and quality of life. With these needs in
mind, the Diabetic Retinopathy Clinical Research Network97
was formed in September 2002 and is funded by the NEI. It is
a collaborative network to facilitate the identification, design,
and implementation of multicenter clinical research initiatives
focused on diabetes-induced retinal disorders. It currently
includes over 150 participating sites (offices) with over 500
physicians throughout the United States and will likely head
significant advances in the near future.

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CHAPTER 37
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411
 CHAPTER
38 Epidemiology of Age-related Macular Degeneration
Johanna M. Seddon and Lucia Sobrin
Age-related macular degeneration (AMD) is the leading cause of
irreversible blindness in older individuals in all developed
countries around the world.1,2 It can adversely affect activities of
daily living, rendering it more difficult or impossible to read,
write, and drive, and thus forcing many individuals in their retire-
ment years to lose their independence. The prevalence of AMD
is increasing as the proportion of our elderly population rises.
The dry or nonexudative forms of this disease, which com-
prise about 85% of the cases, are generally not reversible,
although rates of progression to more advanced disease can be
altered. For the remaining 15% of cases with neovascular or wet
disease, new and more effective treatment strategies have been
developed, some of which can improve vision. The established
demographic risk factors including increasing age and a family
history of the disease are not modifiable. Possible methods to
decrease the incidence of this disease to date are to refrain from
cigarette smoking, maintain a normal weight, and get adequate
exercise.3–6 Nutritional factors and eating a healthy diet are also
important.7–10 A multivitamin/mineral supplement reduces risk
of progression by 25% over five years, for individuals with
intermediate disease or advanced disease in one eye. Foods rich
in lutein and zeaxanthin and omega-3 fatty acids are also poten-
tially beneficial. Genetic variants which increase susceptibility
to AMD have been reported.
This chapter reviews the classification and definition of
macular degeneration, its frequency, and the known and
potential environmental and genetic factors associated with the
occurrence of this prevalent condition.
CLASSIFICATION AND DEFINITION
Macular degenerative changes have typically been classified into
two clinical forms: dry or wet; and the latter form is also called
exudative. Both types can lead to visual loss. In the dry form,
visual loss is usually gradual. Ophthalmoscopy reveals yellow,
subretinal deposits called drusen or retinal pigment irregularities
including hyperpigmentation or hypopigmentation changes.
Each of these signs can be further subdivided into various
categories according to the number and size of the lesions.
Drusen, which become confluent, can evolve into drusenoid
retinal pigment epithelial detachments; many of these lesions
progress to geographic atrophy. Geographic atrophy can involve
both the central and non-central regions of the macula. In the
wet or exudative form, vision loss can appear to occur suddenly,
when a choroidal neovascular membrane leaks fluid or blood
into the subpigment epithelial or subretinal space. Serous
retinal pigment epithelial detachments often, but not always,
advance to the neovascular stage. Late AMD includes two
advanced forms of AMD usually associated with visual loss:
geographic atrophy and neovascular disease. This phenotypic
heterogeneity has led to the use of various definitions of AMD,
and, as a result, difficulties with comparisons among studies.
It is important for investigators to standardize definitions of
a disease and its subtypes in order to enhance comparability and
to promote collaborative efforts.11 The Age-Related Eye Disease
Study (AREDS) enrolled participants into four groups ranging
from no disease (Category 1) to late stages (Category 4), which
included central atrophy based on macular appearance and
visual acuity, neovascular disease, as well as AMD due to visual
loss without these late stages.12 The Clinical Age-Related
Maculopathy Grading System (CARMS) classifies individuals
into five groups, with Grade 4 defined as central or non-central
geographic atrophy, and Grade 5 as neovascular disease, and
there are no visual acuity criteria.13 The classification of age-
related maculopathy will no doubt change in the future as
genetic and epidemiologic studies provide further insight into
the pathogenesis of this disease, and subcategories of AMD are
better defined.
PREVALENCE
Several population-based studies have provided information on
the prevalence of AMD: The National Health and Nutrition
Examination Survey (NHANES)14,15 the Framingham Eye Study
(FES),16 the Chesapeake Bay Watermen Study,17 the Beaver Dam
Eye Study (BDES),18 as well as studies outside the United States
including the Rotterdam Study in the Netherlands,19 and the
Blue Mountains Study in Australia.20 Prevalence rates are
quite variable for all types of AMD combined, because of dif-
ferences in definitions of AMD, but are more consistent for
“advanced AMD.”
The BDES18 found that the early forms are much more
common than the late stages of ARM, and both types increase
in frequency with increasing age. The prevalence of late ARM
was 1.6% overall and the prevalence of late ARM rose to 7.1%
in persons who were 75 or older. Total prevalence of AMD in
the USA was also estimated in 2004 using pooled findings from
seven large population-based studies both inside and outside
the USA, and applying those prevalence rates to the US
population.21 This meta-analysis by the Eye Diseases
Prevalence Group calculated the overall prevalence of
neovascular AMD and/or geographic atrophy to be 1.47% of the
US population aged 40 years or older. This is more than 1.75
million individuals affected with advanced AMD in the USA,
with an estimated increase of 50% to 2.95 million by 2020.
INCIDENCE
Incidence rates have been estimated in various populations.22,23
The FES used the age-specific prevalence data to estimate 5-year 413
 PRINCIPLES OF EPIDERMIOLOGY
incidence rates of AMD, according to the definition of AMD in
that study. These estimates were 2.5%, 6.7%, and 10.8% for
individuals who were 65, 70, and 75 years of age, respectively.22
The BDES determined the 5-year cumulative incidence of
developing early and late AMD in a population of 3583 adults
(age range 43 to 86 years).23 Incidence of early AMD increased
from 3.9% in individuals aged 43 to 54 years to 22.8% in
persons 75 years of age and older. The overall 5-year incidence
of late AMD was 0.9%. Persons 75 years of age or older had a
5.4% incidence rate of late AMD. More recently, the
Copenhagen City Eye Study found the 14-year incidences of
early and late AMD to be 31.5% and 14.8%, respectively, in 946
patients who were 60 to 80 years old at baseline.24
PSYCHOSOCIAL IMPACT
Patients with visual loss due to AMD and other medical
problems often report AMD as their worst medical problem and
have a diminished quality of life.25 In a study of well-being,
patients with AMD had lower scores than did those with
chronic obstructive pulmonary disease and acquired immune
deficiency syndrome.26 The AREDS study showed that
progression to advanced AMD had a significant impact on
vision-related quality of life, as did loss in vision of at least three
lines.27 The largest impact on vision-targeted quality of life
occurred in persons who lost vision in both eyes. Such an
impact on the patient’s psychosocial well-being and activities of
daily living underscores the growing importance of this disease
on the expanding elderly population.
SOCIODEMOGRAPHIC RISK FACTORS
AGE
All studies demonstrate that the prevalence, incidence, and
progression of all forms of AMD rise steeply with increasing
age.16–18,23 By 75 years of age and older, 7.1% of the population
have late age-related maculopathy (ARM) or AMD compared
SECTION 5
with 0.1% in the age group of 43 to 54 years and 0.6% among
people aged 55 to 64.
SEX
In the Beaver Dam Eye Study, while controlling for age, there was
no overall difference in the frequency of AMD between men and
women.18 However, exudative macular degeneration was more
frequent in women 75 years or older compared with men of that
age (6.7% vs. 2.6%, P = .02).18 A similar finding was observed in
the FES.16 In NHANES III, men, regardless of race and age, had
a lower prevalence of AMD than did women.15 Incidence rates
within the Beaver Dam population also suggest a gender
difference. After adjusting for age, women 75 years of age or older
had approximately twice the incidence of early ARM compared
with men of that age.23 In the Blue Mountains Eye Study, there
were consistent, although not significant, sex differences in
prevalence for most lesions of ARM, with women having higher
rates for AMD and soft indistinct drusen but not for retinal
pigmentary abnormalities.20 No gender differences were seen in
the Rotterdam Study.19 Residual confounding by age in the broad
age category “75 and older” may partially explain these
differences. However, true gender differences may exist, and
further research is needed to confirm and expand these findings.
RACE/ETHNICITY
In the NHANES III study,15 a higher frequency of early ARM
414 was reported in whites compared with blacks. Rates for late
stages were too low to make statistical comparisons. In the
Baltimore Eye Survey, AMD accounted for 30% of bilateral
blindness among whites and for 0% among blacks.28 Data from
a population-based study in Barbados, West Indies,29 revealed
that age-related macular changes occurred commonly but at a
lower frequency than in predominantly white populations in
other studies. The prevalence of ARM was also compared by
geographic region and ethnicity in Southern Colorado and
Central Wisconsin.30 Late stage AMD was significantly less
frequent among Hispanics in Colorado compared with non-
Hispanic whites in Beaver Dam (odds ratio [OR] of 0.07; 95%
confidence interval [CI] of 0.01 to 0.49). Overall, the literature
to date suggests that early ARM in blacks and Hispanics is less
common than among non-Hispanic whites, and advanced stages
of AMD are much less frequent in these groups compared with
non-Hispanic whites.
EDUCATION AND SOCIOECONOMIC STATUS
Persons with higher levels of education had a slightly reduced
risk of neovascular AMD in the Eye Disease Case-Control
Study (EDCCS), but the association did not remain statistically
significant after multivariate modeling.31 In the Beaver Dam
Eye Study, no association was found between education,
income, employment status, marital status, and the incidence
of maculopathy.32 Furthermore, no associations were noted in
another case-control study33 or in the FES,16 although different
definitions of macular degeneration were used in those reports.
Education was inversely related to AMD in a case-control study
within the AREDS population.34
OCULAR RISK FACTORS
IRIS COLOR
Investigators have postulated that higher levels of ocular
melanin may be protective against light-induced oxidative
damage to the retina. To date, the literature is inconclusive
about the relationship between iris color and AMD. Darker
irides have been found to be protective in some33,35–36 but not
other studies.31,37–39 Differences in studies may be related partly
to the use of different definitions of disease, different number
and types of other risk or protective factors evaluated, and
residual confounding by ethnicity in some studies.
REFRACTIVE ERROR
Several case-control studies have shown an association between
AMD and hyperopia.31,33 The potential problem with some of
these studies is the setting (ophthalmology practices) in which
they were conducted. Because ophthalmology practices tend to
contain higher percentages of myopic patients, controls selected
from such practices would tend to have a higher prevalence of
myopia than would the general population. However, the
population-based Rotterdam Study also showed an association
between hyperopia and both incident and prevalent ARM.40
This association therefore might implicate structural and
mechanical differences which render some eyes predisposed to
maculopathy.
CUP:DISC RATIO
The EDCCS demonstrated that eyes with larger cup:disc ratios
had a reduced risk of exudative AMD. This effect persisted even
after multivariate modeling,31 adjusting for known and
potential confounding factors. Whether this finding, which is
consistent with the association with hyperopic refractive error
 Epidemiology of Age-Related Macular Degeneration
mentioned earlier, is meaningful in terms of the mechanisms
associated with the development of AMD, awaits further study.
LENS OPACITIES
The literature has not shown a consistent relationship between
the presence of cataract and AMD. FES investigators found no
relationship,41 whereas data from the NHANES Study did
support a relationship between AMD and lens opacities.42 In
the Beaver Dam Eye Study, nuclear sclerosis was associated
with increased odds of early ARM (OR of 1.96; 95% CI of 1.3 to
3), but not of late ARM. 43
On the other hand, investigators have postulated that
cataract surgery may increase the risk for AMD, perhaps
because the cataractous lens can block damaging ultraviolet
light. Inflammatory changes after cataract surgery may also
increase risk of progression of early to late ARM. In the
NHANES, aphakia was associated with a twofold increased risk
of AMD (OR of 2; 95% CI of 1.44 to 2.78).14 Another study
evaluated 47 patients with bilateral, symmetric, early AMD,
who underwent extracapsular cataract extraction with
intraocular lens implantation in one eye. Progression of AMD
occurred more often in the surgical eyes compared with the
fellow eyes.44 In the Beaver Dam Eye Study, previous cataract
surgery at baseline was associated with a statistically significant
increased risk of development of late ARM (OR of 2.8; 95% CI
of 1.03 to 7.6).37 The risk for late ARM with a history of
cataract surgery at baseline persisted at the ten year follow-up,
with a risk ratio of 3.81 (95% CI 1.89–7.69).45 The Blue
Mountains Eye Study found a three fold risk in the 10-year
incidence of late stage ARM in nonphakic eyes when compared
with phakic eyes.46 This effect also persisted in an analysis of
pooled findings from the BDES and the Blue Mountains Eye
Study.47 A study of postmortem eyes was suggestive of an
increase in disciform scars in eyes with cataract extraction and
implantation of an intraocular lens.48
However, an analysis of AREDS data showed that there
was no correlation between cataract surgery and advanced
neovascular AMD, although there was a small risk of
advancement of geographic atrophy.49 For geographic atrophy,
cataract surgery was associated with a 50% increased risk, and
the effect was marginally statistically significant (RR of 1.47, CI
of 0.99 to 2.17). Further matched-pair analyses were under-
taken to validate these initial findings comparing 342 patients
with intermediate or advanced AMD who had cataract surgery
after study entry and before developing AMD and those who did
not have surgery. There was no increased risk of progression to
neovascular AMD among those who had cataract surgery.
ENVIRONMENTAL, MEDICAL, AND
NUTRITIONAL FACTORS
SMOKING
The preponderance of epidemiologic evidence indicates a strong
positive association between both wet and dry AMD and
smoking. Two large prospective cohort studies have evaluated
the relationship between smoking and wet AMD and dry AMD
associated with visual loss.3,50 In the Nurses’ Health Study,
women who currently smoked 25 or more cigarettes per day had
a relative risk (RR) of 2.4 (95% CI of 1.4 to 4), and women who
were past smokers had an RR of 2.0 (95% CI of 1.2 to 3.4)
for AMD compared with women who never smoked.3 Risk
increased as pack-years of smoking increased indicating a dose-
dependent relationship. Risk for AMD remained elevated for
many years after smoking cessation. Results were consistent for
various definitions of AMD, including wet AMD, dry AMD
with different levels of visual loss, and for different definitions
of smoking. Among women, it was estimated that 29% of the
AMD cases in that study could be attributed to smoking.3 These
results were supported by a study among men participating in
the Physicians’ Health Study,50 suggesting that smoking is an
important, independent, avoidable cause of AMD.
Recently reported pooled data on 9523 adults from three
populations living in Australia, The Netherlands, and the
United States support the body of evidence indicating that
smoking is related to an increased risk of incident AMD.4 In the
US Twin Study of Age-Related Macular Degeneration current
smokers had a 1.9-fold increased risk of AMD while past
smokers had about a 1.7-fold increased risk.5
Mechanisms by which smoking may increase risk of develop-
ing macular degeneration include its adverse effect on blood
lipids by decreasing levels of HDL and increasing platelet
aggregation and fibrinogen, increasing oxidative stress and lipid
peroxidation, and reducing plasma levels of antioxidants.3
BODY MASS INDEX
Evidence for a role of increased body mass index (BMI) on the
development and progression of AMD is growing. In one
prospective cohort study of the rate of progression to advanced
AMD, higher BMI increased the risk for progression to the
advanced forms of AMD.6 Relative risk was 2.35 (95%
confidence interval [CI], 1.27-4.34) for a body mass index of at
least 30, and 2.32 (95% CI, 1.32-4.07) for a body mass index of
25 to 29, relative to the lowest category (<25) after controlling
for other factors (P = .007 for trend). In that study there was
also about a two-fold increased risk for progression to advanced
AMD for abdominal obesity as measured by both waist
circumference and waist-hip ratio.6 An observational analysis of
a randomized clinical trial found that a significant association
between late AMD and greater body mass index (1.05 per
1 kg/m, 1.001 to 1.10, P = .05)].51 In AREDS, greater BMI was
significantly associated the incidence of central geographic
atrophy (OR, obese vs. nonobese, 1.93; 95% CI, 1.25-2.65).52 In
CHAPTER 38
the Physicians’ Health Study, the relationship of BMI with dry
ARM was J-shaped, with both the leanest individuals and obese
individuals at increased risk.53 In a French population-based
study, obese subjects had a 2.29-fold (CI: 1.00-5.23) and 1.54-
fold (CI: 1.05-2.26) increased risk of late AMD and pigmentary
abnormalities in comparison with lean subjects.54
CARDIOVASCULAR DISEASES
AMD and cardiovascular disease may have common
antecedents.55 The presence of atherosclerotic lesions,
determined by ultrasound, was examined in relation to risk of
macular degeneration in a large population-based study
conducted in the Netherlands.56 Results obtained from this
cross-sectional study showed a 4.5-fold increased risk of late
macular degeneration (defined as geographic atrophy or
neovascular macular degeneration as determined by grading of
fundus photographs) associated with plaques in the carotid
bifurcation and a twofold increased risk associated with plaques
in the common carotid artery. Lower-extremity arterial disease
(as measured by the ratio of the systolic blood pressure level of
the ankle compared with the arm) was also associated with a
2.5 times increased risk of AMD. In addition, a case-control
study found a relationship between AMD and history of one or
more cardiovascular diseases.33 The NHANES-I study reported
a positive association between AMD and cerebrovascular
disease, but positive associations with other vascular diseases
did not reach statistical significance.57 A Finnish study reported
a significant correlation between the occurrence of AMD and 415
 PRINCIPLES OF EPIDERMIOLOGY
the severity of retinal arteriosclerosis.58 However, some studies
found that persons who reported a history of CVD did not have
a significantly greater risk of AMD.31,58,59
BLOOD PRESSURE AND HYPERTENSION
The role of blood pressure in the etiology of AMD remains
unclear. There was a small, statistically significant relationship
between AMD and systemic hypertension in two cross-
sectional population-based studies.57,60 One case-control study
found that persons with AMD were significantly more likely to
be taking antihypertensive medication.61 Also, a significant
relationship was found between AMD and diastolic blood
pressure measured several years before the eye examination
in the FES.62 The Beaver Dam Study reported that systolic
blood pressure was associated with incidence of RPE
depigmentation.59 In the Macular Photocoagulation Study,
there was an increased incidence of exudative AMD associated
with hypertension, in the second eye of individuals with
exudative AMD in one eye at baseline (relative risk of 1.7; 95%
CI of 1.2 to 2.4).63
Cross-sectional56,58 and case-control studies,31 as well as one
prospective study59 in which duration of hypertension was not
taken into account, did not show an increased risk of late AMD
associated with current hypertension or systolic or diastolic
blood pressure. However, in the EDCCS, a trend for an
increased risk associated with higher systolic blood pressure
was evident.31 Evidence suggests a possible mild to moderate
association between elevated blood pressure and AMD.
CHOLESTEROL LEVELS
There is some evidence linking cholesterol level to AMD, but
not all results are consistent. The EDCCS reported a statistically
significant four-fold increased risk of exudative AMD associated
with the highest serum cholesterol level (>4.88 mmol/L), and a
twofold increased risk in the middle cholesterol level group,
compared with the lowest cholesterol level group, controlling
SECTION 5
for other factors.31 No significant association was noted between
AMD and cholesterol level in the FES.62 A study of plasma
cholesterol and fatty acid levels found no difference between
65 cases of exudative AMD and control pairs.64 The Beaver
Dam Study found that early AMD was related to low total
serum cholesterol levels in women and men older than age 75.
Furthermore, men with early AMD had higher high-density
lipoprotein-cholesterol (HDL-C) and lower total cholesterol/
HDL-C ratios.59,65 Slightly, but not significantly, increased risk
of wet AMD was seen with increasing triglyceride level in the
EDCCS,31 but this finding was not confirmed in the Rotterdam
Study56 or the Beaver Dam Study59 (both of which had small
numbers of exudative AMD cases and therefore limited power).
In a case-control study to assess the risk of AMD in patients
who were taking statins, short term and medium term statin
use was not associated with a decreased risk of AMD.66
DIETARY FAT INTAKE
Dietary fat intake was associated with a slightly elevated risk of
exudative AMD in the Dietary Ancillary Study of the EDCCS.
This association was primarily due to vegetable fat rather than
animal fat. For omega-3 fatty acid intake, an inverse associ-
ation, or a protective effect with higher intake, was found in the
multivariate model controlling for other factors.67,68 A pro-
spective study of dietary fat intake and AMD found that total
fat intake was positively associated with AMD.69 A high intake
of fish was associated with a 35% lower risk of AMD (risk ratio
416 of 0.65, 95% CI of 0.46 -0.91). In the Beaver Dam Study,
persons in the highest quintile of saturated fat and cholesterol
intake compared with the lowest quintile had 80% and 60%
increased risk, respectively, for early AMD.70
A prospective cohort study also supported the role of dietary
lipids. Higher total fat intake increased the risk of progression to
the advanced forms of AMD, with a risk ratio of 2.9.71 Saturated,
monounsaturated, polyunsaturated, and transunsaturated fats
increased the likelihood of progression for the highest fat-intake
quartile relative compared to the lowest fat-intake quartile, after
controlling for other factors Higher fish intake was associated
with a lower risk of AMD progression among subjects. Similar
results regarding fish intake were seen in a twin study.5 Increased
intake of fish reduced risk of AMD, particularly for two or more
servings per week. Dietary omega-3 fatty intake was inversely
associated with AMD comparing the highest vs lowest quartile.
Reduction in risk of AMD with higher intake of omega-3 fatty
acids was seen primarily among subjects with low levels (below
median) of linoleic acid intake, an omega-6 fatty acid.
There is consistent evidence that omega-3 fatty acids may
reduce risk of AMD from multiple studies with different designs
and different study populations. AREDS 2 will test this
hypothesis in a randomized trial.
DIABETES AND HYPERGLYCEMIA
Many studies have investigated the relationship between
diabetes and/or hyperglycemia and AMD, and most have found
no significant relationships.31,33,58,62,63 The Beaver Dam Study
found no overall association between early or late AMD and
diabetes or glycosylated hemoglobin, a measure of glycemia,
although a positive association was found between glycosylated
hemoglobin and exudative AMD only in older men. However,
sample sizes in these subgroup analyses were very small.72
Based on the scant literature to date, the association between
hyperglycemia or diabetes and AMD is uncertain. Difficulties
with these studies include the uncertainty of diagnosing AMD
in the presence of diabetic retinopathy and many studies of
AMD exclude persons with diabetic retinopathy.
REPRODUCTIVE AND RELATED FACTORS
The EDCCS showed a marked decrease in the risk of
neovascular AMD among postmenopausal women who used
estrogen therapy.31 The odds of neovascular AMD were 0.3
(95% CI of 0.1 to 0.8) in current users of estrogen therapy.
Former use of estrogen therapy was also associated with reduced
risk (OR of 0.6; 95% CI of 0.3 to 1). In an ancillary study to the
Women’s Health Initiative, 4262 women sixty-five years and
older were randomized to treatment with conjugated equine
estrogens (CEE), CEE with progestin, and placebo.73 Treatment
with CEE alone or CEE and progestin did not affect early or late
stage AMD. They did find that the treatment with CEE and
progestin may reduce the risk of soft drusen or neovascular
AMD. Snow et. al. found that women with age-related
maculopathy (ARM) who had used postmenopausal estrogen
therapy in the past had significantly lower odds of advanced
ARM than nonusers, after controlling for other risk factors (OR
of 0.5, 95% CI of 0.30 to 0.98).74 No relationship was found in
the Beaver Dam Study between years of estrogen therapy and
exudative AMD.75 However, there were few cases of late AMD
in that study (n = 49). The Blue Mountains Eye Study reported
no relationship between AMD and hormone replacement
therapy or early menopause, although there was a small
decrease in risk of early ARM with increasing number of years
between menarche and menopause.76
A nested case-control study within the Rotterdam Study 77
showed that risk of AMD was almost twice that among women
 Epidemiology of Age-Related Macular Degeneration
who had undergone menopause before 45 years of age compared
with those who had their menopause at 45 years of age or later
(OR of 1.9; 95% CI of 1 to 3.8). A protective effect of estrogen
on AMD cannot be ruled out, and further research is warranted.
SUNLIGHT
The literature to date regarding the association between
sunlight exposure and AMD is conflicting. Overall, the data do
not support a strong association between ultraviolet (UV)
radiation exposure and risk of AMD, although a small effect as
well as an adverse effect of blue light exposure is possible.
In a study of 838 Maryland Watermen,78 sunlight exposure
was assessed by detailed interview and field measurements. A
modest, positive relationship between blue light or visible light
exposure over the preceding 20 years and risk of advanced AMD
was seen, with an odds ratio of 1.36 (95% CI of 1 to 1.85) for
each 0.1 increase in “Maryland Sun-Years.” No adverse effects
were observed for UV-A or UV-B exposure. However, only eight
men had advanced AMD (geographic atrophy or exudative
disease). In the Beaver Dam Eye Study,79 no relationship was
seen between advanced AMD or early ARM and UV-B exposure,
but the effects of UV-A or blue light were not assessed. A
twofold increased risk of advanced AMD was associated with
increased time spent outdoors in the summer. With ten years of
follow-up, participants who experienced more than ten severe
sunburns during their youth were more likely than those who
experienced one or no burns to develop drusen with a 250-
micron diameter or larger.80 The risk ratio was 2.52 (95% CI of
1.29–4.94).
The EDCCS also evaluated crude measures of sunlight
exposure.31 No association was seen between exudative AMD
and leisure time spent outdoors in summer. Advanced AMD
was not associated with leisure time spent outdoors in the
winter, occupational sunlight exposure, or the use of sunglasses
or hats with brims. In an Australian case-control study,81 a
greater proportion of people with advanced AMD reported
higher sensitivity to sunburn compared with the control group.
The controls actually had greater median hours of sun exposure
than did the cases. The authors suggested that sun-sensitive
individuals may be at increased risk of AMD, although they
tend to avoid sun exposure.
Conflicting results in these studies exemplify the difficulties
encountered with studying this complex exposure. These include
challenges in measurement of acute and chronic lifetime
exposure and the effect of potential confounding variables, such
as sun sensitivity and sun-avoidance behaviors.
ANTIOXIDANTS
Antioxidants including vitamin C (ascorbic acid); vitamin E
(alpha-tocopherol); and the carotenoids, including alpha-
carotene, beta-carotene, cryptoxanthin, lutein, and zeaxanthin
may be relevant to AMD due to their physiologic functions and
the location of some of these nutrients in the retina. Trace
minerals like zinc, selenium, copper, and manganese may also
be involved in antioxidant functions of the retina.82
Theoretically, antioxidants could prevent oxidative damage to
the retina, which could, in turn, prevent development of
AMD.83 Damage to retinal photoreceptor cells could be caused
by photo-oxidation or by free radical-induced lipid
peroxidation.84 This could lead to impaired function of the
retinal pigment epithelium and, eventually, to degeneration
involving the macula. The deposit of oxidized compounds in
healthy tissue may result in cell death because they are
indigestible by cellular enzymes.83 Antioxidants may scavenge,
decompose, or reduce the formation of harmful compounds.
In the Dietary Ancillary Study of the EDCCS, an inverse
association between exudative AMD and dietary intake of
carotenoids from foods was observed.7 A high intake of green
leafy vegetables containing the carotenoids lutein and
zeaxanthin was associated with a reduction in the risk of
exudative AMD. Intake of vitamin C was associated with a
small but nonsignificant reduction in risk. Intake of vitamin A
or vitamin E was not associated with a reduction in risk.7 In
this cohort, persons with higher serum levels of carotenoids
(sum of serum lutein/zeaxanthin, beta-carotene, alpha-
carotene, cryptoxanthin, and lycopene levels) had a greatly
reduced risk of exudative AMD.8 Persons with higher individual
serum levels of lutein/zeaxanthin, beta-carotene, alpha-
carotene, and crypotxanthin had reduced risks of exudative
AMD. The study did not find a statistically significant
protective effect for serum levels of vitamin C, vitamin E, or
selenium individually, but when these were combined into an
antioxidant index with carotenoids, there was a significant
reduction in risk of exudative AMD with increasing levels of the
index.
A cross-sectional study using NHANES-I data examined the
relationship between the prevalence of any AMD and vitamins
A and C intake. A weak protective effect was seen with
increased consumption of fruits and vegetables rich in vitamin
A.57 The Beaver Dam Study found no effect of supplemental
antioxidant vitamins alone or in combination on risk of early or
late ARM.85 However, in a case-control study nested within that
study, a low serum level of one carotenoid, lycopene, was
associated with presence of any AMD.9 Another study reported
a protective effect for any AMD among those who had higher
serum vitamin E and among those who had higher values for an
antioxidant index of vitamins C, E, and beta-carotene, but no
protective effect was seen for vitamin supplementation.86 A
study of plasma levels of vitamins A and E and five carotenoids
found no relationship with exudative AMD in 65 case-control
pairs.64 Increased blood levels of carotenoids and antioxidant
vitamins were also related to decreased risk of exudative AMD
in other reports.87–88
CHAPTER 38
Overall, results from observational studies suggest that diets
rich in antioxidant-rich fruits and vegetables are related to
a lower risk of exudative AMD. A prospective follow-up of
women in the Nurses’ Health Study and men in the Health
Professionals Follow-up Study found suggests a protective role
for fruit intake on the risk of neovascular ARM.89 The
Carotenoids in Age-Related Eye Disease Study (CAREDS) found
that a diet rich in lutein plus zeaxanthin may protect against
intermediate AMD in healthy women younger than 75 years
old.90 In the Rotterdam Study, a high dietary intake of beta
carotene, vitamins C and E, and zinc was associated with a
substantially reduced risk of AMD in elderly persons.91
A small randomized trial demonstrated less visual loss due to
AMD and less accumulation of drusen in the group of 97
patients assigned to high-dose zinc supplementation, compared
with 84 patients in the placebo group.92 However, another small
randomized trial found that zinc supplementation had no short-
term effect on the course of disease in 112 patients with wet
AMD.93 The Beaver Dam Study found a weak protective effect
of zinc intake on early ARM.85 The EDCCS did not find any
significant relationships between serum zinc levels or zinc
supplementation and risk of exudative AMD.31 A prospective
study of zinc intake, moderate zinc intake, either in food or in
supplements, was not associated with a reduced risk of AMD.94
A supplement containing antioxidant vitamins and minerals
has been shown to reduce the risk of AMD in a large
randomized clinical trial, the Age-Related Eye Disease Study,
sponsored by the National Eye Institute.10 In this study,
supplementation with a high dose of zinc plus antioxidant 417
 PRINCIPLES OF EPIDERMIOLOGY
vitamins C and E and beta-carotene reduced the incidence and
progression of AMD, but not lens opacities. Based on evidence
from observational studies regarding the potential protective
effects of lutein7,88 and omega-3 fatty acids,5,67–71 a new clinical
trial called AREDS 2 is underway to test supplements
containing these nutrients.
ALCOHOL INTAKE
Studies that have examined the relationship between AMD
and alcohol consumption have yielded mixed results. In the
EDCCS, no significant relationship between alcohol intake
and exudative AMD was noted in univariate analyses.31 In a
separate multivariate analysis, alcohol intake appeared to be
associated with a decreased risk of disease in the highest
quartile of intake compared with nondrinkers.95 Another case-
control study found a nonsignificant association between
current daily alcohol intake and AMD, with a suggestion of a
nonlinear trend of higher risk of AMD in persons who had five
drinks or more per day and a slightly lower risk in persons who
had one or two drinks per day compared with nondrinkers.96 In
a case-control study using NHANES-I data, moderate wine
consumption was associated with decreased risk of developing
AMD, although the analysis did not control for the potential
confounding effects of smoking.97
In population-based cross-sectional studies, there is conflicting
evidence for an association between alcohol and AMD. The Los
Angeles Latino Eye Study found heavy alcohol consumption,
particularly beer, was associated with a greater risk of having
advanced AMD (OR 2.9).98 The Beaver Dam Study found a
slightly increased risk for retinal pigment degeneration in persons
who consumed beer in the past year.99 With the ten years of
follow-up, they found that heavy drinking appears to be related to
an increased risk of late ARM, although the exposure and outcome
were infrequent and the effect was based on few exposed cases (RR
of 6.94; 95% CI of 1.85 to 26.1).100 Neither the Beaver Dam Study
nor the Blue Mountains Eye Study found an increased risk for
AMD related to total alcohol intake. Two prospective studies of
SECTION 5
alcohol consumption and the risk of age-related macular
degeneration have failed to find any association between alcohol
intake and risk of AMD.101,102 In summary, evidence suggests that
alcohol intake has little effect on AMD, although the possible
influence of heavy intake requires further study.
GENETICS
It is now known that genetic factors play a role in the etiology
of AMD. The evidence leading up to recent discoveries includes
the demonstration of familial aggregation,103–104 large twin
studies,105,106 a case-control study,33 and a segregation
analysis.107 In one study,103 first-degree relatives of cases with
AMD were compared with first-degree relatives of control
subjects without AMD. The prevalence of medical record
confirmed age-related maculopathy was significantly higher
among first-degree relatives of all case probands (23.7%)
compared with first-degree relatives of control probands (11.6%)
with an age- and sex-adjusted OR of 2.4 (95% CI of 1.2 to 4.7).
When relatives of cases with exudative disease were evaluated,
the OR was 3.1 (95% CI of 1.5 to 6.7) for relatives of cases
compared with relatives of controls. These results suggested
that macular degeneration has a familial component and that
genetic or shared environmental factors, or both, contribute to
its development. In another study,104 20 of 81 siblings of
affected patients had AMD compared with only 1 of 78 siblings
of control subjects. These studies supported the familial
aggregation of this disease. Additional evidence for a genetic
418 component was suggested by a segregation analysis involving
the Beaver Dam population.107 In a case-control study, cases
were twice as likely to report a family history of this disease.33
Several genetic linkage studies have also been performed to
try to identify regions of the genome that would merit further
exploration. While almost every chromosome has been impli-
cated in linkage studies for AMD, the most reproducible linkage
peaks have been on chromosomes 1q and 10q and this has been
recently confirmed in a meta-analysis.108–113 There is also some,
albeit weaker, evidence for linkage on chromosomes 2p, 3p, 4q,
12q, and 16q.108 The most promising developments in AMD
genetics research have occurred recently in the context of
association analyses. A complete review of the genetics of AMD
up to July 2006 was recently reported.114 Following is a sum-
mary of the findings.
Complement Factor H
In March 2005, three separate groups reported in Science on a
common coding variant, Y402H, in the complement factor H
(CFH) gene on chromosome 1 (1q31) that increases the risk of
developing AMD.115–117 The studies estimated the odds ratio
associated with this variant for all categories of AMD to be
between 2.45 to 3.33. The odds ratios were higher, between 3.5
and 7.4, for advanced dry and wet forms of AMD. CFH inhibits
the formation and accelerates the decay of alternative pathway
C3 convertases and serves as a cofactor for the factor-1 mediated
cleavage and inactivation of C3b.118 The Y402H single
nucleotide polymorphism (SNP) is within the CFH binding site
for heparin and C-reactive protein. Binding to these sites
increases the affinity of CFH for complement protein C3b,
which in turn increases the ability of CFH to inhibit
complement’s effects. Previous to the discovery of the strong
association between AMD and this CFH variant, the inflam-
matory cascade had already been postulated as an important
component in the pathophysiology of AMD.55,119–123 The
discovery of the risk allele lends further support to this theory.
Many studies have verified the importance of the Y402H
variant.124–133 In the Chinese population, even though the
frequency of this polymorphism is low, it was still significantly
associated with neovascular AMD.130 In Japanese patients,
while the single Y402H allele was not associated with AMD,
two haplotype blocks in CFH were associated with AMD.131
Other variants within CFH have been discovered, 124,132,133
including the noncoding variant rs1410996.132
One study has found that the G allele of ERCC6
(chromosome 10) is associated with a risk of AMD and possibly
interacts with a SNP in CFH to influence AMD suscep-
tibility.134 Another group reported that CRP (C-reactive protein)
haplotypes conferring high levels of CRP significantly increased
the effect of CFH Y402H.135
LOC387715/HTRAI
The LOC387717 locus on chromosome 10 has been the second
gene to be convincingly implicated in the risk of AMD develop-
ment. The first study reporting on this locus found the strongest
association for SNP rs10490924 within LOC387715.110 The
odds ratio for this allele was 5.03. More recently, two groups
have reported that the AMD LOC387715 signal is narrowed to
a single nucleotide polymorphism in the promoter region of
HTRA1, a serine protease gene on chromosome 10q26.136–137
Preliminary analysis of lymphocytes and retinal pigment
epithelium from three AMD patients revealed that the risk
allele was associated with elevated expression levels of HTRA1
mRNA and protein.137
Factor B (BF) and complement component 2 (C2)
The BF and C2 genes are found on chromosome 6 within the
major histocompatibility complex (MHC) class III region. They
 Epidemiology of Age-Related Macular Degeneration

act in the same pathway as CFH. An association between these
two genes and AMD has been reported.132,138 The initial finding
was that of one common risk haplotype with an odds ratio of
1.32 and two protective haplotypes with odds ratios of 0.36 and
0.45.138 The first protective haplotype contained the L9H
variant of BF and the E318D variant of C2 while the second
protective haplotype contained the R32Q variant of BF and a
variant in intron 10 of C2.
Complement Factor 3 (C3)
Complement factor 3 is the component of the complement
pathway which has been most recently associated with age-
related macular degeneration. The common functional
polymorphism rs2230199 (Arg80Gly) in the C3 gene was
independently reported in two association studies.139,140
Other Potentially Implicated Genes
Prior to the discovery of the CFH gene, there were several genes
that were the subject of repeated studies in AMD. ABCA4 is
mutated in Stargardt disease, a hereditary macular dystrophy, and
codes for the ATP-binding transporter protein involved in
photoreceptor vitamin A transport. Several studies showed an
association with ABCA4 variants, including variants G1961E
and D2177N, and risk of AMD.141–142 while others have
not.143–145 ABCA4 may be involved in a small number of AMD
cases. There have also been mixed results for the apolipoprotein
E (APOE) gene.146–151 The most consistent effect in this gene has
been a protective effect of the epsilon 4 allele, although the effect
has not reached statistical significance in some studies.147–148
Other genes with mixed results include: ELOVL4 (another gene
implicated in Stargardt’s disease), ACE (angiotensin-converting
enzyme), and the fibulin 6 gene (which plays a role in the stability
of extracellular matrix [ECM] complexes).152–156
Many candidate genes which have been investigated because
of a biological hypothesis for their involvement in AMD have
been associated with AMD in one or two studies. VEGF,
vascular endothelial growth factor, is one such gene; it is located
on chromosome 6p and is definitively involved in the
studies.159 The TLR4 (toll-like receptor 4) gene on chromosome
9 is involved in inflammation pathways and phagocytosis of
photoreceptor outer segments by the RPE and one study found
a relationship with AMD.160 All of these associations remain to
be independently replicated.
Gene-Environment Interactions
Since the discovery of the association between the CFH gene
and AMD, there have been a few epidemiological studies
examining the relationships between environmental risk
factors, the Y402H polymorphism and AMD. One group from
the UK found that the association between Y402H and both
geographic atrophy and choroidal neovascularization was
similar in smokers and nonsmokers, although heavier smokers
with the CC genotype appeared to be at particular risk.161
Another study reported that that the susceptibility to advanced
AMD associated with CFHY402H is modified by BMI, and both
BMI and smoking increased risk of advanced AMD within the
same genotype.129
Similarly, another case-control analysis found that current
cigarette smoking and body mass index were independently
related to AMD, controlling for the LOC387715 genotype. 162
Statistical interactions between smoking and either the
CFHY402H or LOC387715 A69S genotypes were not
observed.129,162 A study of patients in the Netherlands found
that elevated erythrocyte sedimentation rate levels, elevated
CRP levels and smoking further increased the risk of AMD
among CFH Y402H homozygotes.134
SUMMARY
Age-related macular degeneration affects a large proportion of
the elderly population and is influenced by both environmental
and genetic factors. Modifiable risk factors include smoking,
body mass index, antioxidants, and possibly omega-3 fatty
acids. The discovery of AMD genetic risk factors has begun
and our understanding of how genes and gene-environment
associations impact AMD onset and progression will expand

pathogenesis of neovascular AMD.157 Two studies have found
associations with AMD.153,158 Given the critical role of the
human leukocyte antigen (HLA) genes in the immune response,
they have also been chosen for candidate gene association
CHAPTER 38
rapidly over the next several years. This will lead to new
insights into the mechanisms involved in development and
progression of AMD and better ways to prevent and treat this
common disease.

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CHAPTER 38
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113. Weeks DE, Conley YP, Tsai HJ, et al: Age-
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114. Haddad S, Chen CA, Santangelo SL, et al:
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115. Edwards AO, Ritter R, Abel KJ, et al:
Complement factor H polymorphism and
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123. Seddon JM, Gensler G, Milton RC, et al:
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127. Zareparsi S, Branham KE, Li M, et al:
Strong association of the Y204H variant in
complement factor H at 1q32 with
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77:149–153.
128. Schaumberg DA, Christen WG, Kozlowski P,
et al: A prospective assessment of the
Y204H variant in complement factor H,
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47:2336–2340.
129. Seddon JM, George S, Rosner B, et al:
CFH gene variant, Y402H, and smoking,
body mass index, environmental
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61:157–165.
130. Lau L-I, Chen S-J, Cheng C-Y, et al:
Association of the Y204H polymorphism in
complement factor H gene and
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131. Okamoto H, Umeda S, Obazawa M, et al:
Complement factor H polymorphisms in
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132. Maller J, George S, Purcell S, et al:
422 Common variation in three genes, including
a noncoding variant in CFH, strongly
influences risk of age-related macular
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133. Li M, Atmaa-Sonmez P, Othman M, et al:
CFH haplotypes without the Y204H coding
variant show strong association with
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degeneration. Nat Genet 2006;
38:1049–1054.
134. Tuo J, Ning B, Bojanowski CM, et al:
Synergic effect of polymorphisms in ERCC6
5’ flanking region and complement factor H
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135. Despriet DDG, Klaver CCW, Witteman JCM,
et al: Complement factor H polymorphism,
complement activators, and risk of age-
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136. DeWan A, Liu M, Harman S, et al: HTRA1
promoter polymorphism in wet age-related
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137. Yang Z, Camp NJ, Sun H, et al: A variant of
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138. Gold B, Merriam JE, Zernant J, et al:
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component 2 (C2) genes is associated with
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139. Yates JR, Sepp T, Matharu, et al:
Complement C3 variant and the risk of
age-related macular degeneration. N Engl J
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140. Maller JB, Fagerness JA, Reynolds RC,
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associated with risk of age-related macular
degeneration. Nat Genet 2007;
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141. Allikmets R: Further evidence for an
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related macular degeneration. The
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142. Shroyer NF, Lewis RA, Yatsenko AN, et al:
Cosegregation and functional analysis of
mutant ABCR (ABCA4) alleles in families
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age-related macular degeneration. Hum
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143. Guymer RH, Heon E, Lotery AJ, et al:
Variation of codons 1961 and 2177 of the
Stargardt disease gene is not associated
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Arch Ophthalmol 2001; 119:745–751.
144. Rivera A, White K, Stohr H, et al: A
comprehensive survey of sequence
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145. Schmidt S, Postel EA, Agarwal A, et al:
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146. Baird PN, Guida E, Chu DT, et al: The
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147. Souied EH, Benlian P, Amouyel P, et al: The
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148. Zareparsi S, Reddick AC, Branham KE,
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149. Schultz DW, Klein ML, Humpert A, et al:
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153. Haines JL, Schnetz-Boutaud N, Schmidt S,
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155. Abecasis GR, Yashar BM, Zhao Y, et al:
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156. Stone EM, Braun TA, Russell SR, et al:
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157. Ferrara N: Vascular endothelial growth
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159. Goverdhan SV, Howell MW, Mullins RF,
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161. Sepp T, Khan JC, Thurlby DA, et al:
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 SECTION 6 CORNEA AND CONJUNCTIVA
Edited by William J. Power and Dimitri T. Azar
CHAPTER
Anatomy and Cell Biology of the Cornea,
39 Superficial Limbus, and Conjunctiva
Ilene K. Gipson and Nancy C. Joyce
The tissues at the ocular surface include the cornea, conjunc-
tiva, and the intervening zone of the limbus; the regions are
shown diagrammatically and histologically in Figure 39.1. The
primary function of the entire region is to refract and transmit
light to the lens and retina. Although the cornea and its surface
tear film constitute the tissue actually performing the tasks, the
limbus and conjunctiva support the cornea in these important
functions. Because the cornea is such a major functional tissue
of the eye and because damage to or disease of the cornea has
serious visual consequences, its structure, function, and
pathology have received much attention. Increased interest in
the limbus and conjunctiva has yielded new information
regarding the important supportive functions of the tissues
surrounding the cornea. This chapter reviews the anatomy and
cell biology of the three regions of the ocular surface, including
some of the recently observed structural and cell biologic
features. In the previous edition of this text, specific chapters
dealt with the cell biology of the corneal epithelium, the corneal
stroma and its connective tissue, and the corneal endothelium.
In this volume, these topics are combined and the publication
space is restricted. Thus, for more complete details regarding
the cell and molecular biology of these tissue regions, refer to a
previous edition of this text.1 More complete details regarding
the gross anatomy of the region also are available.2,3
CORNEA
The cornea is a highly specialized tissue that refracts and trans-
mits light to the lens and retina. In humans, it is about twice as
thick at the periphery than at the center (1 mm compared with
0.5 mm).4–6 The tissue of the cornea appears simple in
composition because it is composed only of an outer stratified
squamous nonkeratinized epithelium, an inner dense connec-
tive tissue stroma with its resident fibroblast-like keratocytes,
and a monolayered cuboidal endothelium bordering the anterior
chamber (see Fig. 39.1). The cornea, however, actually is highly
ordered and complexly arranged in comparison with other
tissues of the body. Its transparency, avascularity, and highly
ordered structure make it unique among all tissues of the body.
Cells of all layers interact with and influence each others’
functions. They do not act alone, but mediators (cytokines)
expressed by one cell type influence cells of adjacent layers.
EPITHELIUM
The surface of the cornea is covered by a stratified squamous
nonkeratinizing epithelium, which in humans, rodents, and
rabbits has five to seven cell layers. The epithelium is 50–52 mm
thick. The corneal epithelium has functions unique to it and
functions that are common to all other epithelia of the body.
Several of its unique functions include light refraction and
transmittance and survival over an avascular bed. The unique
function of light refraction is brought about by its absolutely
smooth, wet apical surface and its extraordinarily regular
thickness. Transparency of the epithelium to light appears to be
brought about by scarcity of cellular organelles and possibly by
high concentrations of enzyme crystallins.7 The epithelium has
specialized metabolic characteristics that allow it to exist over
an avascular connective tissue.8 Protection of these unique and
vital functions is provided by a high density of sensory nerves
that send unmyelinated endings to terminate within the
suprabasal and squamous cells of the epithelium. The density
of nerve endings per unit area appears to be 300–400 times that
of the epidermis.9 The epithelium also has a rapid and highly
developed ability to respond to wounds, and it is maintained by
centripetal movement of cells derived from an adult stem-cell
population located in the basal layer of the limbal epithelium
(see further ahead).
In addition to its specialized functions, the corneal epi-
thelium has the routine housekeeping functions of all epithelia
that border the outside world. The layers of cells provide a
barrier to fluid loss and pathogen entrance and resist abrasive
pressure by tightly adhering to one another and to the
underlying connective tissue stroma.
The stratified epithelium includes three or four layers of
outer flat squamous cells termed squames, one to three layers
of midepithelial cells termed wing cells because of their rounded
cell body and lateral winglike cellular processes, and a layer of
columnar basal cells (Fig. 39.2). The latter secrete and maintain
the epithelium’s basement membrane, which, compared with
that of the other stratified epithelia (i.e., epidermis), is smooth
or planar and nonundulating. This smooth or planar charac-
teristic may support the regular thickness of the epithelium
over the entire cornea.
Like all other stratified epithelia, the epithelium of the cor-
nea is self-renewing, turning over in humans and rats in
~5–7 days.10 Basal cells are the mitotically active layer; as
they divide, daughter cells begin their movement off the base-
ment membrane toward terminal differentiation and desqua-
mation from the apical surface. It was thought that one
daughter cell resulting from a division moved off the basal
lamina, leaving one daughter cell in place to undergo mitosis
again.11 More recent data using bromodeoxyuridine (BrdU)
labeling indicate that the two progeny of a single division move
together toward the apical surface.12
All cell layers of epithelium have a sparse accumulation of
cytoplasmic organelles. Endoplasmic reticulum and mito-
chondria are sparsely distributed around the cytoplasm, with a
Golgi apparatus present in a supranuclear position, particularly
in the basal cell layer (Fig. 39.3). In the apical cell layers, Golgi 423
 CORNEA AND CONJUNCTIVA
c d
SECTION 6
cisternae and small membrane-bound vesicles consistent in
size and structure with Golgi-associated vesicles are especially
prominent (see Fig. 39.3).
Of the three cytoplasmic filament types within all cells, actin
filaments, keratin filaments and microtubules, keratin or
intermediate filaments are the major type within the cytoplasm
of cells of the corneal epithelium. On electron micrographs,
the cell cytoplasm of all layers of the corneal epithelium appears
full of these filaments, and keratin proteins, which polymerize
to form the filaments, are among the most abundant proteins of
the tissue. The keratin family of proteins that form inter-
mediate filaments is a complex family of ~30 polypeptides,
which are of two classes: type I, or acidic; and type II, or neutral
and basic. The intermediate filaments within ectodermally
derived epithelia are formed by the pairing of two specific
keratin proteins, one from each class. In the corneal epithelium,
as basal cells differentiate to apical cells, two keratin pairs are
424 expressed sequentially. First, K5 and K14 are expressed in basal
FIGURE 39.1. Diagram and light micrographs
of ocular surface tissues. Boxes (A–C)
correspond to regions in the light micrographs
(a–c) at the right; all are sections of human
tissue, as is (d), which shows a higher
magnification of conjunctival epithelium.
(a) Section through the central cornea. a,
epithelium; b, Bowman’s layer; c, lamellar
stroma; d, Descemet’s membrane; e,
endothelium. µ120. (b) Section through the
limbus. The large arrow designates the end of
Bowman’s layer and the small arrow the
position of the first blood vessel encountered
outside the corneal stroma. µ48. (c) Section of
bulbar conjunctiva. Note the highly vascularized
connective tissue. µ120. (d) Section of bulbar
conjunctiva demonstrating the presence of
numerous goblet cells (arrows) within the
stratified epithelium and the cellular nature of
the connective tissue of the substantia propria
(arrowheads). µ300.
cells; subsequently, suprabasal cells express K3 and K12.13–15
K12, a 64-kDa keratin, is believed to be cornea specific.16 The
cytokeratin filaments not only increase the tensile strength of
the epithelial cells but also, by keeping the nucleus and other
organelles in their proper positions, affect the overall
organization of the cell. They also provide a scaffold upon which
associated proteins are organized and regulated to control cell
metabolic and homeostatic activities.17 Another major role of
the intermediate filaments of the corneal epithelium is to
provide the cytoskeletal component of the system that anchors
cells tightly to one another and to their substrate through the
desmosome and hemidesmosome (Figs 39.4 and 39.5). Such
tight anchorage is critical to a stratified epithelium that borders
the outside world and is subject to the abrasive pressures from
lid movement and eye rubbing.
Actin filaments, as with all cells, are present throughout the
cytoplasm of cells of the corneal epithelium. They are particu-
larly prevalent as a network along the apical cell membranes
 Anatomy and Cell Biology of the Cornea, Superficial Limbus, and Conjunctiva
FIGURE 39.2. Sections of corneal epithelium as seen by light (inset)
and electron microscopy showing superficial, wing, and basal cell
layers and Bowman’s layer (bl). In the electron micrograph, note the
surface microplicae and interdigitating cell membranes with electron-
dense desmosomes. Electron-lucent profiles of endoplasmic reticulum
are widely scattered, primarily within basal and wing cells. Electron-
dense hemidesmosomes are prominent along the basal cell
membrane of the columnar basal cells adjacent to the basal lamina.
µ300; inset µ2700.
of the epithelium, where they extend into microplicae
(Fig. 39.6), and at the junction of the lateral membranes, where
they are associated with adherens and tight junctions.18 The
actin filament system is particularly important in providing
the cytoskeletal connection of cell adhesion molecules, such as
the integrins and cadherins, and the cytoskeletal component of
adherens and tight junctions in epithelia.
Composed of both a- and b-subunits of the proteins known
as tubulins, microtubules are the third major cytoskeletal
element within all cells.19 Although they are not obvious on
electron micrographs of corneal epithelia, they are obvious
within the spindles of mitotic basal cells, where they provide
the cytoskeletal framework for chromosome segregation. They
do not appear to play a significant role in corneal epithelial
wound healing, indicating that they are not required for epi-
thelial migration and that mitosis is not required for epithelial
wound coverage.20
The corneal epithelium, like all other epithelia, has inter-
cellular junctions that function not only in cell adhesion but
also in cell communication and barrier formation. Four junc-
tion types are present (see Fig. 39.4). Desmosomes, which are
present along the lateral membranes of all corneal epithelial
cells, function in cell-to-cell adherence; adherens junctions,
which are present along the lateral membrane of the apical cells
of the epithelium, function to maintain cell-to-cell adherence
in the region of the tight junctions; the tight junctions are
present along with adherens junctions in apical cell lateral
membranes, where they function to provide a paracellular
permeability barrier; and gap junctions, which function in cell-
to-cell communication, allow intercellular passage of small
molecules up to 2000 Da. The latter are present along lateral
membranes of all cells of the epithelium. Basal cells have gap
junctions with a different molecular composition (connexin 43)
than suprabasal cells (connexin 50). For a more complete
description of the molecular composition of the four junction
types, see Gipson and Sugrue1 and Alberts and colleagues.21
Molecules present along cell membranes also function in cell-
to-cell adhesion. Two types of cell adhesion molecules in mem-
branes of corneal epithelial cells outside specialized junction
regions are cadherins (specifically, E-cadherin)4 and several of
the integrin heterodimers.22
The two surfaces of the corneal epithelium, the apical and
basal surfaces, have specializations indicative of their roles in
the epithelium. The apical surface is specialized to maintain the
tear film and mucous layer23 and, with that layer, provides the
extraordinarily smooth refractive surface of the cornea. To
facilitate this function, the apical cell membrane has short
ridgelike folds, termed microplicae, that form regular undu-
lations of the membrane when viewed in cross-section (see
Fig. 39.6). In addition, microvilli (finger-like projections of the
membrane) up to ~1 mm in length are present. These two
membrane specializations presumably supply an increased
surface area for adherence of the mucous layer of the tear film.
Scanning electron microscopic studies of the corneal surface
demonstrate that apical cells scatter electrons to varying degrees
(see Fig. 39.6). Cells that scatter electrons to a lesser degree are
termed dark cells. Light cells, which scatter electrons to a
greater degree, have a higher density of surface microplicae and
microvilli.24 It has been hypothesized that the dark cells with
fewer surface membrane specializations represent the ‘oldest’
cells of the ocular surface and therefore are about to
desquamate.25 The undulating, specialized apical membrane
bears a prominent glycocalyx that is intimately associated with
the tips of the microplicae and with the mucous layer of the tear
film (see Fig. 39.6). The corneal cells express three membrane-
spanning mucins, designated MUC1, MUC4, and MUC16,
which are present in the apical cell membrane. The latter is a
major component of the glycocalyx and is particularly prevalent
on the tips of microplicae.26
The basal surface of the epithelium is specialized to provide
tight anchorage of the epithelium to the stroma.27,28 A series of
linked structures, termed the anchoring complex, extends from
the cytoplasm of the basal cell, through the basal cell
membrane, then through the basal lamina and into the anterior
of Bowman’s layer at the anterior region of the stroma. The
structures of the anchoring complex visible by electron
CHAPTER 39
microscopy include keratin filaments that insert into the
hemidesmosome plaque; the hemidesmosome, which is the
specialized anchoring junction on the basal membrane;
anchoring filaments, which extend from the hemidesmosome
to the basement membrane; and anchoring fibrils, which extend
from the basement membrane into Bowman’s layer. These
anchoring fibrils form an intertwining network and terminate
distal to the basement membrane in anchoring plaques. The
linked structures and their molecular components are shown
diagrammatically and by electron microscopy in Figure 39.5.
STROMA
The corneal stroma (see Fig. 39.1a) is the connective tissue
located between the epithelial basal lamina and Descemet’s
membrane, the thick extracellular matrix secreted by the endo-
thelial monolayer. The stroma comprises ~90% of the corneal
thickness and includes both Bowman’s membrane and the
lamellar stroma. The major functions of the stroma are to 425
 CORNEA AND CONJUNCTIVA
a corneal epithelia appear similar to those of all
b c
maintain the proper curvature of the cornea as the primary lens
of the eye, to provide mechanical resistance to intraocular
pressure, and to transmit light into the eye without significant
absorbance. Corneal transparency is dependent on the
maintenance of a low level of stromal hydration and on the
orderly arrangement of collagen fibers within the stroma.
SECTION 6
BOWMAN’S MEMBRANE
Bowman’s membrane (Fig. 39.7) is an 8- to 10-mm acellular
zone of randomly arranged collagen fibrils that forms an
interface between the basal lamina of the epithelium and the
subjacent lamellar stroma. Constituents of this layer are
believed to be synthesized and secreted by both epithelial cells
and stromal keratocytes.29,30 Bowman’s membrane contains
several collagen types, including types I, V, and VII,31,32 and
proteoglycans, such as chondroitin sulfate proteoglycan.33 Both
Bowman’s membrane and the lamellar stroma contain fibrils
composed of collagen types I and V; however, the fibrils in
Bowman’s membrane are smaller in diameter (~20 nm) than
those in the stroma (25–30 nm).34 Fibril diameter appears to be
regulated by the relative ratio of type V to type I collagen,
the greater the amount of type V, the smaller the fibril
diameter.35,36 Studies using competitive polymerase chain
426 reaction to quantify messenger RNA (mRNA) from avian
FIGURE 39.3. Electron micrographs
demonstrating aspects of the ultrastructure of
the corneal epithelium of apical cells (a) and
wing cells (b and c). (a) Portion of an apical cell
and the cell immediately beneath it. Note
microplicae (mp) and Golgi vesicles (gv).
µ21 000. (b) Elaborate interdigitation of
membranes of adjacent cells, characteristic of
wing and squamous cells, shows
mitochondrion (m), Golgi apparatus (g), and
rough endoplasmic reticulum (rer). µ21 000.
(c) Higher-magnification electron micrograph
demonstrating that the cytoplasm of epithelial
cells is rich in keratin filaments (kf). µ42 000. All
these micrographs show the presence of the
cell-to-cell adhesion junctions known as
desmosomes (d), which are present along
interdigitating cell membranes. Desmosomes of
other stratified squamous epithelium.
corneal epithelial cells and stromal fibroblasts indicate that the
amount of mRNA for type V collagen relative to that for type I
collagen is higher in epithelial cells than in stromal
fibroblasts.31 This finding suggests that epithelial cells
synthesize and secrete type I and V collagen fibers in Bowman’s
membrane and that the higher ratio of type V to type I collagen
produced by these cells accounts for the smaller fibril diameter
in Bowman’s layer. Type VII collagen-containing anchoring
fibrils connect epithelial hemidesmosomes to anchoring
plaques located 1–2 mm into the anterior portion of Bowman’s
membrane. These anchoring fibrils intertwine with type I
fibrillar collagen, forming a network that stabilizes the
association between the surface epithelium and the underlying
lamellar stroma.18,37 Bowman’s layer is prominent in primates,
including humans, but is thin or nonexistent in other
mammals. The specific function of this layer is not clearly
understood, but its feltwork of collagen fibrils may stabilize the
transition between the epithelial and stromal layers, ensure
adhesion of the overlying epithelial cells to the stromal matrix,
and contribute to the smooth curvature of the corneal surface.
LAMELLAR STROMA
The lamellar stroma is the thick collagenous layer posterior
to Bowman’s membrane. Collagen types I and V are the
 Anatomy and Cell Biology of the Cornea, Superficial Limbus, and Conjunctiva
a b
c d
e f
Intermediate filaments
Keratins
Hemidesmosome
Bullous Pemphigoid Antigen
a6b4 Integrin
Anchoring Filaments
Laminin V
Bsement Membrane
Laminin
H. Sulf. Proteoglycan
Collagen VII Globular Domain
Anchoring Fibril
Collagen VII HElical Domain
Anchoring Plaque
Laminin
Collagen VII Globular Domain
FIGURE 39.5. Electron micrograph demonstrating adhesion complex
of the corneal epithelium. The linked structures of the complex and
their known molecular components are identified. µ165 000.
predominant fibrillar collagens in the lamellar stroma, although
small amounts of other fibrillar collagens, such as type III, also
may be present.38 The stroma contains collagen type XII, which
cross-links fibrillar collagens, and type VI, which forms
microfibril networks.39 Keratan sulfate proteoglycans are the
FIGURE 39.4. Cell-to-cell junctions of the
corneal epithelium as demonstrated by electron
microscopy (a, c, e) and immunolocalization of
cell-to-cell junction components (b, d, f). (a)
Areas of apparent membrane fusion at the tight
junction are obvious. µ66 000. (b) Vinculin, a
component of the adherens junction, can be
seen in the immunofluorescence micrograph on
the lateral membranes of apical cells (arrows).
µ600. (c) Desmosomes are prominent along cell
membranes. µ66 000. (d) Localization of the
desmosome component desmoplakin is
demonstrated in the immunofluorescence
micrograph. µ1000. (e) A gap junction (arrow)
between two basal cells is shown in the
electron micrograph. µ105 000. (f) The
immunolocalization of the gap junction protein
connexin 43 is shown in a section of chick
corneal epithelium. Note the punctate binding,
particularly along the membranes of basal cells.
Antibody against connexin 43 was provided by DA
Goodenough, PhD. (b) Reproduced from Zieske JD,
Bukusoglu G, Gipson IK: Enhancement of vinculin
synthesis by migrating stratified squamous epithelium.
J Cell Biol 1989; 109:571.
predominant proteoglycans within the corneal stroma.40
Lumican and keratocan are the core proteins of keratan sulfate
proteoglycans, with lumican regulating keratocan expression.40
As shown in Figure 39.8a, the constituents of the lamellar
stroma are organized precisely. The basic structural unit of the
fibrillar collagens is tropocollagen, an asymmetric molecule
~300 nm long and 1.5 nm in diameter. Fibrillar collagens are
composed of three polypeptide chains coiled in a triple helix.
These molecules polymerize to form elongated collagen fibrils
with diameters of 25–30 nm. The uniformity of collagen fibril
diameter appears to result from specific interactions between
type V collagen, located toward the center of the fibril, and type
I collagen, on the fibril exterior. As mentioned previously, the
relative ratio of type V to type I collagen appears to regulate
fibril diameter. The interfibrillar distance also is highly uniform
and may be maintained by apposing interactions at the fibril
CHAPTER 39
surface. In the chick cornea, type XII collagen binds to type I on
the fibril exterior and may form lateral ‘bridges’ between fibrils,
thus limiting interfibrillar distance.41 Proteoglycans bind to the
exterior surfaces of collagen fibrils. The polyanionic nature of
the glycosaminoglycan side chains attracts cations and water
molecules and may exert a swelling pressure on the collagen
fibrils, which is balanced by the interactions between collagen
types I and XII. Microfibrils composed of type VI collagen also
associate with type I collagen,42,43 but the specific function of
these fibrils is not known. Collagen fibrils are packed in parallel
bundles extending from limbus to limbus, and the bundles are
arranged in layers, or lamellae. The stroma of the human eye
contains 200–250 lamellae. Lamellae in the middle and
posterior regions of the stroma are arranged at approximate
right angles, whereas those in the anterior stroma are arranged
at less than right angles. The small diameter of the collagen
fibrils and their close, regular packing creates a lattice or three-
dimensional diffraction grating.44 The ‘lattice theory’ of
Maurice45 suggests that the ability of the cornea to scatter 98% 427
 CORNEA AND CONJUNCTIVA

FIGURE 39.6. Micrographs showing

specialization of the apical membrane of apical
cells of the ocular surface. (a) Electron
micrograph of mucin layer preserved on apical
membranes of guinea pig conjunctiva. Note
microplicae (mp) in cross-section and electron
density of the glycocalyx (gc) region at the tips
of the microplicae. Note tight junction (tj)
between adjoining cells. µ56 000. (b and c) Low
(b, µ750) and high (c, µ6200) magnification
scanning electron micrographs of apical cells of
a rabbit cornea. In (b), cells vary in the amount
to which they scatter electrons, leading to a
mosaic with cobblestone appearance. In
(c), this degree of scatter correlates to the
density of microplicae on the surfaces of the
b cells. (d) Immunofluorescence micrograph
demonstrating specific molecules along the
apical membrane. Cells in this section of human
cornea have been labeled with antibody to the
membrane-spanning mucin termed MUC1.
A similar pattern of labeling is seen with
antibodies to MUC16. µ300.

a d

FIGURE 39.7. (a) Bowman’s membrane (BM)

forms an acellular interface between the basal
cells of the epithelium (E) with its basement
membrane (straight arrow) and the lamellar
stroma (curved arrow). Note the relative
thickness and feltwork-like appearance of
Bowman’s membrane. µ5800. (b) The random
arrangement of collagen fibrils (arrowheads) is
SECTION 6

shown. Also note the close association of the

hemidesmosomal structures (large arrow) on
the basal aspect of the epithelial cells, the
highly organized extracellular matrix (small
arrow) of these cells, and Bowman’s
membrane. µ31 000.

a b
428
 Anatomy and Cell Biology of the Cornea, Superficial Limbus, and Conjunctiva
a
b monolayer acts as a ‘leaky’ barrier, permitting the passage of
FIGURE 39.8. Sections of corneal stroma showing collagen bundles
arranged in lamellae (L), which are oriented at different angles.
(a) Micrograph illustrating the stacked lamellae and long, attenuated
processes (arrowheads) of the stromal fibroblasts (F) located between
the lamellae. µ4800. (b) Collagen bundles in the upper lamella (L) are
sectioned crosswise, whereas those in the lower lamella are sectioned
at an angle. Junctions between the cytoplasmic processes of
neighboring fibroblasts form a network of communicating cells. µ13 000.
of incoming light results from equal spacing of the collagen
fibers. Scattered light waves interact in an ordered fashion,
eliminating destructive interference. The lamellar organization
of the stroma also produces a uniform tensile strength across
the cornea, withstanding intraocular pressure and maintaining
appropriate corneal curvature.
The matrix components of the lamellar stroma are secreted
and maintained by stromal fibroblasts, also known as
keratocytes. As shown in Figure 39.8b, these long, attenuated
cells are arranged parallel to the corneal surface and are located
between the collagen lamellae. The keratocyte cell body
contains an elaborate rough endoplasmic reticulum and Golgi
apparatus, reflecting its active synthetic function. Keratocytes
extend slender cytoplasmic processes and can form gap
junctions with neighboring cells, resulting in a network of com-
municating cells.46 An ultrastructural study47 of human cornea
demonstrated the presence in central stroma of unmyelinated
nerve fibers that run parallel to the collagen bundles, pass
through Bowman’s membrane and the basal lamina of the
epithelium, and associate with subepithelial cells. Nerve fibers
were found to invaginate stromal keratocytes as well as corneal
epithelial cells. This finding suggests that nerves may mediate
information exchange between the epithelium and stroma
under certain conditions, such as corneal wounding. Recently,
bone marrow-derived cells were demonstrated in the corneal
stroma.48 These cells were of both monocytic and myeloid
lineages, demonstrating surface markers of the dendridic cell
(antigen-presenting cells) and monocytes/macrophage type. It is
not clear whether these cells function in immunologic defense
or play a role in the induction of tolerance and the immune-
privileged state of the cornea.48
FIGURE 39.9. Low-magnification electron micrograph illustrating the
posterior portion of the cornea. The stroma (S) is closely associated
on its posterior-most aspect with Descemet’s membrane (DM), the
thick extracellular matrix secreted by the endothelial cells (EN). The
endothelium is the monolayer of cells located at the posterior of the
cornea; it acts as a barrier between the aqueous humor and overlying
corneal tissues. µ750.
ENDOTHELIUM
The endothelium (Fig. 39.9) is the single layer of cells located at
the posterior of the cornea that forms a barrier between the
corneal stroma and anterior chamber. The endothelial cell
nutrients from the aqueous humor into the avascular
cornea.49,50 The endothelium is responsible for maintaining the
relatively low level of stromal hydration required for corneal
transparency. The tendency of the corneal stroma to swell is
balanced by removal of excess stromal fluid via the activity of
‘ionic pumps’ located at the endothelial plasma membrane. The
relatively high extracellular ion concentration produced by
these pumps draws water from the stroma, thus maintaining
the highly organized collagen lamellar structure required for
corneal transparency. The endothelium also secretes com-
ponents of the thick basal lamina, termed Descemet’s mem-
brane, which lies between the endothelium and posterior
stroma.
MORPHOLOGIC AND ULTRASTRUCTURAL
CHARACTERISTICS
The average density of corneal endothelial cells at birth is
~4000 cells/mm2.51 Each cell is 4–6 mm thick, ~20 mm wide,
and has a surface area of ~250 mm2. Scanning electron micro-
scopy of the monolayer surface (Fig. 39.10) reveals that cells
assume a hexagonal shape and possess numerous lateral,
interdigitating cellular processes.51–53 These processes increase
the area of contact between neighboring cells and resemble
CHAPTER 39
interlocking fingers. Numerous small microvilli are present on
the posterior (apical) cell surface, which faces the aqueous
humor. This surface appears to be covered by a mucinous layer
~0.5 mm thick.54 MUC1 is at least one component of this layer
and is believed to have a protective function.55 A single, cen-
trally located cilium, ~2–7 mm long, has been observed on the
apical surface of peripheral cells. This cilium exhibits the
ultrastructural characteristics of other primary cilia,56 but its
function in corneal endothelium is unclear.
The ultrastructural features of the endothelium reflect its
functions.57 Numerous mitochondria within the cytoplasm
indicate that these cells are metabolically active (Fig. 39.11).
The cytoplasm also contains extensive rough and smooth
endoplasmic reticulum, numerous ribosomes, and a prominent
Golgi apparatus reflective of a high level of protein synthesis
(Fig. 39.12). A circumferential band of actin-containing micro-
filaments is located beneath the apical plasma membrane at the
cell periphery. These microfilaments help maintain cell shape
and mediate cell movement.58–60 An intermediate filament 429
 CORNEA AND CONJUNCTIVA

FIGURE 39.10. Scanning electron micrograph of the surface of the

corneal endothelium illustrating the hexagonal shape of the cells as
well as the other surface features, including nuclei (N) that bulge from
the cell surface, a single cilium (C), and long lateral projections (BB)
that bridge from one cell onto the body of adjacent cells.
From Svedbergh B, Bill A: Scanning electron microscopic studies of the corneal
endothelium in man and monkeys. Acta Ophthalmol Scand 1972; 50:321.

network comprised primarily of vimentin forms a basket-like

structure that surrounds the nucleus and anchors at the apical
junctions.59–62 This network appears to be responsible for
nuclear centration and, in part, for maintenance of cell–cell
junction stability. Relatively little is known about the molecular
basis of adhesion of the endothelium to Descemet’s membrane.
Focal areas of increased electron density are present on the
cytoplasmic aspect of the basal plasma membrane63 and may
represent a form of adhesion plaque anchoring the endothelium
to Descemet’s membrane. Cytoplasmic processes extend from
the basal aspect of the cells and penetrate Descemet’s
membrane, possibly contributing to increased adhesiveness of
the monolayer.63 Alpha-v beta-5 integrin has been identified in
the endothelium of human corneas by immunocytochemistry64
and may help mediate endothelial adhesion to Descemet’s
membrane.
SECTION 6
FIGURE 39.11. Low-magnification transmission electron micrograph
illustrating the general orientation and ultrastructural features of the
corneal endothelium and Descemet’s membrane. A band of actin-
containing filaments, termed the terminal web (tw), is present in the
anterior aspect of the cells and excludes other cell organelles.
Junctional complexes located on the apical aspect of the lateral
plasma membranes are visible at this low magnification as a terminal
bar (tb). The intercellular border (ic) formed between adjacent cells is
long and sinuous. A, anterior chamber; E, endothelium; n, nucleus;
D, Descemet’s membrane. Bar = 1 mm.
From Iwamoto T, Smelser GK: Electron microscopy of the human corneal

Focal tight junctions (Fig. 39.12) on the apical aspect of the
lateral membranes are small areas in which the outer leaflets
of the plasma membranes of adjacent cells appear to fuse,
obliterating the extracellular space.63,65–67 These junctional
complexes do not form ‘belts’ or rings extending around the cell,
as are found in many epithelia.68,69 Rather, they occur as small
zones of membrane fusion around the cell circumference. There
have been few studies to specifically identify the protein
constituents of tight junctions in corneal endothelium;
however, it is known that ZO-1 (zonula occludens-1)60,70 and
occludin60 are components of these structures. In fact, focal
tight junctions can be visualized by the discontinuous
immunostaining of ZO-1. Electrical resistance across the endo-
thelial monolayer is low (73 ± 6 W/cm2)71 compared to that
across the corneal epithelium (1.6–9.1 KW/cm2), reflecting the
different organization of tight junctions in these two tissues.71,72
Adherens junctions are also located at the lateral plasma
membrane. These appear to be composed of N-cadherin,73
430 alpha- and beta-catenin, and plakoglobin.60 Gap junctions
endothelium with reference to transport mechanisms. Invest Ophthalmol Vis Sci
1965; 4:270.
(Fig. 39.13) are located at all levels of the lateral plasma
membrane below the tight junctions.63,65 These junctions stain
positively for connexin-43,74,75 possess a characteristic penta-
laminar structure, and are the site of electrical and metabolic
coupling, which facilitates cell-to-cell communication.75,76
BARRIER FUNCTION
As an avascular tissue, the cornea receives oxygen mainly from
the tear film,77 but its nutritional requirements are met via the
aqueous humor. As such, the glucose, amino acids, vitamins,
etc., needed by the epithelial cells and stromal keratocytes
must traverse the corneal endothelial monolayer. This nutrient
transport occurs primarily via a paracellular route, i.e., solutes
move between the cells rather than by being actively
 Anatomy and Cell Biology of the Cornea, Superficial Limbus, and Conjunctiva
FIGURE 39.12. Anterior portion of an endothelial cell illustrating the
extensive endoplasmic reticulum (ER) with its associated ribosomes
as well as focal tight junctional complexes (arrows) and apical folds
(AF) where adjacent cells interdigitate. AC, anterior chamber; TW,
terminal web. µ80 000.
From Hirsch M, Renard G, Faure JP, et al: Formation of intercellular spaces and
junctions in regenerating rabbit corneal endothelium. Exp Eye Res 1976; 23:385.
FIGURE 39.13. Electron micrograph of gap junctional complexes
illustrating the characteristic regular spacing of the connexin cross-
bridges that draw adjacent plasma membranes into close apposition.
Inset, Arrowheads indicate areas in which the gap between cell
membranes is clearly visible. µ180 000.
From Leuenberger PM: Lanthanum hydroxide tracer studies on rat corneal
endothelium. Exp Eye Res 1973; 15:85.
transported through them. This form of transport requires that
the endothelial monolayer be ‘leaky’ to substances within the
aqueous humor, but not permit bulk fluid flow into the corneal
stroma. The barrier to bulk flow of fluid from the aqueous
humor to the stroma is formed primarily by the focal tight
junctions of the endothelium. Experiments with molecular
tracers indicate that small molecules do not penetrate the tight
junctions, but enter the intercellular spaces by leaking around
them.63,65,67,69 Gap junctions and the sinuous, elaborate inter-
digitation of the lateral plasma membranes together may form
a secondary barrier to fluid flow.78 Gap junctions narrow the
width between apposing cell membranes from the normal
intercellular gap of 25–40 nm to ~3 nm.65–67 Narrowed inter-
cellular spaces produced by the formation of gap junctions, plus
FIGURE 39.14. The ‘pump-leak’ hypothesis. When the rate of fluid
leakage into the stroma is balanced by the rate of fluid pumped out of
the stroma, normal corneal architecture and thickness are maintained.
Adapted from Waring GO III, Bourne WM, Edelhauser HF, Kenyon KR: The
corneal endothelium: normal and pathologic structure and function.
Ophthalmology 1982; 89:531.
the requirement that fluid must move between the inter-
digitating lateral membranes, helps prevent bulk fluid
movement across the endothelial monolayer.
PUMP FUNCTION
Transparency is essential for the function of the cornea as the
primary lens of the eye. Transparency results from the
uniformity of the tissue elements comprising the cornea and
from the regularity of their spatial organization. Precise
arrangement of the collagen bundles within the corneal stroma
is especially important for corneal clarity.45 This precise
CHAPTER 39
arrangement depends to a great extent on the maintenance of a
relatively low level of stromal hydration. Proteoglycans
associated with the collagen fibrils within the stroma bind
water, producing a natural pressure gradient across the
endothelial monolayer. In addition, loss of integrity of the endo-
thelial cell layer can hydrate the stroma. The disorganization
of collagen fibrils, which results from stromal swelling, causes
light absorbance, corneal clouding, and reduced vision.
The requirement that the endothelium permits passage of
nutrients into the cornea and, at the same time, maintain a
barrier to the free flow of water into the stroma presents an
interesting cell biological paradox. The ‘pump-leak’ hypothesis
has attempted to resolve this paradox. It states that the rate of
leakage of water and solutes into the corneal stroma is balanced
by the rate of pumping of excess water from the stroma back to
the aqueous.5,79 As long as the equilibrium suggested by this
hypothesis is maintained, the corneal stroma remains relatively
dehydrated and corneal clarity is maintained. Figure 39.14
illustrates this equilibrium. Any imbalance between the rate of 431
 CORNEA AND CONJUNCTIVA
fluid leak into and the rate of ionic pumping of fluid out of the
cornea results in corneal swelling and loss of visual acuity.
The endothelium maintains a low level of stromal hydration
by the activity of ionic ‘pumps’, which mediate the transfer of
Na+, K+, Mg+, Cl⫺, and HCO3⫺. Fluid flow from the stroma to
aqueous humor appears to be secondary to electrolyte move-
ment;80 however, the specific mechanism by which movement
of electrolytes is coupled to the movement of water is not
completely understood.80–82 Metabolic energy is needed to
maintain normal corneal thickness, indicating that at least
part of the mechanism regulating stromal hydration involves
an active process.83,84 The fluid ‘pump’ is dependent on the
presence of Cl⫺ and HCO3⫺ and can be slowed by carbonic
anhydrase inhibitors.82,85 A number of anion transport
mechanisms have been identified in corneal endothelium,
including a basolateral Na+–K+–2Cl⫺ co-transporter86 and a
Na+–HCO3⫺ co-transporter.82,87 In addition, the water channel
protein, aquaporin-1 (AQP1), has been localized to the plasma
membrane of corneal endothelium;88 however, it is currently
unclear how it functions in fluid transport in this tissue.80,89,90
MONOLAYER REPAIR
Corneal endothelial cells are capable of normal division during
fetal development; however, the total corneal endothelial cell
reserve is limited, because cell division in adult cells either does
not occur at all or occurs at a rate too slow to efficiently replace
dead or injured cells.51,91–93 At birth, endothelial cell density is
3500–4000 cells/mm2, whereas, in adults it is reduced to
1400–2500 cells/mm2.51 Cell density begins to decrease during
fetal development due to both a rapid growth in corneal size and
the limited mitotic activity that occurs after the second tri-
mester. Once rapid corneal growth subsides, cell density con-
tinues to decrease, but at a slower rate. Beginning at about the
second year of life, decreased cell density is directly related to
endothelial cell loss and the inability of the endothelium to
reproduce in numbers sufficient to keep pace with this loss.91,92
The overall rate of cell loss accelerates if the endothelium is
injured as the result of trauma, disease, or dystrophy.93–95
Polymegathism, i.e., heterogeneity in cell size, increases in
the endothelium with age and as the result of damage caused
by trauma, corneal infection, or disease.96–99 Cell size can
become heterogeneous for several reasons. When the endo-
thelium is injured or when cells are lost due to normal attrition,
repair of the defect in the monolayer occurs mainly through
enlargement and spreading of neighboring cells, causing cells to
be larger in these areas.94,96,100–103 In addition, the number of
multinucleated cells93,104 and cells with more than 4N DNA
SECTION 6
content105,106 increase with age, producing a population of very
large cells. Increased heterogeneity in cell shape, i.e., pleomor-
phism, also occurs with age or trauma.52,107–109 As the number
of cells within the monolayer decreases and the size of cells
enlarges, there is a decrease in the percentage of hexagonal cells
within the monolayer. As polymegathism and pleomorphism
increase, the endothelial monolayer can become destabilized. It
is well known that a regular hexagonal pattern provides the
greatest cellular packing with an optimal cell-to-membrane
ratio. Irregular cell sizes and shapes can increase surface tension
within the monolayer, producing decreased geometric and
architectural stability.
When cell numbers are reduced as the result of aging or
trauma and the remaining cells become larger and more pleo-
morphic, the ability to maintain or restore normal barrier and
pump function can be compromised.110–112 With decreased
monolayer stability, permeability increases and the cornea
can swell. Decompensation, i.e., loss of monolayer integrity
432 and function, can occur when cell density falls below
300–400 cells/mm2 or when the mean cell size reaches
~3000–3500 mm2.49,93 Because of the stressed state of the
endothelial monolayer under these conditions, the leak rate of
fluid into the stroma becomes greater than the pump rate of
fluid flow out of the stroma, producing stromal edema and
corneal clouding. At present, full-thickness transplantation is
the normal recourse for reestablishing corneal clarity and visual
acuity following decompensation of the corneal endothelium.
PROLIFERATIVE CAPACITY
Investigators are currently re-examining the relative prolife-
rative capacity of corneal endothelial cells.113–115 There is some
evidence to suggest that these cells can divide in vivo, but
at a very slow rate;104,116,117 however, the well-established
observation that endothelial cell density decreases with age
strongly suggests that, if endothelial cells do divide in vivo,
the rate of cell division does not keep pace with the rate of cell
loss. The ability to grow human corneal endothelial cells in
tissue culture without requiring viral oncogene protein
expression70,118,119 clearly indicates that these cells retain
proliferative capacity that can be harnessed under appropriate
conditions. Comparative studies of cell-cycle protein expression
in corneal cells suggest that endothelial cells in vivo are arrested
in G1-phase of the cell cycle.120,121 Thus, the limited pro-
liferation observed in this tissue in vivo appears to be due, at
least in part, to microenvironmental conditions that actively
maintain the endothelium in a nonproliferative state.
A number of mechanisms appear to contribute to inhibition
of corneal endothelial cell proliferation in vivo. One is the
apparent lack of positive growth factor simulation. A number of
growth factors have been detected in aqueous humor,122–125 and
corneal endothelial cells themselves both synthesize a number
of growth factors and express growth factor receptors.123,126,127
However, they do not appear to readily divide despite the
potential for autocrine or paracrine stimulation. Another
inhibitory mechanism appears to be suppression of S-phase
entry by transforming growth factor-b2 (TGF-b2). A role for
this cytokine in negatively regulating proliferation of corneal
endothelium is supported by the fact that TGF-b2 is present in
relatively high concentration in aqueous humor128,129 and that
corneal endothelial cells express the receptor types required to
transmit a TGF-b2-induced signal.130 Studies in cultured
corneal endothelial cells have demonstrated that both
exogenous TGF-b2 and TGF-b2 in aqueous humor suppress
S-phase entry.131,132 Contact inhibition is another mechanism
that suppresses proliferation in corneal endothelial cells. This
has been shown using an ex vivo wound model in which
treatment of the endothelium with the calcium/magnesium
chelator ethylenediaminetetraacetic acid (EDTA) releases
cell–cell junctions and promotes cell division.133 The existence
of a connection between cell–cell contacts and growth
inhibition in corneal endothelium is demonstrated by the fact
that expression of p27kip1, a protein that inhibits movement
from G1- to S-phase of the cell cycle, is upregulated when
cultured endothelial cells reach confluence.134,135
There are also intrinsic, age-related factors that affect the
ability of human corneal endothelial cells to proliferate. A
common finding has been that cells cultured from young donors
grow more robustly and can be passaged more times than cells
from older donors.136–138 Results from both ex vivo wound
models139 and cell culture studies114,140 provide evidence that:
(1) Fewer cells from older donors are responsive to mitogenic
stimulation; (2) Those cells that retain the ability to respond to
mitogens generally require stronger stimulation than cells from
younger donors; and (3) Cells from older donors respond more
slowly to mitogens than cells from younger donors. Recent
 Anatomy and Cell Biology of the Cornea, Superficial Limbus, and Conjunctiva
FIGURE 39.15. Micrograph illustrating Descemet’s membrane (DM)
located between the posterior aspect of the corneal stroma (S) and
the underlying endothelium (EN). Two regions of Descemet’s
membrane are apparent in adult corneas. The anterior ‘banded’ region
(A) is secreted by the endothelial cells during fetal development and is
more highly organized than the posterior ‘amorphous’ region (P),
which is secreted after birth. The posterior region increases in
thickness with age as a result of continued synthesis of its
constituents by the endothelium throughout life. µ9600.
studies have shown that gene transfer to ex vivo human corneal
endothelium of E2F2, a transcription factor whose activity is
required for entry into S-phase, is able to induce proliferation
and increase endothelial cell density.141 This gene therapeutic
method was able to induce proliferation in cells from both
young and older donors; however, the kinetics of the induction
appeared to be age-dependent.
Morphometric studies142,143 have documented that there are
differences in endothelial cell density in peripheral versus cen-
tral cornea, indicating a nonuniform distribution of endothelial
cells across the cornea. Cells in the far peripheral region of the
endothelium close to Schwalbe’s line exhibited a higher cell
density than cells in the paracentral or central regions.143
Although cell densities from all regions decreased with age, the
rate of decrease in density was slowest in the peripheral region.
Recently, the intriguing question has been raised whether there
may be a difference in relative proliferative capacity between
endothelial cells located in central cornea and those located in
the periphery. Tissue culture studies by Konomi et al144 and
ex vivo cornea studies by Mimura and Joyce145 have demon-
strated that human corneal endothelial cells cultured from both
the central and peripheral regions exhibit proliferative capacity,
regardless of donor age. In the ex vivo cornea studies, the
relative percent of cells exhibiting replication competence was
significantly higher in peripheral cornea compared with central
cornea, regardless of donor age. In corneas from older donors,
central endothelial cells exhibited the lowest percent of
replication competent cells. Interestingly, staining for
senescence-associated beta-galactosidase (SA-b-Gal)146 demon-
strated an age-related increase in the relative percent of
endothelial cells exhibiting senescence-like characteristics, with
the percent of cells staining positively for SA-b-Gal highest in
the central region of corneas from older donors. Together, these
studies suggest that, with donor age, central endothelial cells
become senescent and die, and that there may be a slow
centripetal movement of cells from the periphery to replace
them. It is not yet clear whether peripheral cells are recruited
from the periphery as the result of slow cell movement and
rearrangement or whether peripheral cells may divide to help
replace cells lost from central endothelium. Recent studies147
have demonstrated telomerase activity in the far peripheral
region of human corneal endothelium, suggesting that this
region contains progenitor (stem-like) cells. It remains to be
clearly demonstrated that stem-like cells could act as a source
of cell renewal for corneal endothelium.
DESCEMET’S MEMBRANE
Descemet’s membrane is the thick extracellular matrix
synthesized and secreted by the corneal endothelium
(Fig. 39.15). In adults this matrix consists of two layers. An
anterior, ‘banded’ layer is formed during fetal development and
consists of highly organized collagen lamellae and proteo-
glycans. A posterior ‘amorphous’ layer is synthesized after birth
and is less organized than the fetal layer. Adult Descemet’s
membrane contains fibronectin, laminin, type IV and type VIII
collagen, as well as heparan sulfate and dermatan sulfate
proteoglycan. How these constituents are assembled to form
the highly ordered lattice of the fetal membrane and the more
randomly organized adult membrane remains unresolved.148
Corneal endothelial cells slowly synthesize and secrete base-
ment membrane material throughout life. In young adults the
posterior layer measures ~2 mm, but increases to ~10 mm in
older individuals. The positive correlation between age and
Descemet’s membrane thickness149,150 suggests that there is
little, if any, destruction of previously formed basement mem-
brane material. This provides a type of historic record of corneal
endothelial function and has been used to study the develop-
ment of endothelial diseases or dystrophies. By comparing the
morphology and thickness of Descemet’s membrane in normal
and diseased corneas, it is possible to determine the relative
CHAPTER 39
point in time in which the ability of corneal endothelial cells to
synthesize and secrete normal Descemet’s membrane is
compromised. Individual endothelial cells can produce excess
extracellular matrix material, resulting in the formation of
focal or nodular thickenings in Descemet’s membrane. These
thickenings, called Hassall–Henle bodies or ‘warts’, are fre-
quently found in cells at the corneal periphery.51 Similar
structures are termed ‘guttatae’ when they are located centrally
within the cornea.151 The number of these focal thickenings
increases with age, in certain endothelial dystrophies, such
as Fuchs’ dystrophy,152–154 and as the result of inflammation.155
LIMBUS
The various anatomic definitions of the limbus include the
anatomists’ limbus, the pathologists’ limbus, the histologists’
limbus, and the surgeons’ limbus.2,156 The various definitions
and the various angles of lines drawn on sections or diagrams of
cross-sections of the region indicate that there are no definite 433
 CORNEA AND CONJUNCTIVA
a (c) Electron micrograph of basal cells of the
b c
reliable boundaries to the zone. The broadest definition of the
limbus is the zone between a line drawn between the termini
of Bowman’s layer and Descemet’s membrane, which forms
the anterior border, and a line that passes parallel but 1 mm
posterior to the anterior line, passing through the posterior end
of Schlemm’s canal. In this definition, both Schlemm’s canal
and the trabecular meshwork are within the limbus. This
section reviews aspects of the limbus relevant to ocular surface
function, specifically the superficial region, including the
epithelium and loose connective tissue overlying the interface
of the connective tissue at the corneoscleral junction (see
Fig. 39.1b). The limbal region has been termed the transition
SECTION 6
zone between cornea, conjunctiva, and sclera, and although that
may be an apt description, the specialized characteristics of the
limbal epithelium and its immediate subjacent connective
tissue indicate that the region has specialized functions sup-
porting the cornea and that it may be a barrier to conjunctival
overgrowth of the cornea.
EPITHELIUM
The epithelium of the limbus has many features common to
corneal epithelium. It is a stratified squamous nonkeratinizing
epithelium but has several more cell layers than corneal
epithelium.2,157 Cell junctions in the limbus are similar to
those in the cornea, and the apical and basal specializations
present in the limbus are the same as those in the cornea. The
basal cells of the limbal epithelium appear unique and are
believed to be stem cells for maintenance of the corneal epi-
thelium. (For review, see Gipson and Sugrue.1) The cells appear
434 smaller and less columnar and have more cytoplasmic
FIGURE 39.16. Micrographs of the limbal
epithelium and subjacent connective tissue.
The basal cells of the region are smaller and
less columnar than those of the cornea. (a)
Light micrograph showing the absence of
Bowman’s layer and the cellular stroma and
blood vessels (bv). µ300. (b) Scanning electron
micrograph of palisades of Vogt (pv); limbal
epithelium was removed with
ethylenediaminetetraacetic acid to demonstrate
the folds or ridges in the connective tissue.
Remnant epithelium (e) and denuded corneal
basement membrane (cbm) are labeled. µ2000.
limbal region. Note their small size and the
hemidesmosomes (hd) present along the
undulating basement membrane. Small, retelike
pegs (pg) of stromal matrix extend into the
epithelium. µ6000.
organelles (Fig. 39.16). The basal cells sit on a basement
membrane that is not flat and planar like that of the cornea;
peglike interdigitations of the epithelium and the stroma are
present (see Fig. 39.16).2 Probably the best indication to date
that the adult stem cells are a subpopulation of limbal basal
cells is their long 3H-thymidine label retention time, indicating
slower passage through the cell cycle than basal cells of the
cornea and conjunctiva.11 Other recent data using GFP mice
show a slow centripetal movement of successive progeny of
the limbal basal cells toward the center of the cornea.158 There
are differences in keratin expression in basal cells compared
with suprabasal cells of the limbus and cells of the corneal
epithelium;16 and they also show enhanced presence of certain
metabolic enzymes and proteins, including a-enolase, cyto-
chrome oxidase, Na+,K+-ATPase, carbonic anhydrase, metal-
lothionein, and glucose transporter.159–162 Recent data suggest
that the stem cell population binds antibodies to the mem-
brane-transporter protein designated ABCG2.163 Another
characteristic of the region is that ocular surface tumors occur
primarily in the limbal area and rarely are found on the cornea.
Taken together, these data and those from experiments
demonstrating centripetal migration of cells from the limbal
region into the cornea over time indicate that the limbal basal
cells are the stem cells of the corneal epithelium. Further
evidence that these basal cells are important to maintenance of
the corneal epithelium comes from clinical data that
demonstrate the effectiveness of limbal transplantation in the
treatment of persistent, nonhealing corneal problems.164,165 In
addition, these basal cells are protected by pigmentation and are
present within deep crypts in the limbal connective tissue,
termed the palisades of Vogt (see Fig. 39.16b).
 Anatomy and Cell Biology of the Cornea, Superficial Limbus, and Conjunctiva
a b
CONNECTIVE TISSUE
The connective tissue underlying the limbal epithelium is loose
and less organized than the corneal stroma, and Bowman’s layer
is not present. Although the molecular composition of the
two matrices appears to correspond, an exception is absence of
the keratan sulfate proteoglycan (lumican).166 Cellular elements
within the limbal stroma are more diverse than in the corneal
stroma. In addition to fibroblasts, melanocytes, mast cells,
lymphocytes, and plasma cells occur routinely. A major
difference between the limbal stroma and that of the cornea is
the presence of blood and lymphatic vessels that loop into the
area of the limbal stroma. These vessels include capillaries,
small arterioles and venules, and large lymphatics. Bundles of
unmyelinated nerves also are present. The palisades of Vogt,
large folds of matrix, are a unique characteristic of this area (see
Fig. 39.16b). The outward folds of connective tissue are large
enough to accommodate small blood vessels, lymphatics, and
nerves, and crypts of limbal epithelium reach down into the
palisades of Vogt. The deep housing of the limbal epithelium
in the folds not only may protect the stem cell population but
also may increase the surface area for accommodating a large
cell population and increase exposure to vascularly derived
nutrients and effector molecules. In addition to the large
macroscopic folds of the palisades of Vogt, tiny rete (peglike
folds or outpockets of stroma) begin in the peripheral stroma
and extend through the limbal region into the conjunctiva (see
FIGURE 39.17. Micrographs demonstrating
regions of bulbar conjunctiva. In the bulbar
region, particularly in the nasal zone, goblet
cells are dense. They can occur in crypts or
groups, which have the appearance of acini, as
demonstrated in the light micrograph (a). µ750.
(b) Electron micrograph of the apical region of
two adjacent goblet cells. Note the microvilli
(mv) on the surfaces of cells and the fibrillar
pattern in the mucin packets (mp). µ21 000.
(c) Low-magnification electron micrograph of
nongoblet cells in the conjunctiva. Note the
vesicles (v) and granules (g) in the apical region
of cells and clumping of keratin filaments into
bundles (k). µ6000.
Fig. 39.16c). These rete may increase the surface area of the
basal cell membrane of basal cells and may provide for
additional anchoring strength in a region where hemi-
desmosomes are not as extensive.167
CONJUNCTIVA
CHAPTER 39
GENERAL CHARACTERISTICS AND
DESCRIPTION OF REGIONS
The conjunctiva is the mucous membrane that covers the inner
surfaces of the upper and lower lids and extends to the limbus
on the surface of the globe. The two major functions of this
tissue, besides connecting the lids to the globe, are provision of
mucus for the tear film and protection of the ocular surface
from pathogens through immune tissue. The ducts of the
lacrimal, accessory lacrimal, and meibomian glands enter the
conjunctival epithelium and deliver their respective products to
the tear film. Three regions within the conjunctiva are
recognized: the palpebral or tarsal region, which lines the inner
surface of the lids; the fornical region, which lines the upper and
lower surfaces of the recess or cul-de-sac known as the fornix;
and the bulbar region, which lines the surface of the sclera
between the fornix and the limbus. The conjunctiva has two
structural components throughout all regions: the surface
epithelium and the substantia propria (Fig. 39.17; see
Fig. 39.1c). 435
 CORNEA AND CONJUNCTIVA
a showed no binding. Bars = 100 mm.
b c
EPITHELIUM
Conjunctival epithelium is unique among stratified non-
keratinizing epithelia in that it has goblet cells intercalated
within it (see Figs 39.1 and 39.17). The goblet cells are the
major producers of mucins for the tear film. Reports of the
number of cell layers in the stratified epithelium vary, especially
regarding fornical and bulbar areas. These variations may result
from different degrees of stretch on the tissue at time of fixation
for histologic study. Cell layers of the palpebral conjunctival
epithelium do not vary as much, perhaps because the substantia
propria is not as loose and contractile at fixation. Reports have
varied from 2 or 3 cell layers to 10–12, the latter number of
layers being present at the lid margin near the junction with the
epidermis covering the external lid. Langerhans’ cells are
present within the conjunctival epithelium.48
Compared with cells of the corneal epithelium, the stratified
cells of the conjunctiva have more cytoplasmic organelles.
Keratin filaments in these cells are not as dispersed as those in
corneal cell cytoplasm and often appear in bundles (see
Fig. 39.17). Keratin proteins expressed by stratified conjunctival
epithelial cells also are different, with the keratin pairs K4 and
K13, and K3 and K19. K7 is expressed by goblet cells.168 Cell-to-
cell junctions and cell-to-substrate junctions appear similar in
corneal, limbal, and conjunctival epithelia, except that gap
junction proteins in the conjunctiva have not been
characterized.169–171
The apical cells of the stratified epithelium have numerous
SECTION 6
small vesicles within their cytoplasm (see Fig. 39.17). It has
been proposed that these vesicles (which bind Alcian blue
and periodic acid-Schiff stains, indicating a highly glycosylated
content) deliver mucins onto the ocular surface and thus
represent a second source of mucus for the tear film.
Reports indicate that the stratified epithelium is expressing
membrane-spanning mucins MUC1,26 MUC4,172,173 and
MUC16.26
The goblet cells that are intercalated within the stratified
epithelium of the human conjunctiva occur as individual cells;
in rodents, they occur as clusters.174 In humans, there is a regional
variation in goblet cell distribution pattern and density,174 the
highest density being in the palpebral region near the tear
drainage punctum and in the midfornix. In some regions,
436
FIGURE 39.18. Distribution of messenger RNA
for the mucin MUC5AC using a 35S-labeled
probe. (a) Low-magnification micrograph
demonstrating dense signal distributed in
patches (arrows) within the conjunctival
epithelium. (b) Higher magnification of the
epithelium demonstrating label specifically over
goblet cells. (c) The control sense probe
especially the temporal bulbar conjunctiva, goblet cell density is
so great that the cells appear to be clustered and arranged in
acini.175 Goblet cells of the conjunctiva are plump and lack the
goblet ‘stem’ – a thin cytoplasmic extension to the basement
membrane, that is obvious in intestinal goblet cells. Mucin
packets that fill the cytoplasm of goblet cells appear electron
lucent; however, a fine filamentous network can be discerned
within the packets (see Fig. 39.17b). Studies have demonstrated
that a major mucin gene expressed by the conjunctival goblet
cell is the large gel-forming mucin MUC5AC173 (Fig. 39.18).
Tight junctions appear to be present between goblet cells and
adjacent stratified cells (see Fig. 39.17).
With the accumulation of data indicating that the basal cells
of the limbal epithelium are the stem cells for the corneal
epithelium, interest has been generated in the location of the
stem-cell population in the conjunctiva. If stem cells are present
within the conjunctiva, do the stratified epithelial cells and the
goblet cells derive from the same stem cell population? Data
suggest that slow-cycling stem-like cells are present in the
fornical region of the rabbit conjunctiva.176 More recently, data
from observation of conjunctival epithelial cells of GFP mice,
and of BRDU-labeled cells suggest that epithelial stem cells
in bulbar conjunctiva are evenly distributed.177 In addition,
clonal cultures of conjunctival epithelium injected subdermally
into nude mice produce cysts that contain both goblet cells and
stratified cells, indicating that stem cells of the conjunctival
epithelium are pluripotent and can give rise to both cell
types.178 It is not known what causes divergence of the
differentiation pathway to give rise to the two cell types.
The connective tissue of the substantia propria of the
conjunctiva is similar to that of the superficial limbus; immune
cells are especially abundant in its loose and highly vascularized
connective tissue. Lymphocytes, mast cells, plasma cells, and
neutrophils are common cell types in its matrix.157 In fact, the
substantia propria has been described as having two layers: an
inner fibrous layer and an outer lymphoid layer. Although the
lymphoid layer has dense accumulations of lymphocytes, these
do not form lymph nodules. The accumulation of the lymphoid
tissue, in addition to the phagocytic abilities of the conjunctival
epithelium, demonstrates the function of the tissue in dealing
with infectious agents.3
 Anatomy and Cell Biology of the Cornea, Superficial Limbus, and Conjunctiva
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 CHAPTER
40 Corneal Form and Function: Clinical Perspective
Stephen D. Klyce, Claes H. Dohlman, and Carlos E. Martinez
INTRODUCTION
The cornea forms the anterior meniscus-shaped transparent
portion of the ocular globe; it serves as the principal refractive
element in the eye, while maintaining a highly impermeable
barrier between the eye and the environment. The cornea is
avascular, meeting its oxygen requirements largely from the
atmosphere by diffusion across the tear film and epithelium;
conversely, it derives most of its additional nutritional
requirements from the aqueous humor arising from across the
corneal endothelium. The epithelium of the cornea provides
the major barrier to tear-borne pathogens, while the corneal
endothelium is principally responsible for maintaining the
hydration and clarity of the corneal stroma.
This chapter reviews aspects of corneal form and function
most relevant to clinical practice to understand the impact of
corneal diseases and surgical interventions on this unique
tissue.
STROMAL ARCHITECTURE – AN
INVITATION TO EDEMA
Most of the connective tissue in the body, including the
sclera, is composed of a dense mat of interweaving collagen
fibers, which limits swelling to some extent. By contrast, the
corneal stroma can swell to several times its normal thickness;
stromal edema can cloud the cornea. This ability to swell is
due in part to the anatomic framework of the corneal stroma
which consists of very long, thin, and striated type I collagen
fibrils. These are laid down in a remarkably parallel and
equidistant fashion.1 The fibrils appear to span, without
branching, from limbus to limbus and are organized into
bundles, called lamellae, arrayed so that adjacent layers lie at
acute angles with one another. Between the fibrils, however, and
in close association with them, proteoglycans and other
proteins constitute the ground substance. The carbohydrate
components of the proteoglycans consist of glycosaminogly-
cans (keratan sulfate, chondroitin sulfate, and dermatan
sulfate), which are mainly responsible for the unique water-
holding capacity of the stroma (Fig. 40.1). Glycosaminoglycans
are polyelectrolytes that, when placed in an aqueous environ-
ment, tend to occupy a large molecular volume, resulting in
stromal swelling if they are not restricted. The swelling tend-
ency is caused by the fixed anionic groups of polyelectrolytes,
which promote swelling by long-range electrostatic repulsion,
the Donnan effect, and, to a lesser degree, colloid osmotic
pressure.2,3
The tendency of the stroma to swell has been called the
swelling pressure (SP), so named because when the stroma is
denuded of its limiting cell layers and placed in normal saline,
a mechanical force can be used to prevent the tissue from
swelling. Swelling pressure measured in vitro and in vivo is
~55 mmHg at normal thickness.4,5 If the stroma is allowed to
swell abnormally (edema), the swelling pressure drops rapidly;
conversely, if the stroma is allowed to dry (because of tear
film breakup), the swelling pressure rises exponentially. The
swelling force can also be measured as the force required
preventing saline from being sucked out of a cannula placed in
the stroma. In this case, the expansive tendency develops a
negative pressure (imbibition pressure, IP), which has the
same numeric value as the swelling pressure in vitro. In vivo,
IP is modified by the intraocular pressure (IOP) according to
the following equation6:
IP = IOP – SP [1]
As discussed later, this relation has important consequences:
as long as IP is negative, fluid is not apt to collect within the
highly resistive epithelium to produce edema. When the IOP
rises (as with glaucoma) so that it is equal to or exceeds the
stromal swelling pressure, the IP rises above zero, and epithelial
edema can ensue.7
The swelling pressure is the force that moves fluid from one
place in the stroma to another whenever there is a gradient in
hydration. In fact, in the cornea in vivo, the anterior stroma
normally is at a lower hydration than the posterior stroma as
a result of differences in the permeability characteristics of
the epithelium (low permeability) and endothelium (high
permeability).8 This gradient in stromal hydration is sustained
because of the high viscosity of the ground substance, which
has been measured as the hydraulic conductivity by Fatt and
Goldstick.9 This high viscosity, which retards bulk fluid
FIGURE 40.1. Keratan sulfate is the main glycosaminoglycan
(mucopolysaccharide) of the stroma and is part of proteoglycan
molecules. Because of their polyanionic properties, the
glycosaminoglycans occupy large molecular volumes, which explains
the high water content of the tissue.
441
 CORNEA AND CONJUNCTIVA
movement in the stroma (as well as in the epithelial cells), is
the underlying factor predisposing the cornea to dellen
formation in response to tear film defects.
CONTROL OF CORNEAL HYDRATION
Because the stroma normally is kept in a relatively deturgesced
and, therefore, optically transparent state, a dehydrating mech-
anism must exist to counterbalance the swelling pressure and
to maintain normal thickness. It was originally proposed that
stromal thickness was maintained passively by the imper-
meability of the corneal membranes to salt and an excess of
solute concentration in the aqueous humor and tears over
that in the stroma.10 It was soon shown, however, that no
biologic membrane is truly impermeable to tissue electrolytes.
Both of the corneal cell layers are now known to be permeable
to solutes and, although they act as passive (non-energy-
consuming) imperfect semipermeable barriers, there must be
a mechanism to maintain the ion content of the stroma.
This mechanism is assisted by the barrier properties of the
corneal cell layers – their resistance to electrolytes, which is
highest in the epithelium and ~100 times lower in the endo-
thelium.11 In the epithelium, the superficial squamous cells
provide the major barrier to the ion flow because this is the
locus of the tight junctional complex that completely sur-
rounds the margins of every superficial cell.12 The restriction
of ions means that rapid water movement is similarly restrained
for osmotic reasons. Subsequently, Davson13 and Harris and
Nordquist14 demonstrated that corneas swollen overnight in
the cold – at temperatures that slowed their metabolic processes
– were able to deturgesce the following day when rewarmed
to body temperature. From this observation, it became clear
that either the corneal epithelium or the corneal endothelium,
or both must expend metabolic energy in the process of
maintaining normal corneal hydration and transparency.
Trenberth and Mishima15 developed rabbit corneal isolation
chamber techniques to show that such a mechanism resided in
the endothelium and was shut down by the transport inhibitor
ouabain.
ENDOTHELIAL ‘FLUID PUMP’ – A
MISNOMER
The early experiments showed clearly that the endothelium
uses metabolic energy to transfer fluid from the stroma to the
aqueous humor, and Maurice16 coined the term endothelial
‘fluid pump’. Maurice, however, envisioned this pump, not
as a literal entity that packaged fluid and removed it from the
SECTION 6
stroma, but as a consequence of an active ion transport process
associated with the endothelium. This process would obey the
laws of thermodynamics to move an as yet to be identified ion
and co-ion (to maintain electroneutrality) out of the stroma
to lower the osmotic pressure of the connective tissue. The
consequence of this process in the normal tissue would lead
to a steady-state situation in which a constant solute gradient
would be maintained across the corneal cell layers that would
balance the swelling pressure and prevent the imbibition of
water. The endothelial ‘fluid pump’ terminology was adopted,
however, and was soon accompanied by the term ‘fluid leak’.
This provides an easily understood concept – fluid leaks in and
is pumped out – but confuses the underpinnings of corneal
hydration control because water actually always is close to
equilibrium in living systems, obeying the osmotic force
gradients across membranes developed and maintained by
active ion transport processes.
It was clear that active ion transport is involved in the control
442 of corneal hydration, particularly because the active transport of
water had never been demonstrated. The model for the control
of corneal hydration that held the most promise postulated
that one or more ion pumps located in the endothelium
would transport solute out of the stroma to balance the solute
that leaked in across the imperfect semipermeable corneal
endothelium.
EPITHELIAL TRANSPORT PROPERTIES
Donn and associates17,18 were the first to search for ion trans-
port processes across the cornea. These investigators demon-
strated the active transport of Na+ by the rabbit cornea in vitro.
Curiously, the transport not only was associated with the
epithelium but also was oriented in the wrong direction: from
tears to stroma. This anomaly did not help with the under-
standing of corneal hydration control because, as noted
previously, one could eliminate the epithelium entirely and
demonstrate the control of stromal thickness by the
endothelium.
Later studies with in vitro rabbit corneas showed that the
epithelium also actively transports Cl– in the secretory direction
(from stroma to tears).19 This transport is regulated by the b-
adrenergic receptor–adenylate cyclase complex, with possible
control by sympathetic nerve fibers.20 Under normal resting
conditions simulating the in vivo corneal situation, the out-
ward active Cl– transport process competes with the inward
active Na+ transport process, leading to a net outward transport
of solute and fluid that could account for a corneal thinning
rate of 1.3 mm/h.21
It was concluded that, although the epithelium is capable of
thinning the cornea at a rate perhaps 30 times lower than that
demonstrated for the endothelium, at least the epithelium is
operating in synergy and not compounding the solute removal
task of the endothelium.
ENDOTHELIAL TRANSPORT PROPERTIES
The search for a candidate for the endothelial transport func-
tion was arduous. Because of the leaky nature of the cell layer,
passive unidirectional fluxes of small ions are large and apt to
mask a net ion flux, which is the hallmark of active transport
when all forces across a cell layer are eliminated. Furthermore,
the corneal endothelium generates an electrical potential of
only 500 mV across a membrane resistance of ~50 Wcm2. This
electromotive force was difficult to measure and neutralize
accurately, further obscuring the nature of the active ion
transport process. With refinements in technology, however,
it was possible to show that the corneal endothelium appears
to accomplish hydration control with transport of the bicar-
bonate ion from stroma to aqueous.22,23 This is accompanied
by the transport of Na+ in the same direction.24
For an active ion transport mechanism to drive water
movement osmotically, the activity must lead to a reduction
or increase in osmotic pressure in a sequestered space. With an
intact epithelium, the corneal stroma could provide such a
space. A reduction of solute concentration in the stroma by as
little as 1% (3 mOsm) below that of the aqueous humor is suf-
ficient to counterbalance stromal swelling pressure. The fluid-
pumping ability of the corneal endothelium, however, appears
to remain intact despite removal of most of the stroma.16,25,26
Therefore, it would appear that the sequestered space needed for
the endothelial transport function must be the cell interior or
perhaps the intracellular space. Nevertheless, the corneal endo-
thelium secretes bicarbonate and Na+ into the aqueous humor,
and the direction and magnitude of this energy-consuming
process are adequate for the regulation of corneal stromal
hydration.

FIGURE 40.2. Hydration of the cornea is kept in balance by opposing
forces: the stromal swelling pressure and the endothelial pump. The
epithelium and the endothelium restrict rapid fluid movements (see
text). IOP, intraocular pressure; SP, swelling pressure.
EVAPORATION AND INTRAOCULAR
PRESSURE
When the eye is open, evaporation occurs at a rate of
~2.5 mL/cm2 per hour,27 and this can account for the cornea
thinning some 5% during awake periods compared with periods
of sleep. In a normal eye with ample tear secretion, this
evaporation is of little importance for the overall corneal fluid
balance. In a dry eye or an eye with insufficient blink function,
exposure and evaporation can have devastating consequences.
Also, in the normal eye, the IOP does not have a significant
effect on corneal hydration so long as it is in the normal range.
If the IOP is more than 50–60 mmHg, however, or if the stroma
is swollen from endothelial dysfunction or decompensation, the
IOP becomes a major factor in the development of epithelial
edema and reduction of vision (discussed later).
MODEL FOR THE CONTROL OF CORNEAL
HYDRATION
With the endothelial ion transport process identified, the
opportunity is presented to develop a holistic model of corneal
hydration dynamics that considers all the major flows and
forces underlying homeostasis. A full characterization of cor-
neal hydration dynamics must consider at least cell layer barrier
(passive permeability) properties as well as any active ion
transport processes, the swelling pressure and flow charac-
teristics of the stroma, and external forces, such as evaporation
and IOP (Fig. 40.2). Klyce and Russell8 considered these and
developed and tested such a model applying the formalism of
Kedem and Katchalsky,28 which is based on the thermo-
dynamics of irreversible processes. In the absence of more
detailed information regarding specific cell pathways for ion and
water flow, the corneal epithelium and endothelium were
regarded as thin, semipermeable membranes that embrace the
active ion transport processes identified for these layers. The
corneal stroma, which often is considered a large, well-stirred
compartment with respect to corneal permeability studies, was
modeled with the Kedem–Katchalsky equations as well, with
the addition of the well-characterized relations that associate
stromal thickness to its hydrodynamic properties that change
reversibly with edema. These concepts show that water
movements in the stroma are greatly retarded compared to free
solution, and the viscous nature of the stromal ground
substance permits large gradients in hydration to develop under
abnormal circumstances. However, irreversible stromal changes
Corneal Form and Function: Clinical Perspective
that might occur with severe corneal pathology leading to
scarring and opacification are beyond this framework.
Because the normal corneal stroma swells only in thickness,
its diameter and anterior curvature remaining relatively
constant with edema, there is a linear relation between
thickness, q, and water content or hydration, H:
H = 8q – 0.7 [2]
As noted previously, stromal swelling pressure, SP, is the
driving force for water movements in the stroma and has been
shown to be related to hydration, H, as follows:
SP = g e–H [3]
where g is a constant. This exponential relation is the reason
that swelling pressure drops rapidly with hydration. The next
equation relates the flow conductivity for water within the
stroma to local stromal hydration. This is known as the
hydraulic conductivity, Lp, and changes even more rapidly than
swelling pressure with changes in hydration:
Lp = r H4 [4]
where r is a constant. Fluid flow from point to point, Jv, in the
stroma is the product of the driving force, SP, and the flow
conductivity, Lp. Hence, one can write the following:
Jv = Lp µ SP [5]
The consequence of this relation favors corneal stromal
homeostasis with regard to hydration. When the corneal stroma
swells above normal hydration, the swelling pressure (its
gradient drives fluid flow) falls rapidly (see Eqn [3]). When the
corneal stroma thins below normal hydration, its hydraulic
conductivity (permissivity to water flow) falls rapidly (see Eqn
[4]). We can combine the overall effect in Eqn [5] to indicate
that corneal thickness changes are most dynamic when the
stroma is near normal hydration and less dynamic as it either
swells or thins. Clinically, this is the reason that steep
hydration gradients can exist from place to place in the stroma,
as with dellen or focal edema.
The model for corneal hydration control proposed by Klyce
and Russell8 was able to achieve a constant corneal thickness
and mimic the corneal response to several well-documented
observations. For example, the model matched the rate at which
the cornea thinned after cold swelling and the rate of stromal
swelling that occurred when the endothelial transport system
was inhibited by ouabain. The model was also used to show
that the cornea swells in a predictable fashion during hypoxia,
such as occurs under a tight-fitting contact lens due to the
accumulation of lactate within the stroma.29
CHAPTER 40
CORNEAL EDEMA
While the above formalism is developed to understand the
normal regulation of corneal hydration, this knowledge can be
extended to learn the corneal hydration response to pathology.
Chronic corneal edema develops as a consequence of endo-
thelial dysfunction, regardless of whether the original clinical
condition was dystrophy, inflammation, or trauma. The
increased permeability of this cellular layer, its decreased ion
transport function, or both can lead to the subsequent corneal
changes. In mild cases, increased stromal thickness occurs,
with initially little consequence to vision. In advanced cases,
epithelial edema ensues, which rapidly decreases visual acuity.
Late in the course of the disease, painful bullous changes can
develop (bullous keratopathy). If the natural clinical course is
not interrupted by keratoplasty, a thick subepithelial pannus
eventually develops, leading to the disappearance of the bullae
and of the discomfort. Vision at this stage usually is reduced to 443
 CORNEA AND CONJUNCTIVA

FIGURE 40.3. Natural history of progressive

corneal edema. (a) Cornea guttata of Fuchs’
dystrophy often begins in young adulthood and
progresses slowly over decades. Some edema
(thickening) gradually ensues for a long time
without affecting vision. (b) Epithelial edema
begins in midlate life, first as fine microcysts
that distort the surface and cause reduction of
vision. (c) Frank epithelial edema with visible
blebs, opacity, and gross surface irregularities.
(d) End-stage chronic edema after many years
of often painful bullous epithelium. A
connective tissue pannus has formed between
a b the epithelium and Bowman’s layer. At this
stage, the cornea is opaque, but the epithelium
has scarred down, and the pain is gone.

c d

the hand-movement level because of epithelial and stromal
scar formation (Figs 40.3 and 40.4).
Acute corneal edema, which can result from contact lens
wear or angle-closure glaucoma, follows a different path of
physiologic development and is usually reversible.
DEVELOPMENT OF EDEMA
Endothelial Changes
The endothelium under stress changes in a few nonspecific but
characteristic ways. Thus, in acute inflammation or in trauma,
rapid cell degeneration and cell death can occur in a focal
manner that is then repaired by sliding and rearrangement of
neighboring cells. The resulting endothelium is characterized by
decreased cell number and enlarged and irregularly shaped cells
(polymegathism and pleomorphism).30
In chronic inflammation, endothelial cells can undergo
fibrous metaplasia31; this can result in a fibrous membrane bet-
ween Descemet’s membrane and the endothelium. In Fuchs’
SECTION 6
in the posterior direction (corneal anterior curvature and
diameter remain normal), its thickness increases, especially
centrally, because the peripheral corneal swelling appears to be
limited somewhat by structural restriction imposed by the
limbus. This flattening of the posterior surface can throw
Descemet’s membrane into multiple folds that become visible
as striae on slit-lamp microscopy. Usually, there is little tissue
reaction to the swelling; it is only in massive, chronic edema
that scarring of the tissue eventually develops, more markedly
in the posterior layers and especially in the folds created by
Descemet’s membrane.
Epithelial Edema
Epithelial edema resulting from endothelial dysfunction,
elevated IOP, or both is predominantly extracellular.34 Thus,

dystrophy, the cells exhibit a change in form and show vacuoles,

phagocytized pigment, and periodic acid-Schiff stain-positive
material that is eventually deposited on Descemet’s membrane.
These irregular depositions become visible as the characteristic
warts (guttae) over which the endothelial cells eventually
become attenuated. Even in advanced cases of Fuchs’ dystrophy,
however, the endothelial surface appears intact.32 For a review
of this subject, see Waring and colleagues.33

Stromal Edema
When the endothelial cell density falls below a critical level
(200–400 cells/mm2), the ability of the endothelium to main-
tain stromal hydration begins to falter, and stromal edema
develops gradually. The two opposing forces – the osmotic pres-
sure developed by the endothelial ion transport and the stromal
swelling pressure – remain in balance, but the osmotic gradient
established by the endothelial pump must diminish with
reduced transport function and possibly greater ion leakage
444 across the endothelium. Because the stroma can swell only FIGURE 40.4. Massive bullous keratopathy after cataract extraction.
 Corneal Form and Function: Clinical Perspective

fluid begins to accumulate in the space between the basal

epithelial cells, stretching the bridging desmosomes. Later in
the process, these fluid-filled spaces enlarge to form fine
blisters, visible as microcystic edema in the slit lamp. Finally,
larger bullae develop, characteristic of bullous keratopathy.
Epithelial edema rarely involves the anterior-most squamous
cells of the epithelium (Fig. 40.5).
The underlying pathophysiologic mechanism appears to
involve a forward movement of stromal fluid and aqueous,
generated by the IOP. Thus, if the endothelial functional
reserve falls below a certain level, leading to edema and a
reduction in stromal swelling pressure to below the value of
the IOP, fluid from the aqueous can collect.8 Because the other-
wise healthy epithelium has such a high resistance to
electrolytes and to the flow of water, the fluid can be trapped
within the epithelium, resulting in the formation of cysts and
bullae. The anterior-most wing cells generally are unaffected, FIGURE 40.5. Development of epithelial edema. When the intraocular
suggesting that the resistance to this anterior fluid movement pressure (IOP) overpowers the endothelial pump, fluid is pushed into
is situated primarily in this layer. the epithelium, resulting in edema. This can occur with a normal
endothelium and very high pressure (acute glaucoma). In chronic
The IOP can be higher than the stromal swelling pressure,
edema, the IOP is normal, but the endothelium is severely
resulting in epithelial edema, in several different clinical set- dysfunctional (see text).
tings. On one end of the spectrum, poor endothelial function,
even with normal IOP, is sufficient to cause epithelial edema
(e.g., Fuchs’ dystrophy, aphakic or pseudophakic edema). On
the other end of the spectrum, even with normal endothelium,
very high IOP (such as occurs in angle-closure glaucoma) also
can cause epithelial edema (Fig. 40.6). Between these two
extremes, edema can result from various combinations of
endothelial dysfunction and elevated pressure8 (Fig. 40.7).
The concept that the IOP is the driving force behind the fluid
movement in epithelial edema is supported particularly by the
fact that in phthisis with marked hypotony, epithelial edema
does not occur, no matter how damaged the endothelium or
how thick the stroma.
Evaporation can be a balancing factor in borderline epithelial
edema. Commonly in early edema, vision is blurred in the
morning but clears as the day progresses. The lack of evap-
oration when the lids are closed during the night allows fluid
to accumulate in the epithelium. After opening the eyes, evap-
oration results in slight hypertonicity of the tear film, which
in turn extracts water from the epithelium, clearing the vision.
This state can last for months, rarely years, but eventually the
edema worsens.

Edema with Contact Lens Wear

The edema that occasionally is seen as a result of contact lens
FIGURE 40.6. Corneal thickness and IOP as they relate to epithelial
wear differs in many respects from the forms of edema
edema. Such edema is expected to the right of the solid line. The
described previously. The symptoms and signs usually are

attributable to hypoxia, hypercapnia (elevated CO2 tension), or
both under the contact lens. Therefore, gas permeability of
the lens and tear fluid exchange are the most important
parameters in maintaining normal fluid balance in the cornea.
CHAPTER 40
circle indicates normal values.
Data from Ytteborg J, Dohlman CH: Corneal edema and intraocular pressure. II.
Clinical results. Arch Ophthalmol 1965; 74:477; and Klyce SD, Beuerman RW:
Structure and function of the cornea. In: Kaufman HE, Barron BA, McDonald
MB, eds. The cornea. 2nd edn. Boston: Butterworth-Heinemann; 1998.

FIGURE 40.7. (a) and (b) Epithelial edema. The

fluid is pushed into the epithelium, resulting in
distention of the intercellular spaces. (a) µ500;
(b) Electron micrograph, µ2000.
Courtesy of Toichi Kuwabara, MD.

a b
445
 CORNEA AND CONJUNCTIVA
FIGURE 40.8. Irregular Placido’s mires in chronic edema, indicating
the substantial role of the surface abnormalities in reduction of vision.
Mild stromal edema is common in soft contact lens wear.
Epithelial hypoxia causes lactic acid buildup in the stroma35
and reduced pH,36 which in turn may affect endothelial
performance. The lactic acid accumulation, however, raises
stromal osmotic pressure, drawing in water for osmotic reasons
noted previously, and pH effects on the corneal endothelium
may not occur in acute situations.30 This thickening of the
stroma usually is clinically acceptable because it has little
influence on visual acuity or contrast sensitivity.37 Epithelial
edema, on the other hand, resulting from hard contact lens
overwear (Sattler’s veil, epithelial bedewing) can be debilitating
visually, although it is readily reversible on removal of the lens.
Histologically, the location of the fluid collection is primarily
intracellular, in contrast to the primarily extracellular edema in
Fuchs’ dystrophy or similar conditions, and may be related to
induced abnormalities caused by hypoxia and lactate
accumulation.29 For a review of corneal pathophysiology and
contact lens wear, see Bruce and Brennan.38
SECTION 6
Visual Acuity in Edema
Because of its surface smoothness and its transparency, the
cornea normally allows a remarkably sharp image to be
focused on the retina. In general, these optical qualities can
be reduced by opacities within the tissue (stroma or epithelium)
or by surface irregularities in the form of gross astigmatism
(e.g., keratoconus) or minute central irregularities (e.g., bullous
keratopathy).
Normal stromal transparency has been difficult to explain
in view of the internal fluctuations in refractive index between
the stromal components. It has been proposed, however, that
as long as the collagen fibrils are parallel and equidistant and
the distance between them is less than half the wavelength of
light (~2000 Å), light scattering should be at a minimum and
transparency preserved.39,40 In the normal corneal stroma, the
collagen fibrils are spaced some 600 Å from center to center and
are closer together than half the wavelength of light, which
446 explains the optical qualities of the tissue. Transparency is still
well preserved in mild or moderate stromal edema,41 and
backscattering toward the source is minimal.42 In more
advanced and long-standing edema, however, irregular fluid
accumulations occur in the stroma that can reduce
transparency.43 Later, stromal scarring and posterior irregular
astigmatism from folds in Descemet’s membrane reduce
vision further.
In Sattler’s veil, reduction in vision usually is minor and is
caused by a diffraction phenomenon that occurs within the
slightly swollen epithelial cells.44 True epithelial edema, in
contrast to Sattler’s veil and stromal edema, can reduce vision
early and profoundly. The fluid accumulations between or
within the epithelial cells markedly increase light scattering.
Even more important are the minute surface irregularities
from the edema that break up the smoothness normally
provided by the corneal tears and consequently lead to blurring
of the retinal image (Fig. 40.8). In general, in a patient with
vision reduced by corneal pathology, there often is a tendency
for the clinician to overestimate the contribution of opacities
within the tissue and to underestimate the role of surface
irregularities. A hard contact lens refraction or corneal
topography analysis should settle the question of the influence
of surface irregularities on visual acuity.
CLINICAL EVALUATION OF EDEMA
To evaluate a case of corneal edema fully – its cause, extent,
and prognosis – it is advisable to conduct the investigation in
a sequential and systematic manner.
History
As with any other ophthalmic condition, a detailed patient
history is mandatory. Because most cases of chronic edema
result from endothelial malfunction, it is important to find
out whether there has been a family history of dystrophy or
trauma or whether inflammation with a red eye has been
experienced by the patient in the past. Other issues that should
be covered include the degree and duration of visual loss,
whether vision is worse in the morning but clears during the
day (possibly indicating Fuchs’ dystrophy), whether episodes of
blurred vision have been separated by long periods of normal
vision (possible herpes virus infection), whether the symptoms
are unilateral or bilateral, and the results of previous
examinations by other physicians. A detailed history is usually
strongly suggestive of the cause of the condition.
Slit-Lamp Microscopy
The introduction of the slit lamp in 1911 contributed enor-
mously to our ability to diagnose corneal disease, especially
edema. The epithelium should be intact and free of fluorescein
or rose bengal stain. In early epithelial edema, this layer appears
more gray or ‘full’ than the normal, optically empty cell layer.
Later, a microcystic appearance may ensue – patchwise, central,
or uniform – that initially may be visible only in retro-
illumination. In full-blown bullous keratopathy, the variably
sized cysts are obvious to inspection, as is the degree of opacity
within the thickened epithelium. In end-stage severe edema, a
pannus layer of connective tissue may be visible between the
epithelium and Bowman’s membrane.
The stroma usually is thickened somewhat, as indicated by
posterior ‘striae’ from folds in Descemet’s membrane. As men-
tioned previously, the edematous stroma can be optically clear
for a long time, but in severe chronic edema, scar formation
gradually develops, creating haze. Guttate appearance of the
posterior surface, often with pigment (the hallmark of Fuchs’
dystrophy), is apparent in specular reflection using a wide angle
between the light source and the observer. Stromal vas-

b
FIGURE 40.9. Specular microscopy of the corneal endothelium.
(a) The endothelium in an adult, 3200 cell/mm2. (b) The endothelium
in a graft that has suffered a rejection episode but is still clear,
500 cell/mm2, large, and irregular.
cularization and keratic precipitates indicate previous or present
inflammation and, if unilateral, often are suggestive of herpes
virus infection. The corneas should be photographed, with
and without slit beam, for documentation.
Specular Microscopy
The technique of observing and recording the morphology of
the corneal endothelium with high magnification in vivo was
introduced by Maurice in 1968.45 The subsequent develop-
ments of contact and noncontact specular microscopes,
applicable in the clinic, as well as the various types of analyses
of cell density and cell shape, are covered in Chapter 60. This
technique allows the clinician to follow the status of the
endothelium in dystrophy, in degeneration, before and after
surgery, after trauma, and in donor corneas, among other
situations.
It generally is agreed that human endothelium has little or no
ability to divide after birth. Therefore, in aging or in an accel-
erated fashion after injury or disease, the endothelium loses
cells without replacement. The normal endothelial cell count
is 3000–3500 cells/mm2 in young adults, decreasing to about
two-thirds that value in old age.46 After injury, the endo-
thelium heals by sliding, rearrangement, and irregular enlarge-
ment of adjacent cells, usually from a large surrounding
area.47 The result is decreased cell count, often only regionally
rather than uniformly across the whole back surface of the
cornea, rendering specular microscopy variable. The degree of
pleomorphism and polymegathism of the endothelial cells
usually is as indicative of the endothelial health and reserve as
is the cell count (Fig. 40.9).
In clinical practice, it has proved difficult to predict physio-
logic function on the basis of cell density or morphology.48
Cell counts down to only a few hundred per square millimeter
have been observed in edema-free corneas and grafts, whereas
many cases of frank edema have had much higher counts. This
discrepancy at times can be explained by the variability of
morphology across the cornea; at other times, there may be
factors not detectable with a specular microscope.
Corneal Form and Function: Clinical Perspective
FIGURE 40.10. Specular microscopy of the endothelium in a cornea
with guttae but no overt edema. The black dots indicate areas where
the endothelium has been lifted posteriorly and out of focus by the
excrescences on Descemet’s membrane.
TABLE 40.1. Endothelial Cell Count (per mm2)
4000–1500
1500–1000
1000–500
<500 Normal
Low
Borderline
Usually edema
Fuchs’ dystrophy can be diagnosed early with specular
microscopy, even before guttae become visible in the slit
lamp.49,50 The excrescences from Descemet’s membrane cause
the overlying endothelium to bulge posteriorly out of focus,
resulting in multiple round black areas of different diameters
(Fig. 40.10). It can be argued, however, that with our present
state of knowledge, very early diagnosis of Fuchs’ dystrophy is
not essential because the condition takes decades to develop
and because no preventive treatment is available. The visible
endothelial cells in Fuchs’ dystrophy are usually enlarged
and have irregular cell borders.
Specular microscopy has become a useful tool in the evalu-
ation of surgical procedures with respect to their trauma
to the corneal endothelium. Thus, by following the cell counts,
the value of various techniques of cataract surgery and different
CHAPTER 40
models of intraocular lenses has been determined with much
greater precision than was possible when relying only on stat-
istics on the incidence of edema after years of postoperative
follow-up. Thus, switching from an intracapsular to an extra-
capsular cataract extraction technique has not been
accompanied by increased cell loss. In one study done in 1981,
99 consecutive cases of intracapsular cataract extraction
resulted in a 17% cell loss, whereas the same number of extra-
capsular cases had a 17% loss, both series with lens implan-
tation.51 Phacoemulsification in the anterior chamber proved
more traumatic, with a cell loss of up to 30%.52 The protective
effect of sodium hyaluronate during anterior segment surgery –
a milestone in ophthalmic surgery development – has also been
demonstrated with exactness by specular microscopy.53
Although the correlation between endothelial cell count and
frank corneal edema is poor, there remains a linear relation
between the two54; therefore, specular microscopy can have con-
siderable predictive value before and after surgery (Table 40.1).
The technique is gaining increasing popularity in clinical 447
 CORNEA AND CONJUNCTIVA
TABLE 40.2. Indications for Simultaneous Keratoplasty in
Cataract Surgery
Frank epithelial edema, or
Blurry vision in the morning, or
Corneal thickness above 0.70 mm, or
Endothelial cell density less than 500 cells/mm2
practice, especially when the endothelium can be expected pre-
operatively to have borderline function. This is particularly
pertinent when cataract extraction is contemplated in Fuchs’
dystrophy or after trauma. With a very low cell count and
abnormal corneal thickness, a simultaneous keratoplasty might
be indicated (Table 40.2).
Specular microscopy also is a valuable tool in keratoplasty.
Postoperatively, several investigators noted 15–20% cell loss
within the first 3 months, and attrition continued for several
years.33 Mean cell counts in clear grafts were 1000–2000
cells/mm2.55,56 From a practical point of view, specular
microscopy probably has become most useful in the evaluation
of donor corneas, especially for screening of donor material for
Fuchs’ dystrophy. Mild forms of this dystrophy are common,
and such donor grafts would be expected to have shorter
survival times, especially if grafted into patients with edema.
Slit-lamp examination alone of donor corneas with Fuchs’
dystrophy is of limited use because the tissue is usually swollen
when harvested, which makes guttae hard to detect. Most eye
banks in the United States now routinely perform specular
microscopy on donor corneas.
Corneal Confocal Microscopy
The corneal confocal microscope has become a useful diag-
nostic tool for evaluating the cornea with capabilities that
surpass those of the specular microscope. While the magnifi-
cation of the confocal microscope is similar to that of the
specular microscope, the resolution of the confocal is higher.
In the confocal mode, the narrow illumination and viewing
paths are at an angle to one another. This reduces the impact
of contrast loss from tissue light scatter along the illumination
pathway. The early development work was done largely through
the pioneering efforts of Lemp, Masters, Cavanagh, and
Jester,57,58 who demonstrated the potential for the use of con-
focal microscopy in corneal research. Today the confocal micro-
scope is available for clinical application. Designed specifically
for imaging the cornea, scanning spot and scanning slit models
SECTION 6
have been developed. The scanning spot models applanate the
cornea and provide imaging throughout the cornea. The
scanning slit model has a noncontacting objective with a 2 mm
working distance that is optically coupled to the cornea by
means of a globule of gel. This reduces the intense specular
reflection from the epithelial surface. The confocal microscopes
provide high-resolution images at various corneal depths with
optical sections of less than ± 10 um.
Corneal confocal microscopes can provide other useful
clinical information.59 Applications have included evaluation of
corneal wound healing responses after refractive surgery
(e.g., amount and localization of haze after PRK, thickness of
stromal bed and interface artifacts after LASIK), endothelial cell
analysis (e.g., cell density, polymegathism, and pleomorphism),
and identification of corneal pathology (e.g., differentiation
between herpetic keratitis and Acanthamoeba infections. In the
past this technology has had limited success in the clinical
setting, because of the high purchase cost, lack of high-contrast
448 images, and difficulty in manually aligning the axis of the
instrument with the patient’s eye. As with most technology,
costs to purchase have been reduced and major improvements
have occurred in the quality of both live and stored images.
Measurement of Corneal Thickness
As mentioned previously, the thickness of the stroma (or the
whole cornea) is directly related to the dehydrating function
of the endothelium.60 Therefore, the functional status of the
endothelium and its reserve capacity can be measured by
pachometry, the technique of recording corneal thickness
in vivo. The normal thickness of the central cornea is
0.51–0.52 mm according to most investigators.61 As mentioned
previously, the exact relation between stromal thickness and
stromal swelling pressure (assumed to be equal to opposing
endothelial pump pressure) has been determined.4 Pachometry
used to be primarily a laboratory tool in studies of corneal
physiology. Because of the need to determine the status of the
endothelium in a number of clinical situations, however, several
different types of pachometers have been developed for clinical
use. The Haag–Streit optical pachometer is attachable to its
slit lamp and employs an image-splitting principle. This
technique can be exact (±2% error), but reading the endpoint is
difficult, and substantial practice is required to achieve reliable
data.62
Both the specular microscope and the corneal confocal micro-
scope also can be used to measure corneal thickness. When
a contact microscope is focused on the endothelium, the dis-
tance between the applanating lens and the endothelium is
displayed automatically in some models. This technique is
exact and is easily learned by a photographer. Nonapplanating
lens confocal microscopy measurement of corneal thickness is
also possible with ancillary apparatus that measures the
distance between the lens and the globe.
Ultrasound pachometers are most commonly used not only
for measuring edema, but also for measuring corneal thickness
prior to performing refractive surgery.63 They are easy to handle,
but the ultrasound beam must be directed perpendicularly to
the cornea. The machines are designed to record only those
reflections received from within a few degrees of the
perpendicular.
Automatic slit scanning corneal topographers are also in use
for measuring corneal thickness. These not only provide central
corneal thickness, but can present a thickness profile that
extends out to the limbus. These data are useful for detecting
the distribution of thinning in keratoconus.
Clinically, pachometry has been used to follow endothelial
function after cataract surgery59 and after penetrating
keratoplasty.64,65 More important is the use of pachometry in
evaluating the functional reserve of the endothelium in a clear
cornea in which some stromal edema is suspected but no
epithelial edema has yet appeared. This has particular
importance for the prognosis of Fuchs’ dystrophy when cataract
surgeries is contemplated but when there is uncertainty about
whether the endothelial layer could withstand further
manipulation (see Table 40.2). A central reading close to the
normal 0.5–0.6 mm measurement is reassuring, but a value of
~0.7 mm or above measures borderline decompensation with
risk of frank epithelial edema (discussed later).
Endothelial Permeability
Because the ‘leaky’ corneal endothelium maintains corneal
deturgescence by virtue of its transport and permeability
properties, study of its permeability may yield clinically useful
information. Fluorescein has been chosen for the test
substance, and the passage of dye across the endothelium
can be measured with a sensitive fluorometer.66,67 Fluorescein
can be driven into the cornea by iontophoresis and its

diffusion into the aqueous followed. It is safer and simpler, how-
ever, to ingest the dye systemically and measure the forward
diffusion across the endothelium.68 Results of this test have
reconfirmed the key role of the endothelial barrier function in
corneal deturgescence. Also, in this way, fluorophotometry
promises to be a useful diagnostic technique in the clinic.69
For instance, after keratoplasty, the endothelial transfer
coefficient has been found to be increased initially but to return
gradually to near normal, parallel to the reduction of thick-
ness.70 In early Fuchs’ dystrophy, fluorophotometry has
indicated that the swelling is due primarily to a decrease in
the endothelial pump function rather than to a breakdown of
the barrier function.71 Mishima has provided a review of the
theoretical background and clinical applications.69
CLINICAL CONDITIONS LEADING TO EDEMA
The clinical disease entities that can cause corneal edema are
covered fully elsewhere in this text and are not reviewed here.
Therefore, the diagnoses are merely listed, and the reader is
advised to turn to the appropriate section for further
information.
The most common conditions are the following:
1. Inflammation (particularly after infection)
2. Graft rejection
3. Endothelial dystrophy
a. Fuchs’ dystrophy
b. Congenital hereditary endothelial dystrophy
c. Chandler’s syndrome and similar conditions
4. Dysgenesis
5. Keratoconus (hydrops)
6. Trauma
a. Intraocular surgery
b. Other trauma (mechanical, chemical)
7. Acute glaucoma
8. Contact lens overwear
MEDICAL TREATMENT OF EDEMA
Some forms of corneal edema respond to nonsurgical measures,
but most cases do not. The cases in which medical treatment is
effective are usually caused by inflammation – particularly
postinfection – and steroids can be useful in this setting. In
chronic noninflammatory edema, some amelioration of
symptoms can be achieved by a soft contact lens or hypertonic
agents or, rarely, by reducing the IOP. In most patients with
advanced edema, however, only keratoplasty is curative.
Suppression of Inflammation
If given early enough in the disease process, corticosteroids
can be highly effective in reversing corneal edema resulting
from inflammation. Infections, particularly herpetic but also
bacterial or fungal, are often severe enough to affect the
function of the endothelium immediately. The microbes may
be eliminated promptly with appropriate antibiotics, but the
subsequent cascade of postinfectious inflammatory events can
result in edema of such severity that it becomes irreversible
unless corticosteroids are instituted. Energetic efforts to reverse
inflammatory edema are important because severe, irreversible
edema is not easily treated surgically. The long-term prognosis
after keratoplasty is poor for an eye that has been severely
inflamed.
Ophthalmologists are often reluctant to give steroids to
patients with acute infections as long as microorganisms may
still be alive, considering the steroids’ lowering of the host’s
resistance. These fears are often exaggerated, and it can be
much worse to allow inflammatory destruction to advance
Corneal Form and Function: Clinical Perspective
FIGURE 40.11. Common gestalt of topical corticosteroid treatment in
corneal inflammation, especially in stromal herpes. Frequent
instillation of a steroid preparation rarely is necessary for many days,
particularly in cases of lymphocytic response. The drug then can be
tapered quickly, but a subsequent long trial of very low dose or
infrequent treatments may be necessary.
unchecked for a long time than to give steroids early. In patients
with bacterial infections, it is usually safe to start topical
steroids simultaneously with appropriate antibiotics or to
wait 2–3 days at most. In patients with stromal herpes and
developing edema, steroids should be given promptly unless an
active dendritic epithelial process is also present. Fungal
infections such as fusarium keratitis should not be treated with
steroids.
In patients with corneal edema caused by inflammation,
steroids should be administered topically and only rarely
systemically. Steroids given by mouth, even in high doses, result
in only a low concentration in the endothelium – much lower
than what can be achieved by topical treatment. Only in severe
keratouveitis may steroids be added systemically and with
caution, owing to the side effects. The most commonly used
steroids in drop form are 0.1% dexamethasone (as alcohol or
phosphate) and 1% prednisolone (as acetate). One-eighth
percent prednisolone and 0.1% fluorometholone have a weaker
effect. The acetate form penetrates the epithelium more readily
than do the other derivatives.72 The general treatment principle
should be to ‘hit hard’ (administer a strong concentration of
steroids four to eight times per day) for a few days and then to
taper rapidly. Small doses of a weak steroid solution may then
be necessary for months (Fig. 40.11). It is important not to give
more than is clinically necessary. The latter goal certainly is not
always obvious in the clinical setting, and in general, steroid
treatment of corneal diseases requires considerable experience.
In corneal edema caused by dystrophy, corticosteroids are
without value. Thus, in Fuchs’ dystrophy, steroid treatment has
CHAPTER 40
no effect on corneal thickness.73 In obscure cases, especially in
unilateral edema with no signs of guttate lesions, an
inflammatory component (sometimes herpes) is possible, and a
shorter trial of topical steroids is reasonable. Steroid drops given
three to four times per day for 2 or 3 weeks should settle
whether the edema is reversible. Small doses of antiviral or
antibiotic agents may also be indicated prophylactically.
Corneal edema of a graft caused by immunologic rejection
constitutes a special case of inflammation. This topic is covered
in more detail in Chapters 65 and 77, but a few points deserve
mention here. When sensitized lymphocytes attack the graft
endothelium, destruction often is rapid, and recognition and
treatment often late. Therefore, the patient should be taught to
respond to symptoms of decreased vision, redness, or
discomfort by contacting the surgeon within 24 h. Topical
steroids should be instituted as rapidly as possible (e.g., a
strong-concentration solution every hour while awake for a few
days). Systemic steroids rarely are indicated. After a few days,
the medication can be tapered fairly quickly, even if frank 449
 CORNEA AND CONJUNCTIVA
edema remains. The lymphocytes are quickly eradicated, but it
takes some time for surviving endothelial cells to recover and
fill in defects. If the patient still has a clear lens, long-term use
of steroids will eventually result in a cataract. It was shown that
after keratoplasty to treat keratoconus, it takes only ~800 drops
of 0.1% dexamethasone to initiate posterior subcapsular
cataract in half of cases.74 Obviously, the IOP must also be
followed during such steroid therapy. If discovered early, most
graft rejections can be reversed.
Lowering of Intraocular Pressure
Because epithelial edema and the concomitant reduction of
vision are the result of a situation in which the IOP overpowers
the stromal swelling pressure, it seems logical to try to reduce
the IOP. In most cases of chronic edema (e.g., Fuchs’, aphakic,
and pseudophakic edema), however, the tension is normal,
and it is not possible to lower the pressure safely to a stable
single-digit level that would be necessary to reduce the edema.
In acute glaucoma with high pressure and epithelial edema,
prompt lowering is mandatory. In most cases, the endothelium
is normal, and the epithelial edema disappears rapidly once
the pressure has been reduced to the level of 50 mmHg or less.
Only in situations of moderate tension elevation combined
with a marginally decompensated cornea can long-term pres-
sure lowering be of clinical value. This scenario rarely occurs,
but it can be seen in grafts bordering on failure and also in some
dystrophies, such as Chandler’s syndrome.75 The usual anti-
glaucomatous medication can be employed. In the long term,
however, edema tends to worsen gradually, and manipulation
of the pressure becomes increasingly futile.
Hypertonic Agents
When hypertonic drops or ointments are instilled in eyes with
early epithelial edema, some clearing of vision often can be
achieved.10 Enough water is extracted temporarily out of the
epithelium to smooth the surface and reduce the diffraction of
light by the microcysts. Five percent sodium chloride drops for
daytime use and ointment of the same strength at night are
commonly used. Sucrose at 40% concentration (sticky),
anhydrous glycerol (painful), and a hair dryer (dusty) have
also been recommended but have no advantage over sodium
chloride. Similarly, a dry climate causes more rapid evap-
oration of the tear film than does humid air. These measures
are often effective in morning edema in Fuchs’ dystrophy,
when they help to speed up the clearing of vision. Hypertonic
agents thus can be effective for months, rarely years, until the
edema has become constant and irreversible. Even when in
doubt about the efficacy of hypertonic agents, these agents are
SECTION 6
almost always harmless to try.
Stromal edema, in contrast to epithelial edema, is not
affected by the topical use of hypertonic agents. The volume of
stromal water is simply too massive and too rapidly replenished
across the leaky endothelium to be influenced.
Soft Contact Lenses
Chronic corneal edema, once it has reached the bullous stage,
is not only blinding but also painful. The tugging of the blebs
and their corneal nerve endings by the lids during blinking
made life miserable for many patients before keratoplasty
became successful. In cases in which there is likely to be little
or no recovery of vision with transplantation but in which
comfort is important, a soft hydrophilic contact lens often is
an excellent tool. Vision is likely to decrease somewhat but
marked comfort is restored in about three-quarters of patients.76
I prefer the thick, high-water-content lenses that are left in
place around the clock for months at a time. Antibiotics are
450 probably not necessary but can be given in low doses for the first
few weeks. The incidence of infection is unknown but probably
low. Soft contact lenses have become a valuable therapeutic
modality in cases of painful bullous keratopathy in which
keratoplasty is not indicated.
SURGICAL TREATMENT
Before the 1950s there was simply no cure for chronic edema,
and patients often had to live out their lives in blindness and
frequently in pain. The first successful transplantations for
edema were reported in 1952,77 and others followed rapidly.78
Since that time, progress has been rapid because of greater care
in handling the endothelium, larger grafts, the availability of
steroids, and the development of finer suture material.79 Today,
most patients with corneal edema can be helped by keratoplasty
– a remarkable surgical success story (see Fig. 40.10).
CORNEAL TOPOGRAPHY
As an optical component of the visual system, the swelling
properties of the cornea discussed above relate to light scatter.
In summary, mild epithelial edema can produce the symptoms
of halos around bright lights or Sattler’s Veil, while moderate
stromal edema can also decrease visual acuity primarily
through light scatter, although this does not become significant
until swelling of 70% is achieved. The most critical element
in preserving corneal optics is the status of the corneal surface
and tear film. Disruption of the tear film or irregularities in
the corneal epithelial surface such as caused by basement
membrane dystrophy, bullous keratopathy, infectious keratitus,
trauma, ectatic degenerative disease (keratoconus, pellucid
marginal degeneration, terriens marginal degeneration, and
keratoglobus), and keratectasia and other complications sub-
sequent to refractive surgery can all cause significant visual
loss. Corneal topographers have emerged as a powerful tool
with which to assess the etiology of factors that degrade vision
by producing irregularities on the corneal surface that lead to
optical aberrations.
The corneal tear film/air interface provides about two-thirds
of the vergence of the eye. Thus, it plays a critical role in the
quality of the optics of the eye. Furthermore, because of this
property, even small amounts of surface distortion can greatly
reduce the quality of the retinal image. Direct examination of
the corneal surface with the biomicroscope does not provide
enough resolution to detect vision-reducing irregular astig-
matism. Although retinoscopy provides a greater sensitivity to
irregular astigmatism, the distortion seen in the retinal reflex
(e.g., scissoring and distorted shadows) does not always indicate
the nature or the location of the irregular astigmatism. The
interpretation of retinoscopy is, therefore, subjective and details
of the origin of image blur are absent.
Nevertheless, it has been known for more than 300 years
that one can study the corneal curvature through observation of
reflected geometric patterns from the corneal surface.80
Reflection techniques, such as the Placido disk, keratometry,
photokeratoscopy, and corneal topography all arise from this
principle. However, it was not until the development of corneal
topography that clinicians were provided with easily under-
stood color-coded maps of corneal curvature as well as quanti-
tative indices of irregular astigmatism that correlate with
potential visual acuity. This allows the clinician to evaluate the
entire cornea both qualitatively and quantitatively.
A thorough understanding of the fundamentals and appli-
cations, as well as limitations of corneal topography, is of great
importance to the anterior segment surgeon. The remainder of
this chapter explores the background and fundamentals of cor-
neal topography analysis appropriate to the clinical audience.
 Corneal Form and Function: Clinical Perspective

This is followed by a concise pictorial essay of corneal shapes

that are commonly seen clinically. Finally, a brief discussion of
future developments is presented.

BACKGROUND
The study of corneal topography dates back to 1619 when
Father Christopher Scheiner realized that one could estimate
corneal curvature by comparing the reflection of a window on
the corneal surface to that on a series of different sized
marbles.80 The Placido disk, introduced in 1880 by Antonio
Placido, consists of a circular target of alternating white and
black rings or mires with a central aperture through which
one can view its virtual image. This image is formed by
reflection of the target from the surface of the tear film. The size
and the shape of the image features depend on the fact that
convex mirrors will produce a magnification that varies directly
with their radius of curvature. A highly curved surface with
a short radius of curvature will have a low magnification,
making the virtual image of a Placido target appear small. In
contrast, a surface with less curvature will have a greater mag-
nification, making a virtual image appear large. Corneas are
more complex than convex mirrors in their shape; therefore,
the curvature and thus magnification can vary considerably
from one segment to another. However, it is important to note
that all corneas are convex in shape; this includes both normal
prolate corneas as well as oblate corneas that may have
undergone refractive surgery.
In the late nineteenth century, Helmholtz developed the
ophthalmometer.81 This instrument was extremely difficult to
FIGURE 40.12. The NIDEK PKS-1000 photokeratoscope provided a
use; however, out of this invention grew the first clinical Placido disk photograph of the corneal surface.
keratometer, which was introduced by Javal and Schioetz for
the measurement of anterior corneal curvature. The modern
keratometer measures corneal curvature along the orthogonal that such patients could lose more lines of best spectacle
steep and flat principal meridians by manual rotation of a corrected vision than one could account for by increased
dial; the autokeratometer generally improves the repeatability of cylinder alone. A contact lens over refraction would often
the measurements between observers. The sites of measure- provide the clue that irregular astigmatism in the graft might
ment are four positions on the corneal surface ~3–4 mm apart, be more debilitating to visual acuity than induced cylinder.
depending on the underlying curvature of the cornea. Using To examine irregular astigmatism, a larger portion of the
the standard keratometric index, the radius of curvature for corneal surface had to be analyzed than could be measured
two orthogonal meridians is then converted into dioptric with the keratometer. For this, a Placido disk/camera system
powers (see later, Eqn [6]). Because the keratometer is designed was developed – the photokeratoscope (Fig. 40.12). Two of the
to make its measurements from only four positions on the most widely distributed photokeratoscopes were the Nidek
surface, this device can be used to accurately reproduce the PKS 1000 (Nidek Co.) and the Corneascope (Kera Corp.).
curvatures of only spheres and ellipsoids, and it does this with These devices produced a rapid print of a Placido disk image
an accuracy better than 0.25 D. However, because it can from the patient’s cornea. Interpretation was accomplished by
measure only spherocylindrical surfaces, the keratometer visual inspection of the mires. Mires became closely spaced in
cannot be used to detect the myriad of shapes that corneas the areas of the cornea that had a high curvature such as

can exhibit. The assumption that the cornea can be modeled as
an ellipsoid is at best an approximation even for normal
corneas. For corneas with irregular astigmatism, only gross
amounts can be appreciated with the keratometer and then
described with the general sign ‘irregular mires’. Nevertheless,
keratometry remains very useful for anterior segment appli-
cations such as intraocular lens (IOL) calculations and contact
lens fitting for corneas that are not diseased or affected by
surgery or trauma.
Despite the surgical success enjoyed with corneal transplan-
tation, the development of ever-improving methodologies for
the preservation of donor eyes, and the ever-expanding network
of eye banks worldwide, the transplant surgeon is still faced
with one major hurdle: eliminating or reducing the post-
operative astigmatism of the graft. To meet this challenge,
Troutman82 and other pioneers had to rely on the keratometer
to measure induced regular astigmatism, and a ‘good’ result
was a graft with less than 4 D of regular astigmatism and cor-
rected vision better than 20/40. However, it was often observed
CHAPTER 40
the region of the cone in keratoconus, more broadly spaced in
areas of lower power, and irregular near areas of tight sutures
(Fig. 40.13). The reading of photokeratoscopy was indeed
subjective, nevertheless the information was clinically useful.
Photokeratoscopy did, however, have another limitation; that
is, the devices did not cover the central area of the cornea well,
leaving much of the corneal area important to visual acuity
unanalyzed. Additionally, the devices were not able to cover
the corneal periphery, which limited usefulness in the contact
lens fitting area.
With the increased practice of refractive surgery in the early
1980s, the entire corneal surface topography had to be
accurately and objectively evaluated. Doss and associates83,84
were among the first to publish a method for the automatic
scanning and calculation of corneal power from a
photokeratoscope. Klyce extended this approach to the Nidek
photokeratoscope and explored methods that might be used for
the quantitative presentation of corneal topography to the
clinical audience (Fig. 40.14).85 This work culminated in the 451
 CORNEA AND CONJUNCTIVA
a b
a b
SECTION 6
introduction of the color-coded contour map for the
presentation of corneal surface powers by Maguire and
associates86 (Fig. 40.15), which has now become the standard
display for corneal topographers.
The initial work with manually analyzed photokeratoscope
photographs demonstrated the clinical and research value of
corneal topography and led to several observations on normal
and abnormal corneas (see Fig. 40.15).87–90 However, it was
not until the advent of the affordable personal computer that
452 this technology was commercialized. The first commercial
FIGURE 40.13. Photokeratographs with the
NIDEK PKS-1000. (a) Mild keratoconus. Note
the characteristic pear-shaped inner mires.
(b) Penetrating keratoplasty. Although the
central mires look fairly circular, there are large
amounts of cylinder and irregular astigmatism
in this early postoperative examination. Note
also that the early Placido disk
photokeratoscopes do not provide good central
corneal coverage.
FIGURE 40.14. Wire mesh stereo pairs to
represent corneal topography – an initial
approach to displaying shape.6 The heights are
amplified; a normal cornea would have a flat
appearance with this method. (a) Cornea after
radial keratotomy. Note the bowl shape created
by amplifying the corneal height. (b) Stereo
pairs of mild keratoconus.
FIGURE 40.15. The color-coded contour map
of surface powers for a normal cornea
introduced by Maguire and associates (left-
hand panel).7 This early version used manually
traced enlargements from the NIDEK
photokeratoscope and predated widespread
use of color monitors and printers. A numeric
map is shown in the right-hand panel.
device available was the Corneal Modeling System (CMS)
(Computed Anatomy, Inc, New York) in 1988, which at a cost
of $80 000 was accessible only to major clinical research
centers. This device used image analysis techniques to capture
and process both Placido disk images from the corneal surface
as well as cross sectional slit images that could be used to
model both surfaces of the cornea and to provide pachometry.
The scanned slit images constituted a feature that was dis-
continued in order to reduce the costs and make the product
clinically accessible.

FIGURE 40.16. Influence of mire spacing on spatial resolution in
videokeratoscopy. Dotted line, model surface of a central island of
elevated power, simulating an unwanted feature that is sometimes
seen in refractive surgery. In this test, there is a sharp transition
between curvatures (40.08/41.91 D). Solid line, response of a
videokeratoscope that samples every 180 µm on a 40-D surface.
Dashed line: response of a videokeratoscope that samples every
270 µm on a 40-D surface. Note that sparse sampling can smooth out
irregular astigmatism in the corneal surface.
After Belin MW, Ratliff CD: Evaluating data acquisition and smoothing functions
of currently available videokeratoscopes. J Cataract Refract Surg 1996; 22:421.
BASIC PRINCIPLES
Instrumentation
The CMS was the first of a growing number of devices for
measuring corneal topography, and this class of machine
employing the videocapture of Placido disk images has become
known as a videokeratoscope. The more generic term for
devices that measure corneal shape is ‘corneal topographer’.
This device displays results in the form of a color-coded contour
map. There can be differences in the results obtained with the
various videokeratoscopes. The area of the analyzed cornea
varies for the different machines depending on the type of
Placido target used. Those devices with a long working distance
have a large diameter and part of the target is always masked by
eclipse of the brow and nose, whereas those with a compact
cone and short working distance do not suffer from peripheral
data loss to shadows. Automatic alignment and focus or com-
pensation for misalignment is critical for corneal topographers
with short working distances. The ability for a corneal
topographer to show fine detail is somewhat variable owing to
differing resolutions (spacing between mires or mire edges). An
example of this is shown in Figure 40.16, where wider than
optimal spacing of mires results in smoothing of the device’s
response to curvature change.91
In addition, reconstruction algorithms vary among the
devices, and this can degrade the nominal accuracy of 0.25 D,
particularly in the corneal periphery.86,92 With Placido disk
reflection corneal topography, there is no exact equation or
set of equations that can be used. Each device using this prin-
ciple must make a series of approximating calculations, which
can be quite accurate93; in early implementations, this has on
occasion produced misleading results such as the presentation
of a keratoconus pattern where none exists.94 Abnormalities
of the tear film can result in poor quality mires and misleading
results as well, calling for scrutiny of the mire processing for
accuracy assurance. Despite its limitations, Placido disk-based
systems remain the most successful methodology for corneal
topography analysis because of their sensitivity and
reproducibility.
As mentioned earlier, the use of slit beam technology can
provide the opportunity to analyze both the outer and inner
Corneal Form and Function: Clinical Perspective
surfaces of the cornea. Since both of these refracting surfaces
as well as corneal thickness come into play when calculating
total corneal power, measurement of the position of the surfaces
directly would appear to provide an advantage. Additionally,
because each surface can be measured directly with slit beam
technology, no approximation errors should arise as with the
Placido disk-based devices. There are, however, disadvantages to
this approach as well. Because the cornea is in motion from
muscle tremor, pulse, and fixation nystagmus, the entire image
must be captured in a minimum time of 30 ms. Capturing
successive slit images over a period of time longer than this
requires either an eye tracking system or a post capture image
registration technique to avoid movement artifact. The former
is too expensive for routine clinical use and the latter has
proven to be impractical because of the absence of registration
landmarks on the clear cornea. The second limitation of the slit
image technique is that measurement of the position of the
surface directly cannot lead to the same measurement sensi-
tivity as measurement of the position of a reflected image.
Notwithstanding these comments, slit scanning has proved to
be a valuable adjunct to obtain corneal thickness profiles, while
corneal topography can be provided with the traditional
Placido disk approach.
Another approach that avoids the uncertainties in the calcu-
lations of corneal topography is rasterstereography.95–97 With
this approach, fluorescein is first instilled in the tear film, and
a grid or raster pattern is projected with cobalt blue light onto
the anterior surface of the eye. Images are then captured simul-
taneously from two directions and processed using triangu-
lation methodology to reconstruct the shape of the cornea. This
method is also less sensitive than corneal topography for the
reason given earlier, and this factor along with the incon-
venience of having to instill fluorescein to make the measure-
ment reduces its usefulness. Nonetheless, rasterstereography
can supply corneal shape data of very irregular corneas, such as
corneal transplants, over a broad area extending well out past
the limbus.
Perhaps the most accurate methodology that can be used to
measure shape is interferometry.98–100 Interference techniques
are used in the optical industry to detect aberrations of lenses
and mirrors to subwavelength accuracies. In essence, a refer-
ence surface (or its hologram) is compared with the measured
surface, and interference fringes are produced as a result of
differences in the two shapes. With respect to the measurement
of corneal shape, there is such a wide variation in the shapes of
corneas, even among those that are normal, that it is difficult
for a single interference device to represent all the variations.
Examples of interference devices include a phase modulated
CHAPTER 40
laser holography-based device101,102 and an acoustic holographic
technique.103 This approach has not yet been found practical
for the measurement of corneal topography.
Methods of Power Calculation
Corneal topography devices measure the shape or curvature of
the corneal surface. A corneal topographer does not measure
beyond the surface, and therefore the corneal power reported by
the device is based on a series of geometric calculations and
assumptions. The convention that has been adopted is the
same as that used for decades, when only the keratometer (which
like the corneal topographer does not measure beyond the
corneal surface) was available for the measurement of corneal
curvature. This convention leads to the following expression:
P = 0.3375 / Rc [6]
where P is the corneal power in diopters; Rc is the local radius
of curvature in meters; and the keratometric constant, 0.3375,
is the difference between the refractive index of air and the 453
 CORNEA AND CONJUNCTIVA
refractive index of an equivalent cornea with the thickness
and back surface effects for the average cornea considered.
Although this relationship has been adopted because of its
widespread clinical use, it should be remembered that it will not
accurately reflect changes in corneal power after refractive
surgery since this may alter corneal thickness and may pre-
serve the curvature of the endothelial surface. The effect
usually leads to an overestimation of the refractive change by
11.4%. If this method of power calculation is not appropriate in
a given situation, it is possible to change the settings in the
corneal topographer to report radius of curvature in millimeters,
rather than power in diopters. The surface radius of curvature
measurement can then be used with the measured corneal
thickness and endothelial curvature to obtain a more correct
approximation of corneal power after refractive surgery.
However, note that keratometry will not provide accurate
readings from the central area of the surgical cornea; alternative
methods must be used (see further ahead).
Corneal power, when calculated from front surface curva-
ture, might best be called keratometric power, because it derives
from the keratometric index. However, there are other con-
siderations in the estimation of corneal power (and curvature)
that will affect the result. Because the keratometer measures the
curvature at only two points on each of two meridians, the
meridians can only be interpreted as circular arcs. For this
reason, keratometers were calibrated with reference spheres and
the results reported were very accurate for these spheres.
Calibrating corneal topographers with the same approach leads
to representation of corneal power as axial or spherically based.
Although this representation is preferred for routine clinical
diagnosis, details of corneal topography that are important to
understand certain aberrations that occur after corneal surgery
can be made more apparent with other methods.
Corneal power can also be calculated from local curvature
data.85 However, a more elegant and precise method for calcu-
lating local corneal curvature has been shown to be through
the use of the instantaneous radius of curvature.104 This
method may be preferable for evaluating shape changes after
refractive surgery, for example, but it suffers from system noise
due to its extreme sensitivity to small changes in radius of
curvature over short distances.
A final method that may be used to calculate corneal power
is that of refractive power, which is calculated from ray tracing
and Snell’s law. This has the effect of showing the residual
spherical aberration of the corneal surface.93,105 This
information is useful to the optical scientist, but is of little
value to the clinician, because it displays an optical aberration
that is believed to be further compensated for if not eliminated
SECTION 6
by the native lens of the eye and by neural processing.
A final word about power calculation methods in corneal
topography is in order. The reconstruction algorithms among
corneal topographers may vary, and measurement accuracy
generally is lower in the corneal periphery.106 However, in the
central 3 mm diameter of the cornea, all of the methods provide
nearly the same result for the same cornea; it is this portion of
the cornea that is most important to the formation of the image
on the retina. Clinically, the axial method for power calculation
is preferred. It is a direct representation of corneal shape
without the confusion of spherical aberration or measurement
noise.
Presentation Methods
Devices that map the cornea collect information from
thousands of data points. In order for these devices to be useful,
the data must be distilled into a form that is unambiguous and
rapidly interpreted. Initially, a common presentation technique
454 was numeric, with powers displayed on a geographic rep-
resentation of the collection site (see Fig. 40.15). Another
approach,85 is a three-dimensional wire mesh model of surface
powers presented as stereo pairs (see Fig. 40.14). Although this
technique conveyed some topographic information, the
information content was disappointingly low and difficult to
appreciate by the clinical audience.
A major breakthrough in the clinical use of corneal top-
ography analysis came with the idea of the color-coded contour
map of corneal powers (see Fig. 40.15).86 A color spectrum was
chosen so that cool colors were associated with low corneal
powers and warm colors were associated with high corneal
powers. Only a few distinct colors were chosen over the
central range of corneal powers so that a specific power range
could be easily identified. Normally occurring corneal powers
were assigned color values in the green part of the spectrum
to further assist in the identification of normal versus abnor-
mal with this emerging clinical test. It was rapidly apparent
that along with color association, the contours of the color
maps provided diagnostic capability through pattern recognition
and this combination of factors combined with the appropriate
scale (see later) would achieve wide acceptance and use.
Additional options have enhanced the utility of corneal top-
ography. Plotting the contour map directly on the video image
of the patient’s eye was helpful to convey scale and position of
topographic features and can be particularly useful when
evaluating post-penetrating-keratoplasty or post-cataract-
surgery corneas. Plotting metric scales (rectangular or polar) on
the display is likewise helpful to locate the position and merid-
ian of salient features. The ability to view multiple patient
examinations simultaneously can permit a comparison of sev-
eral eyes with a similar disease or can present the time course
of a diseased (Fig. 40.17) or postsurgical cornea (Fig. 40.18).
Difference maps are quite useful to demonstrate the early
postoperative effect of a surgical procedure, to examine the
effects of wound healing over time, or to watch for progression
(keratoconus; see Fig. 40.17) or regression (central island after
excimer laser; Fig. 40.19) of specific topographic features.
Power displays of the corneal surface are useful to under-
stand corneal optics, but there are a number of situations in
which height or actual corneal shape would provide useful
information. For example, in the sculpting of corneal tissue
with the excimer laser, a true height map would be essential
to detail the effect of the removal of tissue by photoablation.
Heights can be presented directly by the corneal topography
units that do not use reflection keratoscopy; corneal
topographer data can also be used, but the algorithms used to
calculate height must be carefully validated.
Finally, ray tracing diagrams can be used to subjectively
visualize the optical quality of the cornea that is being
examined. Maguire and associates107 used such techniques to
calculate the modulation transfer function of surgical and
nonsurgical corneas and showed the effects on the blur of eye
chart symbols. Such routines are currently available on corneal
topographers to help assess the optical quality of the corneal
surface (Fig. 40.20).
Standardized Scales
The clinical utility of corneal topographer technology depends
to a large extent on how well the color-coded maps can be
interpreted. Two aspects are involved: (1) color association, with
the cool, blue end of the spectrum representing low powers
and the warm end of the spectrum representing high powers;
and (2) pattern recognition, with the contours representing
specific topographic entities. Despite initial efforts at
standardization in corneal topography by national and inter-
national organizations, none have been finalized or adopted.
Since standards are essential for the comparison and sharing

of information, those that have been proved and published in
the peer reviewed literature are set forth in the following.
A number of different color scales have been used since the
original introduction of the International Standard or Absolute
Scale (Fig. 40.21).7 This scale spanned a range of corneal
powers from 9 to 101.5 D, with 1.5 D intervals in the middle of
the range and 5 D intervals at each end. Wilson and co-workers
introduced a more practical scale (the Klyce/Wilson scale),
which ranges from 28 to 65.5 D in equal 1.5 D intervals.108
With the advent of refractive surgical corrective procedures
for high myopia, it was important to make the lower range of
the scale have uniform intervals to prevent masking of irregular
astigmatism in the central, surgically flattened cornea. This
scale has been revised slightly to form a universal standard
seeking adoption by the American National Standards
Institute.109
Corneal Form and Function: Clinical Perspective
FIGURE 40.17. Progression of keratoconus in
a young male patient. Upper left, 8/88; lower
left, 7/89; upper right, 1/90; lower right, 7/92.
FIGURE 40.18. Videokeratograph of the left
eye of a myopic patient after laser in situ
keratomileusis (LASIK). Upper left, preoperative
eye. Follow-up: lower left, 2 weeks; upper right,
2 months; lower right, 6 months after surgery.
This format is useful to study topographic
stability.
CHAPTER 40
Even with this alteration, it was often argued that the 1.5 D
interval was so wide that important features in corneal top-
ography may be hidden between contours. The diagnostic
adequacy of the Klyce/Wilson scale was evaluated in a clinical
series that included normal corneas, contact lens-wearing
corneas, early to moderate and advanced keratoconus, pen-
etrating keratoplasties, extracapsular cataract surgery, excimer
laser photorefractive keratectomy, radial keratotomy, aphakic
epikeratoplasty, and myopic epikeratoplasty. It was found that
the correct interpretation for all cases could be made with the
1.5 D scale without resorting to a 1 D or lower interval scale.108
Additionally, the 1.5 D scale proved broad enough to cover the
full range of powers encountered in the study. The routine use
of a fixed standard scale showing only adequate detail and not
redundant information or extraneous noise is essential for
efficient and accurate clinical interpretation. 455
 CORNEA AND CONJUNCTIVA
c
b
It has been tradition that corneal topographers provide
SECTION 6
adaptable scales that are self-adjusting to the range of powers
found for a given cornea. The use of such scales runs counter to
standardization in corneal topography and is misleading in
interpretation. Such scales make grossly irregular corneas look
uncomplicated and quite normal corneas look complex with
extensive amounts of irregular astigmatism. Such adaptive
scales should be avoided, except as an adjunct to examine
details of corneal topography.
User adjustable scales are often also available on corneal top-
ographers. These may be necessary to suit a specific application
or device, but it is recommended that in the learning phase a
user become accustomed to a single fixed scale.
Quantitative Indices
The color-coded contour map of corneal power distribution
provides a powerful method for diagnostics through the
association of particular colors with specific corneal powers and
the recognition of patterns from the contours. Although these
456 maps are based on measured data, they do not by themselves
FIGURE 40.19. Difference map of the right eye
of a patient after LASIK. (a) Note the
astigmatism in the preoperative cornea. (b) One
month after surgery, the cornea shows a central
steep area ‘central island’ within the ablated
zone. (c) Difference map. Difference maps
should always be used when diagnosing a
central island because the preoperative
astigmatism may appear as a central island in
the postoperative cornea. In that case, a
difference map would show no central island.
FIGURE 40.20. Ray tracing is being used to
evaluate corneal optics from topography. The
figure represents the intensity of light focused
on the retina as two point sources move from
convergence to separation. The distance of
separation at which the two peaks in the
distribution can be resolved by the eye is
related to visual acuity.
easily yield numeric measures such as keratometry. Therefore,
in order to complement the information provided by the color
maps, a number of quantitative indices have been developed
and are derived from the corneal topography data files. In
addition to corneal power(s), each topographer will have
calculated the three dimensional shape of the cornea, generally
as a set of heights from a plane normal to the corneal vertex
corresponding with measurement sites on the mires. Most
often, 256–360 sites are measured for each mire along
semimeridians, like spokes of a wheel, giving denser coverage
in the central cornea than in the periphery. These data can thus
be accessed, and indices can be derived to supplement the
corneal maps.
Keratometry
Simulated keratometry (SimK) was one of the first indices to be
broadly available.87 This index aims to simulate the readings
that a keratometer would yield; that is, the maximum power of
the surface along any axis and the power orthogonal to that
power. These were designated SimK1 and SimK2 and were

written in standard notation as: 42.5 µ 85°/43.25 µ 175°, for
example, with the power units given either in diopters or
optionally in millimeters. These measurements are collected
from the data present on corneal topographer mires that
represent positions on a cornea that would be similar to those
positions of the keratometer mires on the same cornea, a
separation distance of 3–4 mm. Cylinder and spherical
equivalent are easily calculated from the SimK values and are
often provided with the printout of the color map.
Several investigators have pointed out that keratometry may
only be valid on normal corneas that do not have irregular
astigmatism, since taking measurements along only two axes
makes the assumption that the measured surface is either
spherical or ellipsoidal. Keratometry has long been used in
refractive power calculations for IOLs and has been shown to be
adequate for normal corneas.110,111 However, studies have
shown that the predictability of standard IOL power calcu-
lations can be reduced in patients who have irregular astig-
matism or who have undergone radial keratotomy.112,113 By its
design, simulated keratometry based on corneal topography
data will suffer the same consequence as its progenitor, and
better estimates of central corneal curvature seemed necessary
to improve the predictability of IOL calculations.
Maloney114 introduced methodology to find the best fit
spherocylinder to the corneal topographer central mires. A more
direct approach was taken by Celikkol,115 who found a good
correlation between the average power of the third corneal
topographer mire and refractive accuracy after IOL implantation
in eyes that had undergone refractive surgery. Fourier analysis
has also been used to separate corneal power into spherical,
cylindrical, and irregular astigmatic components,116 and this
sophisticated approach may find practical application with
clinical testing. As mentioned earlier, the density of sampled
data points is greater in the center of the cornea than in the
periphery owing to the usual procedure of radial sampling from
the center to the limbus. In fact, over sampling occurs in the
innermost mire region, because the same pixel may be sampled
multiple times owing to overlapping scans. In order to
compensate for this fact and to provide a good estimate of
refractive power, an algorithm was developed117 that produced
an area compensated average of corneal power from the central
cornea demarcated by the apparent entrance pupil. This
Corneal Form and Function: Clinical Perspective
FIGURE 40.21. An eye with 2.5 D of regular
oblique astigmatism shown on different scales.
Upper left, absolute scale. Note that the two
ends of the range are in 5-D intervals. Lower
left, Klyce/Wilson scale. All the contour intervals
are 1.5 D; because of the narrow range of
powers in this cornea, this map and the
absolute scale map are identical. Upper right,
normalized scale. This is a self-adapting scale
that makes clinical interpretation difficult
because it can overemphasize small changes in
a nearly normal cornea, as it does here. This
scale can also deemphasize topographic
details in a cornea with large amounts of
irregular astigmatism. Lower right, adjustable
scale. This allows the user to develop a special
purpose scale.
parameter, called the average corneal power (ACP), was
compared with simulated keratometry values for normal
corneas, astigmatic corneas (cylinder ≥ 1.5 D), and corneas that
had undergone radial keratotomy (RK) or photorefractive
keratectomy (PRK). No disparity was found between the SimK
readings and ACP values for normal or astigmatic corneas, but
a disparity of 0.5 D or more was found for significant numbers
of RK (7%) and PRK (25%) eyes.117
Therefore, where keratometry readings are used for refractive
power calculations, better measurements can be obtained by
appropriate calculations using corneal topographer data to
estimate the average curvature of the central cornea. These
values can be particularly important to improve the accuracy of
IOL calculations for certain cases after keratorefractive surgery.
Measures of irregular astigmatism
Irregular astigmatism is the remainder after subtracting sphere
and cylinder from a corneal power map. With reference to
optical analytic tools, irregular astigmatism is equivalent to the
higher-order (HO) terms in the surface fitting Zernike
polynomial series; hence, irregular astigmatism is also referred
to as the HO aberrations, which include the familiar
components: spherical aberration and coma. In this discussion,
CHAPTER 40
‘irregular astigmatism’ will be the preferred terminology, since
the HO aberrations obtained with the Zernike method do not
capture all of the corneal aberrations associated with visual
function, particularly in aberrated eyes.118 On the other hand,
with the very sensitive topographers available, some of the
irregular astigmatism may be clinically insignificant; indeed, a
certain amount is present in even normal corneas (see later).
Clinically the locus of irregular astigmatism and its impact on
vision are assessed with a contact lens over refraction. Reducing
the power of the irregular corneal surface to a thin tear
meniscus beneath a contact lens proves the etiology of reduced
acuity but does little to display the nature of the aberrations.
Initially, the clinician could instill fluorescein under a rigid
contact lens, which is helpful to depict gross shape anomalies
such as keratoconus, but the specific character of the irregular
astigmatism in an individual cornea needs to be known in order
to manage cases of reduced acuity. Corneal transplants, ocular
trauma, cataract surgery, and even scleral buckles can produce
vision impairing irregular astigmatism. Keratorefractive surgery 457
 CORNEA AND CONJUNCTIVA
can produce aberrations unique to that procedure, such as
central islands with excimer laser area ablations, decentered
treatment areas, and undesired spherical aberration.
Viewing the color-coded contour maps has permitted
classification of corneal shapes in both normal and abnormal
eyes. However, a most useful complement to corneal top-
ography analysis has been the development of indices that
are predictive of the potential visual acuity (PVA) of an eye
based on the topographic character of the analyzed corneal
surface. The first such index that was developed along these
lines was the surface regularity index (SRI).86,119 SRI is
calculated from a summation of local power fluctuations along
256 equally spaced semimeridians on the central 4 mm of the
cornea. SRI increases with increasing irregular astigmatism and
approaches zero for a smooth corneal surface. There was a
statistically significant correlation found between the SRI and
best spectacle corrected visual acuity in a prospective clinical
study that included eyes of normals, keratoconics, and
transplants (r = 0.80, P <.001). The SRI and the PVA that
is derived from SRI have been useful clinically as a guide to
the optical performance that might be associated with a
particular irregular astigmatism and as a quantitative index
for monitoring the effect of refractive surgical procedures in
clinical studies.
Another useful quantitative descriptor is the surface asym-
metry index (SAI). The SAI is a centrally weighted summation
of differences in corneal power between corresponding points
180° apart on 128 equally spaced meridians crossing the
corneal topographer mires.86,119 SAI approaches zero for a
perfectly radially symmetric surface and increases as the corneal
shape becomes more asymmetric within specific meridians.
Since the normal cornea usually has a high degree of central
radial symmetry, the SAI is a useful quantitative parameter for
monitoring changes that occur in patients following refractive
surgery. For example, decentration of a refractive procedure will
cause an increase in the SAI value. In addition, since the
steepening that occurs with keratoconus is generally located off
center, SAI increases greatly in these cases. As SAI increases,
there is an associated decrease in vision, although the
correlation is not as strong as with SRI.
Since the early work on topographic indices, a number of
additional variables have been used for the clinical assessment
of irregular astigmatism. Seiler has calculated the spherical
aberration from corneal topography examinations of post-PRK
corneas. He showed that corneal topography can be used to
calculate spherical aberration and that after PRK, the amount of
spherical aberration correlates well not only with measured
glare visual acuity but also with best spectacle corrected visual
SECTION 6
acuity.120 Similar results have been found using the Zernike
polynomial method to examine spherical aberration after
LASIK.
Because of the lack of standardization in the field, an index
developed with one topography unit will not be directly com-
parable to an index developed on another unit, even if the same
equations are used for its calculation. However, this limitation
can be overcome using a Fourier filtering technique, which has
been demonstrated using a wide mire corneal topographer and
a fine mire corneal topographer.121 Hence, it is instructive to
review the indices developed for the TMS 1, and some of these
are discussed here. The coefficient of variation of corneal power
(CVP) is a measure of the distribution of corneal powers in a
topography examination over the entrance pupil. This measure
and the standard deviation of corneal power (SDP) are related
in that the CVP is equal to the SDP divided by the average
corneal power. They both increase as the range of powers
increases in the measured topography. Examples of high CVP
458 and SDP are keratoconus corneas, transplants, and trauma
cases. Studies show that CVP, which is calculated from the
powers limited to the parts of the cornea ahead of the entrance
pupil, is often the strongest correlation to best spectacle
corrected visual acuity in refractive surgical eyes.
Some have pointed out the potential benefit of a fortuitous
bifocal cornea following refractive surgery122 in patients who are
provided with near and distance vision. In reality, more often
surgical corneas are multifocal, or more accurately, varifocal and
this may lead to problems with decreased contrast sensitivity
and visual acuity. Furthermore, with a decentered procedure
untreated peripheral cornea can intrude upon the entrance
pupil producing annoying visual symptoms. Varifocality has
been found to correlate with best spectacle corrected visual
acuity; it may be expected to be a sensitive correlate to contrast
sensitivity as well due to increased image blur. With better
understanding of physiological optics, some procedures such as
conductive keratoplasty are offering simultaneous functional
near and far vision.123
Another measure of irregular astigmatism is the elevation
depression magnitude (EDM). Whereas the SRI is a measure of
high-frequency distortion, EDM is a measure of low frequency
distortion. In essence, it is a measure of the size and power of
the bumps and pits in the topography. It has been used as a
measure of ‘central islands’ after refractive surgery.
The irregular astigmatism index (IAI)124 is an area com-
pensated average summation of inter ring power variations
along every meridian for the entire corneal surface analyzed. It
is analogous to the SRI, but, whereas the SRI is calculated for
the central cornea to be more representative of Snellen acuity,
the IAI is calculated from the whole analyzed surface to be
more representative of overall corneal irregular astigmatism.
IAI is particularly high in corneal transplants shortly after
surgery; persistence often heralds suboptimal best spectacle
corrected vision.
The analyzed area (AA) gives the fraction of the corneal area
covered by the mires that could be processed. AA is lower than
normal for corneas with gross, irregular astigmatism, which
causes the mires to break up and not be resolved. A lower than
normal AA is found with early postoperative corneal trans-
plants, advanced keratoconus, and trauma. AA can also be
artifactually low when the eyes are not opened wide.
The corneal asphericity index (CAI) is a quantitative des-
criptor that indicates the eccentricity of the central cornea. CAI
is calculated by fitting an ellipse to the average curve obtained
from the 256 semimeridians out to the twenty-fifth mire. The
CAI for 22 control corneas was reported to be 0.33 ± 0.26 (SD),
which corresponds with the prolate shape of the normal central
cornea.125 This value is useful in contact lens fitting and for
differentiating between normal corneas and corneas flattened
by myopic refractive surgery.
CLINICAL APPLICATIONS OF CORNEAL
TOPOGRAPHY
Topography of the Normal Cornea
The normal cornea tends to exhibit a great deal of variation
from one individual to another as well as asymmetry from one
area of the cornea to another. A thorough understanding of
the topography of the normal cornea is of fundamental import-
ance to distinguish them from those corneas affected by
trauma, surgery, or disease. In addition, the topography of the
normal cornea must be understood in relation to vision in
the design and planning of corneal surgery.
For almost 100 years, it has been known that the normal
cornea is aspheric with the central cornea being steeper than
the periphery (Fig. 40.22). This change in curvature compen-
sates somewhat for spherical aberration in the eye. In 1989,

Dingeldein and Klyce126 studied eyes with uncorrected vision
of 20/20, no history of contact lens wear, and no evidence of
other corneal abnormalities. They found that the average
central corneal power to be 42.84 D and that the corneas did
flatten progressively toward the limbus. However, the degree
and the rate of flattening as well as the location of the area of
shortest radius of curvature varied widely from one subject to
another. Curiously, in none of these normal eyes did the cornea
flatten more rapidly temporally than nasally. Additionally, when
viewed with the color-coded contour map, each normal cornea
shows a unique pattern, like a fingerprint. Moreover, the
topography of fellow eyes tends to be mirror images of each
other (enantiomorphs). A final characteristic of normal corneas
is that even with these variations between individuals, normal
corneas are relatively smooth in keeping with their optical
performance requirements.
Corneal Form and Function: Clinical Perspective
FIGURE 40.22. All the characteristics of
normal corneas are illustrated in this pair of
maps from the same person. The corneas are
steeper centrally than peripherally; the contours
are relatively regular; the left and right corneas
have a similar mirror image symmetry, and the
specific color and contour pattern of this
individual are quite unique, like a fingerprint.
FIGURE 40.23. With-the-rule cylinder has a
characteristic bow-tie pattern. Again, note the
mirror image symmetry.
CHAPTER 40
Corneal Topography and Astigmatism
Regular astigmatism
Naturally occurring regular astigmatism reveals itself in cor-
neal topography as a bow-tie pattern (Fig. 40.23). When Bogan
and associates examined the fine detail of normal corneal
topography with an expanded (0.4 D contour interval) scale,
they found that 22% of the corneas had round patterns, 21%
had oval patterns, 7% had irregular patterns, and 50% had
bow-tie patterns.127 Corneas that had measurable amounts of
keratometric cylinder also exhibited the bow-tie pattern,
which confirms the use of corneal topography to detect
cylinder. If one were to make a contour map of a sphere with a
small amount of cylinder, a fan shaped figure would result;
however, as noted earlier, the cornea is naturally a prolate
ellipsoid with cylinder added to that geometry. The 3 to 4 D
of flattening occurring between the corneal center and the 459
 CORNEA AND CONJUNCTIVA
periphery turns the fan shape into the bow-tie configuration
seen in the contour maps.
Corneal topography can provide quantitative measures of
astigmatism. Dingeldein and co-workers showed that there is a
high degree of correlation between the weighted average
powers from photokeratoscopes and the average keratometric
powers (r = 0.96, P <.001).87 Later Wilson and Klyce showed a
good correlation between a similar measure, the simulated
keratometry from corneal topography, to the keratometric
values.119 Sophisticated Fourier decomposition techniques116 as
well as polynomial fitting methods can also be used to measure
corneal astigmatism. To correlate astigmatism to refractive
cylinder, such calculations are usually performed only for the
portion of the cornea over the entrance pupil. However, it is
important to note that the magnitude of refractive astigmatism
does not always agree with corneal astigmatism; the lens and
the macula can occasionally be responsible for all or a part of
refractive astigmatism.
Irregular astigmatism
Irregular astigmatism takes many forms and has many causes,
and it can be defined as any aberration that diminishes vision.
It was noted earlier that Bogan and associates found some
topographic patterns in normal 20/20 eyes that exhibited
irregular contours.127 Irregular contours seen with nonstandard,
high-sensitivity topography scales is not pathognomonic for
irregular astigmatism, although the structural detail seen with
this magnification is unique from one person to another.
However, using a standard scale, such the 1.5 D contour
interval suggested for use with corneal topographers, irregular
contours become appreciable only when there is a concomitant
visual deficit and, as mentioned earlier, the extent of irregular
astigmatism can be measured with a topographic quantity
such as the SRI.
Irregular astigmatism rarely occurs naturally, although it is
often a component of the corneal ectasias. Irregular astig-
matism is associated most often with trauma and ocular sur-
gery. Irregular astigmatism that is radially symmetric, such as
spherical aberration, has much less impact on vision than
corneal asymmetries such as coma. The patient with central
keratoconus will achieve functional spectacle vision much
longer than a patient with a similarly advanced cone in the
more typical inferior position. Likewise, a patient with contact
lens warpage consisting of central flattening may tolerate
this without complaint, whereas a patient with asymmetric
contact lens warpage will often complain of spectacle blur.
Significant irregular astigmatism can often be corrected with
rigid contact lens wear as long as the lenses are tolerated.
SECTION 6
Beyond this, corneal transplantation may be required, except
where topographic analysis suggests that asymmetric astigmatic
keratotomy (AK) may improve corneal shape. Phototherapeutic
keratectomy (PTK) which uses the excimer laser and a
smoothing agent has had some success smoothing highly
irregular corneas. Customized ablation with an excimer laser
has made strides toward improving vision in eyes with smaller
amounts of irregular astigmatism.
Corneal Topography in Ectasias
Keratoconus
Keratoconus is the most prevalent of the ectatic corneal
dystrophies128 and has a reported incidence in the general popu-
lation as high as 0.6%. It is characterized as a noninflamma-
tory localized thinning disorder that can lead to anterior
protrusion of the cornea and the development of visual impair-
ment through irregular astigmatism and stromal scarring over
the visual axis. This condition can be classified into two groups:
460 keratoconus suspect129 (a local area of mild corneal steepening)
and clinical keratoconus (an area of corneal steepening with
one or more of the classical clinical signs: corneal thinning,
scissoring of the light reflex on retinoscopy, Vogt’s striae, or
Fleischer’s ring). Somewhat arbitrarily, one can further divide
clinical keratoconus into mild, moderate, and advanced (see
Fig. 40.17) based on variables such as the change in corneal
power from the base of the cone to its apex. However,
keratoconus is often a continuously progressive disease and,
therefore, discrete classification may only be appropriate until
quantitative measures of corneal involvement are agreed upon.
Keratoconus suspects are detected most easily with corneal
topography: it is the most sensitive means for screening.130
Previously, clinicians used distortion of keratometer or
keratoscope mires and scissoring of the light reflex during
retinoscopy as signs of the irregular astigmatism often present
with preclinical (suspect) keratoconus. Relatively rapid changes
in refraction were often noted to accompany keratoconus in
the progressive phase. However, with corneal topography, the
keratoconus suspect cornea is easily identified using the
standard 1.5 D interval scale (see Fig. 40.17), even though by
definition there are no other clinical signs and the only visual
symptom may be spectacle blur from associated irregular
astigmatism. The earliest topographic signs are recognized by
a localized area of corneal steepening some two or more
contour intervals above the surrounding topography. Although
‘atypical inferior steepening’131 is a common finding with
keratoconus suspect cases, the steepest part of the cone in
clinical keratoconus may be found in any quadrant; indeed, it
may be located centrally or superiorly.132 Keratoconus is
almost always bilateral, although one cornea is almost always
more involved than the other (Fig. 40.24). In a few cases
(2.4–4%),133,134 where one eye has moderate to advanced
involvement, keratoconus is unilateral in its topographic
appearance. It is likely that a fraction of these cases will develop
keratoconus in the eye that appears normal at a later time. It is
important to note that the metabolic underpinnings leading to
ectasia in one eye are certain to be present in the contralateral
eye even when clinical signs are not currently evident. When
keratoconus is bilateral, the cone apex seems to be located in
the two eyes at corresponding positions. If the cone is
inferotemporal in one eye, it is inferotemporal in the other eye;
if the cone is central in one eye, it is central in the other eye.
Pseudokeratoconus
The presence of evidence from corneal topography alone is
not sufficient to make the diagnosis of clinical keratoconus
because of the possible confounding influences of contact lens-
induced warpage (Fig. 40.25), misalignment artifact (not a
consideration with modern corneal topographers),94,135 tear
meniscus artifact from excessive tearing or the addition of
a viscous artificial tear solution, or inadvertent external
pressure on the globe. All of these situations can lead to a
‘pseudokeratoconus’; that is, a topographic pattern and some-
times retinoscopic findings similar to those seen in clinical
keratoconus. The latter three artifacts can be eliminated by
repeated corneal topography, but contact lens warpage can
persist for weeks or months (see later).
Pellucid marginal corneal degeneration
Whereas keratoconus produces an ectasia from a focal thinning
of the corneal stroma, pellucid marginal corneal degener-
ation,136 which is much less common, is associated with a 1 to
2 mm wide band of inferior corneal thinning usually near the
limbus from the 4 to the 8 o’clock meridian. Because of the
difference in thinning pattern from keratoconus, pellucid mar-
ginal corneal degeneration produces a characteristic arcuate
band like ectasia of the inferior cornea with marked flattening

of the central cornea along the vertical meridian (Fig. 40.26),
which is quite different from the conical protrusion seen with
keratoconus. Usually, against-the-rule cylinder is present. This
topographic pattern can be present before the classic inferior
thinning pattern of pellucid marginal corneal degeneration is
detectible.137 While the perilimbal band-like thinning usually
occurs inferiorly, the degenerative thinning with pellucid
marginal corneal degeneration can appear in other quadrants as
well, with a concomitant change in cylinder axis.
Terriens marginal corneal degeneration
Terrien’s marginal corneal degeneration is also uncommon
and involves perilimbal corneal thinning that can lead to
topographic changes depending on the extent of thinning
involved. The most frequent topographic pattern is the presence
of a high amount of against the rule astigmatism, which is
present when there is superior or inferior thinning (Fig. 40.27).
Corneal Form and Function: Clinical Perspective
FIGURE 40.24. Keratoconus is almost always
bilateral, and one cornea is more involved than
the other (in this case OD). Note that, while OS
at first impression looks fairly normal, the ‘lazy
eight’ astigmatic pattern is a common
characteristic of keratoconus.
FIGURE 40.25. This patient has corneal
topography that might be misinterpreted as
keratoconus. However, this person wears
contact lens and has contact lens warpage.
CHAPTER 40
However, in some patients, central corneal topography may
appear relatively normal when the peripheral area of thinning
is small or when the thinning extends around the entire
circumference of the cornea.138
Keratoglobus and Posterior Keratoconus. Both keratoglobus
and posterior keratoconus can produce corneal astigmatism,
but the rarity of these conditions has limited topographic
characterization.
Automated Screening for Keratoconus
Because of the widespread application of refractive surgery for
the correction of ametropia, corneal topography has been able
to provide an important role in preoperative evaluation. As
noted below, a high incidence of keratoconus has been found
in the population of patients who elect refractive surgery. This
situation provided the first opportunity to characterize and
recognize a corneal topographic abnormality with artificial 461
 CORNEA AND CONJUNCTIVA
FIGURE 40.26. Pellucid marginal corneal degeneration is
characterized by the central flattening in the vertical meridian and the
inferior band of steepening.
intelligence techniques. With the variety of topography instru-
ments now available and in the absence of universally adopted
standards, it is not possible to use the color-coded map by itself
to differentiate between the normal aspherical and sometimes
asymmetric topography and the abnormal cornea. Keratoconus
often first appears on corneal topography as a localized area of
steepening, inferiorly displaced. It would seem useful to provide
a quantitative method to discriminate corneas with such
steepening from those with clinical keratoconus.
Rabinowitz and McDonnell139 were the first to use a numeric
method to detect keratoconus systematically with data from
corneal topography. They examined dioptric power differences
between the superior and inferior paracentral corneal regions,
which were designated I-S values, the central corneal power or
MaxK, and the differences in power between the two eyes. They
considered that if the central corneal power is greater than
47.2 D or if the I-S value is greater than 1.4 D, then the cornea
could be considered keratoconus suspect. Further, if the central
corneal power is greater than 48.7 or the I-S value is greater
than 1.9, then the cornea could be classified as keratoconus.
Although these criteria are able to distinguish the topography
of keratoconus corneas from normal corneas, its specificity
was not optimized. The numeric approach has been extended
with the use of topographic indices calculated from corneal
SECTION 6
powers and areas, which are used as the input to an expert
system classifier.124 The eight topographic indices are: SimK1
and SimK2 (simulated keratometric steep and flat axis powers),
the surface asymmetry index (SAI), the differential sector index
(DSI), the opposite sector index (OSI), the center/surround
index (CSI), the irregular astigmatism index (IAI), and the
analyzed area (AA). The system was trained with examinations
of 22 clinical keratoconus (a mixture of mild, moderate, and
advanced stages) and 78 nonkeratoconus corneas (normals,
regular astigmatism, keratoplasty, epikeratoplasty (EPIK),
photorefractive keratectomy, radial keratotomy, contact lens-
induced warpage, astigmatic keratotomy, scarred corneas,
postretinal detachment surgery, postcataract surgery, and
keratomileusis). The purpose of including the abnormal corneas
in the nonkeratoconus group was to permit the detection
system to discriminate these from clinical keratoconus.
Analysis yielded the keratoconus prediction index (KPI), which,
in turn, was introduced to a binary decision tree to differentiate
462 between central and peripheral keratoconus. Validation of this
FIGURE 40.27. Terriens marginal corneal degeneration. The
prominent feature here is the marked against-the-rule astigmatism.
approach was done with a second set of topographic exam-
inations consisting of 28 keratoconus corneas and 72 normal
and abnormal nonkeratoconus corneas.
The results of Maeda and associates124 showed a sensitivity
of 100% in the training set and 89% in the validation set. Three
keratoconus corneas that were diagnosed with clinical
keratoconus (based on the medical records) were not confirmed
by the classification scheme; however, because two of these
maps resembled contact lens-induced corneal warpage and one
resembled pellucid marginal degeneration, this discrepancy
seemed acceptable. Specificity was 96% in the training set and
99% in the validation set. All of the false positive results that
were classified as keratoconus involved eyes that had under-
gone keratoplasty and that had the corneal steepening charac-
teristic of keratoconus. The occurrence of these false negative
and false positive results in the validation set seemed reason-
able given the fact that the classification of the validation set
was done with no more information than was available from
the data supplied by a single corneal topographer examination.
This method has been compared with the Rabinowitz/
McDonnell method as well as with a simple method based on
keratometry readings alone using a sample of examinations
independent from the groups upon which any of the methods
were based.140 The sensitivity for keratometry was 84%, for
the Rabinowitz McDonnell method was 96%, and for the expert
classifier system was 98%, whereas for specificity the three
tests had values of 86%, 85%, and 99%, respectively. The per-
formance of the expert classifier system in terms of specificity
was significantly better than either of the two other methods
(P = 0.001).
The aforementioned methods have used indices derived from
the surface power values available from corneal topographers.
Schwiegerling and Greivenkamp141 proposed the use of Zernike
polynomials to fit the actual three dimensional shape data to
detect keratoconus. Although the method showed some merit,
it did not appear to be as sensitive or specific to detect
keratoconus as the discriminant analysis approach mentioned
earlier.
The automatic detection of keratoconus is a good first step
in the development of a topographic classification system. The
approach has been be extended with the use of a neural net-
work that is able to classify a number of categories of corneal
topography in addition to keratoconus, including normals,
transplants, and astigmats.142,121

Corneal Topography in Cataract Surgery
Corneal topography has a wide range of applications in the
preoperative as well as the postoperative management of the
cataract surgery patient.
Preoperative Uses. Studies using corneal topography have
shown that for phacoemulsification, smaller, temporal, and
scleral incisions cause less induced astigmatism.143–148 Clear
cornea incisions tend to produce astigmatism 90° from the
incision and peripheral corneal flattening. Thus, superior clear
corneal incision is only recommended for patients with signifi-
cant preexisting with-the-rule astigmatism. Planned extracap-
sular cataract extraction (ECCE) may yield different results
in that the superior approach may result in less astigmatism,149
and surgically induced corneal cylinder occurs most frequently
along the axis where the incision is placed.150 Small incision
techniques have greatly reduced the impact of the surgery on
corneal topography.
Corneal topography data have also been used in the calcu-
lation of IOL power. In the normal ‘spherical’ or spherocylindrical
corneas, the simulated keratometry values obtained from
corneal topography are in agreement with standard keratometry
values.110,151 Nevertheless, the usefulness of corneal topography
in the calculation of IOL power is controversial. Cuaycong110
and Antcliff152 found measures of corneal power from corneal
topography to yield smaller errors in predicted postoperative
refraction than when using keratometry. However, Husain
found that standard keratometry was more accurate than the
corneal topography or keratometric equivalent.111 Nevertheless,
corneal topography is most useful in the calculation of IOL
power for eyes with irregular surfaces, such as diseased or post-
surgical corneas. After refractive surgery, standard keratometry
may overestimate the power of the central cornea and thus
result in significant amounts of postoperative hyperopia. This is
primarily because the central cornea flattens with myopic
refractive surgery and becomes progressively steeper peripherally.
Keratometry readings tend to measure points peripheral to this
central area of flattening and produce artificially greater
keratometric readings (Fig. 40.28).113,153 This problem is not
limited to refractive surgical corneas154,155 and probably affects
all patients after refractive surgery. In the case of RK, Celikkol
has found that use of the mean power from ring 3 of the TMS
1 for keratometric power yields postsurgical refractive results
closer to the ideal than standard keratometry.115
FIGURE 40.28. In this decentered excimer laser PRK procedure,
keratometry may give an erroneous result. The crosses are placed at
the approximate position where keratometer mires would read the
steep axis.
Corneal Form and Function: Clinical Perspective
Postoperative uses
Corneal topography is the most sensitive way to examine
the entire cornea for changes induced by cataract surgery. Vass
and co-workers have shown that corneal topography and
keratometry have comparable sensitivities and specificities in
detecting corneal changes in the paracentral cornea induced by
cataract surgery.156 Nevertheless, they showed that cataract
surgery frequently results in peripheral corneal changes, irregu-
lar astigmatism, and asymmetric regular astigmatism that
cannot be detected or measured using keratometry. Thus,
corneal topography should be performed in every patient with
decreased visual acuity after cataract surgery with an otherwise
normal examination in order to rule out irregular astigmatism
as the cause.
Postoperatively, corneal topography can be used to detect
irregular astigmatism as well as direct suture removal. The
aforementioned quantitative indices can be used to measure as
well as follow the amount of astigmatism present after
surgery.150 In the future, it may be possible to perform corneal
topography intraoperatively during combined procedures as
well as planned cataract surgery in order to maximize the
refractive benefits of cataract extraction and clear lens
extractions.
Corneal Topography in Penetrating Keratoplasty
Greater tissue availability, improvements in grafting tech-
niques, and better tissue preservation have greatly increased
the success rate of penetrating keratoplasty (PKP). However, the
post-PKP cornea frequently shows excessive amounts of
regular as well as irregular astigmatism. Irregular astigmatism
can result from wound configuration abnormalities such
as ovality/overcut or dehiscence, a thin recipient cornea, graft
elevation, or uneven tension on interrupted sutures
(Fig. 40.29).157 Efforts to improve the refractive results after
PKP have included using contact lenses to mold the corneal
surface after PK in order to reduce astigmatism and increase
the regularity of the cornea,158 adjustable running sutures,159
and selective suture removal (Fig. 40.30).160,161 Nevertheless,
sometimes each of these measures can lead to a further increase
in astigmatism. Thus, corneal topography should be used to
direct these interventions in an effort to maximize the
reduction in astigmatism. Furthermore, in those cases where
the previous measures failed, corneal topography has been used
CHAPTER 40
FIGURE 40.29. Irregular astigmatism after penetrating keratoplasty
due to uneven suture tension. 463
 CORNEA AND CONJUNCTIVA
FIGURE 40.30. Selective suture removal can improve topography.
Top, Presuture removal. Bottom, Reduced astigmatism after removal.
to guide the placement of arcuate incisions and compression
sutures for the reduction of post-PKP astigmatism.162
Corneal Topography in Refractive Surgery
General considerations
Corneal topography should always be performed before
refractive surgery in order to detect preexisting corneal
abnormalities such as irregular astigmatism, ectasias such as
SECTION 6
keratoconus, and contact lens-induced corneal warpage. These
abnormalities are frequently undetected without corneal
topography. If undiagnosed, surgery on corneas with these con-
ditions can lead to devastating consequences, such as exacer-
bation of astigmatism and loss of best corrected vision with
the development of kerectasia. In addition, if a keratoconus
pattern is seen preoperatively in a contact lens wearer, corneal
topography should be evaluated over time to differentiate true
keratoconus from contact lens-induced changes.163 This
becomes more important when one considers the fact that these
abnormalities may be overrepresented in the refractive surgery
population owing to self-selection.164 Postoperatively, corneal
topography has been used to evaluate decentration of the
refractive surgery,165–167 fluctuating vision,168 multifocality,169
regression,164,170 induced astigmatism, and central islands
that can result after refractive surgery; such undesirable fea-
tures can go undetected with other clinical diagnostic
modalities.86,88,159,171,172 This information has been used to
464 explain complaints such as glare, halos, and difficulty with
night vision in keratorefractive surgery patients.120,173–178 These
corneal topography studies have contributed to further the
development of keratorefractive surgery and have helped in the
understanding of the optical performance of the postoperative
cornea.179
The topography of the different refractive procedures and
their complications and also the role that corneal topography
has played in the development of each procedure are now
described.
Radial keratotomy
While RK was rarely performed after the successful intro-
duction of excimer laser techniques, it is instructive to recant
the history of its development for what was learned about the
optics of the cornea. In 1981, 3 years after Bores performed the
first radial keratotomy in the United States, a multicentered
clinical trial of a single standardized technique of radial
keratotomy was initiated. This became known as the
Prospective Evaluation of Radial Keratotomy (PERK) study.180
After 4 years, using data from manifest refractions, the PERK
study group found that the older the patient and the smaller
the clear zone, the greater were the refractive effects of the
surgery.181 Concurrently, they reported a 2.5% incidence of
loss of two lines or more in best spectacle corrected visual
acuity as well as patient complaints of glare and problems
with night driving; they speculated that these complaints
were linked to irregular astigmatism. Atkin and associates182
showed decreased glare contrast sensitivity after RK. Further
studies evaluating the topography of the post-RK cornea were
clearly needed.
Computer assisted corneal topographic analysis was not
commercially implemented until 1988.183 Thus, Rowsey and
co-workers,184 using a nine-ring photokeratoscope, keratometry,
and refraction, showed that a greater amount of flattening
was achieved in eyes with smaller clear zones and in older
patients. Later, they examined the influence of the preoperative
topography on the refractive change after RK.185 They found
that all preoperative corneas had a prolate (steeper centrally
than peripherally) shape and became oblate (steeper periph-
erally than centrally) postoperatively. They also demonstrated
that other factors were also involved in the prediction of refrac-
tive change after RK, namely, the preoperative corneal curva-
ture (less effect with steeper preoperative corneas) and
horizontal corneal diameter (less effect for smaller diameter
corneas).
Although Rowsey did not comment on the etiology of the
vision loss or on the shape of the mires for those patients, it is
well known that RK can introduce irregular astigmatism.186–188
Corneal topography of patients from the PERK study has
shown various degrees of irregular astigmatism with incision
sites being evident in some cases even a decade after surgery.
Other refractive problems after radial keratotomy include
glare, halos, diurnal changes of refraction and vision, regular
and irregular astigmatism, and early as well as late progressive
hyperopia.186,187,189–191
The problem of diurnal fluctuations in vision and refraction
has been reported by many authors.100,168,192,193 It occurs in
1.9–60% of RK patients and may involve a myopic or a
hyperopic shift.168 This change in refraction is thought to be
related more to a change in corneal hydration and not to a
change in intraocular pressure.100,194–198 However, the mech-
anism is not entirely understood. Most studies based on stan-
dard keratometry have not shown any correlation between
variations in refractive error and changes in keratometry.
However, this is most likely because keratometry does not accu-
rately reflect the average corneal curvature over the entrance
pupil. McDonnell and associates168 used corneal topography to

show that most people with diurnal fluctuations in visual acuity
had postoperative corneal topographies that had dumbbell
shaped or split optical zones. Patients with large, round central
optical zones proved to be largely immune to the problem of
diurnal fluctuations. With the use of corneal topography, Lucci
and associates199 examined the diurnal fluctuations in refrac-
tion and average corneal power of a subset of patients in the
PERK Study at their 10-year follow-up and confirmed that the
continued morning to evening increase in myopia was due to
central corneal steepening.
On a more long term basis, progressive hyperopia has been
reported and confirmed by the 10-year follow-up on the patients
in the PERK study.200 Corneal topography has been used to
analyze the specific regional changes that occur in the post-RK
cornea.191 It seems that early on after surgery, the central cornea
appears steeper than the midperipheral cornea whereas the
peripheral cornea is steeper than both. In time, the central
cornea flattens faster than the midperiphery so that it is no
longer relatively steeper.
In order to look at the possible etiology of glare and halos
after RK, Applegate and co-workers used corneal topography to
show an increase in the amount of spherical like as well as
coma like aberrations produced by RK.175 Several reports have
described the multifocality of the cornea after RK.122,169,201 This
multifocality can result in increased depth of focus and ameli-
oration of presbyopia for some people and in decreased contrast
sensitivity in others.202,203 This multifocality may be the result
of the regional changes in curvature with time after surgery as
explained earlier.112 In addition, the increased spherical
aberration that can result from the gradient in curvature from
the treated area to the untreated area has been implicated in the
loss of contrast sensitivity after radial keratotomy.202
Astigmatic keratotomy
Symmetric astigmatism of a magnitude and axis that matches
the magnitude and axis of refractive cylinder and which is too
great in magnitude to be corrected with excimer laser tech-
niques can be treated with the methodology developed by
Thornton and others.204 Semiradial, transverse, and trapezoidal
incisions have been used to neutralize different amounts of
astigmatism.205 However, in cases where the astigmatism is
asymmetric, corneal topography should be used to direct the
relaxing incisions (Fig. 40.31). Software based on corneal
FIGURE 40.31. Asymmetric oblique regular astigmatism induced by
cataract surgery. Keratometry would not be sufficient to detect this
asymmetry; corneal topography was used to plan the procedure that
involved asymmetric relaxing incisions.
Corneal Form and Function: Clinical Perspective
topography can be used to determine the best position and
configuration of the relaxing incisions in order to achieve the
desired result as long as refractive and topographic cylinder are
equivalent.206 Furthermore, astigmatic keratotomy can be
combined with radial keratotomy in the treatment of myopic
astigmatism, but higher amounts of irregular astigmatism may
result from this combined procedure than if each procedure is
done alone.207 Other methods to evaluate the topographic
results of AK include finite element modeling of the eye.208
Using this technique, Hanna and co-workers evaluated the
incision variables and their effect on the curvature of the incised
and unincised meridians: length (longer incisions cause more
steepening of unincised meridian), distance from the center of
the cornea (incisions further from the center cause less
flattening of the incised meridian), and depth (deeper incisions
cause more effect).208
In the surgical management of symmetric astigmatism, accu-
rate determination of the axis of the steep axis is important,
because a small angular error will result in a relatively large
reduction of the anticipated induced cylinder. There are at
least three sources of error in measuring the steep axis with
keratometry, the first of which is eliminated with corneal top-
ography. Having been taught that eyes are most likely to exhibit
with the rule astigmatism, there is a natural tendency when
using the manual keratometer to report axis 90° rather than, for
example, axis 85°–95°. Small errors in axis alignment produce
significant undercorrection in cylinder. Use of the objective
measure of the SimK provided with corneal topography removes
this error of bias. However, the corneal topographer does not
eliminate the error in steep axis angular measurement caused
by head rotation in the head rest or incyclotorsion that can
occur from the stress of the clinical environment. Therefore,
surgeons must adopt strategies to carefully mark the cylinder
axis directly on the bulbar conjunctiva prior to astigmatic
keratotomy. Again, it is important to ensure that the corneal
cylinder matches the refractive cylinder (as it usually does)
before the surgical plan is implemented.
Epikeratoplasty
Epikeratoplasty was first intended for the correction of aphakia
but was later applied to the correction of myopia and utilized
as an onlay lamellar patch to flatten the keratoconus cornea.
Computerized corneal topography was used to study the effect
of this procedure on the surface of the cornea when applied
to each of the three types of patients mentioned earlier. When
used for keratoconus, it causes compression of the cone and
flattening of both the anterior and posterior aspects of the
cornea, whereas when used in myopia, it causes flattening of
CHAPTER 40
the anterior refracting surface of the cornea only.172 For aphakia,
the lenticle results in a steeper anterior surface.172 In addition,
it was through the use of corneal topography of eyes that had
undergone epikeratoplasty for myopia that decentration was
recognized as a complication of keratorefractive surgery.154 In
addition, this type of analysis revealed that it was important to
increase the size of the optical zone in order to maximize
refractive results.154
Corneal topography was helpful in identifying both regular
and irregular astigmatism after this procedure. Corneal top-
ography was also used to show that moderate amounts of
astigmatism were compatible with good Snellen visual acuity209
and to study the effects of this type of astigmatism on optical
performance after epikeratophakia.210
Hexagonal keratotomy
Hexagonal keratotomy was aimed at achieving a steeper cornea
for the treatment of hyperopia, presbyopia, and the over-
correction of RK. However, the procedure was plagued with 465
 CORNEA AND CONJUNCTIVA

FIGURE 40.32. Myopic excimer laser PRK with uniform central

corneal flattening.
FIGURE 40.33. Hyperopic excimer laser PRK showing steepening of
the central cornea.
complications, including corneal ectasia, glare, photophobia,
polyopia, fluctuation in vision, overcorrection, irregular
astigmatism, corneal edema, corneal perforation, bacterial
keratitis, cataract, and endophthalmitis.211 Because of this,
hexagonal keratotomy has been largely abandoned.

Photorefractive keratectomy
In 1983, Trokel ablated the cornea of freshly enucleated, bovine
eyes using an argon fluoride 193 nm excimer laser and showed
that this laser might be used for radial keratotomy because it
resulted in sharp grooves without thermal damage to adjacent
structures.212 At that time, Trokel also suggested that the laser
could be applied using a circular mask with a graded intensity
from center to edge in order to steepen or flatten the cornea.
Subsequent studies demonstrated its superiority over longer
wavelength excimer lasers in the production of corneal incisions
and its ability to flatten the cornea in order to correct myopia
(Fig. 40.32).213–222 Today, photorefractive keratectomy has also
been used in the treatment of hyperopia (Fig. 40.33),223,224 as
FIGURE 40.34. Myopic excimer laser PRK showing moderate
well as combined myopic225 and hyperopic astigmatism.226 decentration (~0.7 mm from the pupil center). The pupil is indicated by
Initial ablations performed on rabbits were difficult to evalu- the black dotted outline.
ate topographically227; however, improvements in laser tech-
nology and data collection allowed the evaluation of important
topographic data in primates.228 This led to further refine- performed.165 Surface ablation (PRK) became more widely
ments in both the surgical approach and in laser technology. practiced with the development of devices with which to
Corneal topography has subsequently been used to evaluate remove the epithelium as a clean sheet. With the introduction
regression, decentration, multifocality, and the optical perform- of Mitomycin C to block keratocyte activation, the incidence of
SECTION 6

ance of the post-PRK cornea.
Regression is a drift over time of the postoperative refraction
toward the preoperative refraction. In the early studies, it was
found that the post-PRK cornea was more likely to regress for
higher attempted corrections than for lower ones.229 This
regression is believed to occur secondary either to thickening
of the epithelial layer or to deposition of stromal collagen or
to both. For PRK corneas, stabilization tends to occur at
~ 6 months166 but can take up to 1 year.165,229 In some corneas,
regression is such that it leads to complete loss of the ablation,
but this is rare. It appears that communication between the
epithelial cells and the stroma is important in regression,230 and
there is growing evidence that the release of cytokines for the
epithelium as it is being scraped from the stromal surface may
be the cell signal that leads to anterior stroma keratocyte
apoptosis and subsequent stromal remodeling.231 In procedures
where Bowman’s layer is not ablated (e.g., laser in situ
keratomileusis (LASIK)), regression is not as great a problem
466 and stabilization may occur within weeks after the procedure is
postsurgical stromal haze formation has been greatly reduced.
Mitomycin C was first proposed to block scar formation after
PRK in the rabbit model.232
Decentration of the ablation is another problem encountered
after PRK. It was first recognized as a complication with the
topographic analysis of epikeratophakia233 but can affect almost
all keratorefractive procedures. Uozato and Guyton suggested
that the center entrance pupil should be used as the center of
the ablation.234 Since then, decentration has been defined as the
distance of the apparent center of the ablation to the apparent
center of the pupil as viewed with corneal topography. When
severe, decentration can result in monocular diplopia, glare,
ghost images, astigmatism, poor visual acuity, and poor con-
trast sensitivity, particularly for small diameter ablations.235
Decentration is difficult or impossible to detect with traditional
tools,90,171 but it can be seen easily and can be measured with
topographic analysis (Fig. 40.34).170,236–238 Using corneal
topography, Wilson and associates170 found that the average
decentration after PRK for the LSU phase IIA study was

0.79 ± 0.11 mm (range of 0.03–2.1 mm, 79% < 1 mm). With
improvements in technique, decentration was reduced to
0.47 ± 0.06 mm for LSU phase IIB, but since that early report,
considerable improvements have been made in technique as
accurate alignment is particularly important for hyperopic
refractive corrections as well as for correction of higher-order
aberrations.
Multifocality of the postoperative cornea can reduce the
optical quality of the cornea. Seiler and co-workers have shown
that spherical aberration resulting from the gradient in refrac-
tion at the edge of the treated zone correlates highly with best
spherical corrected visual acuity in normal eyes and with
measured glare visual acuity in patients with PRK.120 Martinez
and co-workers have shown that both coma and spherical
aberration are increased by PRK, and this is dependent on pupil
size and attempted correction.173 These changes in higher-order
aberrations may account for problems with night driving, halos,
and loss of contrast sensitivity experienced by some patients
after refractive surgery.
Multifocality can also result from unequal ablation within
the treated area. In some cases, areas of local contiguous elev-
ated power within the ablation zone and 2 mm or more in
diameter are seen in the postoperative topography and have
been called central islands.237,239 Clinically, central islands can
cause decreased vision, monocular diplopia, or decreased con-
trast sensitivity or create apparent over and undercorrections.
They occur rarely with the latest generations of excimer laser
but have been observed infrequently with LASIK procedures in
addition to the surface ablation techniques. Their etiology
remains controversial but may be due to degraded laser optics,
beam blockage by the plume of photodisrupted tissue, and
external hydration that results in unequal laser delivery to the
cornea.240 Corneal topography can be used to diagnose as well
as follow central islands after PRK. When diagnosing central
islands or other irregular astigmatism, one should use
difference maps, because preexisting irregular astigmatism may
appear accentuated after PRK (Fig. 40.35). True central islands
tend to resolve by 18 months after PRK (Fig. 40.36), although it
is tempting to remove these earlier with an estimated
enhancement procedure to improve visual performance.
Corneal Form and Function: Clinical Perspective
FIGURE 40.35. Irregularities in the treated area
after excimer laser PRK for myopia. A
difference map must be used to document
induced irregular astigmatism. Here the
preoperative cornea (upper left) is subtracted
from a postoperative examination (lower left).
The difference map (right panel) shows no
significant induced irregular astigmatism; the
treatment was uniform despite the topographic
appearance of the ablation.
Phototherapeutic keratectomy
Partial thickness corneal scars treated previously with lamellar
keratoplasty can now be treated with phototherapeutic
keratectomy. Lamellar keratoplasty may induce large amounts
of irregular astigmatism. PTK can remove the scar noninva-
sively without as great a risk of inducing irregular astigmatism.
In addition, PTK can be used to treat recurrent erosions241 or
treat corneal irregularities after EKC or as a result of a corneal
dystrophy. In general, PTK leads to an improvement in corneal
topography but can result in a decrease in best spectacle cor-
rected Snellen visual acuity.242 As noted above, customized cor-
neal ablations may improve corneal optics when the aberrations
are not too severe.
Automated lamellar keratotomy
Automated lamellar keratotomy (ALK) originated from myopic
keratomileusis (MKM) developed by Barraquer.243 The
technique for MKM was very difficult, and the predictability of
the final refraction was a problem. Furthermore, it was clear
that even patients with excellent postoperative visual acuity
could show significant amounts of irregular astigmatism244 and
CHAPTER 40
complain occasionally of glare and image distortion.90 Maguire
and associates90 reported a patient after MKM who experienced
marked visual distortion despite a normal slit lamp exam-
ination and a smooth corneal surface. Color-coded maps gener-
ated by computer analysis were used to show the degree of
irregular astigmatism, which was not evident from simple
inspection of the keratoscope photographs.90
The modern automated microkeratome for ALK has led to a
more regular treated zone. However, large amounts of irregular
astigmatism can occur (Fig. 40.37). Although strides in the
development of microkeratomes continue to be made, the
complication rate and the introduction of significant amounts
of irregular astigmatism can limit its use.
Laser in situ keratomileusis
When PRK was first introduced, patients could undergo unpre-
dictable amounts of regression accompanied by prolonged
periods of rehabilitation and corneal haze. Myopic corrections
above 6 D were problematic, although some degree of success 467
 CORNEA AND CONJUNCTIVA
FIGURE 40.37. Irregular astigmatism induced by ALK. Dislocated flap
or jamming of the microkeratome during the procedure can reduce
best-corrected spectacle visual acuity.
SECTION 6
was achieved by successive myopic treatments with several
optical zone diameters (multizone, multipass). Furthermore,
reepithelialization requires several days during which vision
was reduced and many patients experienced discomfort. This
helped to promote LASIK, which is a combination of PRK and
ALK.230 During this procedure, a flap of the anterior cornea is
created with the microkeratome, and the underlying stroma is
ablated to produce the myopic correction; following this, the
flap is repositioned. Recovery of good to excellent visual acuity
is often fairly immediate and less discomfort is generally
experienced. While LASIK has had its share of complications
related to the mechanical microkeratome (buttonholes, free
caps, and wrinkles) and to interface opacities due to epithelial
ingrowth, debris, and haze formation, the introduction of the
femptosecond laser for the creation of the LASIK flap has
reduced many of these problems.
Using topography to measure the average central corneal
power, we have found that LASIK may result in greater stability
468 of refraction than PRK (see Fig. 40.18). In particular, refraction
FIGURE 40.36. Central island formation and
resolution after excimer laser PRK. Upper left,
Preoperative examination. Lower left, 1 month.
Upper right, 7 months. Lower right, 10 months.
tends to stabilize within weeks. Solomon has shown that wound
healing in LASIK is very rapid, whereas PRK has a prolonged
response that lasts 4–6 months.232 This might explain the
difference in the amount of regression.
Irregular astigmatism can be a significant problem after
LASIK, but its incidence varies widely.245,246 LASIK increases all
quantitative measures (CVP, EDM, and SRI) of irregular astig-
matism.247 The increase in these indices can be comparable to
PRK. The amount of irregular astigmatism has been deter-
mined by the quality of the microkeratome cut. Moreover,
wrinkles on the flap and misalignment in repositioning the
flap may introduce some additional irregular astigmatism.
However, with the improvements in flap creation noted above,
significant irregular astigmatism after LASIK or surface ablation
is rare. It is noted that spherical aberration and coma are
specific representations of irregular astigmatism, and it is known
that decentration leads to the induction of coma, and small
diameter treatment zones lead to the induction of unwanted
amounts of spherical aberration.
Others
The intracorneal ring, introduced by Kera Vision Corporation,
was originally applied to the correction of refractive error in
ametropes.248,249 Induced astigmatism and long term com-
patibility with corneal functional anatomy are issues of con-
cern, but the potential for reversibility of the procedure is a
definite advantage. As well, intracorneal rings are being used to
improve vision in keratoconus patients. Intracorneal implant
technology has included devices such as deep stromal poly-
acrylate lenses, midstromal hydrogel lenses, and epistromal
collagen gels. Concerns with these devices include anterior
corneal nutrition, long term stability, and long term bio-
compatibility. Tissue necrosis can produce scarring, opacity, and
severe irregular astigmatism. Nevertheless, opaque annular
corneal inlays that have fenestrations for nutritional concerns
and a small 1.6 mm central clear zone are being explored to
improve near vision in presbyopes.
Corneal Topography and Contact Lenses
Contact lenses continue to be a good cosmetic alternative to
spectacles. Despite the incidence of complications that can have

devastating consequences to vision (e.g., amoebic, fungal, and
microbial keratitis), they can be a safer and more efficacious
modality than refractive surgery. As materials used in their
manufacture have improved in oxygen permeability (Dk),
biocompatibility, and wear comfort, adverse events associated
with contact lens wear are on the decline.
Effect of contact lens wear on corneal topography
Contact lenses embed themselves in the tear film and are
held in place by the force of capillary attraction. The primary
resting position is a function of the relationship between the
shape of the contact lens and the shape of the cornea. Gravity
and the action of the lids act to displace the contact lens from
the primary resting position. Owing to thermodynamics, in the
absence of friction, the lens would always center itself in the
same position on the cornea–the position of minimum entropy
where the overall space between the lens and the cornea is at a
minimum. Capillary attraction is a surface tension effect and
provides a force of negative pressure between the contact lens
and the cornea. This force plus the additional force of the lids
can cause the contact lens to alter the shape of the cornea.
When this contact lens-induced shape change is unintended, it
is generally referred to as corneal warpage (Fig. 40.38). There
are, however, situations in which this shape change is
intentional and these will be discussed later.
As mentioned earlier, contact lens-induced corneal warpage
can produce corneal topography that is indistinguishable from
preclinical and mild clinical keratoconus, and this has been
called pseudokeratoconus. Patients who have with the rule
corneal astigmatism and who wear decentered contact lenses
appear to be at risk. This combination of factors can flatten the
area of the cornea in the semimeridian under which the contact
lens is displaced and steepen the opposite semimeridian; the
result can resemble keratoconus.94 Such a case was presented in
Figure 40.25. These patients can complain of spectacle blur,
because asymmetric cylinder in the corneal surface cannot be
corrected with eye glasses. Whereas one management approach
is to discontinue contact lens wear altogether, a more satis-
factory approach for patients with rigid gas permeable (RGP)
contact lenses has been to refit with a daily wear high water
content lens that can allow the corneal topography to return to
normal.250 This strategy did not work well for patients who
were wearing polymethylmethacrylate contact lenses and were
switched to RGP lenses.251
Contact lens-induced warpage is a concern in the pre-
operative screening of refractive surgical patients, not only
because warpage can resemble keratoconus but also because
contact lens-induced warpage can destabilize the refraction of
the eye. Contact lenses, both rigid and soft, can alter corneal
curvature. After discontinuation of contact lens wear, the
average length of time for corneal curvature to stabilize in
patients that were symptomatic for rigid contact lens-induced
warpage can be almost 15 weeks and for soft contact lens-
induced warpage, the average time for stabilization was
5 weeks.163 The change in corneal power was often more than
1 D, and there were examples of both flattening and steepening.
Hence, to maximize predictability in a refractive surgical
procedure it is clear that, for patients with a history of contact
lens wear-even those who are asymptomatic252 -one should
obtain repeated refractions or, better still, repeated corneal
topography examinations until normal topography and
stabilization of refraction have been achieved.
Contact lenses have also been used to intentionally mold
the cornea, both to correct refractive error (orthokeratology
253–255
) as well as to improve the optics of the cornea in the
presence of irregular astigmatism. Although there is renewed
interest in orthokeratology,256 it appears that, while contact
Corneal Form and Function: Clinical Perspective
FIGURE 40.38. Large amounts of irregular corneal astigmatism can
be induced with contact lens wear.
lenses can be fitted to flatten the cornea in the case of myopic
correction, this change of shape is generally not permanent in
the absence of wearing a maintenance lens.257 In the presence
of irregular astigmatism, contact lenses are particularly useful
not only to reduce the visual distortion from the corneal
surface but can in some cases reduce the extent of irregular
astigmatism. Examples of this include the effect of contact
lens wear with clinical keratoconus, where the force of the
lens can reduce the ectasia and the use of contact lenses
after penetrating keratoplasty. It has been suggested that
irregular astigmatism can be reduced by fitting corneal
transplant eyes with rigid lenses.158 Subsequent studies have
concluded that it is safe to fit such eyes with contact lenses
after penetrating keratoplasty and that the topography remains
stable with time.258,259
Using corneal topography to fit contact lenses
The use of keratometry readings to fit contact lenses is tra-
ditional and is usually sufficient for successful wear. However,
as shown earlier, the normal cornea is neither spherical nor is it
completely symmetric. The amount that the corneal shape
deviates from an ideal geometric shape is related to the
adequacy of the fit with conventional lenses. It has become
CHAPTER 40
clear with the availability of computerized corneal topography
analysis that there is a new capability that can be used to
improve contact lens fitting. Corneal topographers math-
ematically reconstruct the shape of the cornea; these data can
be used to evaluate the relationship between the contact lens
and the corneal surface by simulating the familiar fluorescein
examination (Fig. 40.39).260 With this facility it is possible to
observe the fit of a number of trial lenses without testing all of
these directly in the patient’s eye.
Contact lens fitting has been constrained to an art form in
the past, because with only the keratometer to measure corneal
shape, there was insufficient information available. To utilize
corneal topography more fully in this area, a number of contact
lens fitting programs have been developed. Whereas early
software programs have not been very successful,261,262 sub-
sequent versions include expert fitting systems with a wide
variety of commercially available lenses that have met with a
measure of clinical success in both normal and pathologic
corneas.263,264 469
 CORNEA AND CONJUNCTIVA

FIGURE 40.39. Simulation of the fluorescein pattern of a trial contact
lens for a cornea with mild keratoconus.
CONCLUSIONS
It has been over a century since Javal and Schioetz introduced
their keratometer to the clinical practice of ophthalmology.
Now, owing to the computer revolution, the extensive analysis
of corneal curvature with corneal topography has become a
common diagnostic test and the clinical applications of this
technology are numerous. It is not often that a medical advance
answers more questions than it poses, but when used
appropriately, corneal topography can provide a clarity of
perception that makes classification of corneal topography
nearly intuitive. Modern corneal topography had its beginnings
in the refractive surgery clinical research laboratories; it has
succeeded beyond expectations in its initial goal to provide
topographic analysis for keratorefractive surgery. As the field
has matured, more accurate, smaller, and less expensive corneal
topographers have appeared. Finally, by combining slit beam
and Placido technology, we are getting close to being able to
measure separately the shape and refractive properties of both
surfaces of cornea and lens in order to compare these data to the
aberrometry measurements made from the entire eye!

ACKNOWLEDGMENT
Supported in part by the National Eye Institute, Bethesda, Maryland
(R01EY003311 and P30EY002377).

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topography software method for fitting rigid
gas permeable contact lenses. CLAO J
1994; 20:231–236.
262. Donshik PC, Luistro AE, Reisner DS: The
use of computerized videokeratography as
an aid in fitting rigid gas permeable contact
lenses. Trans Am Ophthalmol Soc 1996;
94:135–143.
263. Szczotka LB: Clinical evaluation of a
topographically based contact lens fitting
software. Optom Vis Sci 1997; 74:14–19.
264. Srivannaboon S, McDonald MB, Doubrava
M, Klyce SD: A prospective clinical trial
comparing a topographically guided
artificial intelligence software system
versus clinical expertise for fitting normal
and pathological corneas with contact
lenses. Invest Ophthalmol Vis Sci 1997;
38:S1089.
 CHAPTER
Ocular Surface Epithelial Stem Cells and Corneal
41 Wound Healing Response to Injury and Infection
Leonard P. K. Ang and Dimitri T. Azar

INTRODUCTION corneal epithelium could be replenished from the adjacent

conjunctival epithelium.6–8 Conjunctival transdifferentiation,
The ocular surface is a complex biological continuum respon- in which conjunctival epithelial cells differentiate into a corneal
sible for the protection of the cornea and maintenance of epithelial cell phenotype, was proposed as a mechanism to
corneal clarity. The precorneal tear film, neural innervation and explain replenishment of the corneal epithelium.6–8 Subsequent
the protective blink reflex help sustain an environment studies have argued against the concept of conjunctival
favorable for the epithelial cell layers. The ocular surface, transdifferentiation, as conjunctival tissue rarely resulted in
comprising corneal, limbal and conjunctival epithelia, is self- complete corneal epithelial replacement.9–20
renewing. Ocular surface stem cells are responsible for the The idea that limbal epithelial cells are involved in
maintenance and regeneration of the ocular surface epithelium. regeneration of epithelial cells of the cornea was proposed by
These play an important role in the wound healing process, and Davanger and Evensen in 1971.21 In heavily pigmented
in the regeneration of the epithelium following injury from eyes, they observed pigmented epithelial lines migrating from
accidental trauma, surgery or infection. the limbal region to the central cornea during healing of cor-
neal epithelial defects. Limbal basal epithelial cells appeared to
STEM CELLS be the least differentiated cells of the corneal epithelium.
Schermer et al found a 64 kDa keratin, called K3, among
Stem cells are present in all self-renewing tissues of the body.
Stem cells are a small, quiescent subpopulation of cells within
a given tissue.1,2 Stem cells are highly proliferative and self-
renewing, and are responsible for the continued replacement
and regeneration of tissues, thereby maintaining a steady-state SC
population of healthy cells within tissues during the lifespan Self renewal
of the organism. At steady state, stem cells remain fairly
dormant and replicate infrequently, but when the need for
tissue regeneration arises, proliferation may be rapidly induced. TAC3
Relative dormancy minimizes the possibility of replication
errors during cell division, which can result in mutations.
Stem cells give rise to transient amplifying cells that proliferate
rapidly, ensuring prompt regeneration of the tissue
(Fig. 41.1).3–5 Transient amplifying cells in turn give rise to
postmitotic cells, and finally to terminally differentiated cells. TAC2
These progenitor cells have a long life span, potentially exceed-
ing that of the organism, and show little evidence of aging.
These properties make stem cells also more prone to developing
neoplastic lesions.
Adult corneal and conjunctival stem cells represent the TAC3
earliest progenitor cells responsible for the homeostasis and
regeneration of the ocular surface. An intricate balance of
intrinsic and extrinsic factors modulates stem-cell proliferation
and differentiation, eventually resulting to terminally
differentiated cells that bear the phenotypic characteristics of
PMC
the tissue.

LIMBAL STEM CELLS

Differentiated cells located superficially in the corneal
epithelium are constantly lost, and are replaced by basal cells
entering the differentiation pathway.3–5 Previous reports
suggested that conjunctival and corneal epithelial cells arose
from a common progenitor cell type, and that depletion of the
TDC
FIGURE 41.1. Schematic diagram showing hierarchy of stem cell
(SC), transient amplifying cell (TAC1, TAC2, and TAC3 ), postmitotic cell
(PMC), and terminally differentiated cell (TDC). A self-renewal process,
possibly by asymmetric division, maintains the stem cell population. 475
 CORNEA AND CONJUNCTIVA
differentiated corneal epithelial cells.22 This cornea-specific
keratin was expressed in differentiated cells in the suprabasal
limbal layer, and throughout the corneal epithelium, but was
essentially absent in limbal basal cells, suggesting that they
represented a more primitive, nondifferentiated subpopulation
that did not express this cytokeratin. Kurpakus et al demon-
strated that the cornea-specific keratin K12, expressed in the
suprabasal cells of the limbus and throughout the entire corneal
epithelium, was also absent from the limbal basal cells.23,24
They also demonstrated that stem or stem-like cells found
throughout the basal layer or the limbal and corneal epithelium
during embryonic development were later sequestered in the
limbus.23–25
Current evidence indicates that corneal epithelial cells arise
from specific progenitor cells located in the basal cell layer
of the limbus (Fig. 41.2).3,5,13–20 Upon a demand for tissue
regeneration, for example, following injury, limbal stem cells
are stimulated to divide and differentiate into transient
amplifying cells.3,5 These transient amplifying cells migrate
superficially to the suprabasal limbus, as well as centrally to
form the basal layer of the corneal epithelium. Transient
amplifying cells increase rapidly in number to replace injured or
dead cells within the tissue. These cells differentiate into
postmitotic cells, which in turn differentiate further into
terminally differentiated cells. These eventually migrate
superficially and take on the final phenotypic characteristics of
the tissue. As their names imply, postmitotic and terminally
differentiated cells are incapable of cell division.
The observation that slow-cycling cells were restricted to a
subset of limbal epithelial basal cells provided strong support for
the limbal stem cell hypothesis.26–28 One of the most reliable
ways to identify epithelial stem cells takes advantage of their
slow turnover or slow cycling nature, which can be identified
experimentally as label retaining cells.26,27 Continuous
administration of tritiated thymidine for a prolonged period
labels replicating DNA in all cells that undergo cell division,
including slow-cycling cells. During a prolonged chase period in
the absence of tritiated thymidine, radioactive label in the
DNA of rapidly dividing cells is diluted by incorporation of
nonradioactive thymidine. Slow-cycling cells, presumably stem
cells, retain most of the previously incorporated isotope after
a 4–8 week chase period.26,27 Using this technique, Cotsarelis
et al observed retention of tritiated thymidine in limbal basal
cells, suggesting that these may represent corneal stem cells.28
This small subpopulation of normally slow-cycling limbal
basal epithelial cells demonstrated a greater proliferative
response to wounding and to stimulation by tumor promoting
SECTION 6
FIGURE 41.2. Schematic diagram showing the location of corneal
epithelial stem cells (SC) in the basal layer of the limbus. Solid arrows
denote the centripetal (horizontal) migration of limbal-derived TA cells
which progresseively lose their proliferative potential; dashed arrows
denote the (vertical) migration of cells into the suprabasal
compartment to become terminally differentiated (TD). C: cornea;
CC: central cornea; Cj: conjunctiva; DM: Descemet membrane;
L: limbus; PC: peripheral cornea.
Adapted from: Lavker RM, Sun TT: Epithelial stem cells: the eye provides a
476 vision. Eye 2003; 17:937-942. Figure 1.
compounds compared to cells of the peripheral or central cornea
(Fig. 41.3).13,16,28 The limbal basal cells’ proliferative response
was maintained over a prolonged period, demonstrating a
significantly greater proliferative reserve than cells in the central
corneal epithelium. The label-retaining cells present in the
limbus exhibited properties expected of stem cells.
Exactly how a population of stem cells is maintained is
unclear. A stem cell may divide symmetrically, giving rise to a
transient amplifying cell and producing a daughter stem cell
which replenishes the stem cell pool. Alternatively, regeneration
of stem cells could occur by de-differentiation of early transient
amplifying cells back to stem cells.
Stem cells have the highest growth potential under in vitro
cell culture conditions, and regions enriched in stem cells
display greater colony-forming ability. They can continue to
divide in vitro for at least 120–160 generations.29,30 Limbal
epithelial cells display greater in vitro proliferative capacity than
central and peripheral corneal cells, consistent with the
presence of stem cells in the limbus.31–41 Culture conditions
in vitro do not entirely mimic the original microenvironment of
these cells, as indicated by their eventual senescence. Therefore,
the true proliferative reserve of stem cells relative to the lifespan
of the organism is impossible to determine at present.
Clinical evidence also supports the limbal region to be the
site of corneal stem cells.36,42–45 Destruction of the limbal
epithelium by physical or chemical insult induces a stem cell-
deficient state. Clinical features of limbal stem cell deficiency
include abnormal wound healing with persistent or recurrent
epithelial defects, conjunctivalization (conjunctival epithelial
ingrowth), vascularization, loss of corneal clarity and chronic
inflammation. Additionally, the limbus is the most common
site of ocular surface neoplasias. They likely arise from altered
growth behavior of undifferentiated progenitor cells, suggesting
that a corneal intra-epithelial neoplasm is essentially a stem-
cell tumor.
Transient amplifying cells play an important role in wound
healing. When slow-cycling limbal stem cells are activated by a
demand for tissue regeneration, such as wounding, they give
rise to daughter transient amplifying cells that migrate centrally
or superficially to replenish the population of corneal epithelial
cells.5 Transient amplifying cells have shorter cell cycle times,
resulting in rapid cell division, and have a limited proliferative
capacity. They probably undergo a predetermined number of
cell divisions before differentiating into postmitotic cells which
in turn terminally differentiate and replenish the diminished
epithelial cell population.
A hierarchy of cells extends from the limbus to the central
cornea. Early transient amplifying cells, located adjacent to
limbal stem cells, have a greater proliferative capacity than later
transiently amplifying cells which are migrating from the
periphery toward the center of the cornea. Cells in the central
cornea are mainly postmitotic cells with no capacity for cell
division. These findings are consistent with growth responses
in vitro, where limbal and peripheral corneal cells generate large
colonies and are easily serially cultivated, whereas central corneal
cells are less clonogenic, and cannot be subcultured more than
once.31,32,34,36
UNIQUE PROPERTIES OF THE LIMBUS
Since the corneal epithelium must provide a transparent
medium for vision, it is devoid of pigmentation, and has a
smooth stromal–epithelial junctional structure. As such,
corneal epithelial cells are vulnerable to shearing injury because
of their poor adhesion to the underlying stroma, as evident in
patients with recurrent corneal erosions following relatively
minor corneal injuries.
 Ocular Surface Epithelial Stem Cells and Corneal Wound Healing Response to Injury and Infection
a b
c d
e f
Stem cells in the body are usually located in deeper tissue
layers, presumably for protection. The anatomical structure of
the limbus is significantly different from the adjacent cornea
because it need not be transparent. It is well suited to harbor
and protect the corneal stem-cell population. The limbal
epithelium is 8–10 cell layers thick, compared with five layers
in the corneal epithelium. The limbus tends to be heavily
pigmented, especially in pigmented races; this may protect
basal cells from the carcinogenic effects of ultraviolet
radiation.28,46 In addition, the palisades of Vogt have an
undulating epithelial–stromal junction, which provides greater
adhesion properties, thereby rendering the limbal epithelium
resistant to shearing forces. These folds also greatly increase the
surface area of the basal cells. The stromal component of the
limbus is well innervated, and is supplied by a rich vascular
network, allowing regulation of limbal stem-cell growth and
proliferation through various cytokine- and neural-mediated
pathways. An appropriate stromal micro-environment (stem
cell niche) is important for correctly regulating stem cell
activity.
CONJUNCTIVAL STEM CELLS
The conjunctival epithelium extends from the corneal limbus
to the lid margin, where it gradually merges with the
keratinized, stratified squamous epithelium of the eyelid. The
conjunctival epithelium provides a mechanical and immuno-
logical barrier to injury and infection, and its numerous mucin-
secreting goblet cells contribute to the production and stability
of the tear film.
Corneal and conjunctival epithelia are now believed to arise
from different stem-cell populations.47 Evidence shows that
transdifferentiation of conjunctival epithelial cells in a corneal
FIGURE 41.3. Autoradiograms of corneal (a, c,
and e) and limbal (b, d, and f) epithelia that
have been exposed to a single (c and d) or a
2-day treatment (e and f) of phorbol ester. The
response of corneal and limbal epithelia to
petrolatum treatment is shown in a and b. Note
the low level of [3H]thymidine ([3H]TdR)
incorporation (arrow) in unperturbed corneal
epithelium (a) and limbal epithelium (b). A single
exposure of phorbol myristate (TPA) results in
marked increases in [3H]TdR incorporation in
corneal (c) and limbal epithelia (d). After 2 days
of TPA treatment, both regions show a decrease
in [3H]TdR incorporation.
Adapted from: Lavker RM, Wei ZG, Sun TT: Phorbol
ester preferentially stimulates mouse fornical
conjunctival and limbal epithelial cells to proliferate in
vivo. Invest Ophthalmol Vis Sci 1998; 39:301-307.
Figure 2.
stromal environment is rarely complete.10,48 Conjunctival
epithelium transplanted onto the cornea of limbal stem cell-
deficient patients retained many characteristics of conjunctival
tissue, such as its glycogen content and goblet cells. 49,50
The evaluation of cytokeratin expression under identical cell
culture conditions provided direct evidence for separate lineages
of conjunctival and corneal cells.47 Conjunctival epithelial cells
expressed K4 and K13 cytokeratins, whereas corneal epithelial
cells expressed K3 and K12.36,47 Conjunctival and corneal epi-
thelial cell suspensions were injected subcutaneously into the
flanks of athymic mice.37,47 Cysts resulting from injection of
limbal and corneal epithelial cells retained features of normal
corneal epithelium, a stratified squamous epithelium without
goblet cells, whereas cysts derived from conjunctival epithelial
CHAPTER 41
cells displayed normal conjunctival morphology, a stratified
epithelium interspersed with numerous goblet cells. Current
evidence suggests that conjunctival epithelial stem cells are
bipotent, and can give rise to both nongoblet epithelial cells and
goblet cells.35,37,47
LOCATION OF CONJUNCTIVAL STEM
CELLS
Conjunctival stem cells are likely to be scattered throughout the
various regions of the conjunctiva (i.e., bulbar, forniceal and
palpebral), although the forniceal conjunctiva appears to be a
site that is enriched in conjunctival stem cells.35,36,49 A greater
number of slow-cycling cells (a property of stem cells) were
found in the forniceal epithelium compared with the bulbar and
palpebral conjunctiva (Fig. 41.4).49 Forniceal basal cells also
displayed a greater and more sustained proliferative response
than cells from other regions following injury or stimulation
with a tumor promoting compounds (Fig. 41.5). Further 477
 CORNEA AND CONJUNCTIVA

evidence was provided by in vitro studies which showed that

FIGURE 41.4. Schematic diagram showing the relative densities of
label-retaining cells in the palpebral, forniceal and bulbar conjunctiva
in the mice model. The highest concentration is noted in the forniceal
conjunctiva, which is believed to be the site enriched in conjunctival
stem cells. E: epidermis; T: transitional zone between palpebral
conjunctiva and epidermis (muco-cutaneous junction); P: palpebral
conjunctiva; F: fornix conjunctiva; B: bulbar conjunctiva; L: limbus;
C: cornea.
forniceal conjunctival cells had greater proliferative capacities
compared to the other regions.35,36 The mucocutaneous
junction at the lid margin might also be a site enriched in
conjunctival stem cells, which may be important for the
replacement of the palpebral and forniceal conjunctival
epithelium.51
The fornix is well suited to house and protect conjunctival
stem-cell populations from extrinsic injury, as it is located well
within the upper and lower recesses created by the closely
apposed eyelid and globe, and further from the external
environment than the other conjunctival regions. The network
of collagen and elastic fibers in the stroma protects the
epithelial cells from shearing and mechanical forces. The fornix
is also the most richly vascularized and innervated region of the
conjunctiva, allowing prompt response to cytokine or neural
stimuli.
EPITHELIAL–STROMAL INTERACTIONS
AND THE STEM-CELL
MICROENVIRONMENT
Both intrinsic factors (inherent to the cell), and extrinsic factors
(environmental factors surrounding the cell) are thought to be
involved in the regulation of stem cells.52,53 Schofield proposed

a b c
SECTION 6

d e f

g h i

FIGURE 41.5. Autoradiograms of the response of the bulbar (a, d, and g), fornical (b, e, and h), and palpebral (c, f, and i) epithelia to a single
exposure (d, e, and f) and a 2-day exposure (g, h, and i) of phorbol ester. Response of bulbar, fornical, and palpebral epithelia to petrolatum
treatment is shown in a,b and c. Note the low level of [3H]thymidine ([3H]TdR) incorporation (arrows) in unperturbed fornical epithelium compared
with bulbar and palpebral epithelia (a, b, and c). A single exposure of phorbol myristate (TPA) (d, e, and f) results in marked increases in [3H]TdR
incorporation in all three conjunctival epithelia, most notably in the fornical epithelium (e). Note the marked decrease in [3H]TdR incorporation in
bulbar and palpebral epithelia after 2 days of TPA treatment (g and i), whereas the fornical epithelia (h) has a higher proliferative profile.
Adapted from: Lavker RM, Wei ZG, Sun TT: Phorbol ester preferentially stimulates mouse fornical conjunc-tival and limbal epithelial cells to proliferate in vivo. Invest
478 Ophthalmol Vis Sci 1998; 39:301-307. Figure 1.
 Ocular Surface Epithelial Stem Cells and Corneal Wound Healing Response to Injury and Infection
that stem cells existed in a microenvironment that helped
maintain their undifferentiated state.53 Limbal basal cells
express higher levels of epidermal growth factor receptor (EGFR)
levels compared to the more differentiated cells of the central
cornea, which may serve to allow these cells to respond more
rapidly to various growth factors during development and
following perturbations, such as wounding.54
Limbal basal cells have been found to express intermediate
filaments, cytokeratin 19, vimentin, a6b4-integrin, metallo-
thionein, transferrin receptor, and a protein bound by mono-
clonal antibody AE1.55–57 Intermediate filaments are involved in
maintenance of cell cytoarchitecture, and may play a role in
anchorage of these cells to the underlying tissues. This expres-
sion profile is unique to limbal basal cells, and differs from that
of the surrounding basal cells. In addition, limbal basal cells
express higher concentrations of metabolic enzymes, such as
Na–K–ATPase, cytochrome oxidase, and carbonic anhydrase,
reflecting the different physiologic properties of these cells.3,58,59
Stromal–epithelial interactions are believed to be extremely
important in supporting normal corneal function, and
regulating the limbal stem-cell population. Intercellular
communications between the corneal stromal and epithelial
cells that are critical during early development, homeostasis,
and wound healing, are mediated by a variety of cytokines and
growth factors, such as transforming growth factor-b (TGF-b),
platelet-derived growth factor B (PDGF-B) and interleukin-1
(IL-1).60–62 Hepatocyte growth factor (HGF), expressed by
corneal fibroblasts, and keratinocyte growth factor (KGF),
expressed mostly by limbal fibroblasts, play important roles in
the regulation of proliferation, motility and differentiation
during epithelial stem-cell division in wound healing. 61–63
IDENTIFICATION OF EPITHELIAL STEM
CELLS
One of the most reliable ways to identify epithelial stem cells
takes advantage of their slow turnover or slow cycling nature,
which can be identified experimentally as label retaining
cells.26,27 This may be determined by a continuous adminis-
tration of tritiated thymidine, followed by a prolonged chase
period, and identifying the slow-cycling label-retaining cells that
retained the previously incorporated isotope. The in vitro
proliferative capacity of cells has also been used to distinguish
stem cells from other cells.29,30 Three types of keratinocytes
with different capacities for proliferation have been identified
from the human epidermis: holoclones, meroclones, and
paraclones. The holoclone, which has the highest proliferative
capacity and is able to undergo 120–160 divisions with less
than 5% terminally differentiated colonies, is considered a stem
cell. Although no definite stem-cell marker currently exists,
various putative markers for limbal stem cells have been
proposed. These include the nuclear protein p63,64 alpha-
enolase,14,65,66 high levels of a6-integrin in combination with
low to undetectable expression of transferrin receptor (CD71),67
the absence of connexins 43 and 50,68 and more recently, the
ABCG2 transporter.69 Slow-cycling label-retaining cells in the
mouse cornea limbus were also found to be enriched in cells
that expressed high levels of b1 and b4 integrins and little a9
integrin.70,71
MAINTENANCE OF THE CORNEAL
EPITHELIUM
The corneal epithelium is maintained by a constant cycle of
shedding of superficial cells, proliferation of cells in the basal
layer, as well as the slow migration of basal cells toward the
centre of the cornea.72 It has previously been demonstrated that
FIGURE 41.6. X, Y, and Z hypothesis of corneal epithelial
maintenance
X= proliferation of basal cells
Y= centripetal movement of cells
Z=cell loss from the surface
From: Thoft RA, Friend J: The X, Y, Z hypothesis of corneal epithelial
maintenance. Invest Ophthalmol Vis Sci 1983; 24:1442-1443.
India ink particles phagocytosed by basal cells of normal
corneas migrated centripetally from the limbal region to the
central area, at ~123 mm/week.73 The limbal basal cells, the site
of corneal stem cells, give rise to basal cells that migrate onto
the cornea, constantly renewing the supply of basal cells. These
cells, which do not originally express the 64 KD keratin, slowly
migrate across the corneal basement membranes and upward,
and begin to express the 64 KD keratin.
Thoft first proposed the X, Y, Z hypothesis of corneal
epithelial maintenance (Fig. 41.6).72 He suggested that the
maintenance of the corneal epithelium could be viewed as a
result of three separate, independent mechanisms. X repre-
sented the proliferation of basal epithelial cells, Y represented
the proliferation and centripetal migration of peripheral cells,
and Z referred to the epithelial cell loss from the surface.
Corneal epithelial maintenance, which involved a balance of
these processes, was defined by the equation: X+Y=Z. It is
estimated that the corneal epithelium is constantly renewed
every 7–10 days. Following corneal injury with resultant epi-
thelial cell loss, the regenerative mechanisms designed to
replace the corneal epithelium are set into motion, with
resultant centripetal movement of the cells from the periphery
to the central area. Other investigators have also demonstrated
this migration of epithelial cells from the peripheral cornea and
limbus.33,73–75 The corneal epithelium is therefore maintained
by a balance of cell shedding, basal cell division and renewal of
basal cells by centripetal migration of new basal cells from the
limbal stem cells.
CHAPTER 41
CORNEAL WOUND HEALING RESPONSE
The primary function of the corneal epithelium is to form a
barrier to invasion of the eye by pathogens and for uptake of
excess fluid from the stroma. Injury to the cornea may be
accidental or iatrogenic in origin. Various surgical procedures
may result in corneal wounds or abrasions. Excimer laser
refractive surgery is another important cause of iatrogenically
induced corneal wounds. The process of wound healing
involves a complex cascade of events that eventually results in
wound repair and reestablishment of the normal structure and
function of the cornea.
EPITHELIAL WOUND HEALING
With the advent of refractive surgical procedures, such as
photorefractive keratectomy (PRK) and laser in situ
keratomileusis (LASIK), there has been strong interest in the
study of healing of corneal epithelial wounds. Accidental injury 479
 CORNEA AND CONJUNCTIVA

FIGURE 41.7. Scanning electron micrograph of corneal epithelial cells

migrating to cover an epithelial abrasion.
From Pfister RR: The healing of corneal epithelial abrasions in the rabbit: a
scanning electron microscope study. Invest Ophthalmol Vis Sci 1975;
14:648.

or abrasion of the corneal epithelium results in a prompt

healing response to cover the exposed basement membrane
with cells.
After abrasion, mitosis ceases and the cells at the wound edge
retract, and lose their hemidesmosomal attachments to the
basement membrane. During the first 4–6 h after an epithelial
injury, there is an initial latent phase where no appreciable
decrease in size of the wound occurs. The basal and squamous FIGURE 41.8. Corneal wound healing following a 4 mm diameter
cells in the vicinity of the wound show thickening and abrasion. Cross-sectional view of the wound margin, and the
separation. Neutrophils accumulate along the wound edge ~3 h microphotographic appearance of the defect at the indicated times
after wounding.
after injury, as does thinning of the epithelium to a single layer
Adapted from: Beuerman RW, Thompson HW: Molecular and cellular responses
of flattened cells.76 The leading edge of the migrating cells is of the corneal epithelium to wound healing. Acta Ophthalmol Suppl 1992; 7–12.
only one cell thick. The cells enlarge, and the epithelial sheet Figure 1.
begins to migrate by ameboid movement across the defect until
it is completely covered. The edges of the cell membranes ruffle
and send out filopodia and lamellipodia toward the center of
SECTION 6

the wound (Fig. 41.7).77
Corneal epithelial defects, irrespective of the nature of injury,
result in a fairly consistent pattern of re-epithelization.36,76
A circumferential migration of three to six convex leading fronts
of migrating epithelial sheets from the limbus continue to
advance and progress towards the center.76 The advancing
fronts of epithelium eventually meet and merge imperceptibly
to repopulate the entire surface.74 The wound is covered by a
multilayered sheet made up of both basal and squamous
cells.78,79 After wound closure, mitosis restores the epithelium
to its normal configuration (Fig. 41.8). The healing process
occurs rapidly. An experimental epithelial wound 6 mm in
diameter is closed within 48 h, and the rate of epithelial cell
migration is 60–80 mm/h.80,81
The basal epithelial cells play a key role in proliferating and
covering the epithelial defect. The ultimate source of these cells
arise from the limbal basal stem cells that are activated to help
regenerate and repopulate the surface.6 Lavker et al suggested
480 the mechanism of centripetal migration was the inward draw-
ing of cells by preferential desquamation of central corneal
epithelial cells, rather than the cells forcing their way toward
the center.46 It is interesting to note that the healing rates for
larger (8 mm diameter) corneal epithelial defects were more
rapid (mean rate 0.91 mm2/h) than for smaller (4 mm diameter)
defects (mean rate 0.37 mm2/h). This is attributed to a greater
proliferative response of cells in the peripheral cornea and
limbus than in the central corneal.81 The histological appear-
ance of regenerated limbal epithelium resembles corneal and
not conjunctival epithelium.82
An important aspect of wound healing is the reformation of
adhesion complexes to the underlying connective tissue.
Wounding of the epithelium results in disassembly of the
hemidesmosomes of the remaining epithelial cells, which
allows these cells to migrate over the wounded surface. The
leading edge of the migratory cells form focal linkages from
cytoplasmic actin filaments to extracellular matrix proteins like
fibronectin, fibrinogen–fibrin, laminin, tenascin and integrins.83
Reformation of the adhesion complexes gradually occurs from
 Ocular Surface Epithelial Stem Cells and Corneal Wound Healing Response to Injury and Infection
the periphery toward the center.84 After wound healing, the
adhesion of the epithelium is re-established by formation of
new hemidesmosomes in the basal cell layer. The location of
these hemidesmosomes corresponds precisely to the locations
of anchoring fibrils in the basement membrane.
In corneal wounds where the basement membrane is not
damaged, a normal epithelium with adhesion complexes is
formed soon after. In the situation when the basement
membrane is removed, the epithelium must lay down new
basement membrane after healing and development of normal
adhesion complexes may be delayed for more than 12
months.85,86 This is particularly relevant in excimer laser
procedures such as PRK, where destruction of the basement
membrane and superficial stroma occurs, which results in
delayed corneal healing.
STROMAL WOUND HEALING
Keratocytes are responsible for the maintenance and regener-
ation of the corneal stroma. After injury, keratocytes are capable
of phagocytosis of collagen fibrils and synthesis and secretion
of collagen, glycosaminoglycan ground substance, collagenase,
and collagenase inhibitors.87–89 Stromal wound healing involves
resynthesis and cross-linking of collagen, alterations in
proteoglycan synthesis, and gradual wound remodeling, leading
to restoration of tensile strength.
A cascade of responses of cytokines leads to important
changes in the stroma that contribute to wound healing. Within
hours, polymorphonuclear cells appear around areas of cellular
necrosis in a corneal wound, followed thereafter by monocytes.
Immediately following injury, initial keratocyte apoptosis and
necrosis occurs. Within 12 h, proliferation and migration of
residual activated keratocytes occurs. The proliferating kera-
tocytes are believed to give rise to activated keratocytes,
fibroblasts, and myofibroblasts that repopulated the depleted
stroma.90 Fibroblasts and myofibroblasts have the ability to
establish and maintain intercellular communication with
themselves and nonactivated keratocytes, which may be critical
in the wound healing process.91 Stromal keratocytes lose their
interconnections and undergo morphologic changes, including
hypertrophy, proliferation, and finally reformation of cellular
processes and connecting gap junctions.92 TGF-b has been
found to significantly reduce corneal fibrosis. These early
changes contribute to other responses associated with stromal
remodeling, epithelial healing, production of altered extra-
cellular matrix and wound contraction.93
Corneal wound healing is a complex cascade mediated by
cytokines, growth factors, and chemokines. These complex
functions may be modulated by cytokines from the epithelium,
inflammatory cells and other keratocytes.94,95 The healing
process is initiated immediately after injury through the
release of multiple cytokines and growth factors, such as IL-1,
tumor necrosis factor-a (TNF-a), bone morphogenic proteins
2 and 4 (BMB), epidermal growth factor (EGF), and PDGF
from the corneal epithelium and epithelial basement
membrane.96–98
The early phases of wound healing also involve degradation
and removal of damaged tissue orchestrated by the plas-
minogen-activator/plasmin system, collagenolytic metallopro-
teinases and other enzymes.99 Activation of the plasmin–
plasminogen system is needed for the progression of normal
healing.100 An increase in polymorphonuclear leukocytes in the
cornea often coincides with enhanced production of matrix
metalloproteinases (MMPs). MMPs may be involved in normal
epithelial migration, the initial stromal degradation during the
inflammatory response, and the ultimate remodeling of the
extracellular matrix.
MMPs are responsible for the initial rate-limiting cleavage of
collagen molecules, and changes in expression of these
collagenolytic/gelatinolytic enzymes occur in healing or
ulcerating corneal wounds.101,102 Following corneal wounding,
MMP-2 expression is increased and much of it appears in the
active form. These changes persist for at least 7 months,
suggesting that MMP-2 is involved in the prolonged process of
collagen remodeling in the stromal repair tissue. MMP-9 is
expressed in the epithelial layer of the repair tissue and is
believed to be involved in the degradation of the epithelial
basement membranes that precedes corneal ulceration, as well
as in controlling resynthesis of the basement membrane.102 It is
possible that these proteolytic enzymes may play a role in the
short-term and long-term stromal remodeling in the normal
cornea. The MMP/tissue inhibitor of metalloproteinase (TIMP)
systems may play an important role in the early stages of
corneal wound healing as well as in scar formation and clearing
after excimer laser keratectomy.103,104
Among the many mediators involved in regulating wound
healing, IL-1a appears to play a special role in orchestrating
wound healing by modulating many key processes involved
in stromal healing after its release triggered by epithelial cell
injury or death.105
Return of normal structure and function may take months,
or even years, in some eyes, depending on the nature of injury
or surgery.
WOUND HEALING RESPONSE TO
CORNEAL INFECTION
RISK FACTORS FOR CORNEAL INFECTION
Because an intact corneal epithelial surface, with its tight
junctions formed by desmosomes and hemidesmosomes, is the
main line of defense against microbial infection, an important
complication arising from a breech in the integrity of the
corneal epithelium is infectious keratitis. There are several
other mechanisms that protect the surface of the eye from
infectious agents. The eyelid provides a physical barrier to
protect against organisms gaining direct access to the eye. The
tear film contains antimicrobial enzymes, secretory immuno-
globulins and complement components, such as lysozyme,
lactoferrin, and betalysins. The normal ocular flora provides a
balance to prevent overgrowth of exogenous organisms. The
conjunctiva contains subepithelial mucosal-associated lym-
phoid tissue with a collection of lymphoid cells. These factors
serve to protect the ocular surface against infection.
Any alteration of the local or systemic defense mechanism
CHAPTER 41
may predispose the eye to infection. Disruption of the corneal
epithelium may be caused by trauma, contact lens wear or
from chronic bullous keratopathy, which creates a portal of
entry for microbial organisms. Other predisposing factors
include eyelid abnormalities (e.g., trichiasis, entropion, ectro-
pian or lagophthalmos), tear-film abnormalities (e.g., Sjögrens
syndrome), exposure keratopathy, neuropathic keratopathy,
ocular surface disorders (e.g., Stevens–Johnson syndrome,
chemical injury), and chronic steroid use. Systemic conditions
that may predispose to corneal infection include diabetes
mellitus, and systemic immunodeficiency.
CORNEAL WOUND HEALING FOLLOWING
INFECTION
In bacterial keratitis, entry of organisms results in diffusion of
toxins and enzymes. Polymorphonuclear leukocytes arrive at
the corneal wound site. Stromal damage from bacterial and
neutrophil enzymes facilitates progressive bacterial invasion of 481
 CORNEA AND CONJUNCTIVA

the cornea. There may be progressive tissue necrosis resulting
in sloughing of the epithelium and stroma, which varies with
the virulence of the organism and toxin production. The necrotic
base of the ulcer is surrounded by heaped-up tissue. The host
cellular and humoral immune defense mechanisms retard
bacterial replication, promote phagocytosis of the organism and
cellular debris, and halt destruction of stromal collagen.
In the healing phase, the epithelium resurfaces the central
area of ulceration and the necrotic stroma is replaced by scar
tissue produced by fibroblasts. The reparative fibroblasts are
derived from histiocytes and keratocytes that have undergone
transformation. New epithelium slowly resurfaces the irregular
base. The physiologic processes involved in corneal wound
healing are similar to what are described above. Bowman’s layer
does not regenerate but is replaced with fibrous tissue. New
blood vessels are directed toward the area of ulceration to
deliver humoral and cellular components to promote healing.
These gradually disappear and may leave ‘ghost vessels’. The
fibrous scar tissue results in corneal opacity, which may
gradually fade over time.

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CHAPTER 41
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SECTION 6
484
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 CHAPTER
Corneal Examination, Specular and Confocal
42 Microscopy, UBM, OCT
Ula V. Jurkunas and Kathryn Colby
The cornea is a transparent, superficial tissue, and various
examination techniques readily identify corneal pathology. The
use of different light wavelengths, illumination and magnifi-
cation modes in the instruments described below allows the
examiner to delineate both morphologic and functional changes
in corneal tissue.
PENLIGHT EXAMINATION
Useful information about the cornea can often be gathered
using a simple penlight in an illuminated room before the slit-
lamp (SL) examination. For example, inflammation of the
eyelids, conjunctiva, sclera, episclera, or anterior uvea are often
better noted with this exam. Penlight examination may also
reveal a localized or general opacification or vascularization of
the cornea. Corneal surface irregularities, such as abrasions,
can be diagnosed by scanning the cornea with a penlight,
looking for an irregular light reflection (Fig. 42.1).
SL MICROSCOPY
The techniques of anterior segment examination were crude at
best until the development of the first SL by Gullstrand in
1911.1 The combination of Gullstrand’s focal illumination
FIGURE 42.1. Irregular corneal reflex indicating a corneal abrasion.
source and the Czapski microscope was the first successful
system that allowed the light beam to be projected at various
angles and focused at different depths of the eye.1 Since then,
refinement of SL biomicroscopy has led to universal acceptance
of this modality. Current widely used models of SL include
those made by Haag–Streit, Zeiss, Bausch & Lomb Thorpe, and
Nikon.1 The SL is a compound binocular microscope which
provides variable magnification for delivery of the brightest
possible image. It allows maximum magnification of 40µ and
resolution of 20 mm (Table 42.1).2
The SL uses corneal properties to transmit and reflect light.
Since the cornea is translucent, most of the light encountered is
transmitted, but some is scattered and reflected back, enabling
the visualization of the tissue which is not ‘crystal clear’. Due
to the difference in refractive indices (RI) between two
interfaces, such as air–tear or stroma–aqueous, the light that is
reflected back and/or scattered forms an image, such as seen
during SL examination. When angle of incidence of light is
equal to the angle of reflection, the incident light forms a bright
reflex called specular reflection.2 In clinical practice the two
most commonly used images are the surface corneal light reflex
from the epithelial surface and the specular reflex from the
corneal endothelium. Light can also be reflected in a diffuse
manner when the angles of incidence and reflection are not
equal, as seen in light scattering. Surface irregularities such as
scarring or epithelial edema reflect light in a variety of direc-
tions giving the tissue its opaque appearance. The more dense
the opacity the greater the scattering of the reflected light. The
terms nebula, macula and leukoma represent the continuum of
opacity density with the latter denoting a most opaque white
scar.2 The internal corneal reflection of light from the epithelial
basement membrane or Descemet’s membrane is called
sclerotic scatter. The light can also be transmitted back through
the cornea after it has been reflected from surrounding ocular
structures such as the iris and lens, highlighting corneal guttae
or epithelial edema by retroillumination.3
TABLE 42.1. Comparison of Different Instrument Optical
Properties2,7,9,24
Instrument Maximum Resolution View
Magnification
Slit Lamp 40µ 20 mm Transverse
Specular 500µ 2–5 mm Lateral
microscope Axial
Confocal 600µ 1–2 mm – lateral Lateral
microscope 5–10 mm – axial Axial
485
 CORNEA AND CONJUNCTIVA
CLINICAL TECHNIQUES
The SL examination is a dynamic process that allows an in vivo
three-dimensional view of the structures of the cornea. By
varying the illumination technique and the aperture of the slit
beam, one can achieve different methods of examining the
cornea. Two particularly useful techniques include specular
reflection and retroillumination.
Diffuse Illumination
This technique is used as the means of initial overview of the
anterior ocular structures and provides a general survey and
localization of abnormalities. The slit beam should be opened
to the maximal width. The intensity of the light beam can be
adjusted to minimize patient discomfort. The initial inspection
should be started with low magnification. The light beam can
be rotated at various angles in order to delineate the shape and
the extent of any abnormalities.
Direct Focal Illumination
In order to zoom in on the area of interest, the slit beam is
narrowed and placed at an angle to produce a well illuminated
and magnified optical section. By rotating the illumination
arm, the examiner can obtain vertical, horizontal and oblique
sections. When the illumination arm is placed at an angle of 45°
from the observation axis, the information from the particular
optical section is maximized (Fig. 42.2). The depth of the
abnormality can be determined and, therefore, localized to a
particular corneal layer. Abnormal depressions and elevations,
corneal thinning or changes in corneal shape or contour can be
elucidated by observing differential bending of the thin beam of
light. By moving the slit systematically across the cornea, serial
optical sections are viewed, and a mental construct of corneal
pathology is created.
Indirect Proximal Illumination
This technique aids in identifying the details of the pathology
within the opacity. The beam is shortened to 2–3 mm,
broadened and focused to the area adjacent to the opaque area.
The light beam will undergo internal reflection within the
cornea and will be scattered as it hits the opacity. The reflected
light will readily highlight the internal structure of the opacity,
and will aid in identifying the details that could be missed
SECTION 6
FIGURE 42.2. The slit-lamp beam strikes the cornea in an oblique
fashion. The scattering properties of the cornea give the viewer an
optical cross-section. In this case, folds in Descemet’s membrane can
486 be seen.
otherwise. For example, a small foreign body can be identified
within a dense area of corneal inflammation when viewed by
this method.
Sclerotic Scatter
When the slit beam is directed at the limbus, the opaque sclera
scatters the light, directing part of it inside the corneal stroma.
Based on the principle of total internal reflection of light, the
light travels through the entire stroma and is reflected back and
forth from the anterior and posterior corneal surfaces. When no
opacity is present, the halo of light emerges back 360° around
limbus. Any opacity impedes the spread of light inside the
cornea and reflects the scattered light back to the examiner. By
this method faint opacities such as subepithelial infiltrates or
mild epithelial edema due to Fuchs’ endothelial dystrophy can
be readily observed. This technique may be used early in the
examination, to acclimatize the patient to the light before it is
directed into the pupil.
Specular Reflection
The microscope is placed directly in line with the angle of
reflection, so angle of incidence of the slit beam is equal to the
angle of microscope observation.3 The posterior specular
reflection is at the interface between the endothelium and the
aqueous and is formed due to difference in RI between two
structures. Because the difference in RI between the aqueous
and endothelium is small, only 0.022% of total incident light is
reflected, thus forming only a faint image.2 Since the cornea is
curved, narrowing the beam to ~0.5 mm eliminates the sur-
rounding glare from other tissues. If the beam is too narrow, not
enough corneal surface is illuminated to examine the mosaic
pattern. To start, the beam of light is projected to the central
cornea from the temporal side, then moved towards the
periphery. The slit-beam width should be constantly adjusted
to avoid bright reflections from the tear film. The magnifi-
cation can be adjusted for maximum identification of cellular
structures.1–3
Retroillumination from the Iris
By placing the slit beam at an angle of 60° from the microscope,
transmitted light through the cornea is reflected back from the
iris or surrounding structures. It allows objects whose RI differ
from the surrounding medium to be observed. Subtle anterior
basement membrane changes or endothelial guttae are often
best visualized using this technique (Fig. 42.3).1,2
ADVANTAGES AND DISADVANTAGES
SL biomicroscopy is the most commonly used and readily
available method of examining the cornea. It is a fast screening
technique that does not require additional technical training.
The utilization of dyes, most commonly fluorescein, readily
provides the examiner with additional information about the
different patterns of surface disruption differentiating, for
example, exposure to keratopathy from superior limbic kerato-
conjunctivitis. Rose Bengal dye, which stains devitalized
epithelial cells or cells lacking mucin, will stain the areas
affected from tear deficiency states when the epithelium is still
intact and fluorescein staining is negative.
SL biomicroscopy is limited in its resolution of structures.
The light reflected from other corneal structures anterior to the
area of interest can obscure fine detail. Also, other corneal pathol-
ogy, such as edema or scarring can altogether block the image.
The SL has a number of disadvantages for examining the endo-
thelium, including low intensity of reflected light, low magni-
fication of the cells and illumination of only a small area at any
one time.2 In general these disadvantages are far outweighed
 Corneal Examination, Specular and Confocal Microscopy, UBM, OCT

of the endothelium. Wider slit illumination also causes morpho-

logical distortion of endothelial cells.7 Technological improve-
ment in the design of the objective lens has led to the use of
wide-field SMs without compromising the quality of the image.
In contrast to the SL, the modern SM is designed with separate
illumination and light-viewing pathways, so that reflection from
anterior corneal structures is reduced.7,12,14 Also, a scanning
system developed by Koester captures the entire endothelial
mosaic without overtly compromising resolution.8,10,14

CLINICAL ANALYSIS
Qualitative
The parameters evaluated are cell morphology, cell boundaries
and intersections, posterior corneal surface, and additional
structures.

FIGURE 42.3. Illumination from the fundus provides an orange
background for the early signs of lattice dystrophy of the cornea.
Cell morphology
Normal corneal endothelial cells are hexagonal in shape and
form a regular pattern of contiguous cells. Normally, the cells
are of same size: cell-side lengths are equal and the intersection
between all cell sides is ~120° (Fig 42.4).7 With age cells
become larger, there is more variation in cell wall intersection
angles, with overall tendency towards pleomorphism, or variation
in cell shape (Table 42.2).15 Cell shape can also change from

by the easy use and ready availability, making SL biomicroscopy

the most common corneal examination technique.

SPECULAR MICROSCOPY
It was David Maurice in 1968 who first photographed the
posterior corneal surface of an enucleated rabbit eye, and
published a specular image of the endothelium, captured by the
instrument he called a ‘specular microscope’.4,5 Liang and
colleagues subsequently published photomicrographs with
improved resolution that showed individual endothelial cell
boundaries and intracellular structures.6,7 In the 1980s Koester
developed a wide-field specular microscope (SM) that captured
the entire endothelial mosaic with good resolution by scanning
narrow endothelial layer zones and projecting them on the a b
same film.8 Since then specular microscopy has gained wide-
spread clinical acceptance as a method for evaluating endo- FIGURE 42.4. (a) SM image of corneal endothelium with
thelial cell density and morphology.5,7,9 morphometric analysis. This is a normal measurement of endothelial
cell density (CD) and coefficient of variation (CV). SD, standard
The advent of SM has greatly improved the study of human
variation. (b) Specular microscopy image of corneal guttae
corneal endothelial morphology and allowed quantification of represented as hyporeflective areas (arrow).
endothelial changes. Both types of SM, contact and noncontact,

CHAPTER 42
produce an image superior to the one that is obtained by the SL.
Specular microscopy provides a larger overlapping image of
endothelial cell layer, higher magnification and less interference TABLE 42.2 Specular Microscopy Parameters7,15
from patient’s eye movement (Table 42.1). The latter advantage Qualitative Quantitative Normal Values Quantitative
is mostly seen in contact SM, although it can also be seen with Parameter Parameter Equations
noncontact SM, if the final image alignment is automated.7,10
Computer-assisted morphometric analysis is a powerful tool Cell size Cell density Children – 106/mean
present in most SMs that standardizes cell counting and (cell 3500 cells/mm cell area
count) Adults – 106/mean
analysis, image and data management and provides data Cell area 2400 cells/mm cell
storage.7,10–12 Some machines have a pachymeter attached for density
measurement of corneal thickness.13
Variation in Coefficient Less 0.3 Mean cell
SM captures the specular reflection of light formed at the
cell size – of variation area/SD
optical interface between the endothelium and the aqueous polymega- of mean
humor. By increasing the angle of incidence of the illuminating thism cell area
source, the width of the slit beam can also be increased to image
Variation in cell Percentage 100% N/A
a wider area of endothelium. By increasing the width of the slit
shape – in hexa-
beam, scattering of light from structures anterior to endothelium pleomor- gonal cells
produces more diffuse illumination of the surrounding ocular phism
tissues and a consequent decrease in the contrast and definition 487
 CORNEA AND CONJUNCTIVA

hexagonal to elongated, as seen in the apex of keratoconus.
Also, rounded, square and triangular cells have been noted,
without clear clinical significance of such changes.7
Cell boundaries and intersections
The variability in cell boundary intersections (such that angles
between the walls deviate from 120°), signifies thermodynamic
instability of the endothelium.15 Such boundary formation is
usually formed by cells in transition brought by loss of nearby
cells.15
Posterior corneal surface
The examination of boundary between endothelium and
aqueous humor depicts a silhouette of the posterior corneal
surface which can be smooth or irregular. Hyperreflective
excrescences on Descemet’s membrane due to guttae can be
depicted against the dark background.7,16
Additional structures
Corneal guttae appear as hyporeflective oval-to-round areas
with central highlight at the apex (Fig. 42.4). The surrounding
endothelial cells are hyperreflective in relation to guttae since
the endothelium overlying the guttae is out of plane of focus
and appears as dark or absent.16 Evaluation of endothelial
mosaic and structures around it can distinguish most common
corneal endothelial disorders (Table 42.3).15,17
Quantitative
Wide-field SBM permits evaluation of both central and
peripheral cornea and studies of regional variability.18 The mor-
phologic parameters can be quantified and are summarized in
Table 42.2.
ADVANTAGES AND DISADVANTAGES
The SM provides a detailed picture of both the cell density and
morphology of the corneal endothelium. The wide-field view
provides an image of the entire mosaic permitting a study of
regional variability. In clinical practice the SM is useful for
diagnosis of patients with unilateral corneal edema and no
corneal gutta visible on SL exam of the contralateral eye.19 If
reduced cell counts and abnormal morphology are evident on
specular microscopy, the diagnosis of endothelial cell dys-
function can be made (Table 42.3).19
The main disadvantage of the SM is still image resolution,
which is limited by light scattering of the surrounding corneal
SECTION 6
tissue. In advanced corneal edema or scarring, the endothelial
mosaic can not be visualized with the SM. In order to discern
the cellular detail, the endothelial layer has to be smooth and in
the same plane of focus.16,20 Otherwise the cells are seen as
hyporeflective images with no additional information on their
morphology. As described above, corneal guttae manifest as
confluent reflex-free areas (Fig. 42.4). By changing a plane of
focus it is possible to discern endothelial cells on top of these
excrescences, but these cells are not accounted for in the
measurement of the cell count.16,21 Also, SM has disadvantages
of cost, availability and a need of image interpretation by an
expert or a corneal specialist.
CONFOCAL MICROSCOPY
The confocal microscope (CM), invented in 1957 by Marvin
Minsky, began to be used for imaging the cornea in vivo in
the mid 1990s.9,22,23 The improved optics of CM allow the
magnified imaging of all corneal layers in x,y,z axes over time,
rendering a truly novel four-dimensional in vivo microscopy.9,23
The improvement in lateral (x, y) and axial (z) resolution with
the CM was achieved by eliminating scattered light that is not
in the focal plane of the imaged object (Table 42.1). Diffraction-
limiting pinhole or slit apertures focus the light source and the
image on the same focal plane, thus creating a ‘confocal’
image.24 As a result, out-of-focus reflected signals are excluded.
Because such apertures create a very small field of view of only
a single spot on the cornea, the instrument has to scan the
whole sample by moving both the illuminator and the detector
in a synchronous fashion. By simply varying the plane of focus,
the source and detector scan the tissue along z axis and provide
magnified coronal sections at a variable depth.5,14,24 The speed
at which a single field is scanned at a constant depth provides
the time resolution feature of the microscope. In order to recon-
struct a real-time view on the screen5,8,24 rapid image capture
systems are necessary during scanning to avoid interference
from a patient’s pulse, respiration and ocular microsaccades.24
The newest models contain optical pachymetry, video recording
and automated cell analysis features (Fig. 42.5).22
5. Cornea
2. Nipkow disk
4. Objective
3. Beam lens
1. Light splitter
source

TABLE 42.3 Specular Microscopy Finding in Posterior Corneal

Disorders15–17,20

Specular Fuchs’ PPMD ICE

Findings

Cells Decreased Pleomorphism Enlarged ICE cells

Mosaic endothelial Irregular large White reflections
cell density cells with in center of
6. Front surface
Polymegathism scalloped dark cells
Mirror
Pleomorphism edges as ‘dark-light Direct
Disrupted by dark rings reversal’ Video
viewing
guttae around No disruption camera
lighter from craters or
center guttae FIGURE 42.5. A disk with pinholes arranged with conjugate
Vesicles/pits symmetry is used to provide confocal point source and point
in DM detectors. A full field of view is obtained in real time (i.e., >30
Disrupted by frames/sec) by rotating the disk at high speed to scan the specimen.
crater-like The image can be viewed directly or imaged using a video camera
focal lesions and recorded on videotape or continuously displayed on a monitor, or
488 both.
 Corneal Examination, Specular and Confocal Microscopy, UBM, OCT
CLINICAL APPLICATION
Two types of CM are available, which differ in the type of
scanning system employed. Tandem-scanning CMs (TSCMs)
and slit-scanning CMs (SSCMs) scan the sample either by a
plate of pinholes in the former or by thin optical slits in the
latter.24
SSCM is particularly well suited for imaging transparent
tissues such as cornea because the slit height can be varied to
achieve optimal image clarity.9 The adjustment of the slit adjusts
the depth of focus at z axis and minimizes the surrounding
noise. Also, the slit provides greater signal and higher image
clarity for a particular video frame. For example, using this
device imaging of the poorly reflecting epithelial wing cell layer
is possible.25 In a commercially available SSCM (the Confoscan
3 (NIDEK Technologies), Fig. 42.6), the distance immersion
principle is applied and a noncontact floating probe is utilized.
No alteration in tissues and improved patient comfort render
this technique safer and potentially more popular.9 The major
disadvantage of this SSCM is the inability to quantify the z axis
when a noncontact probe is utilized. Since a fixed ‘zero’ point is
not available, the assessment of depth of the image becomes
arbitrary. In clinical situations, the assessment of keratocyte
count post-refractive surgery or detection of post-LASIK flap
Cell density and area statistics (N =124)
Cell Count: 1960 [cell/mm2]
Normal: 2015-3552 [cell/mm2]
Polymegathism: 34.4%
Normal: <30%
Cell sides statistics (N =124)
Pleomorphism: 60.5%
Normal: >59.6%
ConfoScan 3 Imaging system rev. 3.1 Nidek Technologies s.r.i.
depth might be better performed by the contact method of
a TSCM.26–28
TSCM scans the cornea using Napkow disk containing
~64 000 diffraction-limited pinholes of 20 mm arranged in
spirals. This arrangement allows a z axis resolution of 9 mm and
x, y axes resolution of less than 1 mm. Since the light trans-
mission though the pinholes is low (less than 1%), the loss of
luminance limits the resolution of low-contrast structures. The
TSCM that is commercially available (Advanced Scanning, Ltd.)
utilizes dipping cone objectives. Since the position of the focal
plane relative to the objective lens can be varied, recorded and
converted to z axis position, the depth of the tissue focal plane
can be accurately calibrated.25 In order to reduce surrounding
noise of micromovements, a real-time digitizing system has
been developed that averages sequential frames and saves the
best-quality images. Confocal microscopy through focusing
(CMTF) is a powerful tool which enables imaging of entire
cornea in a highly rapid manner (in ~8 s). By moving the focal
plane of the objective lens through the cornea and capturing
the image focal plane in digitized manner, hundreds of images
are created with available on-screen three-dimensional
reconstruction.25
By measuring the distance between the intensity peaks on
the CMFT intensity curve, tissue and sublayer thicknesses can
FIGURE 42.6. ConfoScan 3 (Nidek
Technologies) image of corneal endothelium.
Automated cell count is performed after the
region of interest (ROI) position and dimension
is selected by the user. By default, the cells are
filled with different colors, depending on their
dimensions and cell side numbers. Percentages
of polymegathism and pleomorphism are
calculated.
Courtesy of Jose de la Cruz, MD. New York Eye and
Ear Infirmary, New York, New York.
CHAPTER 42
489
 CORNEA AND CONJUNCTIVA

be assessed.25 In pre- and post-LASIK patients the effects of
laser ablation on wound healing can be quantified by observing
flap thickness, tissue loss and regrowth, and keratocyte mor-
phology and density.25,27,28
The role of CM in diagnosis and management of atypical
corneal ulcers such as Acanthamoeba, Microsporidium and
fungal keratitis has received widespread attention, but has not
gained widespread utilization.29 TSMF has been showed to be
particularly promising in indentifying the location and depth of
Acanthamoeba trophozoites and cysts when employed by
experienced observers.25,29–32 Due to complexity of result
interpretation, CM use remains in the hands of a few academic
centers.
One of the main advantages of the CM over the SM is its
ability to image the endothelium through a hazy cornea, as is
seen in corneal edema.33,34 Also, earlier detection of Descemet’s
membrane alterations have been reported with CM as opposed
to SM and SL.35,36 On the hand, most of the studies comparing
confocal and specular microscopy find both instruments to be
equal in their clinical application.24,12,14,35,37 The comparison of
SM and CM on evaluation of corneal endothelium is given in
Table 42.4.
Newer advances in in vivo microscopy have combined
Heidelberg retina tomography (HRT II) and the Rostock cornea
module (Heidelberg Engineering GmbH) (HRT II/RCM) into a
digital confocal laser scanning microscope.38 After a poly-
methylmethacrylate (PMMA) plate contacts the patient’s ocular
surface, layer-by-layer three-dimensional images are created and
displayed in a computer monitor. Preliminary studies of HRT
II/RCM have confirmed superior image contrast in evaluation
of corneal and conjunctival layers on a cellular level, including
visualization of corneal keratocytes, Langerhans’ cells, meibomian
glands and goblet cells.38,39
ADVANTAGES AND DISADVANTAGES
In summary, the optics of the CM allows improved resolution
and magnification of the human corneal structures. Both cost
and result interpretation in diagnostic use of CM preclude its
widespread utilization in clinical practice, rendering CM
primarily as a research tool at present.24 On the other hand,
ever-evolving technological aspects in confocal imaging will
most likely turn this technique into a routine practice in
anterior segment evaluation in the future.
OPTICAL COHERENCE TOMOGRAPHY
The cross sectional imaging by optical coherence tomography
SECTION 6
segment since 1990s. First described by Huang, and later
developed into SL-adapted OCT system by Izatt and colleagues,
this new technique led to the novel and noninvasive diagnostic
evaluation of the anterior segment in 1994.40–42
The OCT utilizes an infrared diode light source (wavelength
of 830 or 1310 nm) and a Michelson-type interferometer that
detects differential light backscattering from the tissue micro-
structures. The amplitudes and delays in tissue reflections are
scanned by the reference mirror and the interferometric signal
is simultaneously recorded.40 The OCT’s false-color images
denote the regions with strongest reflection with red and white
colors and the regions with weakest reflection with blue and
black.43 The OCT imaging of transparent corneal tissues in
cross section is comparable to histopathologic sectioning, and it
provides resolution of 10–20 mm (Fig. 42.7).44 Additional quan-
titative information, such as structural dimensions and back-
scattering amplitudes, are readily available by this noninvasive,
noncontact technique.41 The clinically relevant measurements
possible by OCT include corneal thickness, iris thickness, lens
thickness, anterior chamber depth, anterior angle-chamber
angle dimensions and anterior and posterior radii of corneal
curvature with subsequent calculation of corneal refractive
power. In order to image the cornea at a close-up view, higher
spatial frequencies are used to sample the eye. The reflectivity
profile in the longitudinal scan direction can be numerically
fitted and extrapolated to the cornea and its substructure (i.e.,
epithelial layer) thickness measures by measuring the distances
between the optical signals. Because the image contrast is high
between the cornea and the surrounding media, the strong
reflections between anterior and posterior corneal surfaces are
recorded and the corneal thickness measurements are esti-
mated in submicrometer scale (Fig. 42.7).41,45 A relatively low
contrast between the corneal epithelium and Bowman’s layer
reduces the accuracy of thickness estimates and the epithelial
layer measures might vary by 10 mm between the images.41 In
order to determine true corneal thickness, the OCT’s pachy-
metric measurements have to be corrected by factoring in the
refractive index of the cornea, assumed to be 1.3853.42
CLINICAL APPLICATION
OCT has been shown to be a useful tool when examining a
normal cornea. Similarly to ultrasound biomicroscopy (UBM),
but in a less invasive manner, OCT allows identification and
monitoring of intraocular masses and tumors.41 The
relationships between the cornea, anterior chamber angle and
lens can be assessed. At this point, intraocular lenses can not be
visualized in vivo with OCT because they consist of homo-

(OCT) has been available for in vivo examination of posterior genous material with smooth surfaces. On the other hand, the

TABLE 42.4 Comparison of Specular Microscopy and Confocal Microscopy12,14,24,35,37

Advantages Disadvantages Clinical use

Specular Wider field of view Cornea has to be transparent Most cases of endothelial cell
More accurate endothelial cell counts assessment
Easier to use
Quality of images does not depend on
patients movement
Confocal Higher image resolution (axial and Eye movement interferes with exam Earlier diagnosis of dystrophies
lateral) and contrast Narrower field of view (can visualize DM thickening)
Higher magnification Less accurate endothelial cell count in Use in severe cases of corneal edema
Can visualize endothelium in presence of guttae Other corneal structures assessed at
edematous cornea Cellular organelles not routinely seen the same time
Real-time endothelial cell assessment Expensive – not routinely used for
clinical practice yet
490
 Corneal Examination, Specular and Confocal Microscopy, UBM, OCT
a movements during data acquisition might cause image resolu-
b
FIGURE 42.7. OCT image of cornea with scan width of 12 mm and
scan depth 4 mm. (a) Contour of normal cornea is clearly identifiable
against the dark background. The amount of backscatter from within
the nominally transparent cornea decreases from central to peripheral
cornea. The intensity of the signal increases at the corneoscleral
limbus as it approaches highly scattering opaque sclera. Central
corneal thickness is measured to be 561 mm. (b) Image of the corneal
edema. The backscatter intensity is increased in the central region.
Central corneal thickness is measured to be 811 mm.
Courtesy of David Huang, MD, PhD. Doheny Eye Institute, Los Angeles, CA.
capsular bag can be identified, especially in the setting of
posterior capsular opacification. Although lens densitometric
analysis for the objective grading of cataract formation has been
explored, that model still remains experimental and has not
been widely utilized.41,42
The availability of highly accurate biometric measures makes
OCT an invaluable tool in intraocular implant power calcula-
tions and for fitting of contact lenses.
In refractive surgery realm the OCT’s pachymetric analysis
has been utilized for intraoperative and perioperative corneal
thickness measurement.45 The flap-interface reflectivity enables
the measurements of flap thickness up to 15 months post-
operatively.46 The intraoperative measurements of residual
stromal thickness by online OCT coupled with excimer laser
(Online OCP, 4Optics AG) are advantageous due to the employ-
ment of the noncontact method and the ability to obtain con-
tinuous measurements of central corneal thickness throughout
the procedure, thus improving the intraoperative safety of
keratorefractive surgery.47,48
Some authors claim that changes in corneal shape and con-
tour following refractive surgery, as well as wound healing effects
that alter light scattering characteristics of collagen fibrils and
keratocytes, can be reliably imaged and quantified by OCT.42,46
In pathologic conditions, OCT can identify and delineate the
extent and depth of calcified lesions, dystrophic opacities, and
lesions whose accurate assessment is precluded by corneal
edema or haze (Fig. 42.7).43,49 In the diffusely hazy cornea, a
descemetocoele or corneal perforation can be detected. Cross-
sectional images in postoperative patients after deep lamellar
keratoplasty may be used to monitor graft–host junction in
initially edematous grafts.43
ADVANTAGES AND DISADVANTAGES
OCT provides direct quantitative measurements of ocular
tissues in cross section. It does not require immersion or direct
contact with the ocular surface and does not disrupt the tissues
under investigation. Patient discomfort is also minimal. Optical
sectioning of the ocular surface encompasses most of the
anterior segment and provides a general view of the structural
relationships between anterior segment components. The
resolution is comparable to the CM. Still most images are not
able to distinguish Bowman’s and Descemet’s membranes
when no pathological thickening is present.42 Similarly to the
previously discussed instrument limitations, patient micro-
tion degradation requiring averaging of serial measurements.41
When performing biometric analysis, strictly axial measure-
ments should be obtained, as off-axis images contain optical
distortions arising from surfaces crossed by a nonperpendicular
beam of the OCT.42 In summary, OCT provides an optical
biopsy of the anterior segment structures and supplements
corneal examination with a unique and noninvasive method.
UBM
The first use of ultrasound in ophthalmology was in 1956 by
Mundt and Hughes.50 Since then, standardization of both A and
B-scan instruments has led to the widespread utilization of
ultrasound for intraocular and orbital disorders.51 In 1990s
Pavlin and associates popularized the use of UBM, which
greatly enhanced the resolution of anterior segment structures
and lesions.52 In UBM observation of living tissues at a micro-
scopic level is similar to optical biomicroscopy, hence the term.
The essential components of UBM are identical to a conven-
tional B-mode imaging system except for the significantly
higher operating frequencies and subsequently short wave-
lengths (less than 0.2 mm). The resulting longitudinal ultra-
sound waves carry properties similar to light rays since they can
be refracted and reflected. The reflected waves are referred to as
an echo when they hit back the source of the emitted energy
(i.e., the transducer or probe).51–53
UBM utilizes high-frequency transducers in 40–100 MHz range
(frequencies greater than 20 KHz are inaudible to humans) to
provide resolution of 20–60 mm and depth of tissue penetration
of ~4 mm. The lateral resolution of the ultrasound system can
be related to the full width of the ultrasound beam at half-
maximum amplitude (FWHM) and expressed by the equation:
FWHM = cf/(vd)=lambda (f-number)
where c is the speed of sound (a speed of 1 640 m/s is generally
used for cornea and sclera), f is the focal length of the trans-
ducer, v is the frequency of ultrasound, d is the diameter of the
focused transducer, lambda is the wave length, and f-number is
the ratio of the focal length to the diameter of the transducer. By
selecting appropriate frequency and an f-number, the variably
CHAPTER 42
high resolution can be achieved. For example, 60 mm resolution
can be achieved by operating frequency of 60 MHz and an
f-number of 2.0. By increasing the resolution, tissue penetration
is compromised, due to tissue ultrasound attenuation
coefficients that increase with frequency. Therefore, for the
60 MHz frequency, penetration is ~5 mm. The optimization of
transducer parameters is essential in creating the best image
quality, and it is achieved by a compromise between resolution,
contrast and depth of field (range of depth over which the beam
remains well focused).51
CLINICAL APPLICATION
The utilization of UBM in anterior segment examination is
most applicable when corneal opacification or total internal
reflectivity precludes the visualization of the ocular structures.
In the normal cornea, three highly reflective surface echoes are
produced by the epithelium, Bowman’s membrane and the
Descemet’s membrane/endothelial complex.53 Corneal stroma 491
 CORNEA AND CONJUNCTIVA

has low regular reflectivity and it is lower than that found in the
more irregular collagen distribution of the sclera. The difference
of reflectivity between the corneal stroma and the sclera allows
for definition of the corneoscleral junction.
In corneal edema, the separation of the corneal lamellae by
fluid enhances the stromal reflectivity. In bullous keratopathy
the epithelial echo becomes more irregular, and the separation
of epithelium from the stroma becomes readily visible. Other
causes of increased stromal reflectivity are due to deposition of
higher reflectivity material in-between the corneal lamellae and
disruption of their regular (usually weakly reflective) structure,
as seen in scarring, inflammation and dystrophic material
accumulation. Areas of calcification are highly reflective, and
produce complete shadowing of structures behind it.53
When corneal opacification is present it is difficult to assess
the underlying cornea and the anterior chamber. UBM allows
the assessment of corneal anatomy despite corneal edema as
seen in Descemet’s detachment (Fig. 42.8).53,54 In corneal trans-
plantation, UBM aids in examination of graft–host junction, FIGURE 42.8. UBM image demonstrating Descemet’s detachment.
evaluation of wound gaping, apposition of Descemet’s mem- Epithelial layer and Bowman’s membrane create two highly reflective
brane between graft–host junction and presence of iris adhesions lines. Highly reflective image of Descemet’s membrane and
endothelium is separated from corneal stroma. Edematous cornea has
or angle closure.55 The intraocular lens haptic position can be
thickened stroma with higher than usual stromal reflectivity.
identified by UBM and can facilitate the preoperative planning Courtesy of Lois Hart. Massachusetts Eye and Ear Infirmary, Boston, MA.
for lens exchange.56
UBM creates a qualitative representation of iris, ciliary body,
lens and anterior chamber structures, as well as accurately mea-
sures these structures (Fig. 42.9).51,52 The utilization of short
wavelengths and improved resolution of UBM over the conven-
tional ultrasound led to a more accurate measurement of small
diameter structures, such cornea, iris, ciliary body, sclera. The
differential reflectivity of corneal layers enables the measure-
ment of stromal thickness, epithelial thickness, and depth of
intracorneal incisions.57 After LASIK and the photorefractive
keratectomy, the alteration in epithelial layer, Bowman’s layer
and stromal layer reflectivity can be picked up by UBM and
can aid in assessing results and complications of refractive
surgery.57,58

ADVANTAGES AND DISADVANTAGES

UBM produces cross-sectional images of the anterior globe and
allows observation of living tissues at a magnified level. In UBM
the integrity of the imaged structures and their relationship to
one another is preserved. The vast majority of clinical applica- a
tions that UBM can potentially be employed were beyond the
scope of this text. Still, the new advances in transducer sen-
sitivity are in evolution to improve the tissue penetration and
image resolution of UBM technology for corneal and anterior
SECTION 6

segment examination.

KERATOMETRY (OPHTHALMOMETRY)
In 1916, Scheiner59 noticed that shiny glass balls of different
radii produce reflected images of different sizes. This prompted
him to make a series of balls of progressively larger curvatures.
To perform keratometry, Scheiner would match the size of the
image of the window frame reflected form a subject’s cornea
with that produced by one of the calibrated balls.
The next major advance in keratometer was a magnification
system introduced by Ramsden.60 Ramsden also introduced the
doubling device, in which the examiner matches the corneal
reflection to itself, thus eliminating annoying eye movement. b
The cornea acts as a convex mirror and produces an erect and
virtual image of the illuminated target placed near the patients’ FIGURE 42.9. UBM image showing iris and ciliary body cyst (c). Note
cornea. Keratometry allows the operator to measure the size of cyst walls and lack of internal echoes indicating fluid inside the cysts.
the reflected image precisely. The device then converts image (a) Axial view. (b) Transverse view.
492 size to corneal radius using the following relations: Courtesy of Lois Hart. Massachusetts Eye and Ear Infirmary, Boston, MA.
 Corneal Examination, Specular and Confocal Microscopy, UBM, OCT
Corneal radius = (2[cornea-to-mire distance] µ [corneal image
size])/mire size
Corneal refractive power = 03375/corneal radius in meters
The range of most keratometers covers all patients, except
those with extreme keratoconus and cornea plana. The tech-
nique used to extend the range of the keratometer to include
these special patient groups uses a spherical lens mounted over
the central aperture of the keratometer mire. The cornea is then
measured in the usual way, and the value is multiplied by a
constant unique to the auxiliary lens.
The examiner can utilize the keratometer to evaluate the
quality of the corneal surface as well as the dioptric curvature of
the anterior cornea. The keratometric mires that are reflected
from the cornea fall on an area of 3.0–3.5 mm and the resulting
keratometric measurement does not represent the curvature of
the entire cornea.61 The quality of mire overlap can distinguish
between regular and irregular astigmatism. When the mires do
not overlap perfectly, and/or have irregular shape, one should
suspect an ocular surface irregularity or keratoectasia. In
keratoconus, there is steepening and thinning of the paracentral
cornea, that manifest with irregular astigmatism and high
keratometric values.61
TOPOGRAPHY (VIDEOKERATOSCOPY)
Modern video techniques can freeze a reflected corneal image
and use the information in that image to approximate the
corneal shape. Once the image is captured on a video screen, a
computer can measure the image and calculate the radius of
curvature. Corneal topography creates a ‘map’ representation of
the corneal surface. Most commonly used topographers are
based on mire arrangement similar to a Placido disc. A series of
illuminated rings is projected onto a cornea and the reflected
images are captured on the video screen. A computer analysis
reports the radius of curvature in any portion of the cornea and
produces color-coded dioptric maps of the corneal surface.62 A
standard topogram gives a clear cylinder axis and amount of
corneal astigmatism in diopters (e.g., simulated keratometry
values (SIM K) (Fig. 42.10). A reasonably accurate assessment
of irregular astigmatism can be achieved by observation of color
map or by using numerical indices (e.g., surface regularity index
(SRI) or surface asymmetry index (SAI)) provided by some
analyzers.62
In slit-scanning corneal topography (SSCT), the machine
projects a series of slit beams across the cornea. Each of the slit
images is captured and analyzed by the computer software. The
information is used to calculate the shape and corneal thickness
between the captured slit sections. The commercially available
SSCT, the Orbscan, creates a graphic data output of anterior
corneal curvature, posterior corneal curvature and regional map
of corneal thickness in addition to mean axial corneal power.63
FIGURE 42.10. Computerized keratometry device, which uses 18
concentric Placido rings that produce a reflected image that covers
almost the entire cornea. The corneal curvature in different parts of
the cornea is color coded.
Courtesy of Tomey Technology, INC., Cambridge, MA.
There is a multitude of clinical applications of corneal topo-
graphy that aid in diagnosis and management of corneal
abnormalities. Irregular astigmatism from corneal scarring,
keratoectasia, trauma, surgery or postinflammatory conditions
can be readily depicted by keratography (Fig. 42.11).62 Topo-
graphy may explain why best-corrected acuity does not improve
with spectacle refraction in patients with irregular corneas.
The detection of irregular astigmatism may herald an early
ectasia that is a contraindication to the refractive surgery.64 In
Orbscan systems the anterior and posterior differences in the
best-fit spheres, mean axial dioptric maps and pachymetry maps
can aid in detection and monitoring of patients with keratoconus
(Fig. 42.12). The representative maps after the LASIK surgery
for myopia show characteristic flattening in the mean axial
power map, as opposed to steepening in keratoconus maps
(Figs 42.12 and 42.13). Topography aids in the determination of
selective suture removal in corneal transplant patients and
surgical planning of astigmatism by both incisional and laser-
assisted surgery (Fig. 42.14).62
In summary, the advent of refractive surgery created a niche
for the advances in evaluation and measurement of corneal
shape and power. Such methods have evolved from keratometry
CHAPTER 42
to keratoscopy to videokeratoscopy and to SSCT. Despite the
advances in this area, the ever-evolving instrumentation is still
needed to combat the inaccuracies and inefficiencies of the
existing technology.
FIGURE 42.11. Orbscan mean axial
keratometric map (left) of oblique against the
rule astigmatism of 13.2 D in a patient with
Terrien’s marginal degeneration located
superiorly. Corneal thickness map (right) shows
normal central thickness of 559 mm (green
color) with marked thinning superiorly (red
color) due to ectasia.
493
 CORNEA AND CONJUNCTIVA

FIGURE 42.12. Orbscan topography of

keratoconus. (a) Early keratoconus of the right
eye with inferior steepening in the mean axial
keratometric map (bottom left). The area of
steepening coincides with area of thinning
(bottom right). (b) Advanced keratoconus of the
left eye of the same patient. Anterior best-fit
sphere float (top left) and posterior best-fit
sphere float (top right) show markedly higher
elevation compared to the right eye (a) with
less advanced keratoconus. Advanced
steepening denoted with red colors in the mean
axial keratometric map (bottom left) coincides
with thinning in the thickness map (bottom
right).

b
SECTION 6

FIGURE 42.13. Orbscan topography of postmyopic LASIK treatment.

Anterior best-fit sphere float (top left) shows concentric elevation in the
paracentral area denoted with yellow color, and central elevation
denoted with red color in the posterior best-fit sphere map (top right).
The pachymetry map shows central thinning (bottom right), while mean
axial keratometric map shows characteristic central corneal flattening
(bottom left). The constellation of the findings above distinguishes this
keratometric map from the one seen in keratoconus.

494
 Corneal Examination, Specular and Confocal Microscopy, UBM, OCT
a b
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 CHAPTER
Corneal Dysgeneses, Dystrophies, and
43 Degenerations
Kenneth R. Kenyon, Tomy Starck, Glen Cockerham, and Peter S. Hersh

Corneal Dysgeneses Noninflammatory corneal ectasias

Abnormalities of size and curvature
Absence of cornea
Microcornea
Simple megalocornea
Anterior megalophthalmos
Cornea plana
Mesenchymal dysgeneses
Posterior embryotoxon
Axenfeld’s anomaly and syndrome
Reiger’s anomaly and syndrome
Posterior keratoconus
Congenital central corneal opacity (Peters’ anomaly)
Sclerocornea
Congenital anterior staphyloma
Congenital hereditary endothelial dystrophy
Corneal Dystrophies
Anterior dystrophies
Epithelial basement membrane dystrophies (map-dot-
fingerprint)
Hereditary epithelial dystrophy (Meesmann, Stocker-Holt)
Lisch corneal dystrophy
Corneal dystrophies of Bowman’s layer
Vortex dystrophy (Fleischer’s)
Anterior mosaic crocodile shagreen (Vogt’s)
Idiopathic band keratopathy
Stromal dystrophies
Granular dystrophy (Groenouw’s Type I)
Lattice dystrophy
Macular dystrophy (Groenouw’s Type II)
Polymorphic stromal dystrophy
Gelatinous Drop-like dystrophy
Central crystalline dystrophy (Schnyder)
Marginal crystalline dystrophy (Bietti)
Central cloudy dystrophy (Francois)
Posterior amorphous stromal dystrophy
Congenital hereditary stromal dystrophy
Posterior mosaic crocodile shagreen
Fleck dystrophy (Francois-Neetens)
Pre-Descemet’s dystrophies
Cornea farinata
Grayson-Wilbrandt dystrophy
Deep filiform dystrophy
Endothelial dystrophies
Congenital hereditary endothelial dystrophy
Cornea guttata
Late hereditary endothelial dystrophy (Fuchs’)
Posterior polymorphous dystrophy
Iridocorneal endothelial syndrome
Keratoconus
Pellucid marginal degeneration
Keratoglobus
Corneal Degenerations
Peripheral degenerations
Corneal arcus (juvenilis and senilis)
White limbal girdle of Vogt
Idiopathic furrow degeneration
Furrow degeneration associated with systemic disease
Postirradiation thinning
Terrien’s marginal degeneration
Mooren’s ulcer
Central or diffuse degenerations
Iron lines
Coat’s white ring
Lipid degeneration
Amyloid degeneration
Spheroid degeneration (climatic droplet keratopathy, keratinoid
degeneration)
Band keratopathy
Salzmann’s nodular degeneration
Corneal keloid
Conjunctival Degenerations
Pterygium
Pinguecula
The dysgeneses, dystrophies, and degenerations of the cornea
account for a broad spectrum of ocular abnormalities, ranging
from clinical curiosities to sight-threatening anomalies. Knowl-
edge of these entities has traditionally accrued through clinical
study and examination of histopathologic specimens. Within
the past decade, discovery of the specific gene mutations for
corneal stromal dystrophies on human chromosome 5 has
advanced the study of corneal disorders into the exciting age of
molecular genetics. (The recent monograph edited by M. Wang1
is especially current and comprehensive in this regard.)
Dysgeneses of the cornea are developmental disorders,
sometimes inherited, resulting in congenital malformations.
Corneal dysgeneses may be unilateral or bilateral and are
nonprogressive. The central, peripheral, or entire cornea, as
well as other ocular structures, may be affected. Occasionally,
associated systemic abnormalities are present.
A corneal dystrophy generally exhibits a familial pattern, is
bilateral if not symmetric, and does not appear to be secondary
to any environmental or systemic factor. Dystrophies tend to 497
 CORNEA AND CONJUNCTIVA
manifest relatively early in life and are variably progressive.
Abnormalities generally affect the central cornea and are non-
inflammatory in origin. Senescence may encourage dete-
rioration of the dystrophic cornea but is not a primary cause of
the disorder. Each unique dystrophy exhibits characteristic
histopathologic features.
Corneal degenerations, in contrast to dysgeneses and dys-
trophies, appear to have no developmental or hereditary pattern
and may be unilateral or bilateral. A degeneration is often a
manifestation of aging, inflammation, or environmental insult
and, therefore, usually occurs later in life than a dystrophy.
Degenerations most often begin in the peripheral cornea,
although central vision eventually may be affected. Inflam-
mation sometimes is involved early in the degenerative process
and may be accompanied by corneal vascularization. In some
cases, these inflammatory processes are associated with
systemic disease (e.g., collagen vascular disorders).
CORNEAL DYSGENESES
ABNORMALITIES OF SIZE AND CURVATURE
Absence of Cornea
Complete absence of the cornea is rare. In such cases, there is
variable absence of other anterior ocular structures derived from
surface ectoderm, and the eye consists of a sclera-like enclosure
lined with neural ectoderm. Ultrasonography should aid in
differentiating this entity from cryptophthalmos. True
cryptophthalmos, also known as ablepharon, occurs when the
lids fail to form. The cornea and conjunctiva are exposed and
undergo metaplastic changes to form skin.2 This condition is
rare and is usually transmitted as an autosomal recessive trait.3
The term cryptophthalmos syndrome has been used to describe
the association of the ocular findings with extraocular
abnormalities, such as craniofacial anomalies, syndactyly, spina
bifida, cleft lip and palate, genitourinary and cardiac anomalies,
and mental retardation.4
Microcornea
The term microcornea (Fig. 43.1, left) implies a corneal dia-
meter of less than or equal to 10 mm. The size of a normal
newborn cornea measures ~10 mm in horizontal diameter,
whereas the size of a normal adult cornea measures ~12 mm in
diameter. The vertical diameter almost always is exceeded by
~1 mm by the horizontal diameter. The cornea usually reaches
adult size by 2 years of age.5 Microcornea can occur either
unilaterally or bilaterally and is thought to occur secondary to
an arrest in corneal growth after the fifth month of fetal
SECTION 6
development. The eye may be otherwise normal, but often
other ocular abnormalities, such as colobomas, are present. Just
as megalocornea is associated occasionally with anterior
megalophthalmos, microcornea often accompanies anterior micro-
phthalmos, with crowding of the anterior segment structures
commonly resulting in angle-closure glaucoma.6 Microcornea
498
can also be seen in nanophthalmos and as part of many other
anterior segment dysgeneses.
The microcornea is generally clear, with normal histologic
architecture, and in the absence of other ocular abnormalities,
vision may be good. Numerous somatic abnormalities have
been described in conjunction with microcornea and anterior
microphthalmos, including dwarfism and Ehlers–Danlos
syndrome.7
Simple Megalocornea
Simple megalocornea (see Fig. 43.1, right) is a nonprogressive,
usually symmetric, inherited condition in which the cornea and
limbus are enlarged without evidence of previous or concurrent
ocular hypertension. The diameter of the cornea is 13 mm or
greater, but the corneal thickness and histologic anatomy are
normal. Although X-linked recessive inheritance is most com-
mon with 90% of all cases found among males, all modes of
inheritance have been reported.8–10 Female carriers may have
slightly enlarged corneas.
The condition has been mapped to the long arm of the X-
chromosome (Xq21.3-q22 and Xq12-q26).11
Simple megalocornea can be differentiated from congenital
glaucoma by the clarity of the cornea and by the normal intra-
ocular pressure and normal optic nerve in simple megalocornea.
Moreover, the megalocornea demonstrates normal endothelial
cell population densities on specular microscopy, whereas in
congenital glaucoma, these are diminished, ostensibly because
of corneal distention.12 Studies have also suggested using
A-scan ultrasonography to highlight the pathognomonic
biometric findings of megalocornea not present in glaucoma:
markedly increased anterior chamber depth, posterior lens and
iris positioning, and short vitreous length.13 Although some
authorities suspect that megalocornea may represent arrested
congenital glaucoma, a single case reporting the histopathology
of megalocornea did not disclose any of the characteristic angle
abnormalities of congenital glaucoma. Both conditions, however,
have been reported in the same family and in the same person.14,15
Simple megalocornea also must be differentiated from
keratoglobus (see section on Noninflammatory Corneal Ectasias).
Anterior Megalophthalmos
In comparison with simple megalocornea, eyes with anterior
megalophthalmos have enlargement of the lens-iris diaphragm
and ciliary ring in addition to the cornea.16 A large myopic
astigmatic refractive error often results from the abnormal
optical architecture. The iris may exhibit transillumination
defects as a result of attenuation of the dilator muscle. Because
of the abnormal spatial relations of structures in the anterior
segment and stretching of the zonules, iridodonesis, phako-
donesis, and lens subluxation or dislocation may occur; the
latter may result in secondary lens-induced glaucoma. The lens,
furthermore, may become prematurely cataractous.
Marfan’s syndrome,17 Apert’s syndrome,18 and mucolipidosis
type II19 have been found in association with this disorder.
FIGURE 43.1. Left, Microcornea. A young child
had a cornea 9.5 mm in diameter and subtle
peripheral sclerocornea. Right, Megalocornea.
Light microscopy of a 62-year-old man with
corneal diameters of 13 mm. Note the anterior
segment with no abnormalities (except beveled
scar of cataract incision and surgical aphakia).
H & E µ3.
Right, From Wood WJ, Green WR, Marr WG:
Megalocornea: a clinico-pathologic clinical case report.
Md State Med J 1974; 23:57–60.
 Corneal Dysgeneses, Dystrophies, and Degenerations
TABLE 43.1. Genetic Linkage Analysis of Corneal Dysgeneses
and Dystrophies
Disorder Gene Location
Cornea plana Chromosome 12
Corneal dystrophy of Bowman’s layer Unknown
type I
Corneal dystrophy of Bowman’s layer Chromosome 5
type II
Granular dystrophy Chromosome 5q22-32
Avellino dystrophy Chromosome 5
Lattice dystrophy I Chromosome 5
Lattice dystrophy II Chromosome 9q34
Macular dystrophy Chromosome 16q22
Congenital hereditary endothelial Chromosome 20
dystrophy
Posterior polymorphous dystrophy Chromosome 20q11
Cornea Plana
In cornea plana, the cornea is flat with a corneal curvature
of less than 43 D. The radius of curvature may reach levels as
low as 20–30 D, similar to that of the sclera.20–22 Peripheral
scleralization of the cornea is almost always present, and the
condition is indistinguishable clinically from peripheral sclero-
cornea. The limbal landmarks are also obscured, simulating
microcornea.
In cornea plana, the anterior chamber is shallow by virtue
of the low corneal dome. Refractive abnormalities vary from
hyperopia of 7 D to myopia of 9 D, depending on the globe
dimensions and corneal curvature.23 This condition also
features concurrent anterior segment abnormalities,24 including
iris colobomas, congenital cataract, and occasional posterior
segment colobomas. The distortion of the cornea, along with
concomitant sclerocornea, leads to a decrease in corneal trans-
parency. This nonprogressive condition is more commonly
bilateral and asymmetric. Most cases are sporadic, with both
dominant and recessive inheritance pedigrees reported.25,26
Genetic linkage analysis has mapped the gene to the long arm
of chromosome 12 (Table 43.1).27 The embryologic explanation
for sclerocornea lies in the absence of the limbal anlage. The
formation of the limbal anlage occurs between the seventh and
tenth gestational weeks, allowing neural crest mesenchymal
cells to differentiate into either sclera or cornea and to induce a
corneal curvature that exceeds the scleral.28 With its absence,
the normal interface between sclera and cornea is disrupted,
and the normal surface curvature is flattened.
Histopathologic studies of sclerocornea have revealed mor-
phologic features resembling scleral tissue. The stroma consists
of irregularly arranged collagen fibrils with an increased dia-
meter anteriorly, in contrast to the normal cornea.29
Treatment is limited to correction of any refractive error30; for
cases with significant central corneal opacification, penetrating
keratoplasty is indicated.31 The prognosis is guarded, however,
because of a high incidence of glaucoma, a common association
with other ocular anomalies, and an increased risk of graft
allograft rejection.32,33
MESENCHYMAL DYSGENESES
The spectrum of congenital eye findings subsumed by the term
mesenchymal dysgenesis historically has been known by a
variety of names, including mesodermal dysgenesis and anterior
segment cleavage syndrome. A number of pathogenetic theories
have been advanced, all based on concepts of anterior segment
embryogenesis. The somewhat archaic term anterior segment
cleavage syndrome, for instance, implies abnormal separation
of developing tissues (e.g., the lens vesicle).34 With increased
knowledge of ocular embryology, however, the more current
term mesenchymal dysgenesis has been devised to reflect a
developmental arrest and incomplete central migration of
neural crest cells and corneogenic mesoderm.35
Neural crest cells migrate into the developing anterior seg-
ment in three waves, contributing to the corneal endothelium36
and trabecular meshwork, stromal keratocytes, and iris, respec-
tively. Arrest at any of these stages may bring about the
recognized clinical dysgenesis syndromes. In addition to this
developmental arrest, secondary anterior displacement of the
lens-iris diaphragm may account for other abnormalities.37,38
Whatever the exact pathogenesis, because corneal and iris
tissues are likely derived at least in part from the neural crest39
rather than from mesoderm, and because tissues of other origin
(e.g., the ectoderm-derived lens) may also be involved, this
heterogeneous group of congenital anomalies may be described
best by the broader term mesenchymal dysgeneses.40 The
mesenchymal dysgeneses may affect the periphery of the ante-
rior segment, manifest only central pathologic changes, or affect
the entire anterior segment. For simplicity, this spectrum of
disorders can be categorized in a stepladder classification scheme
as suggested by Waring and associates (Fig. 43.2).41 Rarely,
however, a case specifically conforms to only one of these entities.
Posterior Embryotoxon
The simplest dysgenesis of the anterior segment periphery
is posterior embryotoxon, the anterior displacement and
enlargement of Schwalbe’s line, appearing as an irregular,
circumferential ridge on the posterior surface of the cornea just
inside the limbus (Fig. 43.3). Gonioscopy shows that it juts into
the anterior chamber, and the adjacent uveal trabecular
meshwork may appear dense.41 Posterior embryotoxon occurs
in 10–15% of normal eyes.42 A prominent Schwalbe’s line may
be associated with other disorders, including primary congenital
glaucoma,43 Alagille’s syndrome (arteriohepatic dysplasia),44
megalocornea, aniridia, corectopia, and Noonan’s syndrome.45
Axenfeld’s Anomaly and Syndrome
Axenfeld’s anomaly results when posterior embryotoxon is
accompanied by abnormal iris strands crossing the anterior
chamber angle to attach to a prominent Schwalbe’s line46 (see
Fig. 43.3). If glaucoma also is present (secondary to angle
CHAPTER 43
abnormality), the condition is called Axenfeld’s syndrome.47
Reiger’s Anomaly and Syndrome
Reiger’s anomaly is present if hypoplasia of the anterior iris
stroma is found with the changes typical of Axenfeld’s
anomaly.48,49 This anomaly is associated with glaucoma in
~60% of patients, which may result from incomplete devel-
opment of the aqueous outflow system.50 Various systemic
associations have been described, such as Down’s syndrome,
Ehlers–Danlos syndrome, Franceschetti’s syndrome, Noonan’s
syndrome, Marfan’s syndrome, oculodentodigital dysplasia, and
osteogenesis imperfecta. Reiger’s syndrome (see Fig. 43.3)51 is
present when the eye anomaly is accompanied by skeletal
abnormalities, such as maxillary hypoplasia, microdontia, and
other limb and spine malformations. Mutations in the PITX2
and FOXC1 genes have been identified both in Axenfeld-Rieger
syndrome as well as in Peters anomaly (Table 43.1).52
An examination that includes gonioscopy and tonometry is
essential to making the differential diagnosis and to determining 499
 CORNEA AND CONJUNCTIVA
whether the intraocular pressure is elevated. The pneu-
motonometer or Tonopen is preferable to other applanation
instruments because the presence of associated corneal
SECTION 6
abnormalities or small radius of corneal curvature may give
false intraocular pressure readings. Assessment of the optic
nerve is critical to determining the overall visual prognosis and
deciding on the course of future treatment.
Medical therapy can be useful when intraocular pressure is
particularly high and temporizing measures are needed. This
disorder has a generally poor surgical prognosis, both for
glaucoma control and for corneal opacities, if present. Achieving
a balance between chronic medications and performing surgery
is uniquely difficult. The advent of effective use of antimeta-
bolites for filtration in children may favor of surgery when the
optic nerve is threatened significantly. Nevertheless, this type of
treatment in children remains a substantial concern as the eyes
mature.
Posterior Keratoconus
Posterior keratoconus53–56 has no relation to anterior kera-
toconus. It consists of a discrete indentation of the posterior
500 cornea with a variable degree of overlying stromal haze and may
FIGURE 43.2. Composite illustration of the
anatomic findings in mesenchymal dysgenesis
of the ocular segment. The stepladder table
demonstrates the spectrum of anatomic
combinations of terms by which they are
commonly known. The markers in the table
indicate the corresponding anatomic
component in the illustration. The central
abnormalities occur because of focal absence
or attenuation of the endothelium.
From Waring GO III, Rodrigues MM, Laibson PR:
Anterior chamber cleavage syndrome: a stepladder
classification. Surv Ophthalmol 1975; 20:3.
represent the mildest variant of Peters’ anomaly. Some attribute
the cause to an abnormal migration or differentiation of the
secondary mesenchyme that normally forms the corneal
stroma.57 Posterior keratoconus tends to be sporadic, unilateral,
and relatively central. In some cases, pigment surrounds the
edges of the posterior depression, suggesting previous contact to
the iris. On histologic examination, Descemet’s membrane may
be thinned, with concomitant endothelial abnormalities in the
focally abnormal area55 (Fig. 43.4).
Although the irregularity of the posterior cornea may affect
vision to some extent, the anterior surface is normal unless
there is sufficient posterior thinning to cause ectasia. Rarely, the
entire posterior cornea has increased curvature.56 Because
vision usually is acceptable, keratoplasty rarely is indicated.
Congenital Central Corneal Opacity (Peters’
Anomaly)
Peters’ anomaly is a congenital central corneal opacity with
corresponding defects in the posterior stroma, Descemet’s
membrane, and endothelium.34,41,58 Most cases of Peters’
anomaly are sporadic, although both recessive and irregular
dominant inheritances have been described. Eighty percent of
 Corneal Dysgeneses, Dystrophies, and Degenerations
reported cases are bilateral. Mutations have thus far been
described in four genes including PAX6 for aniridia, PITX2 and
FOXC1 for Axenfeld-Rieger syndrome, and CYP1B1 for primary
congential glaucoma.52,59
Although Peters’ anomaly generally is characterized by a
central corneal leukoma, two clinical variants have been
recognized.60 Peters’ anomaly type I (see Fig. 43.4) is almost
an extension of posterior keratoconus, showing the typical
posterior nebular opacity in the pupillary axis, with the
additional feature of iris strands that cross the anterior chamber
from the iris collarette to the margin of the posterior defect. The
lens usually remains clear and is positioned normally.
Associated anomalies, such as microcornea, sclerocornea, and
infantile glaucoma, may be present, but for the most part, no
other ocular or systemic abnormalities are present.
In Peters’ anomaly type II (Fig. 43.5), the lens is abnormal
either in position or in transparency, in addition to the central
corneal opacity and iridocorneal synechiae. Centrally, the pos-
terior cornea and lens may be adherent, and there may be an
anterior polar cataract. This type more commonly is bilateral,
and almost every involved case shows severe ocular and
FIGURE 43.3. Top left and right, Posterior
embryotoxon demonstrating an anteriorly and
centrally displaced Schwalbe’s line. Middle,
Axenfeld’s anomaly. Markedly dense and
advanced Schwalbe’s line (left) accompanied
by adherent abnormal iris processes bridging
the anterior chamber (right). Bottom, Rieger’s
syndrome. Left, Multiple facial anomalies such
as telecanthus, low nasal bridge, and maxillary
hypoplasia. Right, This same patient exhibits
posterior embryotoxon, hypoplasia of the
anterior iris stroma, corectopia, and peripheral
anterior synechiae.
systemic malformations.61 Nearly 50–70% of patients with
Peters’ anomaly have concomitant glaucoma. Other associated
CHAPTER 43
ocular abnormalities include microcornea, microphthalmos,
cornea plana, sclerocornea, colobomas, aniridia, dysgenesis of
the angle and iris and persistent hyperplastic primary vitreous.
Systemic associations include developmental delay, congenital
heart disease, external ear abnormalities, central nervous
system structural abnormalities, genitourinary abnormalities,
hearing loss, cleft lip and palate and spinal defects.61
Histopathologic changes are present in all layers of the
cornea.37,38,62–65 Often, the anterior changes, which include
disorganization of the epithelium, fibrovascular pannus, and
loss of Bowman’s layer as a result of long-standing edema, are
secondary to the posterior abnormalities. Fluid lakes are also
present in the affected edematous stroma.
In the peripheral and unaffected areas, the corneal endo-
thelium forms a continuous monolayer, and Descemet’s mem-
brane is of normal, uniform thickness (~5 mm). In the area of
defect, however, endothelium and Descemet’s membrane can
terminate abruptly or be severely attenuated. The affected
Descemet’s membrane consists of multiple laminations of base- 501
 CORNEA AND CONJUNCTIVA
a b
d dimensions and thin fibroblasts (F) (µ12 600).
ment membrane-like material, with interspersed collagen fibrils
and fine filaments. Because such abnormal material is ela-
borated by the corneal endothelium, a fibroblastic metaplasia of
the endotheliogenic mesenchyme is likely, as is thought to
occur in a number of corneal conditions in which the endo-
thelium is similarly disturbed to secrete a posterior collagen
layer.66
The lens abnormalities in Peters’ anomaly are characterized
histologically by a stalk-like connection between the lens and
SECTION 6
the posterior corneal defect, suggesting primary incomplete sep-
aration of the lens vesicle. Alternatively, there may be contact of
a morphologically intact lens to the posterior cornea, suggesting
subsequent anterior displacement of a normally developed lens.
There are several reasonable explanations for a central
corneal leukoma of the Peters’ anomaly variety. One is in-
complete central migration of corneogenic mesenchyme (i.e.,
neural crest cells), accounting for posterior endothelial and
stromal defects.41 This is corroborated by the finding of ab-
normally large stromal collagen fibrils of 360–600 Å in some
patients. A similar abnormality of mesenchymal development
is found in sclerocornea and congenital hereditary endothelial
dystrophy.40 Another explanation of posterior corneal leukoma
of a Peters’ type anomaly is an in utero subluxation of the lens,
either before or after its full development, in either case
interrupting the normal migration or function of the developing
endothelium.
Historically, the internal ulcer of von Hippel has been
502 grouped with Peters’ anomaly, but the former is probably
FIGURE 43.4. Posterior keratoconus. (a) Light
micrograph of a keratoplasty specimen shows
the posterior central stromal defect (between
arrowheads) devoid of Descemet’s membrane
and endothelium. H & E µ15. (b) By scanning
electron microscopy, the posterior central
defect (asterisk) is prominently displayed and
appears lined by fibrous tissue (µ31).
(c) Higher-power scanning electron microscopy
of the posterior fibrous tissue shows the loose
collagenous network of this layer (µ310).
(d) Phase-contrast microscopy of the posterior
cornea discloses only attenuated fibroblastic
cells (asterisk) covering the posterior stromal
surface. PPDAµ250. (e) Transmission electron
microscopy of the area in (d) shows loosely
aggregated collagen fibrils of normal
an intrauterine inflammatory condition rather than a true
developmental defect.40
The clinical management of these patients is complex and
difficult, and the ~35% success of keratoplasty is usually related
to the control of concomitant glaucoma.67
Sclerocornea
In sclerocornea (Fig. 43.6), the limbus is not well defined be-
cause opaque scleral tissue with fine vascular conjunctival
arcades extends into the peripheral cornea. A broad range of
corneal involvement is possible, the most extreme of which is
complete scleralization of the cornea. Ninety percent of cases
are bilateral, although the disorder generally is asymmetric.
Most cases are sporadic; there is no known heredity. Sclero-
cornea is nonprogressive and must be differentiated from
interstitial inflammatory conditions and arcus juvenilis
(congenital peripheral lipid deposition, also known as anterior
embryotoxon). Sclerocornea is associated with cornea plana in
~80% of patients.68 Other related ocular abnormalities include
microphthalmos, iridocorneal synechiae, persistent pupillary
membrane, dysgenesis of angle and iris, congenital glaucoma,
coloboma, and posterior embryotoxon of the fellow eye.69
Numerous sSomatic abnormalities are also associated, in-
cluding mental retardation, deafness, and craniofacial, digital,
and skin abnormalities.68
Ultrastructural studies40,70,71 have shown the involved
stroma to assume the morphologic features of scleral tissue,
with irregularly arranged collagen fibrils of variable and im-
 Corneal Dysgeneses, Dystrophies, and Degenerations
mensely enlarged diameter for corneal tissue (up to 1500 Å,
comparable to normal scleral collagen). The precise lamellar
organization of normal corneal stroma is not present; thus,
optical clarity is not achieved. Various abnormalities of the
endothelium and Descemet’s membrane exist, from atten-
uation to focal absence. Descemet’s membrane generally is
thin, with multilaminar deposition of basement membrane-like
collagen.
Pathophysiologically, sclerocornea may result from devel-
opmental arrest of limbal anlage responsible for both limbal
differentiation and corneal curvature during neural crest
migration, as is seen in the other mesenchymal dysgeneses.40
Congenital Anterior Staphyloma
Anterior staphyloma (Fig. 43.7) is a congenital opacity of one
or both corneas, which become protuberant, are often lined
with iris tissue, and are associated with an extremely dis-
organized anterior segment. As in Peters’ anomaly, the lens may
FIGURE 43.5. Peters’ anomaly. Top left,
Clinical photograph of a typical bilateral case
with large dense central leukomas that was
successfully treated by penetrating keratoplasty
with optical iridectomy of the fellow eye.
Top center, A more diffuse corneal opacity in a
7-month-old infant. Top right, Intraoperative
photograph demonstrates adhesion of the lens
to the posterior cornea as a corneal button
(grasped with forceps) is trephined. No iris
could be identified. Middle left, Light
micrograph of a corneal button showing a
posterior central depression in which lodged
the cataractous lens (L). H & E µ10. Middle
center, Higher magnification of light microscopy
of the posterior cornea adjacent to the central
stromal defect demonstrates fragments of
presumed lens capsule and lens epithelium
(between arrowheads) immersed in the stromal
collagen. PAS µ200. Middle right, Phase-
contrast microscopy of the same cornea
resolves the thin and undulating Descemet’s
membrane, which terminates (arrow) at the site
of keratolenticular apposition. PPDA µ250.
Bottom inset, Phase-contrast micrograph of
central area of the cornea devoid of
Descemet’s membrane and lined by lens
capsule (arrowhead), lens epithelium (LE), and
cataractous lens cortex (asterisk). PPDA µ250.
Bottom, Transmission electron micrograph of
the same area discloses numerous fibroblastic
cells (F) in the posterior stroma, lined by a
uniform, 8-mm-thick lens capsule (LC) and lens
epithelium (LE) (µ4000).
CHAPTER 43
be adherent to the posterior cornea. Anterior staphyloma may
result from intrauterine inflammation or maldevelopment.72 In
the latter situation, there is no histologic evidence of
inflammation, and there is failure of migration of mesenchymal
tissues that would usually form the posterior corneal structures,
iris, and angle. This maldevelopment, probably coupled with
increased intraocular pressure caused by the angle abnormality,
leads to corneal opacity and thinning and to prominent
buphthalmic enlargement of the entire anterior segment.
Hereditary cases have been reported. Prognosis for keratoplasty
as well as preservation of any functional vision is dismal.
Congenital Hereditary Endothelial Dystrophy
Given the neural crest mesenchymal origin of the corneal
endothelium, we consider congenital hereditary endothelial
dystrophy (CHED) to also represent a variant of mesenchymal
dysgenesis. Detailed discussion of the condition is presented
here in the section devoted to Corneal Endothelial Dystrophies. 503
 CORNEA AND CONJUNCTIVA
SECTION 6
CORNEAL DYSTROPHIES
ANTERIOR DYSTROPHIES
The anterior corneal dystrophies (Fig. 43.8) are confined to the
epithelium, basement membrane, and Bowman’s layer.
Epithelial Basement Membrane Dystrophy
(Map–Dot–Fingerprint)
Disorders involving the epithelium and its basement membrane
may have a variable clinical appearance but probably involve a
common pathophysiology and clinical course. Because the
predominant abnormality involves the basement membrane
complexes that mediate the tight attachment between the
epithelium and Bowman’s layer, the clinical manifestations of
these conditions predictably involve frequent recurrent erosions
and occasional persistent defects of the corneal epithelium.
The appellation of map–dot–fingerprint dystrophy is appro-
504 priately descriptive of the biomicroscopically visible features
FIGURE 43.6. Sclerocornea. Top left, Moderate
corneal haze in a partially affected patient. Top
right, In this advanced bilateral case with
multiple congenital abnormalities, the entire
cornea is sclerified, and the fine vascular
arcades extend centrally from the conjunctiva
and sclera. Middle left, Light micrograph of
anterior cornea shows disorganization of the
epithelium, fragmentation of Bowman’s layer
(b), and interstitial vascularization (v). PPDA
µ350. Middle right, Transmission electron
microscopy discloses a disorganized array of
collagen fibrils that measure as much as three
times normal diameter (µ52 500). Bottom inset,
Light microscopy of the posterior cornea shows
irregularly thick and wavy stromal lamellae (S).
Descemet’s membrane could not be clearly
identified. PPDA µ350. Bottom left,
Transmission electron micrograph of the same
area discloses rudimentary Descemet’s
membrane (DM) with notable absence of
endothelial cells (µ4000). Bottom right, Higher-
magnification electron micrograph of the area
circled in bottom left figure reveals multilaminar
basement membrane material interspersed with
fine filaments (µ75 000).
of intraepithelial microcysts (dots), subepithelial ridges
(fingerprints), and geographic opacities (maps)73–90 (Figs 43.9
and 43.10).73–90 Family studies have revealed a probable do-
minant inheritance, with variable penetrance.91 Other clinical
studies are more consistent with a degeneration that is rather
highly prevalent in the general population.80
The symptoms of recurrent erosion can become prominent
in early adulthood through middle age and range from mild
early morning irritation to painful, erosive episodes. Irregular
corneal astigmatism with complaints of distortion or ‘ghost
images’ also occasionally develop secondary to plaque-like
accumulations of subepithelial cellular debris, basement
membrane, and collagen.
The degree of clinical symptoms, however, often does not
parallel the extent of abnormal slit-lamp findings. Because of
the presumed primary abnormality in the epithelial basement
membrane, even minor trauma can cause a major epithelial
breakdown, with impaired subsequent healing. In a patient who
has had a trivially traumatic or seemingly spontaneous erosive
 Corneal Dysgeneses, Dystrophies, and Degenerations
episode, meticulous examination of the symptomatic eye, as
well as the fellow eye, should be performed in an attempt to
disclose an underlying dystrophy. Careful inspection of the
fluorescein-stained tear film for localized irregularity, instability,
or ‘negative staining’ (focal dark areas where epithelial elevation
thins the overlying tear film thereby reducing fluorescence) as
well as retroillumination at high magnification through a
dilated pupil are helpful in uncovering these often subtle abnor-
malities in a patient who complains of spontaneous irritation.
Hykin and colleagues92 prospectively examined 117 patients
with histories of recurrent corneal erosions. They found that 23
had only epithelial basement membrane dystrophy with no
history of trauma. Seventy-five patients had histories of trauma
but no slit-lamp evidence of dystrophy. Williams and Buckley93
FIGURE 43.7. Congenital anterior staphyloma.
Top left, A 1-year-old girl was born with anterior
staphyloma of the right eye and anterior
segment mesodermal dysgenesis of the left.
The right eye shows enormous proptosis of the
enlarged and scleralized cornea. The axial
length is elongated to 24 mm as a result of
disproportionate enlargement of the anterior
segment. Top right, The left eye immediately
after penetrating keratoplasty and anterior
segment reconstruction. Middle left, Light
microscopy of keratoplasty specimen shows
secondary epithelial metaplasia into keratinized
stratified squamous epithelium. Middle center,
Involved stroma of the same specimen
assumes the morphologic features of scleral
tissue with the presence of abundant blood
vessels. H & E µ75. Middle right, Transmission
electron microscopy of the corneal stroma
discloses abnormally thick (440 Å) collagen
fibrils (µ43 400). Bottom inset, Light microscopy
of posterior cornea demonstrates pigmented
epithelium of the iris apposed to Descemet’s
membrane (asterisks). PAS µ75. Bottom,
Transmission electron microscopy of this same
area discloses iris pigment epithelial cells and
stromal tissue lining the posterior corneal
surface (µ6400).
CHAPTER 43
stated that map–dot–fingerprint dystrophy is the most common
cause of recurrent erosion in general practice.
Many ultrastructural studies of map–dot–fingerprint
dystrophy have disclosed a discontinuous multilaminar,
thickened basement membrane under the abnormal
epithelium.73,88,89 This abnormal basement membrane
sometimes contains an admixture of collagenous and cellular
debris suggestive of prior breakdown episodes. More widespread
coalescence of this subepithelial material gives the clinical map-
like picture. Other configurations of aberrant basement
membrane and fibrillar collagen can be found extending in
ridges into the epithelial layers, thus explaining the fingerprint
pattern. Epithelial microcysts actually are pseudocystic
collections of cellular and amorphous debris within the 505
 CORNEA AND CONJUNCTIVA

FIGURE 43.8. Characteristic corneal changes

in various types of corneal stromal dystrophy.
Adapted from and courtesy of Dr A Bron. From Coney
A, Miller J, Krachmer JH: Corneal diseases. In:
Goldberg M, ed. Genetic and metabolic eye disease.
Boston: Little, Brown; 1974:283-285.
SECTION 6

506
 Corneal Dysgeneses, Dystrophies, and Degenerations

FIGURE 43.9. Map–dot–fingerprint dystrophy. Top left and center, Clinical photograph of a 42-year-old woman with nontraumatic erosions
shows characteristics of map dystrophy with superficial geographic haze interrupted by clear areas and few dots. Top right, Light microscopy of
the clinical dot pattern reveals a large debris-containing intraepithelial cyst. PPDA µ400. Bottom left, Enhanced transillumination view of the dot
pattern. Bottom right, Transmission electron microscopy of the evolving cyst that results from cellular dissolution leaving residual nonspecific
cytoplasmic granular debris (asterisks) (µ8000).

epithelial layer. Their shape changes with time, because they
are formed from entrapped cellular material deeper within the
epithelium. As they travel to the surface, they may coalesce
with other cysts and, finally, break through the surface, giving
rise to an erosive episode.
The primary defect in map–dot–fingerprint dystrophy is
presumably the synthesis of abnormal basement membrane
and adhesion complexes by the dystrophic epithelium
(Fig. 43.11). Unable to form proper hemidesmosomes or
anchoring fibrils, the epithelium undergoes recurrent
subclinical or overt episodes of dysadhesion. This periodic ‘lift-
off ’ allows debris to accumulate subepithelially, providing an
even less adequate substrate on which the already abnormal
basement membrane must form. Moreover, intraepithelial
extensions of abnormal basement membrane and collagenous
material may block the normal surface migration of maturing
epithelial cells, allowing the formation of encysted collections
CHAPTER 43
of debris. Thus, the cycle is to a degree self-perpetuating, with
primary faulty epithelial adhesion secondarily causing ab-
normal epithelial maturation that, in turn, exacerbates the ac-
cumulation of abnormal basement membrane and collagenous
debris and leads to further worsening of epithelial adhesion.
Careful débridement of severely aberrant epithelium and, in
some cases, superficial keratectomy to remove subepithelial
debris are aids to conservative therapy with lubricants,
hypertonic saline ointment, patching, or bandage soft contact
lenses. When used as prophylaxis for recurrent erosion, one
study showed no difference between bland ointment and
hypertonic saline ointment.92 McLean and associates94
recommended the use of a needle to perform anterior stromal
reinforcement or puncture. Pathologic studies of anterior
stromal puncture demonstrate activated keratocytes, new
basement membrane with type IV collagen, and production of
new fibronectin and laminin.95 Excimer laser phototherapeutic 507
 CORNEA AND CONJUNCTIVA

FIGURE 43.10. Map–dot–fingerprint dystrophy.

Top left and center, Two variants of fingerprint
dystrophy show subepithelial ridges and appear
refractile against the red fundus reflection. Top
right, Under direct illumination, otherwise faintly
visible fingerprint lines are enhanced with
fluorescein staining and cobalt light. Irregular
corneal tear film and abnormal tear breakup are
evident. Bottom, upper inset, Phase-contrast
photomicrograph illustrates a prominent
intraepithelial fingerprint extension (arrow) from
the subepithelial zone with marked
rearrangement of basal epithelium. PPDA
µ1200. Bottom, Transmission electron
microscopy of this same area discloses
collagenous and granular composition of the
subepithelial material, as well as cellular
elements (asterisk), and an elaborate
multilaminar basement membrane (bracketed
area) loosely apposed to an undulating basal
cell membrane (µ7000). Bottom, lower inset,
Higher magnification of the bracketed area in
bottom figure resolves typical redundant
laminations of the basement membrane
(asterisks), underdeveloped hemidesmosomes
(encircled areas), and absence of anchoring
fibrils. Ep, basal epithelium (µ40 000).
Top left and center, Courtesy of Dr L Hirst.
SECTION 6

FIGURE 43.11. Theorized pathogenesis of epithelial basement membrane dystrophy. Epithelial cells produce an abnormal multilaminar
basement membrane, both in the normal location and intraepithelially. As the intraepithelial basement membrane thickens, it blocks the normal
migration of epithelial cells toward the surface. Trapped epithelial cells degenerate to form intraepithelial microcysts that slowly migrate to the
surface. The abnormal basement membrane produces map and fingerprint changes, and microcysts produce the dot pattern seen clinically.
From Waring GO III, Rodrigues MM, Laibson PR, et al: Corneal dystrophies. I. Dystrophies of the epithelium, Bowman’s layer and stroma. Surv Ophthalmol 1978;
23:71.

keratectomy (PTK) to stimulate diffuse microcicatrization at
the surface of Bowman’s layer is probably the treatment of
choice when the erosion involves the visual axis.96,97
Similar fingerprint, map, and intraepithelial microcyst
changes may develop after traumatic, infectious, or ulcerative
conditions, and particularly in patients with chronic epithelial
508 edema where repeated liftoff of the epithelial sheet allows the
interposition of material that can again thwart the development
of proper basement membrane adhesion complexes.
Hereditary Epithelial Dystrophy (Meesmann,
Stocker–Holt)
The corneal dystrophy of Meesmann98–104 and of Stocker–Holt105
is a dominantly inherited abnormality of the corneal
 Corneal Dysgeneses, Dystrophies, and Degenerations
FIGURE 43.12. Hereditary epithelial dystrophy (Meesmann’s; Stocker-
Holt). Top left, Broad slit-lamp photograph discloses myriad small,
clear to gray-white punctate opacities in the interpalpebral zone. Top
right, The intraepithelial vesicles stand out with retroillumination.
Bottom, Transmission electron micrograph of the corneal epithelium
shows an intraepithelial pseudocyst containing desquamated cellular
debris (µ18 000).
epithelium, first described clinically by Pameijer in 1935.106 A
possibly recessive form also has been reported. Mutations
within the genes coding for epithelial keratins, specifically K3
and K12 located within chromosome 17q12, have been
described.103,104
Clinically, asymptomatic intraepithelial cysts are biomicro-
scopically evident within the first months of life as myriad
small clear to gray-white punctate opacities in the inter-
palpebral zone of the cornea (Fig. 43.12). The cysts are uniform
in size and shape, and few may stain with fluorescein.74 Occa-
sionally, the opacities also are noted at the level of Bowman’s
layer, although histopathologically, Bowman’s layer is not
abnormal. It has been demonstrated that the cysts actually are
accumulations of degenerated cellular material and basement
membrane-like debris surrounded by adjacent cells. Although
cells in Meesmann’s dystrophy contain material that stains
with the periodic acid-Schiff (PAS) stain, they do not contain
excessive glycogen as was believed previously; rather, they
contain a dense intracellular substance of unknown com-
position.102 Older individuals may complain of mild erosive
symptoms and minimally decreased acuity.
In 1955, Stocker and Holt105 similarly described a domi-
nantly inherited condition in patients 7 months to 70 years of
age, characterized by gray, punctate, scattered corneal opacities
that, with focal illumination, appeared as minute droplets.
Histopathologically, PAS-positive thickening of basement
membrane was present overlying a normal-appearing Bowman’s
layer. In some patients, this nodular thickening of the basement
membrane produced an irregular epithelial surface.
Lisch Corneal Dystrophy
Lisch corneal dystrophy (LCD) is characterized by band-shaped
and whorled microcysts within the corneal epithelium.107 The
inheritance pattern is compatible with either X-chromosomal
or pseudoautosomal dominant transmission, and linkage with
chromosome Xp22.3 has been indentified.108 On clinical
examination, opaque grey lesions resembling whorls, bands or
strands separated by clear areas are present within the corneal
epithelium. Densely packed clumps of microcysts within the
opacities are evident on retroillumination. Vision is affected
if the lesions involve the visual axis, but corneal erosions are
infrequent. Light and electron microscopy demonstrate cyco-
plasmic vacuolization of all epithelial cells within the affected
areas. Removal of affected epithelium by superficial keratec-
tomy may improve vision.
Corneal Dystrophies of Bowman’s Layer
Corneal dystrophies primarily affecting Bowman’s layer have
recently undergone reclassification. Previous clinical and
histopathologic reports were reviewed by Kuechle and col-
leagues,109 who proposed that the dystrophy reported by Reis110
in 1917 and subsequently described by Buecklers111 in 1949 be
renamed cornea dystrophy of Bowman’s layer type 1 (CDB1)
The anterior honeycomb-shaped corneal dystrophy described by
Thiel and Behnke112 in 1967 has been classified as CDB 2. Both
CDB types are inherited as autosomal dominant trait. For
CDB 1 a mutation at R124L within the TGF-beta induced
gene on chromosome 5q31 has been reported,113 and for CDB 2
mutation at R555Q locus within the BIG-H3 gene on chro-
mosome 5q114,115 is known (Table 43.1). Both dystrophies are
usually bilateral and symmetric and become evident in the first
or second decade of life as painful recurrent erosive episodes.
Patients develop decreased visual acuity because of anterior
scarring and surface irregularity. It appears that the recurrent
erosions occur earlier and with fairly more marked visual loss in
patients with CDB 1117–118 (Fig. 43.13).
CHAPTER 43
Slit-lamp examination of the cornea shows an irregular
epithelium with diffuse, irregular, patchy geographic opacities at
the level of Bowman’s layer. As time passes, central opacities
develop as a reticulated pattern spreading into the midperiphery
with a diffuse superficial stromal haze. The clinical appearance
of these dystrophies is similar, and differentiation can be made
only with light microscopy or electron microscopy.
On light microscopy, the region of Bowman’s layer is replaced
with a fibrocellular scar tissue that has an undulating sawtooth-
like configuration. This configuration is not specific to CDB
type I or II and may be seen in both variants. CDB type I stains
positively with Masson’s stain, whereas CDB type II is only
equivocally positive.
Transmission electron microscopy of CDB type I reveals rod-
like, electron-dense paracrystalline structures at Bowman’s
layer, similar to those observed in granular dystrophy.118,119
Instead, the 9–15 nm diameter curly fibers,120 once thought
to be characteristic of Reis–Bücklers dystrophy, appear only in
the region of Bowman’s layer in CDB type II. 509
 CORNEA AND CONJUNCTIVA
The pathogenesis of CDB is unknown. The primary lesion of
CDB type II may be due to fragmentation of the collagen fibrils
SECTION 6
of Bowman’s layer, and the epithelial lesion may occur
secondarily. Alternatively, immunofluorescent localization of
laminin and bullous pemphigoid antigen suggests a primarily
epithelial disease.121 Concomitant abnormalities in the
epithelial basement membrane account for recurrent erosive
episodes.73,122–125
Treatment of these dystrophies varies from early conservative
therapy for recurrent erosions to superficial keratectomy, either
mechanical126 or by excimer laser PTK, for corneal scarring and
opacification.127,128 These methods are helpful in managing the
visual aspects of this disorder and always should be attempted
before lamellar or penetrating keratoplasty.126 Recurrences after
keratoplasty and after superficial keratectomy have been
described.129,130
Vortex Dystrophy (Fleischer’s)
The terms vortex corneal dystrophy and cornea verticillata of
Fleischer have been applied to the finding of pigmented, whorl-
510 shaped lines in the corneal epithelium.131,132 Because this same
FIGURE 43.13. (a) Corneal dystrophy of
Bowman’s layer type I. Top left, Clinical
photograph of the eye of a 20-year-old woman
with recurrent epithelial erosions who had been
followed since age 7 after being diagnosed with
Reis–Bücklers’ dystrophy. Top right, Slit-lamp
photograph shows a reticular pattern of gray
ring-like superficial opacities. Middle left inset,
Phase-contrast microscopy of superficial
keratectomy specimen reveals prominent
subepithelial deposits replacing Bowman’s layer
(arrowhead). PPDA µ 375. Middle, Transmission
electron microscopy confirms deposits as rod-
like paracrystalline structures (µ7500). Bottom,
High-magnification electron micrograph of
random electron-dense deposits at Bowman’s
layer and superficial stroma (µ17 750).
Continued
corneal abnormality is evident in Fabry’s disease, it is now
thought that these patients may have been asymptomatic
female carriers of X-linked Fabry’s disease. In general, similar
whorl-like corneal lesions are evident in patients taking
chloroquine, amiodarone,133 phenothiazines, or indomethacin.
Striate melanokeratosis and fingerprint dystrophic changes can
also mimic the vortex pattern. In the absence of these factors,
however, a thorough survey of family members should be made
to exclude Fabry’s disease.
Anterior Mosaic Crocodile Shagreen (Vogt’s)
Anterior mosaic crocodile shagreen appears as bilateral,
polygonal, grayish-white opacities in the deep layers of the
epithelium and in Bowman’s layer.134,135 These opacities are
usually axial and separated by clear cornea. Because visual
acuity usually is not affected, treatment is not indicated.
Limited histologic study has revealed interruptions of Bowman’s
layer and interposition of connective tissue between it and
the epithelium. It is unclear whether mosaic crocodile shagreen
is an actual corneal dystrophy or an age-related process.
A juvenile form of anterior mosaic crocodile shagreen may
 Corneal Dysgeneses, Dystrophies, and Degenerations
b
occur in association with megalocornea, peripheral band kera-
topathy, and iris malformation. Similar changes may also arise
in posttraumatic conditions.
The so-called anterior mosaic pattern is a different entity, in
which a delicate polygonal pattern is seen after topical instil-
lation of fluorescein. The anatomic explanation for this pattern
is not clear.
Idiopathic Band Keratopathy
Band-shaped keratopathy is a deposition of calcium in the
interpalpebral basal epithelium and Bowman’s layer.136 Most
often, calcium deposition is secondary to a chronic ocular dis-
ease, such as uveitis, or to a systemic disease, such as hyper-
calcemia or chronic renal disease. An inherited type of band
FIGURE 43.13 (cont’d). (b) Corneal dystrophy
of Bowman’s layer type II. Top left and right,
Clinical photographs of the eyes of two patients
with recurrent erosions exhibiting a diffuse
superficial haze. Middle left, Light microscopy
demonstrates the sawtooth configuration of
accumulated subepithelial material with an
irregular basal epithelial layer. H & E µ220.
Middle right, Phase-contrast microscopy
reveals a prominent deposit of subepithelial
fibrocellular tissue (asterisk) with a distorted
Bowman’s layer. PPDA µ300. Bottom inset,
Phase-contrast microscopy demonstrates
degeneration of dark-staining basal cells and
fragmentation of Bowman’s layer (asterisk) by
nodular fibrous pannus. PPDA µ800. Bottom
left, Transmission electron microscopy confirms
thin remnants of a disarrayed Bowman’s layer
(B) and apparent continuity (arrowheads)
between basal cell cytoplasm (Ep) and
degenerate cellular debris (D) within Bowman’s
layer. Basement membrane complexes
(encircled area) are discontinuous and lack
anchoring fibrils (µ30 000). Bottom right, High-
magnification electron micrograph of fibrillar
deposits resolves as masses of irregular curled
filaments, 6-8 nm in diameter (µ63 000).
CHAPTER 43
keratopathy with both childhood and senile forms has been
described without obvious associated cause. In clinical ap-
pearance, the inherited form is identical to that which occurs
secondarily (see section on Corneal Degenerations).
STROMAL DYSTROPHIES
Granular Dystrophy (Groenouw’s Type I)
Granular corneal dystrophy manifests in the first decade of life
and is transmitted as an autosomal dominant trait (Table 43.2).
The lesions are sharply demarcated, milky, opaque figures
resembling snowflakes or bread crumbs and are confined to the
axial portion of the cornea, usually beginning in the most
superficial portion of the stroma (Fig. 43.14). During their 511
 CORNEA AND CONJUNCTIVA

TABLE 43.2. Corneal Stromal Dystrophies

Characteristics Granular Lattice Macular

Genetics Autosomal dominant Autosomal dominant Autosomal recessive

Onset Early adolescence First decade of life First decade
Vision Good until middle age Early reduction with obvious clouding Significantly reduced by 30–40 years;
by 20 years; 20/200 by 50 years finger counting by 50 years
Symptoms Minimal inflammation and Severe recurrent erosions Mild recurrent erosive symptoms
irritation
Opacities Grayish opaque granules; Grayish ‘pipe cleaner’ linear, Grayish opaque spots; indistinct borders
‘bread crumbs’; sharp branching, threads; dots and flakes;
borders distinct borders
Intervening stroma Clear Relatively clear Diffusely cloudy
Distribution of opacities Axial only; periphery clear Entire cornea with dots; linear Entire cornea to limbus, but
opacities central; periphery usually most dense centrally
clear; progress to central disciform
by middle age
Histopathology Discrete, hyaline, granulated Large hyaline lesions with scattered Diffuse, granular, nonhyaline material,
fibrillar material; also subepithelial especially associated with keratocytes
Histochemistry Masson: brilliant red Masson: redpurple Colloidal iron: positive
PAS: negative PAS: positive Alcian blue: positive
Congo red: positive
Birefringent
Two-color dichroism
Thioflavin-T fluoro
Electron microscopy Rod-shaped, electron-dense, Random fibrils (80 Å diameter); Diffuse vesicles, fibrillar material in
crystal structure (ª100–500 mm); electron-dense; keratocytes normal; stroma and Descemet’s; keratocytes
keratocytes normal; endothelium endothelium normal and endothelium distended by
normal membrane-bound vacuoles with
fibrillogranular material
Defect Structural protein: hyaline Structural protein: primary Metabolic: defective acid
degeneration of collagen? amyloidosis of cornea mucopolysaccharide metabolism;
localized mucopolysaccharidosis
From Kenyon KR, Hersh PS, Starck T, Fogle JA: Corneal dysgeneses, dystrophies, and degenerations. In: Duane T, (ed.): Clinical ophthalmology, vol 4. Philadelphia:
JB Lippincott; 1981, p 23.

evolution, they may extend more posteriorly. Between the dense
opacities, the intervening cornea is characteristically clear.
Jones and Zimmerman137 noted that the opacities consist of
areas of hyaline degeneration in which stromal fibers appear
‘granular’. Histologically, the deposits stain red with Masson’s
trichrome stain and are less PAS-positive and less birefringent
than the normal stroma. Reticulin stains demonstrate
numerous argyrophylic fibers. Using histochemical techniques,
SECTION 6
transmission electron microscopic findings reveals that they
are likely to represent corneal dystrophies of CDB type I
(see section on Corneal Dystrophies of Bowman’s Layer). The
so-called corneal dystrophy of Waardenburg–Jonkers143 was
later proved by Wittebol–Post and colleagues145 to be identical to
CDB type II. These variants have an earlier onset and more
severely decreased visual acuity than those of typical granular
dystrophy. Most have increased recurrent erosive episodes. On

Garner138 concluded that the deposits consist mainly of noncol-
lagenous protein-containing tryptophan, arginine, tyramine,
and sulfur-containing amino acids and postulated that the
abnormal proteins originated from the epithelium, keratocytes,
and extracorneal sources. Rodrigues and co-workers139 found
immunofluorescent evidence of a microfibrillar protein, a poorly
characterized glycoprotein, and a Luxol fast blue-staining
phospholipid. More recently, keratoepithelin deposits have been
immunohistochemically identified.140 An epithelial origin of
the deposits based on light and electron microscopic studies of
corneas with recurrent granular dystrophy has been suggested.
Transmission electron microscopy demonstrates rod-shaped or
trapezoidal extracellular structures 100–500 µm wide with
faintly visible periodicity. Keratocytes, endothelium, and
Descemet’s membrane appear unaffected.141
Several atypical variants of granular dystrophy have been
distinguished from the classic form. The first group has been
described as the ‘superficial’ variants.142–146 Careful review of
512 the descriptions, clinical photographs, light microscopic, and
clinical examination, large rings and disks within the superficial
stroma with stellate figures extending to deeper stroma charac-
terize granular dystrophy, whereas snowflake-like opacities or
confluent rings confined to Bowman’s layer forming a diffuse
superficial stromal haze characterize CDB type I.
A second variant of granular dystrophy has been described in
a group of patients tracing their ancestry to Avellino, Italy.146–148
These patients exhibit an appearance similar to typical granular
dystrophy along with axial anterior stromal haze and the
presence at midstroma of discrete linear opacities. On histologic
and ultrastructural analysis, two groups of deposits are found.
The first are in Bowman’s layer and superficial stroma and
display a classic granular dystrophy staining reaction for kerato-
epithelin. The second exhibit lattice-like amyloid deposits.
Similar to granular and lattice corneal dystrophies, trans-
mission is autosomal dominant and localizes to the trans-
forming growth factor beta-induced (Big-H3) gene on
chromosome 5q31.
Linkage of granular corneal dystrophy to a locus in the region
 Corneal Dysgeneses, Dystrophies, and Degenerations
5q22–32 on chromosome 5 was first established in an analysis
of 124 blood samples from a single Danish pedigree of seven
generations149 (see Table 43.1). The markers IL9 and D5S436
flanked the disease locus most closely.149 Subsequently, chro-
mosome linkage analysis of families with lattice corneal dys-
trophy type I, CDB type II, and Avellino dystrophy also mapped
these disease-causing genes to the transforming growth factor
beta-induced (Big-H3) gene on chromosome 5q31119,150–157 (see
Table 43.1). This suggests that either a corneal gene family
exists in this region, or that these corneal dystrophies represent
allelic heterogeneity (different mutations within the same gene
manifest as different phenotypes) of the fundamentally same
disease.
Granular dystrophy does not require keratoplasty as often as
the other familial dystrophies because visual acuity may remain
adequate if clear spaces in the stroma coincide with the visual
axis. Recurrent erosions may occur when deposits involve the
basement membrane zone, but this happens less commonly
than in lattice dystrophy. If vision is reduced markedly, the
surgical management varies based on the depth and extent of
the stromal lesions. If the opacities are extremely superficial,
then superficial keratectomy or lamellar keratectomy can be
FIGURE 43.14. Granular corneal dystrophy.
Top, Three different clinical configurations of
granular dystrophy. Top left, Densely axial
nontranslucent gray-white deposits simulating
bread crumbs. Top center, More discrete and
well-defined round and oval shapes with clear
stroma between lesions. Top right, Christmas
tree-like opacities with moderate anterior
stromal scarring. Middle left, Retroillumination
emphasizes the optical clarity of intervening
stroma between granular opacities. Middle
center, Light microscopy of irregularly shaped
hyaline deposits is accentuated with Masson’s
trichrome stain. (µ220). Middle right, Light
microscopy of a patient with severe recurrent
erosion reveals a superficial deposit evolving to
break the epithelial surface. PAS µ220. Bottom
left, Transmission electron microscopy shows
relatively normal epithelium (E) and basement
membrane (arrowhead) anterior to large
electron-dense deposits (asterisk) within
Bowman’s layer and anterior stroma. µ15 000.
Bottom right, Higher-magnification transmission
electron microscopy of granular deposits shows
the characteristic homogeneous rod-shaped
paracrystalline structure (µ50 000).
Middle left, Courtesy of Dr Lawrence Hirst.
performed.158 Excimer laser PTK has been successfully used
to treat superficial granular dystrophy.159–162 A preoperative
myopic refractive error is desirable because a shift toward
CHAPTER 43
hyperopia has been reported after such treatment.162–165 When
deep stromal lesions occur, the treatment of choice is either
deep lamellar or penetrating keratoplasty.
As in the other familial dystrophies, recurrence in the graft
(usually anterior and peripheral) or after superficial kera-
tectomy150 may take place several years later, suggesting that
the granular deposits are either the result of some acquired
metabolic disturbance in the transplanted corneal tissue or the
product of abnormal epithelium.166–169 Interestingly, LASIK is
apparently specifically contraindicated in Avellino dystrophy
due to the postoperative increase of deposits within the
interface and posterior stroma.170
Lattice Dystrophy
Lattice dystrophy (see Table 43.2) is an autosomal dominant
condition characterized by pathognomonic branching
‘pipestem’ lattice figures within the stroma (Fig. 43.15).
Symptoms usually begin in the first decade of life and include
decreased vision as well as recurrent erosions because of 513
 CORNEA AND CONJUNCTIVA
subepithelial and stromal accumulations of amyloid material.
SECTION 6
In time, the condition progresses to involve marked opa-
cification of the axial stroma, as well as of the superficial layers,
leaving the limbus relatively free. At this stage, because the
cornea also shows a superficial haze, it becomes difficult
to visualize typical lattice lesions, and hence examination of
younger affected family members is useful. Amyloid accu-
mulation under the epithelium gives rise to poor epithelial-
stromal adhesion with consequent recurrent erosion
syndrome.73 The dystrophy advances inexorably, and by 40
years of age or earlier, these problems become markedly aggra-
vated, causing considerable discomfort and visual incapacity.
Many published reports have documented the nature of the
corneal deposits in lattice dystrophy. In 1961, Jones and Zim-
merman137 and others suggested that the disorder was due to
amyloid degeneration of the stromal collagen fibers. In 1967,
Klintworth171 confirmed that the disorder was a familial form of
amyloidosis limited to the cornea and showed that the fibrillar
material stained with Congo red and exhibited the birefringence
514 and dichroism typical of amyloid. On transmission electron
FIGURE 43.15. Lattice corneal dystrophy. Top
left and center, Slit-lamp photography
demonstrates pathognomonic branching lattice
figures throughout the stroma. Top right, Light
microscopy of a cornea in a patient with
multiple episodes of recurrent erosions
discloses an irregular epithelial layer, partial
absence of Bowman’s layer (arrowhead), and
predominantly subepithelial amyloid deposits
(asterisk). PAS µ220. Middle left, Congo red
stain of a fusiform lesion that distorts the
normal stromal lamellar architecture (µ55).
Middle center, Corneal amyloid shows
birefringence and dichroism under the
polarizing microscope (µ20). Middle right,
Transmission electron microscopy of basement
membrane complexes reveals basement
membrane irregularity and discontinuity
resulting from underlying amyloid fibrils
(µ21 300). Bottom left, Transmission electron
micrograph of stroma shows normal collagen
fibrils and keratocytes with electron-dense
material abnormally dispersed extracellularly.
µ16 000. Bottom right, High-magnification
transmission electron micrograph resolves
lattice material as masses of fine amyloid fibrils,
80-100 Å in diameter (circled area) (µ43 400).
Top left, Courtesy of Dr W. J. Stark.
microscopy, the fine, electron-dense fibrils, 80–100 Å in
diameter, are similar to those of known amyloid fibrils. Using
fluorescence microscopy, staining with thioflavin-T is helpful in
further characterizing the amyloid material, as are immuno-
fluorescent studies using antihuman amyloid antisera.172
Evaluation of corneas with typical lattice dystrophy has demon-
strated the presence of the amyloid P (AP) component, but
staining for amyloid A (AA) protein has remained con-
troversial.173–175 The corneal endothelium and Descemet’s
membrane are not involved. Moreover, amyloid deposits have
not been found in other excised tissues from patients with
typical lattice dystrophy.171
The specific cause of the amyloid deposits is unclear. They
may be secondary to collagen degeneration, perhaps from
lysosomal enzymes elaborated by abnormal keratocytes. An
alternative theory holds that abnormal keratocytes actually
produce the abnormal amyloid substance, although this process
is not ultrastructurally evident.
Classic lattice corneal dystrophy (LCD type I), granular
dystrophy, and Avellino dystrophy have been independently
 Corneal Dysgeneses, Dystrophies, and Degenerations
linked to chromosome 5q150–151 (see Table 43.1). Folberg and
associates176 suggested that the morphologic distinction bet-
ween LCD type I and granular corneal dystrophy is not as clear
as previously believed. They found evidence of granular deposits
in LCD type I families, and vice versa. This evidence of
histologic overlap strongly suggests that these dystrophies are
caused by mutations within the same gene.
Treatment is symptomatic, depending on visual acuity and
patient discomfort. Recurrent erosions are treated either con-
ventionally or with superficial keratectomy to remove sub-
FIGURE 43.16. Lattice corneal dystrophy and
systemic amyloidosis (Meretoja’s syndrome).
Top left, A 73-year-old woman with typical
mask-like facies, including skin thickening,
prominent blepharochalasis, depressed
eyebrows, and bilateral facial nerve palsies. Top
right, Slit-lamp view of lattice lines beginning at
the periphery and sparing the visual axis.
Middle left, Light microscopy of conjunctival
biopsy shows continuous subepithelial layer
(asterisks) of extracellular material. PPDA µ300.
Middle right, Transmission electron microscopy
of this biopsy demonstrates masses of fine
amyloid fibrils (asterisk) beneath the epithelial
basement membrane (arrows). E, epithelium
(µ8700). Bottom left, Transmission electron
microscopy of skin biopsy reveals deposition of
extracellular material (asterisk) immediately
beneath the normal epithelial basement
membrane (µ8100). Bottom right, Similar
deposits (asterisks) are found associated with
the perineurium and endoneurium of peripheral
nerves (µ12 060).
CHAPTER 43
epithelial amyloid accumulations. Excimer laser PTK has been
described as an optional treatment for recurrent erosions and
superficial opacities.159,160,162 Penetrating keratoplasty in this
condition carries an excellent prognosis, although recurrence of
the dystrophy in the graft may take place.177–178
In LCD type II (also termed Meretoja’s syndrome or amy-
loidotic polyneuropathy type IV, Fig 43.16) first described by
Meretoja in 1969 in a large series of Finnish patients, is
systemic amyloidosis associated with lattice dystrophy.179–183
The onset of clinical corneal changes usually occurs later, and 515
 CORNEA AND CONJUNCTIVA

FIGURE 43.17. Lattice corneal dystrophy variants. Top left and right, Clinical appearance of two patients with axial thick ropy lattice lines and a
dense nodular opacification. Bottom left, Numerous thinner lines are more easily observed by retroillumination. Bottom right, Light microscopy of
a unique large amyloid deposit at the posterior half of the cornea. Bowman’s layer and epithelium are intact. H & E µ170.
SECTION 6

Bottom right, From Hida T, Tsubota K, Kigasawa T, et al: Clinical features of a newly recognized type of lattice corneal dystrophy. Am J Ophthalmol 1987; 104:241–248.
Copyright by The Ophthalmic Publishing Company.

erosive episodes are less common. Systemic manifestations
include progressive cranial and peripheral neuropathy and skin
changes, such as lichen amyloidosis and cutis laxa, resulting
in ‘mask-like’ facies. Other variable features include poly-
cythemia vera and ventricular hypertrophy. Biomicroscopically,
the lattice lines are fewer, thicker, more radially oriented and
involve mainly the periphery of the cornea, with relative central
sparing. Amorphous deposits are fewer and more confined in
distribution than they are in LCD type I. Open-angle glaucoma
and pseudoexfoliation with or without glaucoma are found
commonly.183
Histologic examination of the LCD type II cornea reveals
characteristic amyloid deposits forming a layer beneath a
normal-appearing Bowman’s layer and focally within the
516 stroma. Deposits also may be found in arteries, basement mem-
branes, skin, peripheral nerves, and sclera. The amyloid in this
systemic disorder may differ from classic lattice dystrophy,
showing loss of Congo red staining after treatment with per-
manganate.174
With the aid of immunohistochemistry, LCD type II can be
diagnosed and differentiated from type I in tissue sections, even
retrospectively.184–186 Using antibodies raised to a chymotryptic
fragment inclusive of the carboxy terminal half of gelsolin as
well as adjacent to and inclusive of the codon 187 mutant 7–
11 kDa fragment, immunoreactivity was detected in the skin
and conjunctival amyloid in LCD type II.184 The amyloid within
the cornea in type II reacted nonhomogeneously with the
antigelsolin antibody but not with the antibodies produced to
the amino and carboxy terminals of gelsolin.184 The mutation
involves a guanine-to-adenine substitution at nucleotide 654,
 Corneal Dysgeneses, Dystrophies, and Degenerations

TABLE 43.3. Comparison of Inherited Varieties of Corneal Amyloidosis

Characteristics Lattice Corneal Dystrophy Familial Subepithelial

Amyloidosis
Type I Type II Type III

Usual age at onset <10 years >20 years >40 years <20 years
Visual acuity Markedly impaired by age Usually good until after age Impaired after 60 years Markedly impaired by
40–60 years 65 years age 10–30 years
Systemic amyloidosis No Yes No No
Mode of inheritance Autosomal dominant Autosomal dominant Autosomal recessive? Autosomal recessive
Facies Normal Masklike facial expression, Normal Normal
blepharochalasis, floppy
ears, protruding lips
Nervous system Normal Cranial and peripheral nerve Normal Normal
palsies
Skin Normal Dry, itchy, and lax with Normal Normal
amyloid deposits
Cornea Delicate interdigitating Thick and radially oriented Thick lines Multiple prominent
network of filaments; lines subepithelial nodules
no lines present at early
stage; lines difficult to see
at late stage
Episodic corneal erosion Yes Yes No No
From Hida K, Tsubota Kigasawa K, et al: Clinical features of a newly recognized type of lattice dystrophy. Am J Ophthalmol 1987; 104:241–248, 1987. Published with
permission from The American Journal of Ophthalmology. Copyright by The Ophthalmologic Publishing Company.

resulting in an asparagine-187 variant of gelsolin.182,186–190 The
gelsolin gene (type II) has been localized to the long arm of
chromosome 9 (9q34)189 (see Table 43.1).
Other atypical variants of lattice dystrophy, as well as rare
cases of unilateral lattice dystrophy, have also been
reported191–192 (Fig. 43.17). The former, termed LCD type III,
characterized by a probable autosomal recessive inheritance
pattern, has thicker lattice deposits within the corneal
epithelium, onset later in life without systemic involvement or
episodic recurrent corneal erosions. Histologically, there is
absence of subepithelial deposits with a normal epithelium and
Bowman’s layer. The stromal deposits are larger than in LCD
types I and II, which correlates with the thicker lattice lines
clinically evident in this variant. Immunohistochemical
analysis has revealed positive staining for AP protein but only
weak staining for AA protein.191 Families with LCD types IIIA,
IV, VI, and VII have also been described.193 Confocal microscopy
may be helpful in distinguishing amyloid deposits and nerve
extend throughout the entire stromal thickness. Corneal thin-
ning confirmed by central pachometry has been documented.196
Also, unique to macular corneal dystrophy is primary involve-
ment of the endothelium as evidenced clinically by the presence
of guttate changes of Descemet’s membrane (Fig. 43.19).
The lesions in macular corneal dystrophy stain intensely
with alcian blue and colloidal iron, minimally with PAS, and
not at all with Masson’s trichrome. Birefringence is decreased.
The lesions have been histochemically identified as an abnormal
keratan sulfate-like glycosaminoglycan that accumulates
extracellularly within the stroma and Descemet’s membrane
and intracellularly within keratocytes and endothelium.197
As consistent with an autosomal recessively inherited
condition, macular dystrophy presumably results from
deficiency of a hydrolytic enzyme (sulfotransferase) and may
thus be considered a localized mucopolysaccharidosis.198,199
The effect of altered glycosaminoglycan metabolism is evident
at the cellular level; on transmission electron microscopy,

devenerataion in LCD from other entities, such as infectious
crystalline dystrophy, ananthamoeba and fungal hyphae.194,195
The cornea may also develop secondary amyloid deposits
after various chronic ocular diseases, but such deposits
generally are insignificant clinically (see section on Corneal
Degenerations). The differences between the inherited varieties
of corneal amyloidosis are summarized in Table 43.3.
Macular Dystrophy (Groenouw’s Type II)
Among the classic corneal dystrophies, macular corneal
dystrophy (MCD), unlike granular and lattice dystrophies, is an
autosomal recessive disorder and is much less common (see
Table 43.2). It usually begins in the first decade of life and leads
to progressive visual deterioration as the stroma becomes
generally cloudy, with superimposed dense, gray-white spots
(Fig. 43.18). Unlike granular dystrophy, these macular spots
have indefinite edges, and the intervening stroma is not clear.
Young patients exhibit axial lesions in the superficial layers of
the cornea, but with time, lesions approach the periphery and
CHAPTER 43
keratocytes and endothelial cells exhibit distention of rough-
surfaced endoplasmic reticulum cisternae. With the acridine
orange technique, compensatory generalized hyperactivity of
the lysosomal enzyme system has been demonstrated. Even-
tually, the accumulated undigested storage products engorge
the cells, and the cells ultimately degenerate or rupture. The
derivation of these intracytoplasmic storage vacuoles from
endoplasmic reticulum suggests that the biochemical lesion in
macular dystrophy occurs at a different metabolic location than
in the systemic mucopolysaccharidoses because in the latter,
storage products accumulate within lysosome-like intra-
cytoplasmic vacuoles associated with the Golgi complex.199
Snip and associates200 were able to determine that the storage
phenomenon affecting endothelium and Descemet’s membrane
is likely also primary because the intracellular and extracellular
lesions appear ultrastructurally comparable with those evident
in the keratocytes and stroma.
Two subtypes of MCD have been immunohistochemically
identified.201–203 MCD type I is the most prevalent and is 517
 CORNEA AND CONJUNCTIVA
characterized by the absence of antigenic keratan sulfate (aKS)
from both cornea and serum. In fact, it may represent a more
widespread systemic disorder of keratan sulfate meta-
SECTION 6
bolism.201,202 In MCD type II, the serum aKS level is normal,
and the corneal accumulations react with the antikeratan sul-
fate antibody.204,205 MCD types I and II cannot be distinguished
based on clinical characteristics. Moreover, some genealogic
studies have shown patients of both types sharing common
ancestors.202 Linkage analysis has mapped MCD type I to the
16q22 locus of chromosome 16206 (see Table 43.1). In addition,
a peak LOD score of 2.50 at a recombination fraction of 0.00
was obtained for the type II families by use of the identical
marker. These findings raise the possibility that MCD types I
and II may be due to the same genetic locus.206 Recent studies
have identified a variety of mutations in a carbohydrate sulfo-
transferase gene (CHST6) encoding corneal glucosamine
N-acetyl-6-transferase.207–210
Treatment of macular dystrophy is either deep lamellar
or penetrating corneal transplantation. In Saudi Arabia, MCD
accounts for 87% of penetrating keratoplasties performed for
classic corneal dystrophies,211 indicative of a remarkably high
518 prevalence of the affected gene within a defined population. As
FIGURE 43.18. Macular corneal dystrophy. Top
left, Clinical appearance of cornea features
diffuse stromal haze with a ground-glass
appearance extending to the limbus. Centrally,
superimposed dense gray-white spots with
indistinct edges are observed. Top right, In a
clinical variant, larger, amorphous lesions
involve only the central cornea, sparing the
limbus. Bottom, right inset, By phase-contrast
microscopy, the epithelium is seen to be
irregular. Fibrocellular pannus intervenes
between the epithelium and the unaffected
Bowman’s layer (asterisks). Several extensively
vacuolated keratocytes are evident in the
anterior stroma. PPDA µ250. Bottom, main
figure, Transmission electron microscopy
demonstrates irregular thinning and breaks
(arrowheads) of basement membrane. Within
the fragmented Bowman’s layer, subepithelial
cells are distended by membrane-limited
intracytoplasmic inclusions containing fine
granular and reticular material (µ12 000).
Bottom, left inset, Higher-magnification
transmission electron microscopy resolves the
reticular pattern of accumulated intracellular
material (µ43 500).
with most other stromal dystrophies, recurrence in the graft has
been reported.212,213
Polymorphic Stromal Dystrophy
Polymorphic stromal dystrophy is a manifestation of corneal
amyloid clinically distinct from the lattice dystrophies and
gelatinous drop-like dystrophy. Thomsitt and Bron214 described
patients with a variety of posterior stromal opacities consistent
with the dystrophic changes reported in 1939 by Pillat.215 Axial
polymorphic opacities resembling stars and snowflakes were
noted, as well as branching filamentous opacities in the pos-
terior cornea. The lesions were gray-white and mildly refractile
when viewed with direct light but appeared transparent in
retroillumination. As the intervening stroma is clear, vision is
minimally affected. Histochemistry and electron microscopy
demonstrate typical amyloid deposits within the opacities.216
The late onset, lack of progression, and absence of familial
association distinguish this condition from LCD.
Gelatinous Drop-Like Dystrophy
Gelatinous drop-like dystrophy (Fig. 43.20) is yet another
clinical manifestation of primary, localized corneal amyloidosis
 Corneal Dysgeneses, Dystrophies, and Degenerations

FIGURE 43.19. Macular corneal dystrophy. Top

right inset, By light microscopy, a large
extracellular accumulation of abnormal material
(large asterisk) is shown. Descemet’s
membrane (bracketed area) is enormously
thickened to ~40 µm with additional guttate
excrescences (small asterisk). Endothelial cells
(arrowheads) are greatly reduced in number and
remarkably attenuated. Alcian blue µ500. Main
figure, Transmission electron microscopy
demonstrates extensive Descemet’s membrane
abnormalities. The anterior banded portion
(bracketed area) is of normal thickness and
configuration. Posteriorly, Descemet’s
membrane (DM) is thickened, here measuring
20 µm, and is honeycombed by fine granular
material anteriorly with banded collagen figures
(circled area) and multiple basement membrane
laminations predominantly posteriorly. A
degenerating cellular process is evident
centrally (asterisk) and an attenuated
endothelial cell (E) is shown posteriorly. S,
stroma (µ8900). Bottom left insets, High-
magnification transmission electron microscopy
(left) and scanning micrograph (right) of area
circled in main figure reveal fusiform long-
spacing collagen configurations with ~1100-Å
macroperiodicity (µ27 000; µ10 000).

CHAPTER 43

FIGURE 43.20. Gelatinous drop-like dystrophy. Central mulberry-like opacity has protuberant subepithelial mounds that appear white on focal
illumination (left) and semitransparent on retroillumination (right). Minor stromal neovascularization is present superonasally. 519
 SECTION 6 CORNEA AND CONJUNCTIVA

FIGURE 43.21. Central crystalline dystrophy (Schnyder’s). Top left, Clinical appearance of the eye of an 8-year-old boy includes an axial ring-
shaped opacity formed by densely packed, fine, needle-shaped polychromatic crystals. Associated genu valgum was present in this pedigree.
Top right, In a different variant, more extensive involvement of the central cornea and associated arcus senilis was present. Bottom inset, Frozen
section of keratoplasty specimen of superficial cornea reveals the epithelium (a) unstained, Bowman’s layer (b) with intense lipid deposits, and
stroma (s) with scattered lipid staining. Oil red O stain µ350. Bottom left, Electron micrograph of basal epithelium and Bowman’s layer reveals
vacuolated corneal epithelium (E), thickened basement membrane (arrows), and distorted, vacuolated Bowman’s zone (B) with polygonal profiles
(µ10 000). Bottom right, High-magnification transmission electron microscopy of the same area discloses multiple polygonal spaces (asterisks),
520 typical of cholesterol crystal ghosts (µ25 000).
Bottom, From Burns RP, Connor W, Gipson I: Cholesterol turnover in hereditary crystalline corneal dystrophy of Schnyder. Trans Am Ophthalmol Soc 1978; 76:184.
 Corneal Dysgeneses, Dystrophies, and Degenerations
characterized by deposition of large amounts of amyloid
beneath the corneal epithelium. First reported by Nakaizumi217
in Japanese patients, this dystrophy remains common in Japan
(estimated prevalence 1 in 33 000) but is rare in Western
countries. The disorder is bilateral and noninflammatory and
may exhibit autosomal recessive inheritance.218–220 The res-
ponsible gene has been identified as membrane component,
chromosome 1, surface marker 1 (M1S1) on the short arm of
chromosome 1.221–223
Photophobia and epiphora commence in the first decade of
life with progressive visual deterioration during the teens. The
clinical presentation is bilateral but often asymmetrical as
multiple superficial yellow to milky white mulberry-like focal
nodular depositions elevating the epithelium and superficial
stroma. Other variations include band keratopathy and either
localized or more diffuse opacity. In longstanding cases, corneal
sensation may be decreased, as stromal vascularization and
scarring lead to severe vision loss.
Histopathologic specimens have demonstrated mounds of
amyloid interposed between the irregular epithelium and de-
generated Bowman’s layer as well as fusiform LCD-like deposits
in the deeper stroma.224–225 The specific type of corneal amyloid
remains to be determined, but immunostaining of the deposits
is mildly positive for amyloid AL and AP, as well as lactoferrin,
apolipoproteins J and E and gelsolin but negative for amyloid
AA, AF, AB, and keratin.226
Corneal opacities and scars usually necessitate superficial
keratectomy in mild cases and either lamellar or penetrating
keratoplasty in more advanced conditions, typically by age 30.
However, recurrence is common as subepithelial haze evolves
into nodular deposits.227 Hypothesizing that a dystrophic cor-
neal epithelium is responsible for secretion of the amyloid
accumulations, Shimazaki combined limbal stem cell trans-
plantation with keratoplasty with seemingly improved pro-
longation of corneal clarity.228
Central Crystalline Dystrophy (Schnyder)
Central crystalline dystrophy, an autosomal dominantly
inherited disorder that occurs in early life and is occasionally
congenital, was initially described by Schnyder in 1929229
(Fig. 43.21). Previously considered to be nonprogressive after
childhood, subsequent reports have documented significant
progression.230 The responsible gene is located in chromosome
1 (1p34.1–36)231 and as the B120 gene located within this area
is associated with lipid production within fibroblasts, it is
speculated that lipid production by keratocytes may be
causative.232
The main feature of the disease is a bilateral, axial, ring-
shaped corneal opacity consisting of polychromatic crystals.
The yellow-white opacity is noted in Bowman’s layer and the
anterior stroma. The epithelium is normal, and the uninvolved
stroma also appears normal, although in time a diffuse stromal
haze can develop.233 In some patients, small white opacities
are scattered throughout the stroma. Schnyder’s dystrophy
without crystals is not uncommon and is usually un-
recognized.234 Histologic examination using lipid stains on
frozen sections reveals neutral fats and cholesterol.235 The
clinically apparent crystals correspond to cholesterol accu-
mulations, both within keratocytes and extracellularly. Neutral
fat is distributed within the stroma among the collagen fibrils.
Both the limbal girdle of Vogt and corneal arcus may also be
associated. The disease is presumably a localized defect of lipid
metabolism; systemic hypercholesterolemia, xanthelasma, and
genu valgum can occur. Hence it is important to perform cho-
lesterol and lipid studies to detect elevated serum lipid levels
and concomitant cardiovascular disease.236
Excimer laser PTK may have a potential role in patients with
significant decreased visual acuity consequent to anterior
stromal opacities.237 Some patients with more severe opacities
may require corneal grafting. Recurrence of cholesterol crystals
may occur in lamellar or penetrating grafts.235
Marginal Crystalline Dystrophy (Bietti)
Bietti238 described crystalline deposits in the paralimbal anterior
corneal stroma in conjunction with intraretinal crystals and
retinitis pigmentosa (RP). Inheritance is autosomal recessive.
Mutations within the CYP4V2 gene of chromosome 4q35–4qtel
may adversely affect fatty acid metabolism within ocular struc-
tures.239 In a series of 200 patients, the prevalence of the
dystrophy was 3% in nonsyndromic RP and 10% in autosomal
RP.240
Yellowish-white subepithelial crystals are typically found in
the corneal periphery, especially at the superior and inferior
limbus.241,242 They may be overlooked due to their small size
(less than 15 mm), are asymptomatic and do not affect vision.
Crystals are also located within all layers of the retina with
atrophy of the retinal pigment epithelium and choroidal
sclerosis. EOG and ERG deterioration precede vision loss and
nyctalopia by years. Lipid inclusions and crystals are found
within keratocytes and conjunctival fibroblasts, as well as
within choroidal fibroblasts and circulating lymphocytes.
Currently there is no treatment.
Central Cloudy Dystrophy (François)
François243 described eight patients having polygonal opacities
with intervening clear zones resembling cracks of the axial
posterior stroma superimposed on nebular stromal haze
CHAPTER 43
(Fig. 43.22). Strachan reported involvement in three consecutive
FIGURE 43.22. Central cloudy dystrophy
(François). This 62-year-old man with visual
acuity of 20/30 in both eyes demonstrates
clouding of the central cornea into segmental
areas of opacification with intervening clear
tissue (left). Slit-lamp view (right) features
mainly posterior opacities that extend forward
but become much less dense.
521
 CORNEA AND CONJUNCTIVA
generations and suggested autosomal dominant inheritance.244
Vision is affected minimally, and as patients remain asyp-
tomatic, no therapy is required. Confocal microscopy reveals
refractile keratocytes and extracellular matrices separated by
dark striae.245 Histopathology is limited, but alcian blue-
positive mucopolysaccharides and lipid-like inclusions within
the stroma by light and electron microscopy have been
described.246 The differential diagnosis of such polymorphous
stromal opacities includes posterior crocodile shagreen, macular
corneal dystrophy, icthyosis, fleck corneal dystrophy, lecithin
cholesterol acetyltransferase deficiency, Grayson–Wilbrandt
pre-Descemet dystrophy and systemic mucopolysaccharidosis.
Posterior Amorphous Stromal Dystrophy
This autosomal dominant disorder was first described in 1977
in a family spanning three generations as symmetric gray-white,
sheet-like posterior stromal opacities centrally and extending
peripherally to the limbus.247 Corneal thinning was also present
in the more advanced cases. Findings in other reported pedi-
grees included: (1) both centroperipheral and peripheral forms;
(2) hyperopia with corneal flattening; (3) iris abnormalities,
including glassy sheets on the iris surface, corectopia, and
pseudopolycoria; and (4) iris processes extending to Schwalbe’s
line.248
The sheet-like opacities may be irregular and broken with
clear intervening stroma. Descemet’s membrane and endo-
thelium may be indented by the opacities, and focal endothelial
abnormalities have been observed. Despite high astigmatism
and amblyopia, vision is usually only mildly affected.
SECTION 6
In the keratoplasty specimen from a 5-year-old child,
fracturing of the posterior stromal collagen lamellae, a thin
Descemet’s membrane, and focal attenuation of endothelial
cells were evident.249 Ultrastructural studies showed disorga-
nization of the posterior stromal collagen. Because the cornea is
structurally abnormal and is thin and flat, the opacities appear
stable throughout life, the iris is affected, and the changes have
been found in a child as young as 6 months of age, this disorder
may be more appropriately classified as a mesenchymal dys-
genesis rather than a dystrophy250 (see section on Mesenchymal
Dysgeneses).
Congenital Hereditary Stromal Dystrophy
Congenital hereditary stromal dystrophy is characterized by
bilateral flaky or feathery clouding of the stroma, present either
at birth or within the first years of life.251 Both the peripheral
and the central cornea are affected, the latter more severely. It
is autosomal dominantly inherited. Genome-wide screening of
522 three generations of a family showed linkage to chromosome
FIGURE 43.23. Posterior mosaic crocodile
shagreen (Vogt). Clinical photography (left) of a
55-year-old asymptomatic woman
demonstrates bilateral central opacification
compromising the entire corneal thickness.
Broad slit-lamp photography (right) discloses
multiple small, fluffy, and indistinct grayish
areas in a polygonal pattern separated by clear,
crack-like zones.
12q22. Mutations have been described in the DCN gene which
encodes for decorin, a dermatan sulfate proteoglycan which is
important in collagen fibrillogenesis.252
Electron microscopy has revealed abnormally small stromal
collagen fibrils with disordered lamellae, suggesting a disorder
in collagen fibrogenesis. Penetrating keratoplasty is usually
successful but recurrence is possible.
Posterior Mosaic Crocodile Shagreen
Posterior crocodile shagreen (Fig. 43.23) is a bilateral condition
marked by a series of small gray polygonal patches of various
sizes, separated by dark regions, at the level of Descemet’s
membrane.253 The condition may be a variant of central cloudy
dystrophy of Francois. Transmission electron microscopic
studies have demonstrated the grayish opacities of posterior
mosaic crocodile shagreen to correspond with sawtooth-like
configurations of the corneal collagen lamellae (see section on
Anterior Mosaic Crocodile Shagreen). Because vision is not
compromised, no treatment is required.
Fleck Dystrophy (François–Neetens)
First described by Francois and Neetens in 1957,254 fleck
dystrophy is a rare, autosomal dominant disorder is detectable
early in life and congenital in some patients255,256 (Fig. 43.24).
The genetic defect occurs on chromosome 2q35 where the
PIP5K3 gene, a member of the phosphoinositide 3-kinase
family, regulates the function of multivesicular bodies within
endosomes.257,258 Subtle grayish specks are present in all layers
of both corneas, and some appear as rings with relatively less
opacified centers. As the stroma is otherwise clear, patients
have no visual disability apart from mild photophobia. Confocal
microscopy demonstates highly reflective material within hera-
tocytes. Histopathologic examination has revealed abnormal
keratocytes that on transmission electron microscopy contain a
fibrillogranular substance within intracytoplasmic vacuoles.259
Histochemical staining shows glycosaminoglycans and lipids
within these vacuoles.
PRE-DESCEMET’S DYSTROPHIES
The pre-Descemet’s category of dystrophy has several rare
entities, all generally compatible with good vision and comfort.
A clear pattern of heredity is not always obvious.
Cornea Farinata
Cornea farinata260 is often a routine finding in older people and
therefore may represent a degenerative process rather than a
dystrophic one. Visual acuity is not usually decreased. Small
 Corneal Dysgeneses, Dystrophies, and Degenerations
gray punctate opacities can be seen in the pre-Descemet’s
membrane area of the stroma on retroillumination. Sometimes,
larger and more polymorphous types of comma, circular, linear,
filiform, and dot-like opacities are observed as well. The
opacities may be distributed axially or annularly. Similar pre-
Descemet’s opacities may be found in association with
ichthyosis.261
Grayson–Wilbrandt Dystrophy
Grayson and Wilbrandt262 described asymptomatic opacities
that were slightly larger and more diffusely scattered than those
in cornea farinata and that were distributed axially and
paraaxially (Fig. 43.25). Familial associations were documented.
Curran and associates263 described the ultrastructure of
abnormal keratocytes anterior to Descemet’s membrane as
containing membrane-bound intracytoplasmic vacuoles that
included fibrillogranular material and electron-dense lamellar
lipid bodies.
FIGURE 43.24. Fleck dystrophy (François-
Neetens). Top, Retroillumination (left) and slit-
lamp view (right) demonstrate discrete flattened
white flecks with comma, wreath, or dot
configuration present throughout the entire
stroma. Bottom inset, Light microscopy of the
posterior cornea illustrates positive staining for
acid mucopolysaccharide limited to a swollen
keratocyte (circled area). Colloidal iron µ500.
Bottom, Transmission electron microscopy of a
markedly vacuolated keratocyte filled with
fibrillogranular (F) or lipid (L) substances. There
are no extracellular abnormalities except for an
accumulation of the fine granular material
(asterisk) and occasional foci of long-spacing
collagen (square) (µ14 400).
All, From Nicholson DH, Green WR, Cross HE, et al:
A clinical and histopathological study of Francois-
Neetens speckled corneal dystrophy. Am J Ophthalmol
1977; 83:554–560. Copyright by The Ophthalmic
Publishing Company.
CHAPTER 43
Deep Filiform Dystrophy
The deep filiform dystrophy of Maeder and Danis264 consists of
multiple filiform gray opacities in the pre-Descemet’s area that
affect the entire width of the cornea except for the perilimbal
region. The original case occurred in a middle-aged woman with
keratoconus. The histopathology has not been documented.
This disorder may represent a degeneration rather than a
dystrophy.
ENDOTHELIAL DYSTROPHIES
Congenital Hereditary Endothelial Dystrophy
Initially described by Maumenee265 in 1960, this congenital
disorder of the endothelium has been mapped in its autosomal
dominant form to the pericentromeric area of chromosome 20,
within the same region assigned to posterior polymorphous
dystrophy whereas its autosomal recessive form has been
localized to a distinct area of chromosome 20p266–267 (see Table 523
 CORNEA AND CONJUNCTIVA
43.1). Congenital hereditary endothelial dystrophy (CHED) is
usually characterized clinically by diffuse, bilaterally symmetric
corneal edema (Fig. 43.26).268 The autosomal recessive variety
(CHED 2) is present at birth and is relatively nonprogressive.
Symptoms of discomfort are not prominent despite profound
epithelial and stromal edema. Nystagmus is common. A
dominantly inherited form (CHED 1) is less severe, developing
in the first or second year of life, and in contrast to the recessive
variety, progressive photophobia and tearing are the initial
symptoms. Nystagmus generally is absent. Some CHED
patients have no affected family members and may represent
SECTION 6
autosomal recessive cases or a new mutation. CHED has also
been sporadically associated with sensorineural hearing loss, nail
anomalies, corneal amyloidosis and corpus callosum agenesis.
As in all instances of congenital corneal clouding, it is im-
portant to rule out congenital glaucoma. Corneal diameters and
globe axial length remain normal in CHED, whereas the
corneal thickness is greatly increased, thereby artifactiously
elevating applanation measurements of intraocular pressure.
The combination of congenital glaucoma and congenital here-
ditary endothelial dystrophy may occur and should be suspected
when persistent and total corneal opacification fails to resolve
after normalization of intraocular pressure.
The degree of edematous corneal clouding varies from a mild
haze to a milky, ground-glass opacification. Epithelial micro-
bullae may be obvious, and stromal thickness may be increased
threefold or more. Uniform thickening of Descemet’s mem-
brane sometimes is evident on clinical examination, but no
guttata are apparent. Interstitial inflammation and secondary
524 vascularization are absent. Apart from rare congenital
FIGURE 43.25. Grayson–Wilbrandt dystrophy.
Top left, Slit-lamp photograph shows discrete
pleomorphic opacities in the pre-Descemet
area that have comma-shaped, circular, linear,
filiform, and dot-like configurations. The
intervening stroma is clear. Top right, Phase-
contrast microscopy demonstrates the refractile
vacuolar inclusions (arrows) within a deep
keratocyte. Descemet’s membrane (bracketed
area) is uniformly normal, and endothelial cells
(E) are artifactitiously vacuolated. Toluidine blue
µ1000. Bottom, Transmission electron
micrograph of a keratocyte filled with vacuoles
that have clear to fibrillogranular material (F),
pleomorphic substances (arrowhead), and dark
electron-dense bodies (asterisk). The
surrounding stroma (S) is normal (µ12 000).
Inset, High-magnification transmission electron
micrograph resolves pleomorphism of
accumulated material and the presence of
membranous lamellas (arrow) (µ40 000).
glaucoma, there are no other associated ocular anterior or
posterior segment abnormalities.
Histologic study269–273 reveals nonspecific anterior and
stromal changes consistent with long-standing secondary
edema: basal epithelial cell swelling, basement membrane
thickening and disruptions, and irregularities of Bowman’s layer
with pannus formation. It may be significant, however, that in
some patients, ultrastructural examination discloses greatly
enlarged stromal collagen fibrils sometimes measuring as much
as 600 Å in diameter. Descemet’s membrane is uniform in a
given specimen; it may display diffuse thinning of 3 µm to
massive thickening of 40 µm (normal thickness is 3–5 µm in
neonates and 8–10 µm in adults). The anterior banded layer of
Descemet’s membrane always is present and of relatively usual
thickness; however, the posterior layer consists of multilaminar
basement membrane-like material with fine filaments and of
collagen fibrils with a 550 Å and 1100 Å banded configuration.
With the exception of the lack of guttata, these findings are
similar to those in Fuchs’ dystrophy and thus represent another
example of posterior collagen layer formation by either
primarily or secondarily abnormal endothelium.36,65,273 We
postulate that in patients with thin Descemet’s membranes,
complete endothelial loss occurred in utero such that only the
fetal anterior portion of Descemet’s membrane was secreted.272
In contrast, thickened Descemet’s membranes may be the
product of dystrophic but persistent endothelium having
secreted a hypertrophic posterior collagen layer. The posterior
collagenous layer of Descemet’s membrane in congenital
hereditary endothelial dystrophy contains collagen types I–V
and laminin.274 This distribution of collagen within the pos-
 Corneal Dysgeneses, Dystrophies, and Degenerations
terior collagenous layer supports previous morphologic obser-
vations of fibroblast-like change of the endothelium. CHED
endothelial cells also stain positively for cytokeratin inter-
mediate filaments, typically found in cells of epithelial origin. A
similar pattern is seen in the dytrophic endothelium of
posterior polymorphous corneal dystrophy.275
Evaluation and management of young children with CHED
is challenging and best performed in collaboration with a
pediatric ophthalmologist. Visions are difficult to measure in
the first years of life. Despite the special and multiple
challenges of pediatric keratoplasty, clear corneal transplants
and improved vision are possible, but amblyopia is too often a
limiting factor.276,277 Better surgical and visual outcomes may be
attained if corneal transplantation is delayed until fusion is lost,
as determined by nystagmus or exophoria changing to exotropia.
The frequent finding of enlarged stromal collagen fibrils
suggests some primary developmental abnormality of both
FIGURE 43.26. Congenital hereditary
endothelial dystrophy. Top left, Clinical
photograph of a mildly affected 20-year-old
woman shows diffuse corneal haze and visual
acuity of 20/200. Top center, On slit-lamp
biomicroscopy, diffuse edematous thickening of
the corneal stroma is evident in the same
patient. Top right, Comparison of similarly
prepared survey light micrographs of congenital
hereditary endothelial dystrophy (a) and normal
human cornea (b). Note the extraordinary
increase in the thickness of the stroma in the
former. H & E µ60. Bottom, upper right inset,
Light micrograph of the edematous stroma
demonstrates vesicular water clefts (asterisks).
PAS µ200. Bottom, upper middle inset, Electron
micrograph of the central stroma shows the
cross-sectioned collagen fibrils to have
enlarged diameters (~500 Å) with some at
700 Å (arrowheads) (µ45 000). Bottom, main
figure, Transmission electron micrograph of the
posterior cornea. The anterior portion of
Descemet’s membrane (DM) appears to have
banding of normal thickness, but the posterior
collagenous layer is markedly thickened
(8–15 mm). In addition, an abnormal posterior
collagenous layer is present (asterisk). No
endothelial cells are present. S, stroma; AC,
anterior chamber (µ10 240). Bottom, lower
inset, At higher magnification, the components
of the posterior collagenous layer are visible as
fine filaments (~12 nm diameter) interspersed
with basement membrane-like material
(asterisk) (µ50 000).
CHAPTER 43
keratocytes and endothelium, perhaps qualifying this disorder
as another example of mesenchymal dysgenesis.40
Cornea Guttata
Cornea guttata is usually initially evident as a primary con-
dition in middle to older age groups. Slit-lamp examination
reveals a typical beaten-metal appearance of Descemet’s mem-
brane (Fig. 43.27). These wart-, anvil-, or mushroom-shaped
excrescences are abnormal elaborations of basement membrane
and fibrillar collagens by distressed or dystrophic endothelial
cells. The endothelial cells over these excrescences become
attenuated and eventually die prematurely. The lesions often
are located in the axial areas of the cornea and may be
distributed sparsely. Brownish pigmentation often is seen at the
level of the guttata (clinically misnamed ‘pigmented guttata’),
as this in fact represents pigment phagocytosis by the endo-
thelium. 525
 CORNEA AND CONJUNCTIVA
SECTION 6
Guttata located in the periphery of the cornea may be seen
even in young patients; these are called Hassall–Henle bodies
and are of no clinical concern. If, however, the guttata become
more numerous and central, this may portend functional com-
promise of the endothelial cells to the extent that their barrier
and pump functions become insufficient. In this event, stromal
edema occurs, followed by epithelial edema and bullous kera-
topathy, and the condition may then be appropriately termed
Fuchs’ dystrophy. The presence of central guttata without
edema does vary with age: 3% of patients between ages 20 and
40 years, 10% of patients over 40, and 18% of patients over 50.
However, in individual cases mild to moderate guttata can
remain stationary for years without obligate dystrophic
progression.
Secondary cornea guttata is usually associated with dege-
526 nerative corneal disease, trauma, or inflammation. The corneal
FIGURE 43.27. Cornea guttata. Top left, Slit-
lamp photography shows stromal edema and
folds in Descemet’s membrane with metal-
beaten appearance. Top right, Extensive
endothelial guttae are demonstrated by
retroillumination. Left, upper inset, By light
microscopy, excrescences (arrows) of
Descemet’s membrane are evident with loss of
endothelial cells. PAS µ100. Left, middle inset,
Specular photomicrograph of the endothelial
mosaic represents such guttae as dark holes.
Bottom right, Transmission electron micrograph
features a thickened Descemet’s membrane
with individual guttae (asterisk) (µ3000). Left,
bottom inset, At higher magnification, the
guttata are resolved as fine filaments, multiple
segments of basement membrane material, and
collagen in long-spacing configuration
(arrowheads) (µ40 500).
endothelial cells may be affected adversely by iritis, deep
stromal inflammation or infection, and anterior segment
surgery. In severe inflammation, the endothelial mosaic may be
affected by edema of the endothelial cells,278 a condition
resembling cornea guttata. On removal of the causative agent,
the pseudoguttata subside, whereas the guttae of true cornea
guttata are permanent.
The normal endothelial pattern can be well demonstrated
with nitroblue tetrazolium stain. The cells form a uniform
mosaic. If trypan blue stain is used, the decreasing endothelial
viability is noted by staining of the nuclei. An abnormal endo-
thelial cell population is suggested by abnormally sized and
shaped cells (polymegathism and pleomorphism), numerous
guttata, and areas of Descemet’s membrane that are not covered
by cells. Specular microscopy also can be used to study in vivo
the size, shape, and number of endothelial cells.279
 Corneal Dysgeneses, Dystrophies, and Degenerations
Pachymetry of the corneal stroma is extremely helpful in
monitoring the functional status of the endothelium. Cornea
guttata per se does not require treatment. If serial pachymetry
measurements over time remain stable, then the patient can
be reassured of retaining vision and deferring corneal transplan-
tation. In contrast, if endothelial decompensation and stromal
edema progress to visually incapacitating and/or painful epi-
FIGURE 43.28. Late hereditary endothelial
dystrophy (Fuchs’). Top left, upper and lower,
Clinical photographs of moderately advanced
cases illustrate severe stromal edema and
surface irregularities secondary to epithelial
microcysts and coalescent bullae. Top right
inset, Light microscopy demonstrates
intraepithelial edema, thickening of the
basement membrane, subepithelial bullae
(asterisk), and fibrocellular pannus with an
adjacent break in Bowman’s layer. H & E µ350.
Top right, Transmission electron micrograph of
basal epithelial cells and Bowman’s layer shows
multilaminar basement membrane complexes
(BM), the sequela of chronic epithelial edema
(µ25 000). Bottom, main figure, Transmission
electron micrograph of posterior cornea shows
unremarkable stroma (S) and anterior
Descemet’s membrane (D), but remarkable
thickening of the posterior Descemet’s
membrane to 12 µm with additional
superimposition of large guttae (G). The
remaining endothelial cells (En) are severely
degenerated and attenuated (µ5000). Bottom
inset, By scanning electron microscopy, the
comparable picture shows disconnected and
enormously attenuated endothelial cells (En)
and numerous exposed mushroom-shaped
excrescences (asterisk) projecting from the
posterior collagenous layer (µ1600).
CHAPTER 43
thelial edema, then medical measures or corneal trans-
plantation may be indicated.
Late Hereditary Endothelial Dystrophy (Fuchs)
First described in 1910, Fuchs’ dystrophy (Fig. 43.28)280 usually
is seen in the fifth or sixth decade of life and is more common
in women. It is bilateral and commonly of dominant 527
 CORNEA AND CONJUNCTIVA
inheritance.281–283 The dystrophy has been linked to
chromosome 1p34.3–p32 in the region of the COL8A2 gene,
which encodes the alpha-2 chain type VIII collagen, a
component of endothelial basement membrane.284,285 Serial
analysis of gene expression demonstrates diminished expres-
sion of mitochondrial pump function and antiapoptotic defense
genes.286 The fundamental functional defect is progressive
deterioration of the endothelium. The endothelial cells in
adults lack significant mitotic capability, and as they undergo
attrition, the surviving cell population must enlarge and spread
to maintain an intact monolayer and to remain functionally
competent as a barrier and pump in maintaining corneal
deturgescence. Thus, as in patients with cornea guttata, serial
pachymetry, specular microscopy279 and confocal microscopy
are helpful in following the disease process. Specular
microscopy demonstrates dark areas, corresponding to the
guttata, within the normally uniform endothelial cell mosaic.
Both discrete (guttate) and diffuse thickening of Descemet’s
membrane usually develop, with progressive endothelial
degeneration and dysfunction leading to advancing stromal
edema and decreased corneal transparency.
Clinically significant edema starts axially and spreads peri-
pherally. As stromal edema progresses to involve the
epithelium, microbullous elevations of the epithelium produce
irregularities of the tear film with conseqent decreased visual
acuity, and in time, macrobullous keratopathy erupts with
profound visual compromise. When these epithelial blisters
rupture, patients experience foreign body sensation or pain,
which may be symptomatically relieved by lubricants,
occlusion, or a bandage soft contact lens. On histologic
examination, the sequelae of chronic epithelial and stromal
edema are prominent. Anteriorly, abnormalities of the
basement membrane adhesion complexes develop because of
repeated liftoff of the edematous epithelium.287 There are
occasional breaks in Bowman’s layer, and subepithelial debris
and fibrovascular pannus collect in the zone of bullous edema.
The most striking abnormality (especially vivid with PAS
staining) is diffuse thickening of Descemet’s membrane (often
to 20 mm or more) and posteriorly projecting excrescences,
corresponding to clinically apparent guttata. The remaining
endothelial cells are flattened and attenuated, corresponding to
the clinically observed decrease in cell population density with
compensatory increased cell size and polymegathism.
Histologic evidence of abnormal endothelial cell function is
apparent many years before the clinical signs of cornea guttata
and thickened Descemet’s membrane appear.288
Ultrastructural examination shows the newly deposited
abnormal portion of Descemet’s membrane to consist of
SECTION 6
bundles and sheets of widely spaced banded collagen and
multiple laminations of basement membrane material. This
abnormal posterior collagenous layer of Fuchs’ dystrophy can be
considered analogous to the deposition of excess collagen and
basement membrane material found in other circumstances of
the ‘endothelial distress syndrome’.36,66
A number of surgical considerations arise in patients with
endothelial dystrophy. Excimer laser vision correction should be
undertaken cautiously and only in patients with mild stages of
the dystrophy, as nonresolving corneal edema after routine
LASIK surgery has occurred.289 As both cataract and Fuchs’
dystrophy tend to be progressive disorders of older age, the
approach to such patients must be individualized, favoring
cautious cataract surgery alone (i.e., copious use of viscoelastics,
minimal phakoemulsification energy and duration) in younger
patients with milder stages of dystrophy (i.e., no epithelial
edema and pachymetry < 640 mm).290 Patients with more
advanced corneal changes and denser cataract benefit from the
528 combined or ‘triple’ procedure of penetrating keratoplasty with
extracapsular cataract extraction and posterior chamber IOL
implantation. Fuchs’ dystrophy is a common indication for
corneal transplantation (~15–20% of cases) in predominantly
caucasian patients but is far rarer in the oriental population.291
Keratoplasty is generally very successful as 5- and 10-year graft
survival rates are 97% and 90%, respectively.292 The recent and
ongoing development of deep lamellar endothelial keratoplasty
is especially exciting as this approach allows tissue specific
replacement of diseased endothelium and Descemet’s mem-
brane without surface or stromal incision or sutures, thereby
producing more rapid visual rehabilitation and less post-
operative astigmatism.293 In rare cases for which keratoplasty is
not indicated due to other vision-limiting factors (e.g., advanced
glaucoma or optic atrophy), then anterior stromal puncture,
surface cauterization or amnion membrane transplantation
may afford symptomatic relief.
Posterior Polymorphous Dystrophy
Posterior polymorphous corneal dystrophy (PPCD), originally
described by Koeppe,294 is a bilateral, usually dominantly
transmitted corneal dystrophy that may be stationary or only
slowly progressive, such that affected patients generally retain
normal visual acuity and demonstrate no stromal edema or
vascularization.295–297 Based on recent genetic studies, the
Human Genome Nomenclature Committee has identified
several loci for PPCD284,297,298: PPCD 1 on chromosome 20q11;
PPCD 2 in the COL8A2 gene on chromosome 1p, which is also
associated with Fuchs’ endothelial dystrophy; and PPCD 3 on
chromosome 10. Transcripts of all three identified genes are
present in corneas. However, other unidentified genes appear to
be involved as some cases of clinically and pathologically typical
PPCD do not map to these loci. The co-occurrence of both
PPCD and keratoconus has been reported. Mutations in the
VSX1 homeobox gene may be responsible for both phenotypes.
The condition is characterized by polymorphous opacities,
some of which are vesicular or annular with surrounding halos,
at the level of Descemet’s membrane (Figs 43.29 and 43.30).
Broad peripheral anterior synechiae also is a characteristic
feature, present in up to 27% of patients.296,299 Although careful
biomicroscopy usually is adequate to establish the diagnosis,300
specular microscopy301 and confocal microscopy may be helpful
in differentiating posterior polymorphous dystrophy from other
corneal endothelial disorders (Fig. 43.31). Iris abnormalities
include corectopia, papillary ectropion and rare Desce-
metization of the iris surface by endothelial outgrowth. Elevated
intraocular pressure has been described in 14% of 59 PPCD
patients.295 Elevated intraocular pressures were present in 62%
of patients with PPCD undergoing keratoplasty, all of whom
displayed iridocorneal adhesions. The differential diagnosis of
PPCD includes Fuchs’ endothelial dystrophy, iridocorneal
endothelial (ICE) syndrome, tears in Descemet’s membrane and
interstitial keraitis.
Numerous histologic studies have demonstrated endothelial
cells that morphologically and immunopathologically resemble
epithelium302–304 (see Figs 43.29 and 43.30). These cells contain
epithelial keratin and are connected by well-developed
desmosomes. Scanning electron microscopy of the posterior cell
membrane reveals myriad microvilli, again suggestive of an
epithelium-type cell. Ultrastructural studies have also revealed
some endothelial cells that resemble fibroblasts.295 Descemet’s
membrane is also pathologic as the normal anterior banded
zone is accompanied by a thickened posterior collagenous layer
comprised of disorganized collagen and basement membrane
material. An aberrant developmental differentiation of the
endotheliogenic mesenchyme (neural crest) has been
suggested,39 possibly similar to the pathogenesis of the
iridocorneal endothelial syndromes.
 Corneal Dysgeneses, Dystrophies, and Degenerations
In some cases, endothelial decompensation occurs and
stromal edema develops with decreased vision necessitating
keratoplasty. Preexisting glaucoma and iridocorneal adhesions
are implicated in graft failiure. The dystrophy can recur in
grafts. Short-term followup suggests LASIK is safe and effective
in mildly affected PPCD patients.305
Iridocorneal Endothelial Syndrome
Chandler’s syndrome, essential iris atrophy, and iris nevus
or Cogan–Reese syndrome have been regarded as variations
of a single disease process and pathogenetic mechanism,
FIGURE 43.29. Posterior polymorphous
dystrophy. Top left, Broad slit-lamp illumination
reveals multiple coalescent posterior corneal
vesicles with surrounding halos. Top right, In a
similar case, retroillumination highlights band-
like and polymorphous configurations of the
posterior cornea. Middle left, Scanning electron
microscopy discloses an epithelial-like cell with
characteristic myriad microvilli lining the
posterior corneal surface (µ1000). Bottom,
Transmission electron micrograph illustrates
other features of these multilayered cells, such
as desmosomal attachments (circles), and
bundles of cytoplasmic filaments (arrows). Dm,
Descemet’s membrane; Ac, anterior chamber
(µ19 000). Middle right, Higher-magnification
transmission electron microscopy shows details
of the microvilli as seen in transverse and
longitudinal sections. Note resolution of the
central filamentous core typical of cilia
(µ87 500).
CHAPTER 43
the so-called iridocorneal endothelial (ICE) syndrome
(Fig. 43.32).306–310 These conditions are predominantly
sporadic, almost always unilateral and generally arise in early
adulthood, usually in women. Evidence of herpes simplex viral
DNA or antigens has been detected in ocular tissues of ICE
syndrome patients, although the significance is unclear.
Typical of Chandler’s syndrome is corneal edema secondary
to endothelial abnormality, usually accompanied by ipsilateral,
unilateral glaucoma. The degree of corneal edema is severe
relative to the level of intraocular pressure. The various iris
changes (stromal thinning, full-thickness holes, ‘nevi’, and broad, 529
 CORNEA AND CONJUNCTIVA
SECTION 6
tenting peripheral anterior synechiae) vary, depending on the
subcategory of ICE syndrome. Campbell and colleagues307 have
proposed that the primary abnormality resides in the corneal
endothelium, which, besides malfunctioning and allowing
corneal edema in Chandler’s syndrome, tends to grow across
angle structures and iris surface, elaborating a Descemet’s
membrane-like tissue. Contraction of the membrane then leads
not only to anterior synechiae but also to pupillary distortion
and the iris abnormalities seen to a greater or lesser extent in all
of the ICE syndromes. Glaucoma is common, as high as 77% in
one large series, all accompanied by iridocorneal adhesions.308
Mechanisms for glaucoma include iridocorneal adhesions and
overgrowth of trabecular meshwork with ectopic endothelium.
Chandler’s syndrome must be differentiated from Fuchs’
dystrophy and posterior polymorphous dystrophy. The latter
530 may also be considered within the spectrum of ICE syndromes
FIGURE 43.30. Posterior polymorphous
dystrophy. Top right, Slit-lamp photograph
demonstrates focal thickenings (arrows) of
varying size at the level of Descemet’s
membrane. Upper center inset, Phase-contrast
photomicrograph of guttae changes in
Descemet’s membrane with many irregularly
shaped excrescences and deteriorating
endothelial cells. PPDA µ400. Lower center
inset, Phase-contrast photomicrograph shows
the posterior stromal pit as an infolding of
Descemet’s membrane. Note continuity of the
fibrocellular tissue (filling central cavity) with
posterior collagen layer of Descemet’s
membrane (DM). PPDA µ400. Bottom, main
figure, Transmission electron micrograph shows
Descemet’s membrane to have a normal-
appearing anterior-banded zone (diamond) and
an extremely thickened posterior portion
(asterisk). Normal endothelium is absent;
instead, epithelium-type cells with numerous
microvillous projections, desmosomal
attachments (arrows), and aggregates of
keratofibrils are seen. µ12 800. Middle right,
Scanning electron microscopy discloses a
geographic area of endothelial cell
degeneration exposing a fibrillar posterior
collagen layer. The remaining cells are
configured bizarrely, with extended cytoplasmic
processes (µ540).
Lower center inset, From Henriquez AS, Kenyon KR,
Dohlman CH, et al: Morphologic characteristics of
posterior polymorphous dystrophy. A study of nine
corneas and review of the literature. Surv Ophthalmol
1984; 29:139.
because a similar pathogenic defect in the corneal endothelium
may be implicated, possibly reflecting abnormal proliferation or
induction of embryonic neural crest cells.39 In contrast to the
ICE syndromes, however, posterior polymorphous dystrophy is
familial and bilateral and without similar iris findings, except
peripheral anterior synechiae in some patients. In addition to
posterior polymorphous dystrophy, the abnormal beaten-metal
appearance of Descemet’s membrane in Chandler’s syndrome
resembles that of Fuchs’ dystrophy. Specular microscopy may
prove useful in better describing the in vivo characteristics of
these conditions.311
Histopathologic study of Chandler’s syndrome has revealed
abnormalities in the mesenchymally derived cells lining the
cornea, trabecular meshwork, and anterior iris surface.312–314
The corneas typically exhibit a posterior collagenous layer
containing collagen types III, IV, V, and VIII. The abnormal
 Corneal Dysgeneses, Dystrophies, and Degenerations

FIGURE 43.31. Posterior polymorphous dystrophy. Pathognomonic specular microscopy patterns occurring in the same patient range. From
mild polymegathism and pleomorphism (top left) to discrete geographic areas (arrows) with minimal residual normal-appearing endothelial cells
(asterisks) (top and bottom right). Bottom left, Grouped vesicles make up dark rounded central areas (asterisk) surrounded by halos of abnormal
endothelial cells.

single- or multilayered endothelium extends from the cornea 50 and 230 per 100 000 population.318 Keratoconus occurs in
over the trabecular meshwork and, in some specimens, onto the all races and has a female preponderance. Despite being com-
iris. Elaboration of excessive multilaminar basement membrane mon, its causes and pathogenesis remain poorly understood.

by flat and discontinuous corneal endothelial cells is further
evidence of an endothelial distress syndrome.315
Medical and surgical control of glaucoma in ICE syndrome is
difficult with poorer success rates compared to other types of
glaucoma. Trabeculectomy with 5-fluoruracil or mitomycin-C
and aqueous shunt surgery have been described, with multiple
surgical procedures and revisions frequently required. Pene-
trating keratoplasty is indicated for visually significant corneal
edema. Clear corneal transplants have moderately high prog-
nosis, although chronic intraocular inflammation, glaucoma and
rejection compromise the long term success.316,317 Recurrence
of abnormal endothelium may occur in the grafted cornea.
NONINFLAMMATORY CORNEAL ECTASIAS
Keratoconus
Keratoconus is a bilateral, noninflammatory condition charac-
terized by axial ectasia of the cornea (Fig. 43.33). Reported
estimates of its frequency vary widely, but most range between
CHAPTER 43
Although familial in nature, no exclusive pattern of inheritance
exists. Large studies have suggested that the frequency of
inheritance is 6–8%.319 Feder believes that it is reasonable to tell
patients that the chances of a blood relative developing
symptoms of the disease are less than 1 in 10.320
Subtle irregular astigmatism often is the first clinical finding
in keratoconus, and this is evidenced by a distortion of the
corneal image as noted with the Placido disk, retinoscope,
keratometer, keratoscope, and computerized keratographs.
Among computer assisted corneal topography devices, the
development of the scanning slit combined with Placido disk
instrumentation (Orbscan II, Bausch & Lomb) is especially
useful, particularly in the early diagnosis of subclinical cases
which might by all other criteria appear appropriate for excimer
laser vision correction. Keratoconus manifests at puberty and
can progress either slowly, stabilizing over the course of ~10
years, or relatively rapidly, requiring keratoplasty. Thinning of
the cornea with conical protrusion of the apex occurs, such that
in downgaze, the lower lid is distorted by the cone (Munson’s 531
 CORNEA AND CONJUNCTIVA
SECTION 6
sign; Table 43.4). Ultrasonic pachymetry, especially if performed
serially over time is, in conjunction with topography, an important
objective means of monitoring the progression of the disease.
Two types of cones have been described: a well-demarcated
nipple-shaped cone and a larger, oval or sagging cone.321 The
apex of the nipple cone usually is slightly inferonasal, whereas
the oval-shaped cone often is slightly displaced to the infero-
temporal quadrant and extends closer to the periphery. The
apex of the cone often exhibits subepithelial scarring. Vertical
stress lines (Vogt’s striae) are seen deep in the affected stroma.
Increased visibility of the corneal nerves and Fleischer’s iron
ring are additional diagnostic signs. The latter is caused by a
deposition of hemosiderin pigment deep in the epithelium and
Bowman’s layer at the base of the cone.
An early histopathologic change is focal disruption of
532 Bowman’s layer,322 which is replaced in affected areas with
FIGURE 43.32. Iridocorneal endothelial
syndrome. Top right, Clinical appearance of a
cornea in a 48-year-old woman with unilateral
stromal edema, peripheral anterior synechia, iris
atrophy, and glaucoma. Bottom left, Phase-
contrast photomicrograph illustrates posterior
stroma (S), relatively normal anterior
Descemet’s membrane (D), and the ~10-mm-
thick posterior collagen layer (bracketed area).
The endothelial layer is irregular and
discontinuous. PPDA µ400. Main figure,
Transmission electron micrograph shows the
corresponding area with posterior stroma (S),
ultrastructurally normal Descemet’s membrane
(D), thick posterior collagen layer (between
arrows), and an attenuated endothelial cell
(µ9000). Middle right inset, Higher magnification
of area indicated by asterisk in main figure to
resolve basement membrane-like material, fine
filaments, and long-spaced banding patterns of
the posterior collagen layer (µ50 000). Lower
right inset, Scanning microscopy of
keratoplasty specimen shows an attenuated
endothelial cell (En) extending numerous
cytoplasmic processes (µ2000).
keratocytes and collagenous material. The epithelium is
irregular in thickness and has an abnormal basement
membrane in areas where Bowman’s layer is destroyed.323,324
Stromal changes, even in areas of extreme thinning, are
nonspecific.
Acute hydrops may occur when Descemet’s membrane is
stretched beyond its elastic breaking point. Such a rupture leads
to sudden, profound corneal edema. Endothelium bridges the
gap in 6–8 weeks, with resultant stromal deturgescence and
residual stromal scarring of varying severity. Although the
abrupt onset of often massive corneal edema and profound
visual loss is startling, management remains conservative me-
dical therapy (patching, bandage soft contact lens, lubricants,
hypertonic agents and occasionally topical steroids) plus
reassurance that the situation should resolve spontaneously
within 3 months. Thus there is never urgent indication for
Corneal Dysgeneses, Dystrophies, and Degenerations

FIGURE 43.33. Keratoconus. Top left, Clinical

photograph in lateral projection demonstrates
extreme anterior protrusion of the markedly
ectatic cornea. Top right, Munson’s sign. The
V-shaped conformation of the lower lid is
produced by the ectatic cornea in downgaze.
Upper middle left, Acute hydrops due to a
break in Descemet’s membrane is
accompanied by extreme stromal and epithelial
edema. Upper middle right, Keratoscopic view
of typical egg-shaped appearance of the
central corneal mires caused by inferotemporal
steepening. Lower middle left, Corneal
retroillumination is a useful technique to identify
the position and extent of the cone. Bottom
left, ‘Fish-mouth’ break in Descemet’s
membrane remains after resolution of corneal
hydrops. Bottom right inset, Light micrograph
of a cornea with healed hydrops shows a ledge
formed by detached Descemet’s membrane (D)
and endothelium (e). New regenerated
endothelium (ne) lines the anterior surface of
the ledge and posterior stroma (s). AC, anterior
chamber. Phase-contrast, PPDA µ400. Bottom
right, Electron micrograph of area indicated by
square in inset demonstrates normal
ultrastructure of endothelium (e) and
Descemet’s membrane (D). A thin basement
membrane (BM) is subjacent to the new
endothelium (NE). K, keratocyte (µ8100).

CHAPTER 43

533
 CORNEA AND CONJUNCTIVA
TABLE 43.4. Differential Diagnosis of Noninflammatory Corneal Ectasias
Keratoconus Pellucid Marginal Degeneration
Frequency Most common Less common
Laterality Usually bilateral Bilateral
Age at onset Puberty Age 20 to 40 years
Thinning Inferior paracentral Inferior band 1 to 2 mm wide
Protrusion Thinnest at apex Superior to band of thinning
Iron line Fleischer’s ring Sometimes
Scarring Common Only after hydrops
Striae Common Sometimes
From Feder RS: Noninflammatory ectatic disorders. In: Krachmer JH, Mannis MJ, Holland EJ, (eds.): Cornea. 2nd edn. St Louis: CV Mosby; 2005
keratoplasty, rather elective keratoplasty should be reserved for
cases that persist longer than 3–4 months. Ultrastructural
examination in areas of healed hydrops has shown the torn
edges of Descemet’s membrane to have retracted as scrolls and
the disrupted endothelium to have migrated across the exposed
surface of posterior stroma, depositing new Descemet’s
membrane material and renewing continuity of the endothelial
monolayer.325
Keratoconus can occur in association with a variety of ocular
and systemic diseases, including atopic dermatitis,326 vernal
catarrh,327 Down’s syndrome,328,329 retinitis pigmentosa,330
infantile tapetoretinal degeneration,331 Marfan’s syndrome,332
and aniridia,333 and in patients with blue sclera, Ehlers–Danlos
syndrome, and osteogenesis imperfecta type I. The association
with atopy and vernal keratoconjunctivitis has led to speculation
that frequent, vigorous eye rubbing may aggravate, accelerate, or
even cause keratoconus.334,335 Some investigators, moreover,
have inferred contact lens wear as causative.336
Initial treatment requires astigmatic spectacle correction or
various combinations of rigid gas permeable and/or toric soft
contact lens that compensates for the irregular corneal
astigmatism. When lens fit or comfort becomes a problem due
to a focal elevated pannus over the apex of the cone, superficial
keratectomy may be performed to smooth the corneal surface.
Excimer laser phototherapeutic keratectomy may also be
cautiously applicable in similar circumstances.337 Thermo-
keratoplasty generally is only a temporary measure because
SECTION 6
resteepening, scarring, or persistent epithelial defects usually
ensue.338 and, like epikeratoplasty, is currently seldom per-
formed (except occasionally for persistent hydrops). Contact
lens-intolerant patients with clear central corneas may benefit
from reduction of astigmatism by surgical insertion of
intracorneal ring segments (INTACS).339 Deep lamellar
keratoplasty is highly useful in special situations of clinical
circumstance (e.g., Down’s syndrome) or surgical difficulty (e.g.,
thin corneal periphery requiring large diameter keratoplasty
with consequent high rejection risk). In most cases, however,
penetrating keratoplasty remains a highly successful procedure
of choice for long-term visual rehabilitation of advanced
cases.340 Postkeratoplasty myopia can be reduced by using the
same-sized donor and host, if the anterior lens-to-retina length
is not less than 20.19 mm.341,342
Pellucid Marginal Degeneration
Pellucid marginal degeneration is characterized as a bilateral,
534 peripheral corneal ectasia with an inferior band of corneal
Posterior
Keratoglobus Keratoconus
Rare Least common
Bilateral Usually unilateral
Usually at birth Birth
Greatest in periphery Paracentral posterior
excavation
Generalized Usually none
None Sometimes
Mild Common
Sometimes None
thinning 1–2 mm in width. The area of thinning usually
extends from the 4 o’clock to the 8 o’clock position and is
located 1–2 mm central to the inferior limbus. The protruding
cornea usually is central to the area of thinning and remains of
normal thickness (see Table 43.4). The abnormal contour
usually induces a shift in the axis of astigmatism from against-
the-rule superiorly, to with-the-rule, at the top of the protrusion
(Fig. 43.34). There is no sex or racial predilection, nor does it
appear to be inherited. These corneas are clear, avascular, and
without apical scarring, lipid deposition, or iron ring. Corneal
topography is an especially useful diagnostic aid. There is some
consensus that keratoconus, keratotorus, keratoglobus, and
pellucid degeneration are related because these different
conditions have been found to coexist in families. Histo-
pathologic reports demonstrate similar abnormalities in these
various disorders.
Because of extremely abnormal corneal topography, the
treatment of pellucid zone degeneration is difficult. Contact
lens wear should be attempted initially. If the patient is contact
lens intolerant, a large-diameter, penetrating keratoplasty may
be performed.343 Alternatively, tectonic lamellar grafting of
the thinned periphery may be followed by a central penetrating
keratoplasty.344 Krachmer345 has suggested that thermo-
keratoplasty may be a reasonable alternative.
Keratoglobus
Keratoglobus (Fig. 43.35) is a rare bilateral condition resembling
megalocornea, with the exception that the cornea in
keratoglobus is uniformly thinned, particularly peripherally (see
Table 43.4). Corneal scarring may be seen, but an iron ring is
not observed. No definitive inheritance pattern has been
demonstrated. A familial association between keratoconus and
keratoglobus has been made.346 Acquired keratoglobus has been
described in association with hyperthyroidism and after
unilateral preexisting keratoconus.347,348 Rupture of Descemet’s
membrane may occur, as in keratoconus, but this is not usually
the case.346,349 Especially in cases associated with Ehlers–
Danlos syndrome type VI, patients must be cautious to avoid
even minor ocular trauma because rupture of the globe can
occur easily, and repair is difficult. Unlike keratoconus,
keratoglobus is not associated with atopy.
Spectacle correction may help to achieve functional vision in
addition to providing protection from corneal rupture. Contact
lens fit is difficult, and surgical intervention should be con-
sidered when functional vision cannot be obtained. In general,
surgery should be delayed, when possible. Large-diameter,
 Corneal Dysgeneses, Dystrophies, and Degenerations

FIGURE 43.34. Pellucid marginal degeneration.

Left top and bottom, Clinical photographs
feature corneal ectasia occurring above the
narrow band of clear, thin, nonvascularized
cornea that parallels the inferior limbus
(arrows). Right top and bottom, Slit-lamp view
and corresponding illustration show normal
corneal thickness central and peripheral to the
band of thinning (arrow).
Bottom right, From Krachmer JH, Feder RS, Belin MW:
Keratoconus and related noninflammatory corneal
thinning disorders. Surv Ophthalmol 1984; 28:293.

CHAPTER 43

FIGURE 43.35. Keratoglobus. Left, Clinical photograph of acquired keratoglobus shows globoid protrusion of clear, diffusely thin cornea.
Corneal thickness is one-third normal. Right, Horizontal pupil–optic nerve section of this eye reveals bulging cornea and deep anterior chamber.
The entire cornea is about one-third normal thickness, except in extreme periphery nasally and temporally. H & E µ4.
Right, From Jacobs DS, Green WR, Maumenee AE: Acquired keratoglobus. Am J Ophthalmol 1974; 77:393–399. Copyright 1974, Elsevier Science.

535
 CORNEA AND CONJUNCTIVA
tectonic penetrating or preferably lamellar keratoplasty followed
by smaller-diameter, central penetrating keratoplasty may be
appropriate. Alternative surgical approaches include crescentic
lamellar keratoplasty, thermokeratoplasty, epikeratoplasty, and
wedge or crescentic resection.
CORNEAL DEGENERATIONS
PERIPHERAL DEGENERATIONS
Corneal Arcus (Juvenilis and Senilis)
Corneal arcus appears as a whitish ring of the peripheral cornea
separated from the limbus by a clear zone (Fig. 43.36). Arcus
juvenilis is sometimes called anterior embryotoxon. Both the
juvenile and the adult forms represent paralimbal stromal
accumulations of cholesterol esters, triglycerides, and
phospholipids.350–352 Patients younger than 40 years of age with
corneal arcus are at increased risk of coronary artery disease and
should be evaluated for hyperlipoproteinemia. Hyperlipo-
proteinemia types 2 and 3 are associated with premature arcus
formation. These diseases may be primary or secondary.
Diseases causing a rise in b-lipoproteins include nephrotic
syndrome, hypothyroidism, increased cholesterol intake,
obstructive jaundice, and diabetic ketoacidosis. Rare lipoprotein
disorders causing arcus or generalized corneal clouding include
lecithin cholesterol acyltransferase deficiency and Tangier
disease.
In histologic sections, the deposits appear wedge-shaped and
are most prominent near Bowman’s layer and Descemet’s
membrane. Abnormalities in blood lipids may be concomitant
in younger patients displaying corneal arcus or Schnyder’s
crystalline corneal dystrophy.
White Limbal Girdle of Vogt
The white limbal girdle of Vogt type II is a common finding in
patients older than 45 years. It is a white opacity in the medial
and temporal limbal regions and may be mistaken for corneal
arcus. Fine white lines extend irregularly from the limbus. A
clear interval may be present between the girdle and the limbus.
The limbal girdle is not associated with inflammation, is not
vascularized, and does not progress. The incidence of Vogt’s
limbal girdle increases with age, reaching ~55% at 40–60 years
SECTION 6
FIGURE 43.36. Corneal arcus. Left, The arcus lipoides shows a dense white annular opacity of the peripheral stroma. Right, At higher
536 magnification, an intervening zone of clear stroma separates lipid deposition from the limbus.
of age.353 Sugar and Kobernick353 described the pathologic change
in Vogt’s limbal girdle type II as a subepithelial hyperelastosis
with degeneration similar to that in pingueculae and pterygia.
The type I limbal girdle is likely to be more closely related to
early calcific band keratopathy because it appears, as Vogt
described it, as a white band with clear holes at several points
and separated from the sclera by a clear interval.
Idiopathic Furrow Degeneration
Thinning of the cornea in older people in the area of an arcus
senilis sometimes occurs. There is no tendency for this thinned
area to perforate, and no vascularization develops. Visual acuity
is generally not affected.
Furrow Degeneration Associated with Systemic
Disease
Focal or extensive ring-type epithelial defects and sterile
ulceration near the limbus can accompany certain systemic
diseases, such as rheumatoid arthritis, Wegener’s granulo-
matosis, polyarteritis nodosa, relapsing polychondritis,
systemic lupus erythematosus, and other collagen vascular
diseases (Fig. 43.37). The treatment of such immunogenic
diseases is discussed elsewhere.
Postirradiation Thinning
Noninflammatory corneal excavation at the limbus may occur
after high local doses of b-radiation.354
Terrien’s Marginal Degeneration
Terrien’s marginal degeneration is an uncommon but distinct
variety of marginal thinning of the cornea355 (Fig. 43.38). It is
usually bilateral, although often asymmetric, and is seen
mainly in younger men. The condition progresses slowly over
the course of years and generally starts superiorly as a marginal
opacification. Stromal thinning and ectasia develop with an
intact epithelium, and there is a lucid zone between the
advancing edge and the limbus. A yellow border of lipid is
present characteristically at the advancing edge. Vessels traverse
the furrow and pass beyond it. Difficulties arise from the
induced corneal astigmatism, and minor trauma may result in
rupture if thinning is sufficient. Most cases are non-
inflammatory, although patients with recurrent inflammation
 Corneal Dysgeneses, Dystrophies, and Degenerations

FIGURE 43.37. Furrow degeneration associated with systemic disease. Top left, Clinical photograph of a patient with polyarteritis nodosa shows
a full-ring ulcer and associated lipid deposition near the limbus. Top right, Light microscopy of the same eye illustrates peripheral corneal
thinning (arrows) corresponding to the area of clinical ulceration. H & E µ3. Bottom left, Severe necrotizing vasculitis of a medium-caliber artery
confirms the diagnosis. H & E µ100. Bottom right, Light microscopy of a 47-year-old woman with rheumatoid arthritis who developed a
perforated marginal corneal ulcer shows adherent iris incorporated into a fibrous scar. PAS µ32.

have been described.356 Electron microscopy demonstrates
collagen precursors, stromal ground substance, and possibly
lipid phagocytized by histiocytic cells with high lysosomal
activity.355 Therapy is limited to tectonic grafting to prevent or
to repair perforation of thinned areas.
Mooren’s ulcer, in contrast to typical degenerations, is
characterized by a fulminating, centrally progressive, and
painful inflammation occurring more often in males.362–366 The
leading edge of the ulcerative process often undermines the
more central corneal stroma. Two types of Mooren’s ulcer have

Mooren’s Ulcer
Mooren’s ulcer (Fig. 43.39) is probably best considered a
localized inflammatory ulceration rather than a degeneration of
the corneal periphery. It must be differentiated, however, from
entities such as Terrien’s marginal degeneration. It is a diag-
nosis of exclusion; other serious systemic connective tissue
diseases with generalized vasculitis and collagen destruction
must be excluded before the diagnosis of Mooren’s ulcer is
made.
The disease can be divided into two groups: primary and
secondary. Primary Mooren’s ulcer is the classic idiopathic
variety, whereas the secondary Mooren ulcer may be associated
with different insults to the cornea. A Mooren-like ulcer has
been reported after cataract surgery,357 penetrating kera-
toplasty,358 corneal trauma and chemical burns,359 herpes zoster
virus infection,360 syphilis, and tuberculosis. An association has
been reported between Mooren’s ulcer and hepatitis C
infection.361
CHAPTER 43
been described. A benign type is seen in older patients. This
type is usually unilateral and responds to treatment more often
than the more severe type that occurs in younger patients.365
The latter variety is relentlessly progressive and often bilateral.
Young Nigerians have exhibited a severe form of Mooren’s ulcer,
with a rapid progression to perforation and marked involvement
of the limbal sclera and episclera in a necrotizing process.363
Histologic studies reveal necrosis of collagen tissue, with
vessels and chronic inflammatory cells in the adjacent limbal
margin. Autoantibodies to human epithelium have been
demonstrated.364 Polymorphonuclear leukocytes intensely
infiltrate the zone of active ulceration, suggesting that acute
inflammatory cells play a role in the collagenolytic process.367
The results of treatment have not been encouraging. A
stepladder approach to therapy is recommended.366 Initially,
patients are aggressively treated with topical steroids every hour
in addition to topical antibiotics for prophylaxis. If there is no
evidence of clinical improvement, conjunctival excision is
performed.368 Generally, most patients with unilateral disease 537
 CORNEA AND CONJUNCTIVA

FIGURE 43.38. Terrien’s marginal

degeneration. Top left, Clinical photograph of a
patient with extensive thinning of the peripheral
stroma extending circumferentially from the
9-o’clock to the 2-o’clock position. Top right,
Higher magnification discloses vascularization
of the involved stroma with lipid deposition at
the advancing edge. Bottom inset, Light
microscopy reveals numerous foamy histiocytic
cells and blood vessels (asterisk) within the
anterior stroma. H & E µ300. Bottom,
Transmission electron micrograph shows
histiocytic cells laden with neutral lipid
inclusions (circled area). Several reactive
fibroblasts and chronic inflammatory cells also
are seen (µ5000).

or bilateral nonsimultaneous disease respond to this approach.
In more aggressive cases, perforation may occur from colla-
genolytic processes or secondary infection, especially in the
potentiating presence of topical corticosteroids. Tissue adhesive
and lamellar grafting may be necessary in the event of per-
foration.369 Systemic immunosuppression may be of value in
patients with progressive disease.370–374
SECTION 6
viewed with the slit lamp. It was originally thought to be lipid
in nature but probably contains iron from the foreign body.379
The condition causes no symptoms and requires no therapy.
Lipid Degeneration
Lipid degeneration (Fig. 43.40) is characterized clinically by the
accumulation of a yellow or cream-colored diffuse or crystalline
material in the corneal stroma, which may be abnormally thick

or thin. There is typically a history of prior corneal inflam-

CENTRAL OR DIFFUSE DEGENERATIONS matory episodes with resultant stromal vascularization. The
Iron Lines lipid accumulations are, therefore, of a secondary nature, with
Iron deposition in the cornea occurs secondarily in a number of extravasation of cholesterol and fatty acids from the vessels.
clinical settings375–376: Lipid keratopathy has been reported after hydrops380 and as a
Hudson–Stähli line Normal aging cornea finding with no clear antecedent corneal damage or
Ferry’s line377 Adjacent to filtering bleb vascularization.381,382
Stocker’s line Adjacent to head of pterygium
Fleischer’s ring Base of keratoconus cone Amyloid Degeneration
Histologic examination reveals hemosiderin deposition in Acquired corneal amyloidosis can be associated with intraocular
the basal corneal epithelial cells.377 The pathogenesis of corneal disease or may be secondary to corneal trauma.383–386 Such
iron lines is unclear, although they may be related to chronic amyloid deposition may also occur as a result of long-standing
abnormalities of tear flow. Iron lines do not affect vision and are diseases, such as retrolental fibroplasia, trachoma, glaucoma,
asymptomatic. uveitis, bullous keratopathy, keratoconus, and leprosy.387 These
corneal amyloid lesions consist of salmon-pink to yellow-white,
Coat’s White Ring raised, fleshy masses that create a nodular surface (Fig. 43.41)
This small corneal opacity usually is located in an area that and that may be amenable to treatment by superficial kera-
previously harbored a foreign body.378,379 The opacity, which tectomy. The cornea may be vascularized, depending on other
538 contains iron, appears as a small granular oval ring when factors. The deposits seen in lattice and gelatinous dystrophies
 Corneal Dysgeneses, Dystrophies, and Degenerations
also are amyloid in nature, but those conditions are primary
disorders.
Polymorphic stromal degeneration is another manifestation
of primary localized amyloid deposition in the cornea. Thomsitt
and Bron388 described patients with a variety of posterior
stromal opacities consistent with the type of dystrophic change
reported in 1940 by Pillat.389 They described axial polymorphic
star- and snowflake-shaped and branching filamentous stromal
opacities, some of which indented the anterior surface of
Descemet’s membrane, causing an apparent irregularity of the
posterior corneal surface. Punctate opacities were polymorphic,
FIGURE 43.39. Mooren’s ulcer. Top left,
Clinical photograph of a 55-year-old man with
painful and rapidly progressive ulcerative
keratitis. Top right, Same patient 15 days after
conjunctival resection reveals marked
improvement with decreased inflammatory
response and arrest of ulceration. Bottom inset,
Phase-contrast micrograph of the stroma at the
margin of the ulcerating area includes abrupt
termination of Bowman’s layer (arrow) with
numerous acute inflammatory cells. PPDA
µ800. Bottom, Transmission electron
micrograph of the area in bottom inset resolves
multiple intrastromal inflammatory cells actively
engaged in degranulation and phagocytosis.
Note the remnants of the epithelial basement
membrane (arrowheads). E, epithelium; B,
Bowman’s layer (µ7500).
CHAPTER 43
gray-white, and somewhat refractile when examined directly
but were transparent in retroillumination. Because intervening
stroma appeared clear, visual acuity was not markedly affected.
Histochemical staining and electron microscopy have shown
the deposits to be composed of amyloid.390,391 The late
appearance of the linear opacities, the lack of progression, and
the apparent nonfamilial pattern help to distinguish this
condition from lattice dystrophy. Amyloid degeneration also
occurs in association with spheroid degeneration.392,393
The amyloid material in the cornea is identical to that in
other organs. It stains with Congo red, displays birefringence 539
 CORNEA AND CONJUNCTIVA
SECTION 6
and two-color dichroism with the polarizing microscope, and is
fluorescent with thioflavin-T stain and ultraviolet light. Amy-
loid contains protein, carbohydrate, and polysaccharide com-
ponents as well as a-chain immunoglobulins. Ultrastructural
study reveals short fibrils, 90–100-Å in diameter, in a random
pattern of aggregation within a granular background.
Spheroid Degeneration (Climatic Droplet
Keratopathy, Keratinoid Degeneration)
Keratinoid degeneration,393 climatic droplet keratopathy,394–407
proteinaceous degeneration,398 Labrador keratopathy,400–402 and
540 chronic actinic keratopathy402 are likely all similar nonhereditary
FIGURE 43.40. Lipid degeneration. Top left,
Clinical photograph of a dense white deposition
of lipid with feathery edges occurring in
association with superior limbic pannus. Note
that this eye has previously undergone an
intracapsular cataract extraction and secondary
implantation of an anterior chamber intraocular
lens. Top right, Same patient after keratoplasty
shows a clear graft with residual opaque lipid
deposition at the periphery. Upper middle left,
Light microscopy shows an intact Bowman’s
layer with multiple clear vacuoles within the
stroma (asterisks). µ400. Upper middle right,
Phase-contrast microscopy includes numerous
fine osmiophilic deposits (circled area) within
Bowman’s layer. PPDA µ800. Lower middle,
Transmission electron micrograph of the same
area discloses confluent globular empty spaces
below Bowman’s layer (B) as well as some
electron-dense complex lipid deposits (arrows).
E, epithelium (µ40 000). Bottom right,
Transmission electron micrograph of the
anterior stroma illustrates the same type of
deposits without disruption or other
abnormality of keratocytes (K). µ12 000. Bottom
left, At higher magnification, lipid deposits of
~1-µm diameter have the characteristics of
saturated neutral fats (asterisk (µ40 000).
degenerations related to geographic or climatic conditions.405–407
Spheroid degeneration has been classified into three basic
types. Type 1 occurs bilaterally in the cornea without evidence
of other ocular pathology. Type 2, or secondary, spheroid
degeneration occurs in the cornea in association with other
ocular pathology. Type 3 is the conjunctival form of the degen-
eration and may occur concurrently with types 1 and 2.
This degeneration is characterized by yellow, oily-appearing
subepithelial droplets within the interpalpebral fissure,
generally beginning at the periphery (Fig. 43.42). These droplets
may replace Bowman’s layer or may lie deeper. Types of spheroid
degeneration resulting from a local disease or chronic irritant
 Corneal Dysgeneses, Dystrophies, and Degenerations
FIGURE 43.41. Amyloid degeneration. Left, In a patient with long-standing herpes keratitis and subsequent corneal scarring and vascularization,
superficial irregular amyloid deposits developed. Right, Light microscopy of corneal specimen discloses characteristic birefringent, Congo red-
positive amyloid deposits (µ100).
may be unilateral and involve the central cornea as well as the
periphery. Spheroid degeneration has been described in asso-
ciation with lattice dystrophy.
Electron microscopy reveals that the lesions appear to
develop from extracellular material deposited on collagen fibrils.
Some suggest that this material is secreted by abnormal
fibrocytes, forming collagenous spheroids.403 An interaction
between ultraviolet light and plasma proteins within the stroma
FIGURE 43.42. Spheroid degeneration. Top
left, Clinically, numerous spheroidal deposits
appear over the anterior stroma (arrows). Top
right, Histologic section reveals multiple
densely staining spherules beneath the
distorted epithelium and within the anterior
stroma. H & E µ20. Bottom, Survey
transmission electron micrograph shows
spheroidal deposits as extracellular
accumulations of electron-dense material with
variably crystalline structure. Lipid substances
and blood vessels are also evident (µ5000).
Bottom inset, High-magnification transmission
electron micrograph of a spheroidal deposit
shows variable electron density with a
crystalline fragment similar to calcium
(µ40 000).
CHAPTER 43
has also been proposed to result in the abnormal deposits.408
The deposits are PAS-negative but stain positively with rhoda-
mine B – hence the designation keratinoid, even though keratin
is not present. The condition probably is related to elastotic
degeneration of collagen, as in conjunctival pingueculae.407
The conjunctiva may become involved with spheroid de-
generation, often in association with pingueculae. Patients with
spheroid degeneration do not usually have symptoms, but if 541
 CORNEA AND CONJUNCTIVA
FIGURE 43.43. Band keratopathy. Left, In a 42-year-old woman with chronic uveitis, band keratopathy has resulted in epithelial erosion with a
persistent central defect. Center, Light microscopy discloses dense staining within Bowman’s layer. Alizarin red, µ40. Right, Transmission
electron micrograph resolves the fine crystalline characteristic and extreme electron density of calcium or hydroxyapatite particles (µ70 000).
aggravating local factors exist, the disorder may rapidly progress
and predispose to spontaneous sterile ulceration and secondary
microbial keratitis.409
General guidelines for approaching the rehabilitation of
patients with climatic droplet keratopathy with or without
associated cataract have been proposed.411 If the patient is
aphakic or pseudophakic or has a clear lens, excimer laser PTK
may provide primary visual rehabilitation. Because most
patients usually are older and have significant cataract, the
therapeutic options include cataract extraction without ad-
dressing the corneal opacity; combined penetrating keratoplasty
and cataract extraction; combined lamellar keratoplasty and
cataract extraction; or PTK followed by cataract extraction at a
later date. If postoperative PTK is anticipated at the time the
surgical decision is made, it is preferable to begin with PTK and
then to perform cataract extraction ~3 months later. The
combination of lamellar keratoplasty with cataract extraction
avoids the risk of immunologic rejection but is technically more
difficult. This procedure should be limited to cases in which
the anterior pathology is too deep for effective removal with
PTK and there are major contraindications to penetrating
keratoplasty. Penetrating keratoplasty with cataract extraction
has a high risk of failure because these patients usually have
poor goblet cell function, ocular surface wetting, and abnormal
SECTION 6
lid–globe relations. This combination of procedures should be
reserved for cases with both superficial and deep scarring and
for those in which there is a reasonable tear film function and
in which lid–globe anomalies either are absent or have been
addressed with oculoplastic repair.
Band Keratopathy
Band keratopathy (Fig. 43.43) can arise from localized ocular
inflammation or systemic disease. Hydroxyapatite deposits
of calcium carbonate accumulate in the epithelial basement
membrane, Bowman’s layer, and superficial stroma.411,412
Calcific degenerations, phthisis bulbi, necrotic intraocular
neoplasms, and conditions in which bone is formed in other
parts of the eye are common associations.413
Band keratopathy is confined to the interpalpebral fissure
area. A lucid interval separates the calcific band from the
limbus. Small defects in the band are scattered throughout and
probably represent areas where nerves penetrate Bowman’s
542 layer.
Histopathologically, the earliest changes consist of basophilic
staining of the basement membrane of the epithelium; this
is followed by involvement of Bowman’s layer with calcium
deposition and eventual fragmentation.
The factors that stimulate precipitation of calcium salts in the
interpalpebral region of the anterior corneal layers are thought
to involve gaseous exchanges at the corneal surface, leading to
decreased carbon dioxide levels and elevated pH.412 Anatomic
peculiarities in the basement membrane and Bowman’s layer
invite calcium deposition, as does the decreased content of acid
mucopolysaccharides in an edematous cornea.414 The calcific
deposits caused by local disease usually are extracellular. In
systemic hypercalcemia, the deposits are intracellular.
Band keratopathy can also result from deposition of urates in
the cornea415; these customarily are brown, instead of the gray-
white usually seen in calcific band keratopathy.
The instillation of mercury-containing eye drops, as in
glaucoma and dry-eye states, has a circumstantial relation to the
development of band keratopathy in some patients.416–418 The
dry-eye condition itself, through concentration of tear calcium,
also may encourage its deposition near the corneal surface.
Conditions that can result in band keratopathy include the
following:
• Hypercalcemia419–424
• Sarcoidosis (rare)
• Fanconi’s disease
• Still’s disease (nongranulomatous uveitis)
• Hypercalcemia (uremia, parathyroid adenoma)
• Hypophosphatasia
• Multiple myeloma
• Discoid lupus erythematosus
• Hyperphosphatemia
• Vitamin D toxicity
• Metastatic disease (lung and bone disease with increased
calcium)
• Ichthyosis
• Ocular disease
• Chronic nongranulomatous uveitis (juvenile rheumatoid
arthritis)
• Prolonged glaucoma
• Long-standing corneal edema
• Degenerated globe (phthisis bulbi)
• Spheroid degeneration
 Corneal Dysgeneses, Dystrophies, and Degenerations
FIGURE 43.44. Salzmann’s nodular degeneration. Top left and center, Clinical photographs of two different patients with the classic bluish-gray
elevated paraaxial nodules with sparing of the remainder of the cornea. Right, Higher-magnification slit-lamp photograph emphasizes the
minimal vascularization of the underlying stroma.
• Norrie’s disease
• Toxic and mercury vapors
• Irritants and exposure
• Spheroid degeneration
• Noncalcific band keratopathy (urate deposits)415
• Idiopathic causes
Band keratopathy can be treated by superficial keratectomy
either with or without application of the calcium-binding agent,
ethylenediaminetetra-acetic acid (EDTA). After instillation of
topical anesthesia, EDTA 0.4% may be applied to the de-
epithelialized cornea. Superficial keratectomy is then performed
by carefully stripping the calcific scale with forceps and by per-
forming blunt dissection with dry cellulose sponges.425 Band
keratopathy has also been treated successfully using excimer
laser PTK.426
Salzmann’s Nodular Degeneration
Salzmann’s nodular degeneration is noninflammatory and
creates multiple, blue-white, superficial corneal nodules,
usually in the midperiphery (Fig. 43.44). The nodules may
be related to previous inflammation, especially phlyctenular
disease, vernal keratoconjunctivitis, trachoma, or lues and
interstitial keratitis. It has also been reported in patients with
epithelial basement membrane dystrophy, contact lens wear,
and keratoconus and after corneal surgery.427 Although patients
do not often have symptoms, they may develop recurrent
epithelial erosion or decreased vision.
The nodules represent focal areas of subepithelial fibro-
cellular avascular pannus, replacing Bowman’s layer and
superimposed on a normal stroma. Transmission electron
microscopy has shown reduplication of the epithelial basement
membrane in some patients.427
Treatment may include simple stripping of the focal nodules
by superficial keratectomy. Lamellar or penetrating keratoplasty
rarely is required for visual rehabilitation.
Corneal Keloid
Corneal keloids may be found in either the central or the
peripheral cornea and resemble the nodules in Salzmann’s
degeneration. They occur as hypertrophic scars after corneal
injury, inflammation, or surgical trauma. Keloid-like lesions
have also been reported in early life without antecedent trauma.
Immunohistochemical and electron microscopic studies have
demonstrated the presence of myofibroblasts in these lesions,
differentiating them from Salzmann’s nodules.428
CONJUNCTIVAL DEGENERATIONS
Pterygium
Pterygia are triangular, fibrovascular connective tissue over-
growths of bulbar conjunctiva onto the cornea (Fig. 43.45).
They are located horizontally in the interpalpebral fissure on
either the nasal or the temporal side of the cornea. A pigmented
iron line (Stocker’s line) may be seen in advance of a pterygium
on the cornea. The location of the pterygium is determined by
exposure to ultraviolet energy, the amount of which varies
with the geographic latitude.429 True pterygia are found only in
the interpalpebral fissure. Wearing glasses can decrease their
incidence because the ultraviolet transmission is decreased.
A pterygium may progress slowly toward axial cornea or may
become quiescent. Indications of activity are corneal epithelial
irregularity, opacification of Bowman’s layer, and prominence of
CHAPTER 43
active blood vessels and inflammation.
Histopathologic examination reveals the subepithelial tissue
to exhibit elastotic degeneration of collagen, resulting from
breakdown of the collagen and destruction of Bowman’s mem-
brane.430 The subepithelial material stains for elastin but is not
sensitive to elastase.
Generally, pterygium excision is indicated if the visual axis
is threatened or if the pterygium causes extreme irritation.
A pterygium that recurs after excision does so within several
weeks, starting from the excised conjunctival border. The rate of
recurrence is significant – as high as 40% – when a bare scleral
excision is performed. This rate usually is reduced when surgery
is followed by b-radiation treatment with strontium-90.
Treatment with autologous conjunctival transplantation431–433
has been shown to decrease the incidence of recurrence to ~5%,
as has adjunctive treatment with mitomycin drops.434,435
Pseudopterygia occur after chemical injury, corneal
ulceration, or other inflammatory problems in which the con-
junctiva becomes scarred and drawn on the cornea. A probe can 543
 CORNEA AND CONJUNCTIVA

FIGURE 43.45. Pterygium. Top left, Clinical appearance of a typical interpalpebral pterygium shows extension of the fibrovascular conjunctival
tissue on to clear cornea. Top right, Light microscopy of the limbus features a subepithelial mound of inflammatory tissue invading the cornea
(µ20). Bottom, Histologic sections show elastotic degeneration of collagen fibers (circled area, left figure) and positive stain for elastin (asterisk,
right figure). Phosphotungstic acid-hematoxylin µ375; elastin stain µ40.

be passed between this conjunctival bridge and the sclera, a
feature that distinguishes pseudopterygia from true pterygia.
Pinguecula
Like pterygia, pingueculae likely represent an age-related
degeneration associated with ultraviolet and general environ-
mental exposure. Pingueculae appear as raised, cream-colored,
white, or chalky perturbations of the conjunctiva adjacent to the
limbus and within the palpebral fissure. Occasionally, they
become inflamed but generally do not require treatment. As in
the case of pterygia, pingueculae may represent elastotic
degeneration of the substantia propria of the conjunctiva.430

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SECTION 6

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SECTION 6
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366. Brown SI, Mondino BJ: Therapy of
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368. Feder RS, Krachmer JH: Conjunctival
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369. Kenyon KR: Decision-making in the therapy
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370. Foster CS: Systemic immunosuppressive
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374. Hollan EJ, Olsen TW, Ketcham JM, et al:
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375. Gass JD: The iron lines of the superficial
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376. Barraquer-Somers E, Chan CC, Green WR:
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377. Ferry AP: A ‘new’ line of the superficial
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378. Coats G: Small superficial opaque white
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379. Nevins RC Jr, Davis WH Jr, Elliott JH:
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380. Shapiro LA, Frakas TG: Lipid keratopathy
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381. Fine BS, Townsend WM, Zimmerman LE,
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382. Friedlaender MH, Cavanagh HD, Sullivan WR,
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383. Stafford WR, Fine BS: Amyloidosis of the
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384. McPherson SD Jr, Kiffney GT Jr, Freed CC:
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385. Garner A: Amyloidosis of the cornea. Br J
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386. Ramsey MS, Fine BS, Cohen SW:
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387. Rodrigues MM, Zimmerman LE: Secondary
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388. Thomsitt J, Bron AJ: Polymorphic stromal
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390. Mannis MJ, Krachmer JH, Rodrigues MM,
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391. Matta CS, Tabbara KF, Cameron JA, et al:
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392. Santo RM, Yamaguchi T, Kanai A:
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393. Garner A: Keratinoid corneal degeneration.
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394. Freedman A: Climatic droplet keratopathy.
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395. Garner A, Morgan G, Tripathi RC: Climatic
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396. Anderson J, Fuglsang H: Droplet
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397. Ahmad A, Hogan M, Wood I, Ostler HB:
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398. Christensen GR: Proteinaceous corneal
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399. Freedman A: Labrador keratopathy. Arch
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400. D’Alena P, Wood IS: Labrador keratopathy:
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401. Johnson CJ, Ghosh M: Labrador
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402. Klintworth GK: Chronic actinic keratopathy:
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Corneal Dysgeneses, Dystrophies, and Degenerations
403. Hanna C, Fraunfelder FT: Spheroidal
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404. Fraunfelder FT, Hanna C: Spheroidal
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III. Incidences, classification and etiology.
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405. Young YDH, Finlay RD: Primary spheroidal
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406. Fraunfelder FT, Hanna C, Parker JM:
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407. Rodrigues MM, Laibson PR, Weinreb S:
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408. Johnson GJ, Overall M: Histology of
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416. Kennedy RE, Roca PD, Landers PH:
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418. Lemp MA, Ralph RA: Rapid development
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CHAPTER 43
adjunctive treatment for pterygia and its
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551
 CHAPTER
Keratoconus and Corneal Noninflammatory
44 Ectasias
Elisabeth J. Cohen, MD
INTRODUCTION
Keratoconus is a progressive, noninflammatory, thinning
disorder causing irregular corneal astigmatism of unknown
cause. Pellucid marginal degeneration (PMD) is a less common,
but related condition with peripheral corneal thinning.
Keratoconus, typically, has a teenage onset and is usually bila-
teral, but often asymmetric. It occurs in ~1 in 2000, affecting
on the order of 150 000 people in the USA.1 As it is not rare, it
is important to consider the diagnosis in young adults with
increasing myopic astigmatism, especially if it is difficult to get
20/20 visual acuity with a manifest refraction or the patient
complains of decreased quality of vision due to monocular
diplopia, haloes, or ghost images. It is critical to make the
diagnosis of keratoconus, as it is a contraindication for most
refractive surgery. In addition, in recent years it has been
recognized that patients who are suspects for keratoconus,
i.e., have forme fruste keratoconus, on the basis of abnormal
corneal topography alone with normal slit-lamp biomicroscopy
exams, are not good candidates for refractive surgery. These
patients are at increased risk of postoperative complications,
particularly corneal ectasia.
After a teenage onset, keratoconus typically progresses and
then stabilizes in the fourth decade. However, relatively little
has been published about keratoconus in older patients. In a
study of the demographics of keratoconus, less than 10% of
patients were over age 50.2 It was concluded that either kerato-
conus is associated with premature death or that older patients
are followed locally and not at referral centers, which is probably
more likely. Recent publications regarding patients followed for
more that ten years after penetrating keratoplasty (PK) for
keratoconus have shown evidence of late increases in astigma-
tism and inferior peripheral thinning and steepening consistent
with progressive keratoconus in the host.3 Therefore, it is
possible that keratoconus progresses for a longer time than is
often thought, at least in some patients. Generally, patients with
onset before 18 years of age have worse disease with shorter
time to PK than patients who are older at the time of diagnosis.4
Keratoconus is usually an isolated condition, but it may be
associated with other conditions, including Down syndrome,
atopy, and floppy eyelids. Eye rubbing is a common deno-
minator in all these conditions, which may be why they are
associated with keratoconus.5 Keratoconus can be associated
with Leber ’s congenital amaurosis and connective tissue
diseases including mitral valve prolapse. It may also be
accompanied by rare genetic diseases.6–8 There is a positive
family history of keratoconus in ~10% of patients.7
Keratoconus is routinely referred to as a form of corneal
ectasia,8 but this may not be an accurate term to use. Ectasia is
associated with stretching and should result in increased
corneal surface area, which does not occur in keratoconus.9
There is steepening in the area of the cone and compensatory
flattening elsewhere, usually superiorly, in keratoconus.
CLINICAL SIGNS
Key Features
• Diagnosis of keratoconus is made by the presence of thinning
and protrusion on slit-lamp examination.
• Forme fruste keratoconus, or keratoconus suspect, is
diagnosed by abnormal topography only and a normal slit-
lamp examination.
• Vogt’s striae and Fleischer ring are other frequent findings.
• Hydrops usually resolves over several months and is not an
indication for emergent penetrating keratoplasty.
The diagnosis of keratoconus is based on a careful slit-lamp
biomicroscopy examination. The clinical diagnosis requires the
presence of localized corneal thinning and protrusion, typically
inferiorly or centrally. These early slit-lamp signs can be subtle
and easily missed. It is helpful to observe the shape of the
anterior cornea with a narrow slit beam to detect mild ectasia
where the cornea bulges forward (Fig. 44.1a). In keratoconus the
area of maximal thinning corresponds to the area of maximal
ectasia. After identifying the area of ectasia, one can look
carefully for thinning in that location by comparing the
thickness of the cornea above and below there using a narrow
slit beam. The area of corneal steepening and ectasia is more
obvious on corneal topography than by slit-lamp examination
(Fig. 44.1b). If the topography is suspicious for keratoconus, one
should reexamine the patient by slit lamp to look for minimal
ectasia and thinning that may have been overlooked on initial
examination. The slit-lamp findings are critical: if abnormal,
the patient has keratoconus, and, if normal in the presence of
abnormal topography, the patient is a keratoconus suspect, also
known as forme fruste keratoconus.
Vogt’s striae and Fleischer ring are present on slit-lamp
examination with mild to moderate disease. Vogt’s striae are
relatively frequent. They are vertical folds at the level of the
posterior stroma and Descemet’s membrane in the area of
maximal thinning that are best seen with a wide slit-lamp beam
(Fig. 44.2). These are stress lines that can be made to disappear
with gentle pressure at the limbus. The Fleischer ring of iron
deposition in the corneal basal epithelium surrounds part, or
all, of the cone. It is best seen with a broad slit beam and the
cobalt-blue light, but is not always present (Fig. 44.3).
553
 CORNEA AND CONJUNCTIVA

FIGURE 44.1. (a) Slit-lamp examination of

keratoconus patient shows maximal thinning
which coincides with maximal ectasia. Note
abnormal curvature of cornea is evident by
shape of anterior image of slit beam.
(b) Topography of same patient shows inferior
steepening that is more readily appreciated
than by slit-lamp examination. Superior
changes are artifact.

a b

FIGURE 44.3. The Fleischer ring of iron in the epithelium is seen with
FIGURE 44.2. Vogt’s striae are vertical folds in the deep cornea that Cobalt-blue illumination.
are best seen with a wide slit beam.

With more advanced disease anterior corneal scarring

develops, progresses and reduces vision.10 Scarring may have a
reticular pattern and corresponds on histopathology with
fibrosis in areas where there are breaks in Bowman’s layer
(Fig. 44.4). Elevated scars can develop in the area of maximal
ectasia and limit contact lens tolerance. Significant scarring
can develop in advanced disease, which limits best-corrected
visual acuity with rigid gas-permeable (RGP) lenses, and is an
indication for PK. Scarring usually coincides with the area of
maximal ectasia so will be more visually significant in central
than inferior cones.
Some clinical signs of advanced disease associated with
SECTION 6

keratoconus are less helpful in diagnosing keratoconus than in

the past due to the widespread availability of slit-lamp bio-
microscopes, autorefractors and corneal topography which
make early diagnosis much easier. Munson’s sign refers to the
V-shaped protrusion of the lower lid observed in downgaze
caused by the ectatic cornea. Rizzuti’s sign, describing light
focused on the nasal limbus with lateral illumination, and the
‘Charleux’ oil droplet sign by retroillumination are primarily of
historical interest. Scissoring of the light reflex on retinoscopy
is also rarely observed in the era of autorefractors.
Hydrops is a manifestation of advanced keratoconus in which
there is a sudden loss of vision usually associated with pain
caused by breaks in Descemet’s and acute, marked corneal
edema, often with fluid clefts in the stroma, involving a variable
area of the cornea (Figs 44.5a and b). It is not an indication for
PK. It usually resolves over a period of weeks to months and
results in corneal scarring and flattening, with or without cor-
neal neovascularization. Although hydrops is frequently treated
554
FIGURE 44.4. In advanced keratoconus superficial scarring develops
which can have a reticular pattern and is associated with fibrosis
within areas of breaks in Bowman’s layer.
with topical hyperosmotic agents, aqueous suppressants,
cycloplegia, antibiotics and steroids, it is not clear that any of
these shorten the time until resolution.11 Rarely, hydrops is
complicated by corneal perforation which may be treated with
tissue adhesive or require PK, and even less often by corneal
infection. Sometimes after hydrops resolves there is additional
inferior scarring, the cornea flattens, and the visual acuity
improves.
 Keratoconus and Corneal Noninflammatory Ectasias
a b
FIGURE 44.5. (a) In hydrops there is sudden corneal edema due to breaks in Descemet’s. (b) In the same patient, by narrow slit beam, fluid
clefts within the stroma are present.
CORNEAL TOPOGRAPHY
Key Features
• Corneal topography is very helpful in the diagnosis, evaluation
and management of keratoconus.
• It also is important in research to advance understanding of
the disease.
• The diagnosis of forme fruste keratoconus by topography has
evolved over time.
Computer-assisted corneal topography has revolutionized the
diagnosis, evaluation, and management of keratoconus. Prior to
corneal topography, corneal curvature measured by keratometry
and keratoscopes was used to diagnose and follow keratoconus.
Keratometry measures usually four points on the central cornea
3 mm apart and assumes a regular spherocylindrical shape of
the cornea. Irregular mires and skewed axes of astigmatism
(i.e., the steep axes are not along one meridian and are at an
angle to each other) are signs of irregular astigmatism
suggestive of keratoconus. Inferior corneal steepening measured
by keratometry in upgaze compared to primary position is also
associated with keratoconus. Placido-based keratoscopy pro-
vides qualitative evidence of irregular astigmatism and localized
corneal steepening. Amsler in 1938 used a photographic placido
disk to evaluate very early keratoconus determined by small
amounts of skewed astigmatism where the horizontal axis
deviated by only 1–8°5! The photokeratoscope in the 1970s
provided qualitative information about corneal curvature only
beyond the central 3 mm.
Since 1990 computer-assisted corneal topography or video-
keratoscopy has become the standard way to evaluate corneal
curvature and has vastly improved our ability to diagnose and
treat keratoconus as well as many other corneal conditions
affecting the shape of the ocular surface.5,12 Scanning slit-
topography devices (Orbscan, Bausch and Lomb, Claremont,
CA) provide information on the anterior elevation, posterior
elevation, and pachymetry, but there is some concern about the
accuracy of the posterior elevation or curvature.7 Placido-based
systems are used more commonly. Color coded maps makes
them relatively easy to interpret. There are different scales that
can be used. One is the absolute scale with 1.5 D steps where
each color is always associated with the same corneal curvature
in diopters. Another is a normalized scale where the range of
colors is used to cover the range of powers in the specific cornea
being imaged. The size of the steps varies depending on the
shape of the cornea and can be very small, 0.5 D or less. The
color of a given power varies from one eye and map to another.
There is some controversy as to what scale is most appropriate
to use. In the late 1990s it was thought that the normalized
scale would detect inferior steepening and suspect keratoconus
in too many patients,5 but with the increasing problem of
ectasia after LASIK, more recently 0.5 D intervals have been
recommended specifically to detect forme fruste keratoconus.13
In order to judge the quality of corneal topographic color-coded
maps it is important to review the reflection of the rings on the
cornea imaged at the same time. By looking at the rings one can
determine whether the image was well centered on the cornea
and the number and quality of complete rings (Fig. 44.6). If the
eye is misaligned, for example by the patient looking up, regular
astigmatism will look irregular and can be mistaken for
keratoconus.
There is considerable variation in slit lamp and topographic
findings in keratoconus. Typically, the apex of the cone where
there is maximal thinning, ectasia, and steepening is located
inferocentrally, but it often can be inferotemporal or infero-
nasal. By slit-lamp examination advanced cones have been
analyzed as round, nipple, central and oval, sagging, inferior
cones.14 The location of corneal steepening is more obvious by
topography. Central nipple cones by slit lamp are associated
with central steepening and peripheral flattening by topography
(Fig. 44.7). Low, sagging, oval cones are associated with inferior
CHAPTER 44
steepening and superior flattening (Fig. 44.8). The approach to
contact lens fitting and surgical planning varies depending on
FIGURE 44.6. Example of inferocentral cone. It is important to
examine the image of the reflected rings from the cornea in order to
judge the quality of the topography with regard to the number of
complete rings and the centration of the image on the cornea.
555
 CORNEA AND CONJUNCTIVA
FIGURE 44.7. Paracentral steepening is present in a patient with a
paracentral nipple cone. The flat K is 52 D corresponding to a radius
of curvature of 6.50 mm. The steep K is 65 D. Trial lens fitting with a
Rose K lens was started using a 6.50 mm base curve lens. The lens
prescribed had a 6.70 mm. Base curve, flatter than the flat K.
FIGURE 44.8. Inferior steepening and superior flattening are present
in this patient with an inferior cone.
the location of the cone. In addition many keratoconus patients
and suspects have steepening in an asymmetric bow tie pattern
with skewed radial axes (SRAX) of astigmatism5,13 (Fig. 44.9).
Software has been developed to generate quantitative indices
from topographic images to distinguish normal from
keratoconus suspect corneas. These indices have evolved over
time and continue to do so. In 1995 Rabinowitz indices
included central corneal power more than 47.2 D, inferior
SECTION 6
minus superior (I-S value) asymmetry above 1.2 D, Sim K
astigmatism greater than 1.5 D, and SRAX more than 21°.15 By
2004 Levy concluded that from studies of familial keratoconus
that I⫺S greater than 0.8 D should be considered as abnormal
and suggestive of forme fruste keratoconus.13 Maeda and Klyce
indices distinguish keratoconus from a variety of conditions.16
The KISA% index (K µ (I⫺S) µ Astigmatism (Sim K1⫺Sim K2)
µ SRAX µ 100) has been developed by Rabinowitz and is helpful
for analyzing the evolution of suspect patterns over time.7,17 A
novel approach recently reported uses maps of mean curvature,
determined at each point by averaging the curvature along two
principal perpendicular directions, to topographically charac-
terize, diagnose and follow keratoconus and pellucid without
indices.18 Corneal topography is indispensable in the diagnosis
of forme fruste keratoconus. Various indices may be helpful, but
they must be used in conjunction with a thorough clinical
examination and good clinical judgment in the evaluation of
patients for refractive surgery. Over time, as the problem of post
556 LASIK ectasia has increased, our understanding has grown and
FIGURE 44.9. This is an example of the topography in a case with an
asymmetric bowtie pattern with skewed radial axes of astigmatism
(SRAX).
the threshold for diagnosing forme fruste keratoconus has
decreased and may continue to do so.
Corneal topography is not only helpful in the diagnosis of
keratoconus and contact lens fitting but in many other ways. It
has improved our understanding of the disease by showing it is
more often bilateral than previously thought. Patients with so-
called unilateral disease often have topographic evidence of
forme fruste keratoconus in their ‘normal’ eye. In one large
series, one third of unilateral cases developed bilateral disease,
and abnormal topographic findings of higher I⫺S values and
asymmetric bow tie with SRAX were predictive of developing
keratoconus in the second eye.19 Topography is also helpful in
evaluating disease progression in affected eyes over years. The
disease has a familial predisposition more often than previously
recognized if one includes family members with forme fruste
disease. Topography is also very helpful in patient education.
One can compare the image to a geographic map where the
lines are closer together on the side of hills. The obvious
irregularity makes it easier to explain why vision can not be
corrected with glasses or soft contact lenses and why gas-
permeable lenses are necessary to create a normal-shaped
anterior surface of the eye with tears filling in the space between
the back of the contact lens and the irregular cornea.
ETIOLOGY AND PATHOGENESIS
Key Features
• The cause of keratoconus is unknown.
• Eye rubbing is associated with keratoconus.
• Long-time use of PMMA lenses is at most a very uncommon
cause.
• Biochemical abnormalities and genetics of keratoconus appear
to be complex and are under investigation.
The cause of keratoconus is unknown and probably multi-
factorial. This is subject of ongoing research on a number of
fronts. It is thought that mechanical trauma and chronic
epithelial injury are involved in the pathogenesis of kerato-
conus. Eye rubbing is frequent among keratoconus patients.20
The association of atopy with keratoconus is via eye rubbing.8
 Keratoconus and Corneal Noninflammatory Ectasias
Asymmetric keratoconus has been attributed to asymmetric eye
rubbing.21 Chronic eye rubbing is frequent in keratoconus with
not only atopic disease but also floppy eyelids, contact lens
wear, Down syndrome and Leber’s congenital amaurosis.22 It is
important to ask keratoconus patients if they rub their eyes. If
patients rub their eyes due to ocular itching, topical mast-cell
stabilizers/antihistamines should be prescribed for treatment.
They should be advised to stop rubbing their eyes. Artificial
tears and cool compresses can also be helpful in reducing
eye rubbing.
The floppy-eyelid syndrome is relatively common in
keratoconus, including patients who are not obese. In one series
10% of keratoconus patients had floppy eyelids.23 Keratoconus
tends to be worse on the side the patient with floppy eyelids
sleeps due to the increased mechanical trauma to that side.24
It’s easy to diagnose a floppy eyelid: when one everts the upper
lid in the course of a routine eye examination to check for
papillary changes on the superior tarsus, the lid everts
spontaneously. It is important to recognize this condition so it
can be treated with eye shields or taping the lid shut at bedtime,
or by lid-shortening procedures. Floppy-eyelid syndrome
typically occurs in obese patients who need to be evaluated for
sleep apnea. It is unclear whether or not floppy-eyelid syndrome
in nonobese keratoconus patients is associated with sleep
apnea, but medical referral for possible work-up for this
potentially serious and treatable condition is appropriate.
The role of contact lenses, specifically polymethylmetha-
crylate (PMMA) hard contacts, in the cause of keratoconus has
long been debated. Reviewing the evidence suggests that hard
contact lens use is probably a very uncommon cause of
keratoconus. In 1968 Hartstein reported four patients who
developed keratoconus after wearing hard contact lenses, but
two patients had steep keratometry when they were fit and two
patients were teenagers so the evidence for the hard contacts
causing keratoconus was relatively weak.25 In 1978 Gasset
reported a large series of patients in which 26.5% (43/162) of
keratoconus patients had a history of wearing PMMA contacts
prior to the diagnosis of keratoconus compared to only 1 patient
(of 1248 controls) who wore soft contact lenses.26 Possible
explanations given for the frequent use of PMMA contact lenses
prior to the diagnosis of keratoconus were that many patients
were at the age when keratoconus typically begins and that
often patients become more myopic prior to the diagnosis. This
topic resurfaced when Macsai reported a retrospective study of
keratoconus patients diagnosed in the 1980s and observed that
patients who wore contacts (89% PMMA for an average of
twelve years) prior to the diagnosis of keratoconus were
significantly older than those who had not (age 32 years vs 19
years, p<.0001).27 In addition 75% of patients with a history of
contact wear had central cones, compared to 80% without a
contact lens history who had inferior cones (p<.0001). One
limitation of this study is that most of the patients were not
examined by one of the authors, who were cornea specialists,
prior to contact lens fitting so that subtle slit-lamp signs of
keratoconus could have been missed. In addition, corneal
topography was not available so it was not possible to diagnose
forme fruste keratoconus in the patients prior to contact lens
fitting. However, it is possible that PMMA lenses cause
mechanical trauma contributing to the development of
keratoconus. Many patients who wear contacts also rub their
eyes after removing them. PMMA lenses, however, are a very
uncommon cause of keratoconus, since only 10% of contact
lens wearers in the USA use RGP lenses, and very few wear
PMMA lenses. Concern about this possibility is one reason to
refit people who wear PMMA contacts with gas-permeable
lenses, although a more important reason is to avoid hypoxia.
To further elucidate the possible role of contact lens use in the
pathogenesis of keratoconus it would be helpful to routinely
obtain baseline corneal topography prior to initial contact lens
fitting in all patients.
Biochemical and molecular abnormalities in keratoconus
are the subject of much ongoing research. Thinning is thought
to be due to an increase in degradative enzyme activity and a
decrease in a number of enzyme inhibitors. Kenney proposed
a unifying working hypothesis for the pathogenesis of
keratoconus.28 First, there is abnormal processing of free
radicals and superoxides generated by UV light exposure in
keratoconus corneas. Second, there is a build-up of destructive
aldehydes in the cornea due to reduced aldehyde dehydrogenase
activity resulting in oxidative stress and damage. Third, cells
that are damaged irreversibly undergo apoptosis resulting in
thinning. Fourth, cells that are damaged reversibly undergo
wound healing which involves upregulation of degradative
enzymes and leads to focal areas of thinning and scarring.
Recent work by this group has shown that keratoconus corneal
buttons obtained at the time of PK (with advanced disease) have
more mitochondrial DNA damage than do normal corneas and
hypothesize that this is both due to oxidative stress and may
add to it.29
The genetic basis of keratoconus is another area of active
investigation and great interest given the potential for gene
therapy of corneal diseases.6 In contrast to granular and lattice
stromal corneal dystrophies, abnormalities in the transforming
growth factor beta-induced gene (BIGH3) are not the cause of
keratoconus.30 One rare form of keratoconus associated with
abnormal retinal function and posterior polymorphous
dystrophy has been determined to be associated with a muta-
tion in the retinal transcription factor VSX1 gene.31 The genetic
basis for most keratoconus remains unknown and appears to be
heterogeneous and complex.32 Recent work by Rabinowitz to
create a data base of genes expressed in human keratoconus
corneas obtained at the time of PK has found abundant expres-
sion of a novel gene (designated KC6) of unknown function and
absent expression of another gene, Aquaporin 5, which involves
water channels. The significance of these findings is yet to be
determined. There were many genes expressed involved in
apoptosis. Comparison to genes expressed in normal corneas
has not yet been done.
DIFFERENTIAL DIAGNOSIS
Key Features
• Pellucid marginal degeneration is in the differential diagnosis of
keratoconus and is distinguished from it by the location of
CHAPTER 44
thinning inferior to the area of maximal ectasia.
• Keratoglobus and posterior keratoconus are very different, less
common, nonprogressive congenital disorders.
The major differential diagnosis of keratoconus is PMD, a
similar, but less common noninflammatory ectatic disorder.
Keratoglobus and posterior keratoconus are often included in
the differential diagnosis, but they are very rare, nonprogressive,
congenital conditions that are readily distinguished from
keratoconus. In keratoglobus there is limbus-to-limbus thin-
ning and ectasia with the maximal thinning in the mid-
periphery. Keratoglobus is associated with connective tissue
disorders such as Ehlers–Danlos syndrome type VI much more
frequently than keratoconus. Keratoglobus is a true ectasia with
corneal stretching resulting in increased surface area.9 The
cornea may also be enlarged. Keratoglobus patients, especially
those with blue sclera and systemic connective tissue disease,
are at risk for spontaneous corneal rupture, probably because of 557
 CORNEA AND CONJUNCTIVA
a b
corneal stretching.33 In this series hydrops was very common,
occurring in almost all eyes. The mainstay of treatment for
keratoglobus is spectacles due to the difficulty and risks
associated with contact lenses or surgery. If surgery is necessary
for repair of a perforated cornea, a lamellar tectonic limbus-to-
limbus procedure is usually done first.34 Alternative approaches
to support the thin peripheral cornea in keratoglobus in the
absence of perforation have been tried using an onlay of corneal
tissue.35
Posterior keratoconus is an entirely unrelated condition. It is
a mild form of anterior segment dysgenesis in which there is
localized area of posterior increased corneal curvature resulting
in mild corneal thinning with or without scarring.36 The
anterior surface of the cornea is minimally involved, so vision
is usually not greatly affected.
PMD differs from keratoconus in that the area of maximal
thinning is typically below the area of maximal ectasia whereas
in keratoconus the two coincide (Fig. 44.10a). In PMD there is
a band of thinning usually inferiorly within 1–2 mm of the
limbus, but the thinning can be located elsewhere, even
superiorly. There is a characteristic butterfly or crab-claw
pattern on corneal topography with radial steepening at 4 and
8 o’clock and far inferiorly as well as flattening along the 90°
axis centrally and superiorly (Fig. 44.10b). Low-sagging cones
can have a similar topographic appearance. The distinction
between PMD and a very low-sagging cone is made by careful
slit-lamp examination, although sometimes it can be difficult to
be certain whether the maximal ectasia is above or coincident
with the area of maximal thinning. The term ‘pellucid’ means
clear, and it is generally true that in PMD there are no Vogt
striae or Fleisher ring and deep stromal scarring is mild unless
there is a history of hydrops.37,38
MANAGEMENT OF KERATOCONUS AND
SECTION 6
PELLUCID
Key Features
• Rigid gas-permeable lenses do not slow progression of the
disease.
• Rigid gas-permeable lenses are indicated to improve vision
when it is inadequate with glasses or soft contact lenses.
• Contact lens fitting in keratoconus is an art and a science.
Topography and trial lens fitting are very helpful. Expertise in
lens fitting enhances the success rate.
• When patients are intolerant of contact lenses or have reduced
vision due to scarring they are candidates for surgery.
• Penetrating keratoplasty is highly successful in keratoconus.
• New surgical approaches include intracorneal ring segments
(INTACS) for mild to moderate disease and deep anterior
lamellar keratoplasty.
558
FIGURE 44.10. (a) In pellucid marginal
degeneration the maximal thinning is inferior to
the area of maximal ectasia. This is best seen
with a narrow slit beam. (b) Corneal topography
in pellucid has a butterfly pattern with
steepening at 4 and 8 o’clock in addition to far
inferiorly. Superiorly and inferocentrally there is
flattening. A low sagging cone can have a
similar appearance.
Gas permeable lenses are indicated and the mainstay of treat-
ment to correct irregular astigmatism caused by keratoconus
and pellucid when vision is inadequate with spectacles or soft
contact lenses. When gas-permeable lenses fail due to decreased
vision or contact lens intolerance, then surgery is indicated. PK
has the highest success rate in keratoconus. It is much more
problematic in pellucid due to difficulty getting beyond peri-
pheral thinning. New, promising modalities in the surgical
management of keratoconus and pellucid include intracorneal
ring segments (INTACS) and deep anterior lamellar
keratoplasty (DALK). Despite the achievement of usually
excellent visual acuity in keratoconus, there is good data to
suggest that this condition adversely impacts the quality of life
of patients similar to those patients with grade 3–4 age-related
macular degeneration.39 Further improvements in management
are necessary.
CONTACT LENSES
It is a common misconception that gas-permeable lenses are
indicated to prevent progression of keratoconus. There is no
evidence to support this. They are indicated only when patients
are dissatisfied with their vision with glasses or soft contacts. If
a patient has good vision in one eye due to asymmetric disease,
as is often the case, it is often easier for the patient to adjust to
one gas-permeable lens in the eye that needs it rather than to
fit both eyes at once. Due to the superior comfort of soft
contacts, many patients prefer to sacrifice some vision and not
wear gas-permeable lenses; they are often reassured to learn
that they do not need to wear gas-permeable contacts for
treatment. Advances in frequent replacement toric soft contact
lenses have facilitated their use in patients with mild
keratoconus. Trial lenses can be ordered on the basis of the
patient’s manifest refraction using minus cylinder. Patients who
wear gas-permeable lenses in only one eye may develop
unilateral, reversible ptosis in that eye. There is some evidence
to suggest that gas-permeable lenses may cause disease
progression. If they are fit relatively steep, they can cause
ectasia, and if they are fit relatively flat, they can cause apical
scarring, so they should not be used to prevent disease
progression.40–42 In addition, patients tend to rub their eyes
after removing gas-permeable contact lenses, and this eye
rubbing may aggravate the condition.
Gas-permeable lenses are the mainstay of treatment for
keratoconus. The expertise of the contact lens fitter is an
important factor in the success of contact lenses. Many patients
referred as contact lens failures for PK can often be refit with
contacts successfully.43,44 An attempt at contact lens fitting is
almost always indicated prior to PK, especially if the problem is
contact lens intolerance and not decreased visual acuity with
contacts. Successful lens fitting is time-consuming for both the
patient and the fitter. PK does not always eliminate the need for
 Keratoconus and Corneal Noninflammatory Ectasias
contact lenses in keratoconus patients, since over a third of
patients wear them postoperatively either due to astigmatism,
anisometropia or for convenience because they wear them in
the other eye.43,45
There are many approaches to fitting gas-permeable contact
lenses in keratoconus. There is controversy between the con-
cepts of apical clearance, vaulting and relatively steep fit versus
apical touch, bearing or support and relatively flat fit. Some
degree of apical touch is tolerated and often desirable in
keratoconus.46 Relatively flat-fitting lenses are used most
commonly.47 Corneal topography and in the recent past
keratometry are helpful in lens selection for trial lens fitting.
Corneal topography is less accurate in highly irregular corneas
such as keratoconus than in normal eyes, but it still is very
useful in initial trial lens selection and refitting. The approach
to lens fitting varies depending on if there is a central, round,
nipple cone or a low, sagging, oval cone.14 Multicurve lenses
designed for keratoconus with a steep central curve and wide
flat peripheral curves such as the Soper cone and McGuire
lenses work best in central cones. In recent years, the Rose K
lens is one of these lenses that has been used with success and
improved comfort in patients with advanced central cones.48
Adequate peripheral clearance determined by the pattern of
fluorescein staining has been associated with good contact lens
comfort.49 Even when the base curve of the initial trial lens is
chosen on the basis of the flat peripheral curvature, flatter
lenses are often necessary to obtain a comfortable fit without
central microbubbles or discomfort superiorly suggestive of a
tight fit (Fig. 44.7).
In general, standard spherical lenses are used successfully to
fit many keratoconus patients. They are indicated when there is
corneal steepening inferiorly, as is most commonly the case.
There is corneal flattening superiorly in these patients. The
initial trial lens can be selected on the basis of the flat curvature
above. Larger diameter (usually 9.0–9.6 mm) lenses are used
more often in these patients than in patients with central cones
in whom smaller lenses (usually 8.5–8.8 mm) fit better. In
patients with PMD or low sagging cones with a similar butterfly
pattern on topography large, relatively flat lenses are used. The
special design Dyna intra-limbal lenses (Lens Dynamics Inc.,
Golden, CO), a large 11.2 mm diameter lens designed for
irregular corneas has been helpful in patients with PMD and
similar topography.50
Fitting gas-permeable lenses is both an art and a science.46
Tight lenses are a frequent cause of lens intolerance. They get
predictably more uncomfortable as the day goes on. Patients
often feel the lens more above near the upper lid than below
because the cornea is flatter superiorly and the lens is tighter
there. It is helpful to fit lenses without topical anesthesia,
although one has to wait for reflex tearing to subside in order to
judge the fit. It is important to listen to the patient as to where
he or she feels the lens most and then compare the curvature of
the cornea by topography to the base curve of the lens and make
adjustments accordingly to improve the lens corneal alignment.
Loose lenses cause variable discomfort. Both tight and loose
lenses can cause central superficial punctate keratitis. In
general, moderate- or high-Dk lens materials should be used.
Sometimes the material makes a difference in contact lens
tolerance, and one should consider changing the material if the
fit seems optimal and yet the patient is uncomfortable.
Moderately high-Dk lenses tend to wet better and develop
coating less than very high-Dk lenses.
Softperm lenses (Wesley Jessen Corp, Des Plaines, Il) are a
hybrid lens with a gas-permeable lens center and a hydrogel
skirt. They can be used with good success in keratoconus when
patients are intolerant of gas-permeable lenses or can not be
fit with them due to extreme steepening, but have good visual
acuity.51 They have one diameter (14.3 mm) and a limited range
of base curves (6.50–8.10 mm). They have relatively low gas
permeability and tend to be tight fitting with limited
movement. Use of Softperm lenses has been associated with
corneal swelling and endothelial cell loss due to hypoxia.52,53
Due to concern regarding a tight fit and hypoxia, a relatively flat
lens with some movement and even a small amount of edge
pucker represents a desirable fit, as long as the patient is
comfortable.46 These lenses tighten up quickly, even in the
course of trial lens fitting in the office. Patients must be
followed regularly for chronic hypoxic complications such as
neovascularization. In addition giant papillary conjunctivitis is
relatively frequent.50 Regular enzymatic cleaning can be helpful
to prevent and treat this problem. These lenses are cleaned
using soft contact lens solutions. The lenses are fragile and tend
to break along the junction between the gas-permeable center
and soft hydrogel skirt. They are more expensive than gas-
permeable lenses and need to be replaced more frequently.
Despite these limitations, patients often strongly prefer these
contacts to gas-permeable lenses due to their increased comfort.
SynergEyes (SynergEyes Inc., Carlsbad, CA) hybrid lenses are
new, more gas permeable, and more durable lenses that are
increasing the success of hybrid lenses.
Other approaches to contact lens fitting in keratoconus
include piggyback lenses and scleral lenses. Piggyback lenses
involve the use of a soft lens as a carrier for an RGP lens.
The availability of highly gas-permeable lenses for both
components has decreased the hypoxia associated with this
approach in the past. In addition, a daily-disposable soft lens
decreases the care necessary and probably improves safety.
Nonetheless, people who require this approach are often candi-
dates for surgery. Scleral contacts were used before the
development of corneal lenses, and now with the availability of
RGP scleral lenses there is renewed interest in them. They can
be successful in keratoconus patients who are intolerant of gas-
permeable corneal contact lenses as well as other patients
with severe ocular surface disease. To date their use in the
USA has required lens fitting at the Boston Foundation for
Sight where Perry Rosenthal, MD developed these lenses
(www.bostonsight.org).
PK
Corneal transplantation is the standard treatment for
keratoconus patients who have decreased visual acuity with
RGP lenses due to corneal scarring or who are contact lens
intolerant. Corneal transplantation has the highest success rate
CHAPTER 44
in keratoconus with clear grafts obtained in over 95% of cases.
In the USA it is the fourth most common indication for PK
after pseudophakic bullous keratopathy, regraft and Fuchs’
corneal dystrophy.54 Despite the very high rate of graft clarity
and excellent visual outcome, the recovery of vision is
prolonged, astigmatism is common, graft rejection episodes are
relatively frequent, contact lenses may be necessary, and
patients are at life-long risk for graft rejection and traumatic
wound dehiscence. Other surgical options currently being
evaluated as an alternative to PK for keratoconus include
INTACS, for early and moderate disease, and DALK. In PMD
corneal transplantation is more problematic than in
keratoconus due to peripheral corneal thinning, and a variety of
other techniques have been used.
The frequency of and risk factors for having a PK in
keratoconus following referral to tertiary referral centers have
been studied.4,55–57 The likelihood of undergoing PK was ~20%
in studies with four years of follow-up, but was almost 65% in
559
 CORNEA AND CONJUNCTIVA

a series with a longer average follow-up of almost 11 years, OTHER SURGERY

despite attempted contact refitting prior to keratoplasty.4,55,56
Steep baseline keratometry was a risk factor for PK in all series.
Black race was identified as a significant risk factor for PK in
one series.4 In PMD the majority of patients are managed
medically with spectacles or contact lenses, in part because of
increased difficulty and worse prognosis for surgery, as well as
the absence of central cornea scarring.58,59
Special considerations in performing a phakic PK in
keratoconus include preoperative planning of graft size and
efforts to prevent positive pressure during surgery. The size of
the host trephination is determined by the extent of thinning
and ectasia observed at the slit lamp. One-quarter millimeter
larger or sometimes same-size donor buttons are used to reduce
postoperative myopia. Despite preoperative digital massage and
intravenous mannitol there is often positive pressure during
surgery due to low scleral rigidity and scleral collapse. Reverse
Trendelenburg position is helpful to reduce positive pressure,
especially in obese patients.
A fixed dilated pupil of unknown etiology (Urrets–Zavalia
syndrome) has been reported after PK for keratoconus, but is
rare.60 It is associated with iris ischemia and is more likely
related to intraoperative positive pressure than postoperative
pressure spikes which are uncommon in keratoconus.61,62
In pellucid corneal degeneration, larger grafts, often
decentered inferiorly are necessary to get beyond the area of
inferior thinning.63 When this is not possible peripheral lamel-
lar procedures are used, recently in combination with PK.64,65
Despite the high success rate and excellent visual acuity after
PK for keratoconus, meticulous, indefinite postoperative care
is necessary. Rejection episodes occur in ~30% of patients,
but rarely result in graft failure.45,66–68 Larger graft size (host
greater than or equal to 8.25 mm) is a risk factor for rejection.
There has been concern that bilateral transplantation increases
the risk for rejection, but this has been shown not to be the case
in more recent studies.68,69
Astigmatism after PK for keratoconus is relatively common.
It can usually be managed with RGP lenses, but relaxing
incisions, compression sutures, laser refractive surgery, and/or
wedge resections are sometimes done. Progressive late astig-
matism is also relatively common more than 10 years after PK.
It is associated most frequently with thinning of the inferior
graft–host junction and host periphery due to progressive
disease rather than recurrent keratoconus in the graft. In a
series of keratoconus patients followed for 20 years after PK,
keratometric astigmatism was stable for the first seven years
after suture removal and then increased progressively in
association with thinning of the graft–host junction consistent
SECTION 6
Currently, INTACS and deep anterior lamellar keratoplasty are
alternative surgical approaches for keratoconus, but other
procedures have also been tried. Epikeratoplasty was performed
in the past, but lost favor due to poor visual outcome. Excimer
laser procedures are generally contraindicated in keratoconus,
except occasionally excimer phototherapeutic keratoplasty
(PTK) can be performed to remove elevated nodular scars, when
they can not be readily shaved off with a blade, in order to
improve contact lens tolerance. Riboflavin/ultraviolet-A-induced
collagen crosslinking has been reported to stop progression of
keratoconus and induce regression in some patients.72
Some success with INTACS to reduce myopia and astigma-
tism and improve uncorrected and spectacle corrected vision in
keratoconus without central scarring has been reported.73 The
role of INTACS in keratoconus remains unknown, but patients
with milder keratoconus appear to have a better outcome.74
Complications are relatively frequent in patients with moderate
to advanced disease, especially thinning over the implants
resulting in their exposure.75 Optimal indications and tech-
nique for INTACS for keratoconus remain to be determined.
Deep anterior lamellar keratoplasty, an alternative to full-
thickness PK, is gaining popularity. It has the advantage of
avoiding the risk of endothelial rejection, since the healthy
endothelium of the patient is left in place. Although the
procedure is technically challenging, early reports suggest that
the visual results may be as good as after PK.76
CONCLUSIONS
Keratoconus is a condition affecting ~150 000 people in the
USA. Much is known about the diagnosis and management,
and yet there are many unresolved issues regarding the
pathogenesis and treatment which are areas of current research.
The focus in caring for keratoconus patients is visual rehabili-
tation. In most cases this is achieved by correcting irregular
astigmatism using contact lenses and by surgery when contacts
fail. Patients are often myopic and require comprehensive eye
care. Intraocular pressure (IOP) measurement may be falsely
low due to corneal thinning in keratoconus. However, optic
nerve changes suspicious for glaucoma should be further
evaluated as these patients may be susceptible to progressive
glaucomatous optic neuropathy at low pressures.
Although the prognosis is good for patients with keratoconus,
it is of great concern that the disease appears to have a decidedly
negative impact on the quality of life.39 Studies have tried to
address so-called difficult personality traits associated with

with disease progression in the host.3 This has been observed by
others and should be looked for by careful slit-lamp exam-
ination.70 Late recurrences of keratoconus within the donor
cornea confirmed by histopathology after repeat corneal
transplantation have been reported, but are infrequent.71
keratoconus with variable results.77–80 It is very possible that
personality issues, if they exist, are secondary to the condition.
Improved management of this disease may lessen the burden
for patients and improve not only their vision but also their
quality of life.

REFERENCES
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31. Heon E, Greenberg A, Kopp KK, et al:
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33. Cameron JA: Keratoglobus. Cornea 1993;
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34. Macsai MS, Lemley HL, Schwartz T:
Management of oculus fragilis in
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35. Kanellopoulos AJ, Pe LH: An alternative
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38. Cameron JA: Deep corneal scarring in
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39. Kymes SM, Walline JJ, Zadnik K, et al:
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40. McMonnies CW: Keratoconus fittings.
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41. McMonnies CW: The biomechanics of
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Eye Contact Lens 2005; 31:80–92.
42. Barr JT, Wilson BS, Gordon MO, et al:
Estimation of the incidence and factors
predictive of corneal scarring in the
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Keratoconus (CLEK) Study. Cornea 2006;
25:16–25.
43. Smiddy WE, Hamburg TR, Kracher GP,
Stark WJ: Keratoconus – Contact lens or
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44. Belin MW, Fowler WC, Chambers WA:
Keratoconus – Evaluation of recent trends
in the surgical and nonsurgical correction
of keratoconus. Ophthalmology 1988;
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45. Silbiger JS, Cohen EJ, Laibson PR: The
rate of visual recovery after penetrating
keratoplasty for keratoconus. CLAO 1996;
22:266–269.
46. Lembach RG: Use of contact lenses for the
management of keratoconus. Ophthalmol
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47. Edrington TB, Szczotka LB, Barr JT, et al:
Rigid contact lens fitting relationships in
keratoconus. Optom Vis Sci 1999;
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48. Betts AM, Mitchell GL, Zadnik K: Visual
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49. Edrington TB, Gundel RE, Libassi DP, et al:
Variables affecting rigid contact lens
comfort in the Collaborative Longitudinal
Evaluation of Keratoconus (CLEK) Study.
Optom Vis Sci 2004; 81:182–188.
50. Ozbek Z, Cohen EJ: Use of the intralimbal
rigid gas permeable lenses for pellucid
marginal degeneration, keratoconus, and
after penetrating keratoplasty. Eye Contact
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51. Chung CW, Santim R, Weng WJ, Cohen EJ:
Use of Softperm contact lenses when rigid
gas permeable lenses fail. CLAO 2001;
27:202–208.
52. Owens H, Watters G, Gamble G: Effect of
Soptperm lens wear on corneal thickness
and topography: a comparison between
and keratoconic and normal corneae.
CLAO 2002; 28:83–87.
53. Edmonds CR, Wung SF, Husz MJ,
Pemberton B: Corneal endothelial cell
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54. Cosar CB, Sridhar MS, Cohen EJ, et al:
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associated procedures, 1996-2000. Cornea
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55. Lass JH, Lembach RG, Park SB, et al:
Clinical management of keratoconus.
Ophthalmology 1990; 97:433–445.
56. Sray WA, Cohen EJ, Rapuano CJ,
Laibson PR: Factors associated with the
need for penetrating keratoplasty in
keratoconus. Cornea 2002; 21:784–786.
57. Reeves SWW, Stinnett S, Adelman RA,
Afshari NA: Risk factors for the progression
to penetrating keratoplasty in patients with
keratoconus. Am J Ophthalmol 2005;
140:607–611.
58. Sridhar MS, Mahesh S, Bansal AD, et al:
Pellucid marginal corneal degeneration.
Ophthalmology 2004; 111:1102–1107.
59. Tzelikis PF, Cohen EJ, Rapuano CJ, et al:
Management of pellucid marginal corneal
degeneration. Cornea 2005; 24:555–560.
60. Jastaneiah S, Al-Towerki A, Al-Assiri A:
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keratoplasty for macular corneal dystrophy
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140:484–489.
61. Tuft SJ, Buckley RJ: Iris ischaemia
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Cornea 1995; 14:618–622.
62. Chien AM, Schmidt CM, Cohen EJ, et al:
Acute glaucoma after penetrating
keratoplasty. Am J Ophthalmol 1993;
115:711–714.
63. Varley GA, Macsai MS, Krachmer JH:
The results of penetrating keratoplasty for
pellucid marginal corneal degeneration.
Am J Ophthalmol 1990; 110:149–152.
64. Cameron JA: Results of lamellar crescentic
resection for pellucid marginal corneal
degeneration. Am J Ophthalmol 1992;
113:296–302.
65. Rasheed K, Rabinowitz YS: Surgical
treatment of advanced pellucid marginal
degeneration. Ophthalmology 2000;
CHAPTER 44
107:1836–1840.
66. Olson RJ, Pingree M, Ridges R:
Penetrating keratoplasty for keratoconus:
a long-term review or results and
complications. J Cataract Refract Surg
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67. Javadi MA, Motlagh BF, Jafarinasab MR,
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68. Donshik PC, Cavanagh HD, Goruchoff AS,
Dohlman CH: Effect of bilateral and
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69. Musch DC, Meyer RF: Risk of endothelial
refection after bilateral penetrating
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70. Lim L, Pesudovs K, Goggin M, Coster DJ:
Late onset post-keratoplasty astigmatism
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71. Bourges JL, Savoldelli M, Dighiero P, et al:
Recurrence of keratoconus characteristics.
Ophthalmology 2003; 110:1920–1925.
72. Wollensak G, Spoerl E, Seiler T:
Riboflavin/ultraviolet-A-induced collagen
crosslinking for the treatment of keratoconus.
Am J Ophthalmol 2003; 135:620–627.
73. Colin J, Cochener B, Savary G, et al:
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Ophthalmology 2001; 108:1409–1414.
74. Levinger S, Pokroy R: Keratoconus
managed with intacs. Arch Ophthalmol
2005; 123:1308–1314.
SECTION 6
562
75. Kanellopoulos AJ, Pe LH, Perry HD,
Donnenfeld ED: Modified intracorneal ring
segment implantations (INTACS) for the
management of moderate to advanced
keratoconus. Cornea 2006; 25:29–33.
76. Watson SL, Ramsay A, Dart JK, et al:
Comparison of deep lamellar keratoplasty
and penetrating keratoplasty in patients
with keratoconus. Ophthalmology 2004;
111:1676–1682.
77. Farge EJ, Baer PE, Adams GL, Paton D:
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78. Mannis MJ, Morrison TL, Zadnik K, et al:
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79. Swartz NG, Cohen EJ, Scott DG, et al:
Personality and keratoconus. CLAO 1990;
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80. Giedd KK, Mannis MJ, Mitchell GL,
Zadnik K: Personality in keratoconus in a
sample of patents derived form the
internet. Cornea 2005; 24:301–307.
 CHAPTER

45 Corneal Manifestations of Metabolic Disease

Kristin M. Hammersmith and Christopher J. Rapuano

The optical clarity of the cornea permits visualization of subtle
deposition of metabolites not possible in other tissues of the
body. This chapter concentrates on disorders of metabolism
that have clinically observable changes in the cornea that may
help to establish or confirm a systemic diagnosis. Such dis-
orders may indicate disturbance in aspects of metabolism
involving amino acids, lipids, or complex carbohydrates. Most
metabolic diseases are inherited on an autosomal recessive
basis. Hunter’s syndrome and Fabry’s disease are two notable
exceptions, both of which are X-linked. Generally, the metabolic
disorder is a result of an enzymatic deficiency causing accu-
mulation of substrate either locally or after transport in the
blood stream.
As an organizational device we divide our descriptions into
disorders of metabolism involving amino acids, lipids, complex
carbohydrates, purines, and metals.
features can include variable degrees of mental retardation,
seizures, and multiple congenital anomalies.5
Ocular symptoms and findings occur early in tyrosinemia
type 2 and may even be the presenting manifestation of the
disease. Keratoconjunctivitis with photophobia may appear
before the patient is 2 weeks of age.6,7 Corneal opacities are
bilateral superficial punctate crystalline deposits that may
assume a dendritiform pattern leading to an ulcerative keratitis
and a mistaken diagnosis of herpes simplex virus (HSV) kera-
titis. Peripheral corneal vascularization may develop (Fig. 45.2).
In contrast to HSV, the dendritiform lesions are often bilateral
and do not have terminal bulbs and corneal sensation is
normal.2 The ocular findings may appear months before the
hyperkeratotic skin lesions on the hands and feet, which can be

DISORDERS OF AMINO ACID METABOLISM

TYROSINEMIA (TYROSINOSIS)

Key Features
• Autosomal recessive
• Two forms: type 1, most common, no corneal involvement
type 2, can develop superficial punctate crystalline lesions,
often in a dendritiform pattern, that may mimic HSV keratitis

Tyrosine is an organic amino acid precursor in the metabolic

pathways of amines, which include thyroid hormones and the
neurotransmitters epinephrine, norepinephrine, dopamine, and
tyramine. Tyrosine can be derived from phenylalanine by
hydroxylation or from proteins (Fig. 45.1). Elevated levels of
serum tyrosine can occur in transient neonatal tyrosinemia
as well as in two autosomal recessive conditions: tyrosinemia
type 1 (hepatorenal tyrosinemia) and tyrosinemia type 2
(oculocutaneous tyrosinemia, Richner–Hanhart syndrome).
Tyrosinemia type 1, the more common variant, is caused by
a deficiency of fumarylacetoacetate hydrolase. Corneal
changes have not been reported with this disorder.1 Tyrosinemia
type 2 (Richner–Hanhart syndrome) is due to a deficiency
of hepatic tyrosine aminotransferase, the gene for which is
located on chromosome 16q22.2 In this disorder, serum tyro-
sine levels range from 2.5 to 25 times normal. Urinary tyrosine
and several of its metabolites are found in high concentrations.3
Differentiation from tyrosinemia type 1 can be made by the lack
of hepatorenal disease and the lack of the inhibitor effect of the
patient’s serum on D-aminolevulinic acid dehydrogenase FIGURE 45.1. Points of enzymatic deficiencies in organic amino acid
activity, which is specific to hepatorenal tyrosinemia.4 Systemic metabolic pathways in tyrosinemia type 2 and alkaptonuria. 563
 CORNEA AND CONJUNCTIVA

a b

FIGURE 45.2. Corneal changes in tyrosinemia type 2. (a) The left eye shows
peripheral neovascularization, marked irregularity of the epithelium, patchy
opacities, and loss of corneal transparency. (b) The right eye shows even
more extensive involvement. (c) After 6 weeks of therapy, there is marked
clearing of the lesions.
(a–c) From Goldsmith LA: Cutaneous changes in errors of amino acid metabolism:
tyrosinemia, phenylketonuria, and argininosuccinic aciduria. In: Fitzpatrick TB, Eisen AZ,
Wolff K, et al, eds. Dermatology in general medicine. 3rd edn. New York: McGraw-Hill;
1987:1636. Copyright © 1987 by McGraw-Hill, Inc. Used by permission of McGraw-Hill
Book Company.

painful enough to prevent walking. The skin lesions begin as

bullae and erosions that progress to white-yellow hyperkeratotic
plaques and papules.8 Treatment with dietary restriction of
phenylalanine and tyrosine results in complete reversal of
both ocular and dermatologic abnormalities (Fig. 45.3).2,7,9
Nystagmus, strabismus, conjunctival thickening, and cataract
have also been reported in association with Richner–Hanhart
syndrome, as has variable mental retardation.
Transient neonatal tyrosinemia is a temporary biochemical
abnormality affecting premature infants or infants who ingest a
SECTION 6

high-protein diet such as evaporated milk formula. Crystalline

corneal opacities characteristic of tyrosinemia type 2 have been
reported in transient neonatal tyrosinemia. The subepithelial
crystals can completely reabsorb within 5 days of normalization
of plasma tyrosine concentrations.10

ALKAPTONURIA (OCHRONOSIS)
Key Features
• Autosomal recessive
• The yellow–brown pigmentation that develops in sclera,
cartilage and tendons is termed ochronosis
Alkaptonuria is a rare, recessively inherited disorder of amino
acid metabolism caused by the lack of the hepatic and renal
enzyme homogentisate 1,2-dioxygenase (HGO). The locus for
564 this enzyme is on chromosome 3q21-23.11 Large amounts of
a b
FIGURE 45.3. (a) Diffuse plantar hyperkeratosis in an adult with
tyrosinemia. (b) The hyperkeratosis cleared on a low-tyrosine, low-
phenylalanine diet without topical treatment.
(a and b) From Goldsmith LA: Cutaneous changes in errors of amino acid
metabolism: tyrosinemia, phenylketonuria, and argininosuccinic aciduria.
In: Fitzpatrick TB, Eisen AZ, Wolff K, et al, eds. Dermatology in general
medicine. 3rd edn. New York: McGraw-Hill; 1987:1639. Copyright © 1987
by McGraw-Hill, Inc. Used by permission of McGraw-Hill Book
Company.)
 Corneal Manifestations of Metabolic Disease

homogentisic acid are excreted unchanged into the urine, which

turns dark black if the urine becomes alkaline from standing
exposed to air. There is a low prevalence of alkaptonuria
(1:100 000–250 000) in most ethnic groups, with the exception
of Slovaks, in whom the incidence rises to 1:19 000.12
The normal metabolism of phenylalanine and tyrosine
produces homogentisic acid, which is cleaved by HGO (see
Fig. 45.1). In the absence of HGO, homogentisic acid is excreted
into the urine, where it is oxidized into a melanin-like product.
Ochronosis is the name given to the ochre, yellow-brown
pigmentation that begins to appear in the collagen of the sclera,
cartilage, and tendons toward the end of the second decade
of life (Fig. 45.4). Tracheal, bronchial, laryngeal, costal, and
auricular cartilages are involved as is the dura mater.13,13a Over
a period of time, the pigment deposition increases and takes on
a darker bluish–black appearance that may be mistaken for
melanoma.14 The arthritis, which is the major clinical mor-
bidity, has an insidious, progressive character that may begin
in the fourth decade of life.15,16 It may lead to incapacitating
kyphosis and joint immobility. Cardiovascular disease, athero-
sclerosis, prostatic, and renal lithiasis may also occur.
The cornea and more characteristically the sclera become FIGURE 45.4. Alkaptonuria showing the yellow-brown pigmentation
pigmented in ochronosis without causing a decrease in visual in the paralimbal cornea with a predilection of deposition anterior to
function.17 Corneal involvement, when it occurs, is usually the insertion of the horizontal muscle tendons.
limited to the deposition of fine, brown, oil-like droplets at the From Donaldson DD: Atlas of external diseases of the eye. 2nd edn. Cornea and
level of Bowman’s layer and the anterior stroma near the limbus sclera. St Louis: CV Mosby; 1980:65.
in the horizontal meridians.14,16 By slit-lamp examination,
scleral as well as conjunctival and subconjunctival pigment is
seen in the nasal and temporal interpalpebral area anterior to
the insertions of the horizontal muscles in patches, flecks, or
spots.14 Over a period of time, the patches coalesce to form
triangular, deeply pigmented areas in the same location as
pingueculae.

CYSTINOSIS

Key Features
• Autosomal recessive
• There are three forms of cystinosis: infantile (most severe),
intermediate, and adult (least severe)
• Corneal deposition of multiple fine, needle-shaped refractile
crystals are noted in all three forms
• When advanced, filamentary keratopathy, recurrent erosions
and decreased vision from dense crystals can develop FIGURE 45.5. Cystinosis. Fine, needle-shaped refractile crystals can
• Renal failure develops within the first decade in the infantile be seen within the corneal stroma. All forms of the disease show
form, typically requiring kidney transplantation corneal changes.
From Mandel ER, Wagoner MD: Atlas of corneal disease. Philadelphia: WB

Cystinosis is an autosomal recessive hereditary disorder in
which free cystine accumulates intracellularly within
lysosomes.18 The exact metabolic defect is not yet defined but is
known to involve defective lysosomal cystine transport.19 The
cystinosis gene has been linked to markers on the short arm of
chromosome 17.20 There are three forms of cystinosis based on
age of onset: infantile, intermediate, and adult. All forms of
cystinosis show characteristic corneal deposition of fine, needle-
shaped refractile crystals, which have been described as tinsel-
like opacities pathognomonic of the condition.21 While corneal
findings are the same in the various forms, the systemic clinical
findings vary widely. A routine ophthalmic examination may
lead to the diagnosis (Fig. 45.5).22
Infantile nephropathic cystinosis is the most common and
severe form of cystinosis. Children present toward the end of
the first year of life with recurrent fever and dehydration. By the
first year, Fanconi’s syndrome is established, involving a com-
plex dysfunction of the proximal renal tubules and metabolic
CHAPTER 45
Saunders;1989:47.
bone disease. There is also associated renal rickets, which may
be related to impaired renal conversion of 25-hydroxyvitamin
D3 to 1,25-dihydroxyvitamin D3.23 Failure to thrive is a promi-
nent feature of nephropathic cystinosis. Affected children may
have pale irides24 and hypopigmentation of skin and hair for
their race.25 Over time as crystalline accumulation increases,
glomerular damage occurs, resulting in severe renal failure by
the age of 10 years. Renal transplantation is life-saving but does
not prevent progressive damage to other organs, including
the eyes.26
The characteristic needle-like refractile corneal crystals pre-
sent in the first year of life, usually preceding the full-blown
renal disease. Crystals are initially deposited in the peripheral
and anterior corneal stroma. With age, deposition proceeds pos-
teriorly and centripetally, so that by the age of 7 years, crystals 565
 CORNEA AND CONJUNCTIVA
TABLE 45.1. Clinical Manifestations of the Three Major Forms of Cystinosis
Manifestation Infantile Nephropathic
Presenting signs Failure to thrive; fever; dehydration;
Fanconi’s syndrome; rickets; photophobia
Age at presentation Late infancy
Ocular findings Corneal crystals by age 1 yr; increase with age
Patchy peripheral retinal pigment epithelial atrophy
Growth Third percentile
Free cystine content 80 times normal
of leukocytes
can be found within or on the endothelial surface. The depth
of stromal deposition is greater in the periphery and symmetric
between the two eyes. The crystals appear more dense and
larger in the anterior stroma.27 In a study of children, who had
undergone renal transplantation, all showed full-thickness
corneal involvement that was so dense in one 18-year-old
patient that it resembled mutton fat keratitic precipitates. Of
the same group, two patients had band keratopathy.24 Crystals
can also be seen in the iris and lens.
Corneal thickness is increased in patients with nephropathic
cystinosis, even at a young age.28 This may be an indication of
subclinical dysfunction of the endothelial or epithelial cells.
Corneal sensation is also significantly reduced.29 Photophobia
and sensitivity to glare are common and related to light
scattering by the crystals rather than to retinal problems.30
Glare disability is correlated with age and the density of
clinically observable corneal crystals.31 With time, however,
recurrent erosions may become a major clinical problem
requiring bandage contact lenses or corneal transplant for the
relief of symptoms.30,32,33 Superficial punctate keratopathy and
filamentary keratopathy are more common in older patients.
Band keratopathy, corneal neovascularization, and posterior
synechiae are also seen in older patients.34 Symmetric patchy
depigmentation of the peripheral retina is a constant finding in
nephropathic cystinosis and may be noted even before the
corneal changes are visible.35
Visual acuity is generally normal in the early stages of
cystinosis. As patients are living longer after renal transplan-
tation, impaired visual function may result from abnormal
retinal function, posterior synechiae, glaucoma,36 and
hemorrhagic retinopathy.26,30 Progressive neurologic dys-
function, primarily motor incoordination and hypotonia, may
SECTION 6
occur in young adults.39
a b
566
Intermediate Juvenile Benign Adult
Renal dysfunction Incidental finding on eye examination
18 mo–17 yr Incidental finding at any age
Corneal crystals Corneal crystals
Variable retinal findings None
Nearly normal Normal
30 times normal
An intermediate juvenile form of cystinosis exists with the
onset of renal dysfunction between 18 months and 17 years of
age with corneal crystals and variable retinopathy. Growth is
nearly normal.
The adult form is characterized by the asymptomatic pre-
sence of corneal crystals without retinopathy or renal dysfunc-
tion.37 Diagnosis can be confirmed by conjunctival biopsy.
The diagnosis of cystinosis can be made by measuring the
free cystine content in leukocytes or cultured amniotic cells.
Those with infantile nephropathic cystinosis have values 80
times normal (Table 45.1).38
Treatment is symptomatic, addressing electrolyte and fluid
imbalances caused by the renal disease, vitamin D therapy for
rickets, and thyroid hormone for hypothyroidism. When renal
function fails, dialysis or renal transplantation is frequently
necessary in patients between 6 and 12 years of age. After
corneal transplantation the grafted cornea generally remains
free of crystals.30,33,39 Oral cysteamine is a more specific therapy
to reduce intracellular cystine levels. This therapy has met with
success in maintaining renal glomerular function and improv-
ing growth but does not relieve the symptoms of the Fanconi
syndrome.40,41 Similarly, it has not been shown to retard the
rate of corneal crystal accumulation even after 8 years of
therapy.42 In contrast, topical cysteamine given hourly as eye
drops in doses ranging from 0.1 to 0.5% has been successful in
reducing the number of corneal crystals in treated eyes of young
children.42,43 Reducing the frequency of instillations to four
times a day over a 7-month period was not as successful in
reducing crystal formation, although it did reduce photophobia
in a 21-year-old.44 The higher concentration, 0.5%, was more
effective than the 0.1% concentration in reducing crystals in
older patients. The best therapy may be prophylaxis with the
early institution of cysteamine eye drops (Fig. 45.6).45
FIGURE 45.6. Slit-lamp photograph of a
placebo-treated right eye (a) showing crystals
in the central cornea compared with the
cysteamine-treated left eye of a 26-month-old
child showing no crystals (b).
(a and b) From Kaiser-Kupfer MI, Gazzo MA,
Datiles MB, et al: A randomized placebo-controlled
trial of cysteamine eye drops in nephropathic
cystinosis. Arch Ophthalmol 1990; 108:689.
Copyright 1990, American Medical Association.
 Corneal Manifestations of Metabolic Disease

DISORDERS OF LIPOPROTEIN AND LIPID HYPERLIPOPROTEINEMIAS

METABOLISM
This diverse group of disorders affecting lipoprotein and lipid
metabolism frequently has associated ocular manifestations.
DYSLIPOPROTEINEMIAS
Key Features
Disorders of lipid metabolism such as lecithin-cholesterol
acyltransferase (LCAT) deficiency, Tangier disease, and fish eye
disease are important to recognize as they may be associated with
coronary artery and peripheral vascular disease.
The dyslipoproteinemias are a group of lipid metabolism dis-
orders that include the hyperlipoproteinemias, lecithin-
cholesterol acyltransferase (LCAT) deficiency, Tangier disease
(familial high-density lipoprotein (HDL) deficiency), and fish
eye disease. The recognition of these ocular findings, especially
corneal arcus and xanthelasma, is important, as they may be
associated with coronary artery and peripheral vascular
disease.46 The presence of corneal arcus in men under 50 years
of age may be a harbinger of developing coronary artery disease
(Fig. 45.7).47
Lipoproteins in plasma allow the transport of cholesterol,
triglycerides, phospholipids, and proteins throughout the
systemic circulation. Lipoproteins consist of chylomicrons, very
low-density lipoproteins (VLDLs), LDLs, and HDLs. Dietary
lipids are packaged into chylomicrons during absorption by
intestinal mucosa. They are modified and selectively trans-
ported to specific tissues under the control of lipid-cleaving
enzymes (lipases), plasma lipoproteins, and the corresponding
lipoprotein receptors on tissues. Apolipoproteins on the surface
of lipoprotein particles assist in the directed transport and
uptake of nutritive lipid and protein at specific tissue sites
throughout the body. At least five different electrophoretic
patterns of abnormal elevations of lipoprotein levels have been
described that may be associated with secondary systemic and
ocular defects (Table 45.2).
Type 1 hyperlipoproteinemia (hyperchylomicronemia), a rare
autosomal recessive disorder, is characterized by a massive
elevation of plasma chylomicron levels and a corresponding
increase in triglyceride levels.48 The genetic locus is 8p22. This
disorder may be associated with hepatosplenomegaly, repeated
episodes of abdominal pain, central nervous system dys-
function, recurrent pancreatitis, lipemia retinalis, and palpebral
or diffuse eruptive xanthomas. Early atherosclerosis and corneal
arcus are usually not features.49 Type 2 hyperlipoproteinemia

FIGURE 45.7. Corneal arcus. (a) Note the clear

zone between the corneal limbus and the
peripheral stromal deposition of phospholipid,
cholesterol esters, and triglycerides. (b) The
lipid deposition is limited to the paralimbal
cornea. The central cornea is clear.

a b

TABLE 45.2. Classification of the Major Types of Hyperlipoproteinemia

Characteristic Type I: Type II: Hyperbeta- Type III: Type IV: Type V: Hyperprebetali-
Hyperchylo- and Prebetali- Broad-Beta Hyperprebetali- poproteinemia and

CHAPTER 45
micronemia poproteinemia Disease poproteinemia Hyperchylomicronemia

Elevated Chylomicrons; LDL; VLDL Abnormal VLDL Triglycerides; apoprotein

lipoprotein triglycerides chylomicron abnormalities
remnant removal;
VLDL: triglyceride
>30%
Skin Xanthomas Xanthomas Xanthomas — Xanthomas
Eyes Lipemia retinalis — Lipemia retinalis — Lipemia retinalis
Corneal arcus — Early Early ± —
Atherosclerosis — + + — —
Other findings Hepatosplenomegaly; Hepatosplenomegaly
pancreatitis
Inheritance Rare, autosomal Autosomal Autosomal Autosomal
recessive; dominant or recessive; dominant;
secondary secondary secondary secondary
Abbreviations: LDL, low-density lipoprotein; VLDL, very low density lipoprotein.
567
 CORNEA AND CONJUNCTIVA
(hyperbetalipoproteinemia and prebetalipoproteinemia) can
occur as an autosomal dominant disorder or it may be
secondary to hypothyroidism, dysgammaglobulinemia, and
hepatic and renal disease.50 This disorder results in elevation of
LDL levels alone or in combination with elevated VLDL levels.
Corneal arcus, xanthelasma, conjunctival xanthomas, and
coronary artery disease occur.
Type 3 hyperlipoproteinemia (familial dysbetalipopro-
teinemia; broad-beta disease) is inherited autosomal recessively,
but secondary cases have also been described. This disorder
results from a mutation in the apolipoprotein E gene, linked
to 19q13.2.51 Early atherosclerosis and xanthomas are the
major clinical features of this disorder. Characteristic palmar
(xanthochromia striatum palmaris) or tuboeruptive xanthomas,
typically on the elbows, may develop.52 Ocular findings can
include early corneal arcus and lipemia retinalis.
Type 4 hyperlipoproteinemia (hyperprebetalipoproteinemia)
is characterized by an elevation of VLDL levels and may be
transmitted by autosomal dominant inheritance, although this
disorder can also be related to obesity and diabetes mellitus.53
Corneal arcus and xanthelasma are usually not prominent
clinical features of this disorder.
Type 5 hyperlipoproteinemia (hyperprebetalipoproteinemia
and hyperchylomicronemia), like type 1, results in marked elev-
ation in triglyceride levels, but also often has other associated
apoprotein abnormalities.52 Eruptive xanthomas, lipemia
retinalis, and hepatosplenomegaly may occur. As with type 1,
corneal arcus and vascular disease are not prominent features.
The corneal involvement by types 2 and 3 hyperlipopro-
teinemias is usually limited to premature development of cor-
neal arcus. Histopathologic evaluations demonstrate peripheral
lipid deposition in the corneal stroma, Bowman’s layer, and
Descemet’s membrane. An intervening clear space between the
limbus and the arcus opacity as well as central corneal sparing
is characteristic. The arcus may first appear in the superior
cornea, then inferiorly, and progress to become confluent.
Successful treatment and control of elevated lipoprotein levels
in patients with hyperlipoproteinemias does not reverse corneal
arcus once it has developed (see Fig. 45.7).53
TABLE 45.3. Hypoproteinemias With Significant Corneal Involvement
Characteristic Lecithin Cholesterol
Acyltransferase Deficiency
Accumulated Unesterified cholesterol
metabolite
SECTION 6
Serum cholesterol Free cholesterol:cholesteryl ester
ratio increased
Triglycerides Above normal
VLDL High
LDL Normal or high
HDL Low
Apolipoproteins Low
A-I and A-II
Atherosclerosis Accelerated
Other Proteinuria
Anemia
Cornea Arcus and diffuse stromal haze;
crocodile shagreen appears in
second decade of life
Abbreviations: VLDL, very low density lipoprotein; LDL, low-density lipoprotein; HDL, high-density lipoprotein.
568
HYPOLIPOPROTEINEMIAS
The hypolipoproteinemias are also disorders of lipid catabolism
but result in abnormal reductions of circulating lipoprotein
levels. This group of disorders includes Bassen–Kornzweig dis-
ease (abetalipoproteinemia), familial hypobetalipoproteinemia,
LCAT deficiency, Tangier disease, and fish eye disease.54,55
The ocular manifestations of Bassen–Kornzweig disease and
familial hypobetalipoproteinemia are primarily retinal abnor-
malities and are not discussed further in this chapter, except to
mention a report of moderate diffuse opacification of the cornea
in a case of a possible familial variant of apolipoprotein A.56
Lecithin-Cholesterol Acyltransferase Deficiency
The enzyme LCAT, encoded on chromosome 16q22, is a plasma
enzyme which catalyzes cholesterol ester formation from
cholesterol.57 LCAT deficiency is an autosomal recessive meta-
bolic disorder that results in the accumulation of unesterified
cholesterol within tissues, particularly in blood vessels and the
bone marrow (Table 45.3).58 Levels of HDLs as well as apolipo-
proteins A-I and A-II are reduced in this disorder, and levels of
VLDLs and LDLs are typically elevated. Levels of serum chol-
esterol and triglycerides are often above normal. Other common
systemic manifestations include anemia, renal insufficiency,
and accelerated atherosclerosis.59
The cornea may develop a dense peripheral arcus and diffuse
stromal haze due to the deposition of multiple, fine, grayish
opacities (Fig. 45.8). The visual acuity is relatively unaffected.60
Anterior and posterior crocodile shagreen is present in the mid-
corneal periphery.61 The corneal opacities are usually noted by
the early teenage years. Heterozygous carriers may have an
increased incidence of arcus-like changes, but this association
lacks sufficient sensitivity or specificity to be of value
diagnostically. Although the composition of the opacities is not
known, pathologic evaluation has demonstrated collections of
tiny vacuoles in Bowman’s layer containing many electron-
dense particles.62 A recent case report presented light and
transmission electron microscopic findings of a patient with
LCAT deficiency.63 All stromal layers had extracellular vacuoles
Tangier Disease Fish Eye Disease
Cholesterol ester
Low High
Very high
Normal High
Low Normal
Very low Very low
Low Low
— —
Peripheral neuropathy
Orange tonsils
Fine diffuse clouding may be Diffuse stromal clouding; denser yellow-
detectable in first decade of life gray peripheral opacification appears
but generally in fifth decade in second decade of life; impairs vision

FIGURE 45.8. Lecithin-cholesterol acyltransferase deficiency with a
dense peripheral arcus and diffuse stromal haze. Vision is relatively
unaffected.
Courtesy of Ernst J Schaefer, MD.
with acid mucopolysaccharide contents measuring up to
2.5 µm. Amyloid deposits were also noted predescemetally.
Other ocular findings can include venous dilatations, angioid
streaks, and peripapillary mottling and hemorrhage.64
Tangier Disease (Familial High-Density Lipoprotein
Deficiency)
Tangier disease was initially described in 1961 as a familial HDL
deficiency occurring in two residents of Tangier Island, Virginia.65
The rare disorder is transmitted by autosomal recessive
inheritance and is characterized by a deficiency or complete lack
of plasma HDLs (see Table 45.3). The genetic locus is 9q31.66
Heterozygous carriers of the disease typically have reduced but
measurable levels of HDLs on electrophoretic study.67,68
HDLs in these patients, when detectable, have altered com-
positions such as markedly reduced levels of apolipoproteins
A-I and A-II.69–71 In addition, other lipid abnormalities may
also be present, including reduced plasma cholesterol levels,
mild elevations of serum triglyceride levels, and abnormal
plasma levels of chylomicron metabolites.72
Clinical features include neuropathy, hepatosplenomegaly,
lymphadenopathy, hyperplastic yellow-orange tonsils, and the
late presence of corneal clouding. Lipid deposition, presumably
cholesterol esters, has been identified within histiocytes,
Schwann’s cells, or fibroblasts in various tissues including
skin, nerves, cardiac valves, tonsils, spleen, liver, gastrointes-
tinal tract mucosa, bone marrow, cornea, and conjunc-
tiva.69,73–79 Clinical manifestations of Tangier disease have
been reported in patients as young as 5 years.65
Ocular manifestations described include corneal clouding
and mottling of the retinal pigment epithelium. Orbicularis
oculi weakness, lagophthalmos, and exposure keratopathy as
well as ocular motility disturbances are secondary to the neuro-
pathy.73,74,77,80,81 The corneal opacification appears to be caused
by lipid accumulation, particularly of esterified cholesterol and
phospholipids.82 Corneal clouding most frequently can be
detected by slit-lamp examination in affected individuals older
than 40 years, but it has also been found in childhood.69,73 The
clouding may be diffuse or localized in the stroma, typically
more central than peripheral and more posterior than
anterior.73,79,83 Small posterior stromal dot-like opacities distri-
buted randomly or in a whorl pattern and peripheral corneal
haze along the horizontal meridian may also be seen.69,83
Neither type of corneal opacification significantly reduces visual
Corneal Manifestations of Metabolic Disease
acuity. Confocal microscopy, reported from one affected patient,
demonstrated lipid deposits as small granular bodies, which
were fairly uniformly distributed in the stroma. This also
showed an unaltered sub-basal nerve plexus, which was
contrary to expectation.84
Neuropathy is a common feature of the disease, affecting
peripheral nerves. Corneal exposure due to seventh-nerve palsy
and lagophthalmos may be the most clinically significant ocular
finding with secondary exposure keratopathy (Fig. 45.9). Punctal
occlusion by cautery or plugs ameliorates corneal drying.
There are no reports of coronary artery disease occurring in
homozygous patients younger than 40 years.78 It has been
suggested that other coexisting alterations such as low serum
cholesterol levels, thrombocytopenia, and decreased platelet
adhesiveness may have counterbalancing antiatherosclerotic
effects in Tangier homozygotes.79
Fish Eye Disease
Fish eye disease is an autosomal recessive disorder that was first
described in 1979.85 Unlike Tangier disease and LCAT
deficiency, patients with this disorder are unable to esterify
cholesterol within HDLs in spite of near-normal LCAT activity
as measured by the endogenous plasma cholesterol esteri-
fication rate.86,87 Molecular defects in the LCAT gene have been
associated with fish eye disease, explaining the almost absent
LCAT activity, when measured with exogenous HDL analogs as
substrate.88 Levels of HDL cholesterol and lipoproteins apo A-I,
A-II, and D are dramatically reduced, and LDL triglyceride
levels are strikingly high (see Table 45.3). The effect of these
changes on the incidence of atherosclerosis is not clear.
Characteristically, the cornea is diffusely cloudy with small,
dot-like gray-white-yellow opacities deposited in all layers of the
cornea except the epithelium.85 The peripheral cornea appears
more opaque than the central zone and may contain a thin,
superficial, yellow, ring-shaped opacity ~1 mm from the limbus
(Fig. 45.10). Gradual progression of the corneal changes can
cause marked visual impairment during the second decade
of life.89
LYSOSOMAL STORAGE DISEASES
Disorders of catabolic lysosomal enzymes make up a diverse
group of diseases including the mucolipidoses, mucopolysac-
charidoses, galactosialidoses, sphingolipidoses, gangliosidoses,
mannosidosis, and fucosidosis. These disorders are charac-
terized by their major storage product, although abnormal
accumulation of other metabolic compounds may occur,
e.g., complex lipids may be detected ultrastructurally in muco-
CHAPTER 45
polysaccharidoses.90 All are inherited as autosomal recessive
conditions with the exception of Fabry’s disease and Hunter’s
syndrome, which are X-linked.
Although therapeutic options have previously been limited,
bone marrow transplantation has been successful in modifying
the natural history of lysosomal and peroxisomal storage
diseases.91 Cord-blood transplants of stem cells from unrelated
donors has also proved to be an effective treatment for patients
with Hurler ’s syndrome.92 Targeted treatments for the
lysosomal storage disorders, via enzyme replacement and/or
substrate depletion have also been successfully used for some
clinical serotypes.93
MUCOLIPIDOSES (OLIGOSACCHARIDOSES)
The mucolipidoses are a group of lysosomal storage diseases
characterized by the accumulation of oligosaccharides. The
mucolipidoses may occur sporadically or may be transmitted by
autosomal recessive inheritance.94 569
 CORNEA AND CONJUNCTIVA

a b c

FIGURE 45.9. Tangier disease. (a) Right eye of a 52-year old woman
shows very subtle powdery corneal stromal clouding. Vision is 20/15.
The corneal clouding is subtle in this patient and must be astutely
looked for despite the fact that she has had neuropathy for more than
20 years. (b) Slit-beam photograph shows mild fluorescein staining of
the epithelium at the junction of the mid and lower thirds of the
cornea. (c) Her major ocular problems are due to lagophthalmos from
facial nerve palsy. The irregular corneal light reflex indicates a rough
epithelial surface in the lower third of the cornea. (d) Corneal stromal
opacification is mild, generally only observed on slit-lamp
examination.
(d) Courtesy of Ernst J Schaefer, MD.

retinal and conjunctival vessels.57 Fine corneal epithelial

and stromal opacities can occur but do not typically produce
significant corneal clouding.102 Histopathologic study has
revealed single membrane-bound inclusions similar to those
of mucopolysaccharidoses in corneal epithelium and
keratocytes.57 As in sphingolipidoses, rare intracellular mem-
branous lamellar inclusions may also be present.
Mucolipidosis type 2 (I-cell or inclusion cell disease) is caused
by an abnormality of N-acetylglucosamine phosphotransferase,
the gene for which is located on chromosome 12.103 Clinical
features include dysmorphic facies with gingival hyperplasia,
skeletal deformities, organomegaly, short stature, and mental
retardation. Orbital changes include hypoplastic orbits with
hypoplasia of the supraorbital ridges and prominence of the
globes. Other ocular changes can include glaucoma,
megalocornea, or corneal clouding. The cornea usually remains
clear in early life, but ~40% of patients later develop abnormal
FIGURE 45.10. Fish eye disease. The cornea shows diffuse clouding
stromal granularity and mild opacity.104 Keratocytes and
with moderate reduction in vision due to stromal opacification. There
SECTION 6

is denser yellow-gray peripheral opacification.

Courtesy of Harry Koster, MD, Yves Pouliquen, MD.
Mucolipidosis type 1 (dysmorphic sialidosis, Spranger ’s
syndrome) is caused by a mutation in the gene encoding
neuraminidase, located on 6p21.3.95 There are two subtypes.
Patients with sialidosis type 1 have decreased visual acuity with
an associated macular cherry-red spot, myoclonus, and gait
abnormalities.96–98 Sialidosis type 2, with infantile onset, is
more severe and results in dysmorphic Hurler-like facies
(prominent brow, hypertrichosis, frontal bossing, and saddle
nose), organomegaly, mental retardation, dysostosis multiplex,
sensorineural hearing loss, and progressive neurologic
decline.99–101 Most affected patients with either subtype do
not survive past adolescence or early adulthood.
Ocular manifestations of sialidosis type 2 include spoke-like
570 lenticular opacities, a macular cherry-red spot, and tortuous
fibroblasts of the corneal stroma and conjunctiva have
membrane-bound vacuoles with fine fibrillogranular and
irregular membranous lamellar inclusions. These cells are thus
referred to as inclusion cells or I cells. Such I cells have been
found in mucolipidoses, types 2 and 3.
Mucolipidosis type 3 (pseudo-Hurler’s polydystrophy) is
also caused by a deficiency of N-acetylglucosamine phos-
photransferase, but its features, although similar to those of
type 2, are less severe.102 Fine opacities of the corneal stroma
may also be present, but these do not significantly affect visual
acuity (Fig. 45.11).102 Disk edema, surface wrinkling macu-
lopathy, and hyperopic astigmatism are sometimes seen.105
Mucolipidosis type 4 (Berman’s syndrome) is an autosomal
recessive neurodegenerative disorder, characterized by psycho-
motor retardation and ophthalmic abnormalities due to a
deficiency in the gene encoding mucolipin-1, which is linked to
chromosome 19p13.3–p13.2.106 Patients have prominent
diffuse corneal clouding that is present at birth or appears in
early infancy.107 A mild variant can also present later in child-

FIGURE 45.11. Mucolipidoses type 3, pseudo-Hurler’s polydystrophy.
There is diffuse corneal stromal clouding. The epithelium is regular.
Courtesy of Trexler M Topping, MD.
hood with corneal clouding having the appearance of cornea
verticillata and retinal dystrophy.108 The vacuoles within the
corneal epithelium are either single membrane-bound vesicles
containing fibrillogranular material suggestive of mucopoly-
saccharides or membranous lamellar bodies consistent with
phospholipids.107,109 Conjunctival cytologic studies, which
reveal characteristic lysosomal inclusions on light and electron
microscopy, may help confirm the disorder.110
Mechanical epithelial débridement or penetrating kera-
toplasty does not prevent recurrent corneal opacification after
reepithelialization or replacement of the graft epithelium by
cells of host origin.111 Transplantation of epithelium through
conjunctival allografts, particularly limbal grafts, which
theoretically may allow normal stem cell repopulation, may
offer more prolonged maintenance of corneal clarity.112 Other
features include cataract, optic nerve atrophy, attenuated retinal
vasculature, and electroretinographic abnormalities suggestive
of a retinal degeneration.110,112,113
GALACTOSIALIDOSIS
Galactosialidosis (neuraminidase deficiency with B-galactosidase
deficiency, Goldberg’s syndrome, Goldberg–Cotlier syndrome,
sialidosis type 2 juvenile onset) is an autosomal recessive
disorder that is caused by combined deficiency of the lysosomal
enzymes beta-galactosidase and alpha-neuraminidase and
linked to chromosome 20.114,115 Systemic features include facial
dysmorphism, mental retardation, seizures, skeletal deform-
ities, ataxia, hearing loss, and myoclonus. The characteristic
ocular finding is reduced visual acuity associated with a macular
cherry-red spot, but mild degrees of corneal clouding may
occur.116 An adult form of galactosialidosis, characterized by
fine corneal opacities, a cherry red spot and optic atrophy, has
also been described and may be diagnosed by a conjunctival
biopsy.117
SPHINGOLIPIDOSES
The sphingolipidoses are a group of lipid storage diseases
presumably caused by deficiencies of specific hydrolytic
enzymes that result in the accumulation of lipids within
Corneal Manifestations of Metabolic Disease
various tissues throughout the body. All the sphingolipidoses,
with the exception of Fabry’s disease, are transmitted by
autosomal recessive inheritance. Those disorders with corneal
involvement are included in this chapter.
Fabry’s Disease
Key Features
• X-linked condition
• Cornea verticillata, fine powdery epithelial deposits in a spoke-
like pattern in the inferior cornea, develop in both the affected
males and carrier females
• Cornea verticillata rarely affects vision
• Cornea verticillata can also develop secondary to medication
use, such as amiodarone, indomethacin, chloroquine and
phenothiazines
Fabry’s disease, an X-linked disorder, is caused by a deficiency
of a-galactosidase A. This results in the accumulation of
ceramide trihexoside in all areas of the body, but predominantly
within lysosomes of vascular endothelial and smooth muscle
cells. Renal failure and cardiovascular complications are
common in adult life.
Recurrent episodes of pain in the peripheral extremities,
associated with fever and sedimentation rate elevation, are
typical in affected males beginning in childhood.118,119
Sphingolipid deposition within vascular endothelium results in
the characteristic small dot-like skin lesions, referred to as
‘angiokeratoma corporis diffusum’, that become manifest in the
bathing trunk area around the time of puberty and become
more elevated and hyperkeratotic with time.
Corneal involvement is typical in Fabry’s disease in both the
affected hemizygous males and the female carriers. The fine
powdery opacities of the corneal epithelium or subepithelium
usually develop in early infancy and are best seen by retro-
illumination during slit-lamp examination.120 They can be
observed by the age of 4 years in hemizygotes and by the age of
10 years in heterozygotes, in whom the corneal involvement is
often more pronounced. They occur in a whorl or vortex
distribution (cornea verticillata) like force lines in a magnetic
field (Fig. 45.12). They are usually inferior and typically cream
colored but range from white to golden-brown or appear very
faint.121 They do not affect vision, although vision can become
seriously affected by vascular accidents in both the retina and
the central nervous system. Confocal microscopy demonstrates
the accumulated glycosphingolipids as intracellular inclusions
in the corneal and conjunctival epithelial cells and epithelial
CHAPTER 45
basement membrane.122 Striate melanokeratosis and various
medications, including indomethacin, chloroquine, amiodarone
(Fig. 45.13), and phenothiazines can cause corneal epithelial
changes that may mimic the cornea verticillata of Fabry’s
disease.
Other ocular manifestations include telangiectasia and tor-
tuosity of conjunctival and retinal vasculature, seen in 70%
of affected males as opposed to 25% of carrier females
(Fig. 45.14).123 These changes may precede the corneal mani-
festations. Lenticular changes can include a characteristic
granular anterior subcapsular wedge-shaped or propeller-shaped
lens opacity as well as a posterior linear whitish deposit of
granular material at or near the posterior capsule, which
sometimes resembles a herpetic dendrite.
Characteristic Maltese cross-pattern birefringent intracellular
inclusions can be histopathologically identified after biopsy of
the conjunctiva, skin, kidney, or other blood vessel-containing
tissue.124 Electron microscopy can confirm the typical lamellar
inclusion bodies within these cells. 571
 CORNEA AND CONJUNCTIVA

a c

FIGURE 45.12. (a) Subepithelial whorl pattern of corneal opacities in a man with Fabry’s disease. (b) Corneal involvement in his sister. (c) Fine
whorls may give the appearance of force lines in a magnetic field.
(a and b) Reprinted from Miller CA, Krachmer JH: Corneal diseases. In: Renie WA, ed. Goldberg’s genetic and metabolic eye disease. 2nd edn. Boston: Little, Brown;
1986:350.
SECTION 6

FIGURE 45.13. Amiodarone, a cardiac antiarrhythmic, causes a

vortexshaped keratopathy that is reversible with cessation of the drug FIGURE 45.14. Conjunctival telangiectasia with microaneurysmal
therapy. dilatation of the vessels. Similar changes can be seen in the mouths
of patients with Fabry’s disease. Similar conjunctival vascular changes
have been reported in patients with fucosidosis.184,185
Reprinted with permission from Miller CA, Krachmer JH: Corneal diseases. In:
Renie WA, ed. Goldberg’s genetic and metabolic eye disease. 2nd edn. Boston:
572 Little, Brown; 1986.

The DNA sequence for human a-galactosidase has been
isolated and enzyme replacement therapy, utilizing recom-
binant technology, has been demonstrated to be a safe and
effective treatment.125
Multiple Sulfatase Deficiency
Multiple sulfatase deficiency (MSD) (metachromatic leuko-
dystrophy–Austin’s juvenile form) is a disorder that combines
features of metachromatic leukodystrophy and mucopoly-
saccharidoses. This autosomal recessive disorder results from
a deficiency of arylsulfatases A, B, and C.126 Consequently,
excessive amounts of sulfatide accumulate within tissues.
The genetic locus is 3p26.127 In the classic MSD presentation,
this disorder is characterized by the development of facial
dysmorphism, skeletal abnormalities, ichthyosis, and early
psychomotor retardation. Ocular findings are similar to those
of mucopolysaccharidoses, including rare corneal clouding,
grayish cherry-red spot, optic atrophy, and pigmentary retinal
degeneration.128,129
GANGLIOSIDOSES
Gangliosides are glycosphingolipids that contain sialic acid in
their oligosaccharide chain. They occur in high concentration
in the brain in nerve ending membranes and synaptic
membranes.
GM1 Gangliosidosis Type 1 (Generalized
Gangliosidosis)
GM1 gangliosidosis type 1 is an autosomal recessive disorder
caused by deficiency of the enzyme b-galactosidase, which is
encoded on chromosome 3.130 In GM1 gangliosidosis type 1,
there is an accumulation of ganglioside in the central nervous
system as well as accumulation of the glycosaminoglycan
keratan sulfate in the liver and spleen. The clinical picture is
one of initially normal development, followed by severe
neurologic decline. There are at least five subtypes of GM1
gangliosidosis caused by variable residual activity of the mutant
enzyme against different substrates. Thus some patients have
severe neurologic deficit and early death, whereas others have
severe bony abnormalities, facial dysmorphism, and normal
intelligence.
Ocular findings include macular cherry-red spots, nystag-
mus, strabismus, retinal hemorrhages, and optic atrophy. Some
patients also have mild, diffuse corneal clouding and numerous
histopathologically detectable intracytoplasmic vacuoles within
all layers of the cornea.131
GM2 Gangliosidosis Type 2 (Sandhoff’s
Disease)
The GM2 gangliosidoses comprise three distinct genetic
disorders, Tay–Sachs disease, Sandhoff ’s disease, and the GM2-
activated protein deficiency. In contrast to Tay–Sachs disease,
which is the most common ganglioside storage disease and
occurs from a deficiency of hexosaminidase A, Sandhoff ’s
disease results from a deficiency of both hexosaminidase A and
B. Severe central nervous system dysfunction occurs owing to
the accumulation of GM2 gangliosides in neurons. Membrane-
bound vesicles have been detected within keratocytes by
histopathologic and ultrastructural evaluation, although the
cornea may appear clear clinically.132 Other ocular features may
also include tapetoretinal degeneration, optic atrophy, and the
presence of a macular cherry-red spot. Involvement of white
matter of the optic radiations has been shown by magnetic
resonance imaging.133
Corneal Manifestations of Metabolic Disease
MUCOPOLYSACCHARIDOSES
Key Features
• All mucosaccharidoses are autosomal recessive except
Hunter’s syndrome, which is X-linked
• Corneal clouding is a significant feature of all
mucosaccharidoses except Hunter’s and San Filippo’s
syndromes
The mucopolysaccharidoses (MPSs) result from lysosomal
enzyme deficiencies affecting the degradation of glycos-
aminoglycans (mucopolysaccharides). All these disorders,
except for X-linked Hunter’s syndrome, are transmitted by
autosomal recessive inheritance. The clinical manifestations
vary for each MPS type because of differences in the specific
enzyme defect or in the tissue localization of the involved
enzyme. Ten enzyme deficiencies have been identified that give
rise to different syndromes.134 There are geographical variations
in incidence of the different MPS types.135 The cornea is
frequently affected by abnormal glycosaminoglycan metabolism
because glycosaminoglycans make up the corneal stromal
ground substance and constitute 4–4.5% of the dry weight of
the cornea. Corneal keratan sulfate makes up 50% of the
ground substance; it differs from keratan sulfate in cartilage.
Chondroitin and chondroitin sulfate A each constitute ~25% of
the ground substance. Chondroitin is found only in the
cornea.136 The diagnosis of a disorder of mucopolysaccharide
catabolism is made on the basis of characteristic clinical
findings in association with demonstration of the enzymatic
deficiency or detection of elevated levels of urinary
glycosaminoglycans (Table 45.4).
HURLER’S SYNDROME (MPS I-H)
Hurler’s syndrome, results from deficiency of the enzyme
a-L-iduronidase, which is required for the breakdown of heparan
sulfate and dermatan sulfate. In its severe form as Hurler’s
syndrome, clinical manifestations are usually evident by the
first year of life. Characteristic features include short stature,
dysostosis multiplex, facial dysmorphism (coarse facies, pro-
minent forehead, hypertelorism, anteverted nostrils, hyper-
trichosis, synophrys, and depressed nasal bridge), and progressive
psychomotor retardation (Fig. 45.15). Other findings can
include hepatosplenomegaly, neurosensory hearing loss, joint
stiffness, umbilical hernia, and cardiac defects. Death often
occurs in the second decade of life due to recurrent pneumonia
or heart failure. The diagnosis can be confirmed by measure-
CHAPTER 45
ment of a-L-iduronidase activity in isolated peripheral leuko-
cytes or cultured dermal fibroblasts or amniotic cells.
Diffuse corneal clouding usually becomes apparent by the age
of 3 years and may present with photophobia. The fine, punc-
tate corneal opacities are usually distributed throughout the
stroma (Fig. 45.16a), although they are most pronounced
centrally and are best visualized by slit-lamp examination
(Fig. 45.16b).137 Although penetrating keratoplasty can restore
corneal clarity in severe cases of corneal clouding, visual acuity
is often limited in these patients because of associated optic
nerve or retinal disease. Glaucoma may also be present and may
be difficult to diagnose and monitor because of corneal opacifi-
cation and thickening.135 Progressive retinopathy with vascular
narrowing, hyperpigmentation of the fundus, and later bone
spicule formation occur. Papilledema and optic atrophy are
common.138 Bone marrow transplantation can improve some of
the ocular manifestations such as corneal clouding, optic nerve
edema, and retinopathy.139 Cord-blood transplants from
unrelated donors have been demonstrated to be effective in the 573
 CORNEA AND CONJUNCTIVA

TABLE 45.4. Major Clinical Findings in the Mucopolysaccharidoses

Mucopoly- Clinical Glaucoma Retinal Optic Skeletal Retardation Major Age at Death
saccharidosis Corneal Bone Atrophy Involvement Compound Onset
Clouding Spicules* Stored*†

MPS I-H Diffuse, ± + Late ++ Gargoyle Severe HS, DS 6–12 mo By teens

Hurler’s progressive;
onset age
1 yr
MPS I-S Diffuse; onset ++ + Late – – HS, DS 5–7 yr Normal
Scheie’s after 4 yr
MPS I-HS Diffuse; onset ± + Late + + HS, DS 2–4 yr
Hurler-Scheie 2 yr
MPS II-A – – + Often + + HS, DS Under Before
Hunter’s A 1 yr age
15 yr
MPS II-B ± Late – + Often – – HS, DS 4 yr 30–60 yr
Hunter’s B
MPS III-A, – – + Rare – + HS 2–6 yr 20–30 yr
-B, -C, -D
Sanfilippo’s
MPS IV Morquio Diffuse; after – – – ++ – KS, CS 1–2 yr Varies
age 10 yr
MPS VI Diffuse; – – Rare Dwarf + – DS 2–3 yr
Maroteaux- punctate
Lamy
MPS VII Sly’s ± – – – – + HS, DS
Modified from Lang GE, Maumenee IH: Retinal dystrophies associated with storage diseases. In Newsome DA (ed): Retinal Dystrophies and Degenerations. Philadelphia,
Lippincott-Raven, 1988, pp 320–321.
Abbreviations: HS, heparan sulfate; DS, dermatan sulfate; KS, keratan sulfate; CS, chondroitin sulfate; MPS, mucopolysaccharidosis.
*Night blindness is associated with retinal bone spicules.
†Retinal involvement occurs when HS is stored.

FIGURE 45.15. may be excessive and may be managed by large doses of

Hurler’s syndrome,
mucopolysaccha-
ridosis (MPS) I-H.
Short stature and
facial dysmorphism
are characteristic of
patients with MPS I-H.
Facial features are
coarse, the nares are
anteverted, and the
brows are heavy and
close, with wideset
eyes. The abdomen is
protuberant with an
SECTION 6
atropine in the preinduction period and avoidance of narcotics
in the postoperative period.141
SCHEIE’S SYNDROME (MPS I-S)
Scheie’s syndrome, previously referred to as MPS V, also results
from a deficiency of the enzyme a-L-iduronidase, but it is the
least severe form of MPS. Clinical manifestations include
coarse facies, clawlike hand deformities, carpal tunnel
syndrome, hernias, neurosensory hearing loss, joint stiffness,
and cardiac abnormalities. In contrast to Hurler’s syndrome,
mental retardation, dwarfism, and early death are usually not

umbilical hernia.
Courtesy of Trexler M
Topping, MD.
treatment of these patients, as it favorably alters the natural
history of Hurler’s syndrome.92
Patients with Hurler’s syndrome are at increased risk for
cardiovascular collapse or laryngospasm during general
anesthesia. Difficult or failed intubations are common in
574 children with mucopolysaccharidoses.140 Pharyngeal secretions
features.
Ocular abnormalities include corneal clouding (Fig. 45.17),
optic nerve head swelling or late optic atrophy, and pigmentary
retinal degeneration.135 The corneal clouding is usually pro-
gressive and diffusely involves the stroma, particularly in the
corneal periphery and posterior stromal regions. Ultrastructural
analysis of corneas from patients with MPS I-H and MPS I-S
has found a greater range and size of collagen fibril diameter, the
presence of fibrous long-spacing collagen, vacuolated stromal
cells, and disrupting sulfated glycosaminoglycan deposits com-
pared with normal corneal stroma.142,143 Glaucoma also occurs
more frequently than in Hurler’s syndrome. Success of pen-
etrating keratoplasty for corneal opacification may be limited by
coexisting optic nerve and retinal disease.
HURLER–SCHEIE SYNDROME (MPS I-HS)
The activity of the enzyme a-L-iduronidase and systemic
manifestations in patients with Hurler–Scheie syndrome are

a b
FIGURE 45.16. Hurler’s syndrome, Corneal clouding increases over time with punctate opacification of the stroma (a), which can be best seen
by retroillumination at the slit lamp (b).
FIGURE 45.17. Scheie’s syndrome, MPS I-S. Corneal clouding is fine,
diffuse, and slightly more prominent in the peripheral stroma.
Courtesy of Trexler M Topping, MD.
intermediate between those occurring in Hurler’s syndrome
and those in Scheie’s syndrome.135 Corneal clouding is usually
progressive and typically requires penetrating keratoplasty for
visual rehabilitation within the first decade of life. Other ocular
abnormalities include glaucoma, optic atrophy, and retinal
pigmentary degeneration.
HUNTER’S SYNDROME
Hunter’s syndrome is the only MPS that is transmitted by
X-linked recessive inheritance. It is caused by a deficiency of the
enzyme iduronate-2-sulfatase and results in the accumulation
of heparan sulfate and dermatan sulfate within tissues.
Several allelic variants have been identified, with varying
levels of severity. In the more severe form, Hurler’s-like features
may occur, including deafness, coarse facies, short stature,
mental retardation, hepatosplenomegaly, cardiac disease, and
death within the second decade of life. Hirsutism and smooth,
pinpoint dermal elevations are also frequent.
Ocular findings in either type often include papilledema
(Fig. 45.18), optic atrophy, and pigmentary retinal degenera-
tion. Corneal clouding may be detectable by slit-lamp
examination, but it is not clinically significant. However, muco-
Corneal Manifestations of Metabolic Disease
FIGURE 45.18. Hunter’s syndrome, MPS II. A clinically clear cornea
allows excellent visualization of papilledema in a 13-year old boy with
MPS II.
Courtesy of Trexler M Topping, MD.
CHAPTER 45
polysaccharides have been shown histologically in corneas
that appeared clear clinically.136
SANFILIPPO’S SYNDROME (MPS III TYPES A
THROUGH D)
Sanfilippo’s syndrome is caused by one of four different
enzymatic defects of heparan sulfate catabolism, all of which
have different loci. Type A is caused by a deficiency of the
enzyme heparan sulfate N-sulfatase (heparan-S-sulfaminidase)
and is the most severe form. Type B is due to deficiency of
N-acetylglucosaminidase. Type C results from deficiency of
N-acetyltransferase (acetyl-CoA-a-glucosamide-N-N-acetyl-
transferase). Type D occurs due to a deficiency of the enzyme N-
acetylglucosamine-6-sulfate sulfatase.
Clinical manifestations of Sanfilippo’s syndrome include
deafness, coarse facies, short stature, joint stiffness, mild
hepatosplenomegaly, and severe mental retardation. 575
 CORNEA AND CONJUNCTIVA

Ocular findings are usually limited to pigmentary retinal
degeneration and corresponding electroretinogram changes.144
Optic atrophy rarely occurs. Corneal opacification has been
reported, but is not a prominent feature.
MORQUIO’S SYNDROME (MPS IV)
Morquio’s syndrome results from the accumulation of keratan
sulfate secondary to a deficiency of the enzyme
N-acetylgalactosamine-6-sulfate sulfatase. The gene for Morquio
MPS IV-A maps to 16q24.3.145 Historically, MPS IV was divided
into IVA and IVB; however, MPS IVB is now considered a
variant of GM1-gangliosidosis.135
Skeletal deformities are the most prominent clinical features;
these include dysostosis multiplex, dwarfism, pectus
carinatum, kyphoscoliosis, short first metacarpal bones, genu
valgum, and other deformities (Fig. 45.19). Spinal cord com-
pression may develop owing to vertebral abnormalities. Aortic
valvular disease and recurrent pneumonia are common.
Intelligence is normal.
Ocular findings are limited to papilledema and diffuse,
stromal corneal clouding (Fig. 45.20). It appears as a myriad
of white minute dots in the stroma.146 The corneal opacity,
however, usually does not necessitate penetrating keratoplasty.
Associated lenticular opacities have been described.147
Ocular manifestations include progressive corneal clouding
with increased corneal thickness (Fig. 45.22), papilledema, and
optic atrophy. Retinal involvement has not been reported.
Diffuse corneal opacities can necessitate penetrating kera-
toplasty. A case of repeat opacification of the corneal graft has
been reported.148 However, clear grafts have been reported in a
patient after bone marrow transplantation 13 years
postkeratoplasty.149
SLY’S SYNDROME (MPS VII)
Sly’s syndrome results from deficiency of the enzyme
b-glucuronidase, which is normally encoded for by a gene on
chromosome 7.137 The lack of b-glucuronidase can be docu-
mented in cultured fibroblasts. Metachromatic ‘Alder’ granules
have been identified in leukocytes of affected patients.
Clinical manifestations include mental retardation,
dysostosis multiplex, hepatosplenomegaly, frequent respiratory
infections, and umbilical hernias. Ocular findings can include
mild corneal opacities, papilledema, and retinal pigmentary

MAROTEAUX–LAMY SYNDROME (MPS VI)

Maroteaux–Lamy syndrome is caused by deficiency of the
enzyme arylsulfatase B (N-acetylgalactosamine-4-sulfate
sulfatase). Elevated levels of dermatan sulfate and heparan sulfate
are excreted in the urine. Prominent intracellular inclusions are
often present in circulating leukocytes (Fig. 45.21).
Systemic changes can resemble those of Hurler’s syndrome,
but intellectual function is usually preserved. Clinical features
include cardiac anomalies, dwarfism, and other skeletal
deformities. Spinal cord compression may occur owing to
vertebral anomalies. Meningeal involvement can cause
hydrocephalus.

FIGURE 45.19. FIGURE 45.20. Morquio’s syndrome, MPS IV. Patchy dotlike and
Morquio’s syndrome, diffuse corneal clouding gives the cornea a ground-glass appearance.
MPS IV. Dwarfism, Courtesy of Trexler M Topping, MD.
short neck, pectus
carinatum,
SECTION 6

kyphoscoliosis, and
other skeletal
abnormalities are
characteristic of
MPS IV.
Courtesy of Trexler M
Topping, MD.

FIGURE 45.21. Maroteaux–Lamy syndrome, MPS VI. Circulating

leukocytes often have prominent inclusions.
576 Courtesy of Trexler M Topping, MD.
 Corneal Manifestations of Metabolic Disease

FIGURE 45.22. Maroteaux–Lamy syndrome,

MPS VI. Corneal clouding varies from mild (a)
to moderate (b) in type A disease. (c and d) The
peripheral corneal clouding is much more dense
and visible to the unaided eye in type B
disease.
Courtesy of Trexler M Topping, MD.

a b

c d

degeneration, as well as late optic atrophy.100 Corneal clouding
may be severe enough to require transplantation.150
MISCELLANEOUS STORAGE DISEASES
‘Mannosidosis’ and ‘fucosidosis’ are caused by deficiencies of
a-mannosidase and a-fucosidase, respectively. In patients with
fucosidosis, corneal opacities in a verticillatapattern and con-
junctival and retinal vessel tortuosity (Figs 45.15 and 45.18),
have been noted.151 Cataracts, specifically spoke-like posterior
cortical opacification, have been observed in mannosidosis.152
of the corneal epithelium removes the corneal haze. The
stroma and endothelial layers of the cornea do not appear to
be involved.
XERODERMA PIGMENTOSUM
Xeroderma pigmentosum is an autosomal recessive disorder
caused by a deficiency of DNA repair mechanisms that pre-
disposes affected individuals to radiation-induced damage.

DISORDERS OF NUCLEIC ACID

METABOLISM

GOUT
Gout is a group of diseases of humans that result in a variable
combination of clinical findings including increased con-
centrations of serum urate, deposits of monosodium urate
monohydrate in and around the joints of the extremities
(tophi), renal disease, and uric acid urolithiasis. Hyperuricemia

CHAPTER 45
may result from an increased rate of uric acid production and by
diminished clearance by the kidneys.
Ocular findings in gout are rare, but histological or ultra-
structural features of tophi have been demonstrated in the
conjunctiva, cornea, lateral canthus, brow and orbit.153,154
Recently, urate crystals were reported in the iris and anterior
chamber.154 Ferry et al reported on the ocular abnormalities
in 69 patients with severe gout.155 The most common finding
was red eyes (62%), pingueculae (25%), elevated IOP (14%),
asteroid hyalosis (4%), and corneal crystals (one patient). Band
keratopathy (Fig. 45.23) consisting of urate crystals has been
reported.153 By slit-lamp examination, fine golden-yellow
scintillating crystals were found to be present in the epithelial
and subepithelial regions extending to the limbus; these were
more numerous in the interpalpebral space. On morphologic
examination, hexagonal, octagonal, or cylindrical crystals were FIGURE 45.23. Urate band keratopathy in a 68-year-old man with
found within the nuclei of epithelial cells. In cross-section, the gout. The patient had dry eyes with a Schirmer test with anesthetic of
crystals demonstrated a regular lattice structure. The cyto- 2-mm wetting. Vision was 20/50 and returned to 20/20 after the
plasmic structure of the cells was normal.156 Simple scraping epithelium was scraped. 577
 CORNEA AND CONJUNCTIVA
Various dermatologic manifestations of sun-exposed areas
include hypopigmentation, hyperpigmentation, hyperkeratosis,
and neoplasia (Fig. 45.24).157 Excision of multiple basal or
squamous cell carcinomas of the eyelids often produces
secondary eyelid deformities. Recurrent corneal ulcerations may
develop.158 Fibrovascular pannus of the cornea may develop as
well as squamous cell carcinoma (Fig. 45.25). Avoidance of sun
exposure and the generous use of sunblock are essential to
reduce cumulative actinic damage and subsequent neoplasia.
DISORDERS OF MINERAL METABOLISM
WILSON’S DISEASE
Key Features
• Autosomal recessive.
• The Kayser–Fleischer ring, copper deposition in the deep,
peripheral cornea, can be seen early in the disease process.
As the disease progresses, it becomes more prominent. It can
change in color from a yellow-green and gold hue to deep
brown. It may initially only be seen using gonioscopy, while
later it is obvious with the naked eye.
• Patients typically present with neurological symptoms. The
ophthalmologist may be consulted to evaluate the patient for a
Kayser–Fleischer ring.
• If treatment is started prior to significant liver and neurological
damage, patients often do very well. The Kayser–Fleischer ring
may regress or disappear with treatment.
SECTION 6
FIGURE 45.24. Xeroderma pigmentosum on sun-exposed areas of
skin showed hypopigmentation and hyperpigmentation with varied
intensities of pigmentation. The deformity of the left part of the
patient’s nose is due to surgical excision of a tumor.
From Calonge M, Foster CS, Rice BA, et al: Management of corneal
578 complications in xeroderma pigmentosum. Cornea 11:175, 1992.
FIGURE 45.25. Right eye of the patient shown in Figure 45.29 shows
corneal stromal scarring with peripheral pannus and lipid deposition.
The bulbar and tarsal conjunctiva are injected with notable
telangiectasia of vessels.
From Calonge M, Foster CS, Rice BA, et al: Management of corneal
complications in xeroderma pigmentosum. Cornea 1992; 11:175.
Wilson’s disease, an autosomal recessive disorder, is the most
common genetic disorder of copper metabolism. The gene for
Wilson’s is closely linked to the esterase D locus near 13q14.159
Both the biliary excretion of copper and its incorporation into
ceruloplasmin, the copper transport enzyme, are severely
impaired in Wilson’s disease, leading to progressive accumu-
lation of copper in the liver. Accumulation of copper also
occurs in the brain, especially in the basal ganglia. Neurologic
symptoms of tremor, dysarthria, or choreoathetosis may be
presenting signs, usually after puberty, but these can develop as
late as 60 years of age.160
Kayser–Fleischer rings are copper deposits in Descemet’s
membrane of the cornea (Fig. 45.26). They are first seen by
gonioscopy at the upper and lower limbal edges of Descemet’s
membrane. While they do not affect vision, they are an import-
ant diagnostic sign and management indicator. With time they
extend to the full corneal circumference and change from a
lighter yellow-green-gold color to deep brown, visible to the
unaided eye. They are found in most patients with neurologic
manifestations of Wilson’s disease and in ~95% of all Wilson’s
patients. The absence of a Kayser–Fleischer ring does not
exclude the diagnosis.161 It may be absent in up to 30% of young
patients presenting with acute liver disease and up to 60% of
patients in the presymptomatic stage.162 The rings may fade or
disappear after treatment.162–163 A Kayser–Fleischer ring may
also be seen in primary biliary cirrhosis, familial cholestatic
cirrhosis, neonatal liver disease, and multiple myeloma.164–167
Sunflower cataracts are disk-shaped axial opacities with
spoke-like deposits radiating peripherally. The deposits are
brilliantly colored immediately below the anterior and posterior
lens capsules. They occur in a minority of patients and do not
impair vision. They may disappear within a few years of starting
D-penicillamine therapy.168
The diagnosis of Wilson’s disease is most reliably made by
liver biopsy demonstrating greatly increased copper levels.
Administration of D-penicillamine is a standard treatment for
Wilson’s disease. Other therapies include trientine, zinc, and
tetrathiomolybdate. If therapy is instituted before severe hepatic
and neurologic damage have occurred, patients can enjoy a
normal life span and good health.

a b
c d
HEMOCHROMATOSIS
Hemochromatosis, a condition resulting from the excessive
accumulation of iron in various organs, manifests as cirrhosis,
diabetes mellitus, cardiomyopathy, hyperpigmentation,
arthritis, and hypogonadism. Iron overload can occur as a
consequence of excessive absorption of iron or after repeated
transfusions. Idiopathic hemochromatosis is transmitted by
autosomal recessive inheritance.
With excessive iron deposition, brown pigmentation may
appear at the eyelid margin and in the perilimbal conjunctiva
encroaching on the peripheral corneal limbus.169 The inferior
cornea is usually affected more than the upper. Histopathologic
examination has confirmed the presence of iron in the corneal
epithelium in affected patients.
MISCELLANEOUS DISORDERS
SCHNYDER’S CRYSTALLINE DYSTROPHY
Key Features
• Autosomal dominant
• Deposition of cholesteral and lipid in the cornea
• During young adulthood, the main feature is a prominent
corneal arcus
• During mid adulthood, the entire corneal stroma becomes
diffusely cloudy, greater centrally than in the mid-periphery
• The classic central superficial corneal crystals may only be
noted in half of patients with Schnyder’s. They can be seen as
early as the first decade
Schnyder’s crystalline dystrophy is transmitted by autosomal
dominant inheritance and characterized by bilateral deposition
of cholesterol and lipid in the cornea. The gene has been
mapped to chromosome 1p36.2-36.3. The word’s largest
pedigree of patients has a Swede-Finn heritage.
The dystrophy progresses with age. In the third decade of life,
a peripheral corneal arcus may be prominent. By the fifth decade
the stroma becomes diffusely cloudy (Fig. 45.27). As the patients
age, corneal sensation decreases and visual acuity worsens as
the central cornea becomes more hazy from cholesterol depo-
Corneal Manifestations of Metabolic Disease
FIGURE 45.26. Kayser–Fleischer ring in
Wilson’s disease. (a and b) These patients have
full 360-degree corneal involvement as is
classically described. (c) This patient has
involvement of only the upper cornea. (d) The
slit-lamp view of the patient in (c). The arrow
points to the Kayser–Fleischer ring in
Descemet’s membrane.
(a–d) From Wiebers DO, Hollenhorst RW, Goldstein
NP: The ophthalmologic manifestations of Wilson’s
disease. Mayo Clin Proc 1977; 52:414.
FIGURE 45.27. Cornea of a 78-year old woman with dense arcus and
diffuse stromal lipid deposition.
From Weiss JS: Schnyder’s dystrophy of the cornea. A Swede–Finn connection.
CHAPTER 45
Cornea 1992; 11:98.
sition. The opacification can involve the central cornea in a
disc-shaped pattern or the paracentral area in a ring distribution
(Fig. 45.28). The corneal opacities, even if prominent on
slit-lamp examination, do not greatly reduce visual acuity in
most patients. In a study of 33 patients with Schnyder’s
crystalline dystrophy, only 51% actually had clinical evidence of
corneal crystalline deposits.170 If cholesterol crystals are absent,
the disease may be very difficult to diagnose. A subtle corneal
haze best seen by retro-illumination may be the only sign
(Fig. 45.29).171 Crystals in the subepithelial or Bowman’s layer
may lead to epithelial destabilization and corneal epithelial
erosion. The crystalline opacities may disappear but recur over
a period of years.172
Various systemic lipid abnormalities occur in association
with Schnyder’s crystalline dystrophy, the most frequent of
which is familial hypercholesterolemia.173 The differential 579
 CORNEA AND CONJUNCTIVA
FIGURE 45.28. Schnyder’s crystalline dystrophy. Patient with ring of
fine crystalline deposits.
FIGURE 45.29. Schnyder’s crystalline dystrophy with a circular
pericentral non crystalline opacity
diagnosis includes systemic disorders affecting lipid metabolism,
such as LCAT deficiency, fish eye disease, Tangier disease, as
well as those disorders with corneal crystals, such as cystinosis,
multiple myeloma and gout.
There are no local or systemic treatments that halt the
SECTION 6
progression. Phototherapeutic keratectomy can be used to treat
subepithelial crystals if they are affecting vision.174 Penetrating
keratoplasty can be performed successfully in advanced cases.
Histochemical evaluations of excised penetrating keratoplasty
buttons have demonstrated crystalline deposits of cholesterol
or cholesterol esters within Bowman’s layer, superficial corneal
stroma, and anterior sclera.175 Recurrent deposition of
crystalline lipids may eventually occur in corneal allografts after
penetrating keratoplasty.176
AMYLOIDOSIS
Familial systemic amyloidosis may occur in association with
lattice corneal dystrophy, cranial nerve palsies, peripheral neu-
ropathy, and skin changes. Meretoja’s syndrome, an autosomal
dominant disorder, is further described in Chapter 241
(Vascular Lesions of the Orbit).
580
FAMILIAL DYSAUTONOMIA (RILEY–DAY
SYNDROME)
Key Features
• Autosomal recessive
• Decreased corneal sensation and severe dry eyes can lead to
severe ocular surface disease and even corneal erosion,
ulceration and perforation
• Aggressive treatment with ocular lubricants, punctal occlusion
and possibly autologous serum eye drops may be necessary
Riley–Day syndrome is a rare autosomal recessive disorder
that is characterized by sensory and autonomic nervous system
dysfunction.177,178 Approximately 1 in 3700 patients of
Ashkenazic Jewish or Eastern European ancestry is affected.179
The familial autonomia gene has been mapped to polymorphic
markers in the q31 to q33 region of chromosome 9. A deficiency
of the enzyme dopamine b-hydroxylase results in elevation of
levels of homovanillic acid.180
Clinical manifestations include paroxysmal hypertension,
emotional lability, increased sweating, an absence of fungiform
papillae on the tongue, and coldness of the distal
extremities.181–182 Excessive drooling is common due to salivary
gland hypersecretion. This may be attributable to salivary gland
denervation supersensitivity, a mechanism present in the
cardiovascular system and pupil in familial dysautonomia.183
The characteristic ophthalmic signs are reduced corneal
sensation and a lack of tearing, which may lead to exposure
keratopathy, corneal erosion, and eventual ulceration
(Fig. 45.30) and perforation.184 Early intervention with punctal
occlusion using silicone plugs or cautery can prevent signifi-
cant corneal morbidity and preserve vision. Autologous serum
eye drops may also be helpful. Other features including
blepharoptosis, anisocoria, tortuosity of retinal vasculature,
myopia, and anisometropia may also be present.184,185 Prenatal
diagnosis of familial dysautonomia in families with a previously
affected child can be performed using linkage analysis.
FIGURE 45.30. Familial dysautonomia, Riley–Day syndrome. The
patient is a young girl with a neurotrophic corneal epithelial defect that
resulted in sterile ulceration and stromal scarring.
From Mandel ER, Wagoner MD: Atlas of corneal disease. Philadelphia: WB
Saunders; 1989:48.

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Wilson disease and esterase D in 3
kindreds: detection of linkage for an
autosomal recessive disorder by the family
study method. Genet Epidemiol 1986; 3:201.
160. Danks DM: Disorders of copper transport.
In: Scriver CR, Beaudet AL, Sly WS, Valle
D, eds. The metabolic basis of inherited
disease. New York: McGraw-Hill;
1989:1411–1431.
161. Demirkiran M, Jankovic J, Lewis RA, et al:
Neurologic presentation of Wilson disease
without Kayser-Fleischer rings. Neurology
1996; 46:1040.
162. Lossner A, Lossner J, Bachmann H, Zotter
J: The Kayser-Fleischer ring during long-
term treatment in Wilson’s disease
(hepatolenticular degeneration). A follow-up
study. Graefes Arch Clin Exp Ophthalmol
1986; 224:152.
163. Esmaeli B, Burnstine MA, Martonyi CL,
et al: Regression of Kayser-Fleischer rings
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 CORNEA AND CONJUNCTIVA
systemic manifestations of Wilson’s
disease. Cornea 1996; 15:582.
164. Kaplinsky C, Sternlieb I, Javitt N, Rotem Y:
Familial cholestatic cirrhosis associated
with Kayser-Fleischer rings. Pediatrics
1980; 65:782.
165. Lipman RM, Deutsch TA: A yellow-green
posterior limbal ring in a patient who does
not have Wilson’s disease. Arch
Ophthalmol 1990; 108:1385.
166. Tauber J, Steinert RF: Pseudo-Kayser-
Fleischer ring of the cornea associated with
non-Wilsonian liver disease. Cornea 1993;
12:74.
167. Dunn LL, Annabele WL, Kliegman RM:
Pigmented corneal rings in neonates with
liver disease. J Pediatr 1987; 110:771.
168. Wiebers DO, Hollenhorst RW, Goldstein
NP: The ophthalmologic manifestations of
Wilson’s disease. Mayo Clin Proc 52:409.
169. Davies G, Dymock J, Harry J, Williams R:
Deposition of melanin and iron in ocular
structures in haemochromatosis. Br J
Ophthalmol 1972; 56:338.
170. Weiss JS: Schnyder crystalline dystrophy
sine crystals. Recommendation for a
revision of nomenclature. Ophthalmology
1996; 103:465.
171. Weiss JS: Schnyder’s dystrophy of the
cornea: A Swede-Finn connection. Cornea
1992; 11:93.
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172. Chern KC, Meisler DM: Disappearance of
crystals in Schnyder’s crystalline corneal
dystrophy after epithelial erosion. Am J
Ophthalmol 1995; 120:802.
173. Bron AJ, Williams HP, Carruthers ME:
Hereditary crystalline stromal dystrophy of
Schnyder. I. Clinical features of a family
with hyperlipoproteinemia. Br J Ophthalmol
1972; 56:383.
174. Dinh R, Rapuano CJ, Cohen EJ, et al:
Recurrence of corneal dystrophy after
excimer laser phototherapeutic
keratectomy. Ophthalmology 1999;
106:1490–1497.
175. Weller RO, Rodger FC: Crystalline stromal
dystrophy: histochemistry and
ultrastructure of the cornea. Br J
Ophthalmol 1980; 64:46.
176. Ehlers N, Matthiesson ME: Hereditary
crystalline corneal dystrophy of Schnyder.
Acta Ophthalmol 1973; 51:316.
177. Brunt PW, McKusick VA: Familial
dysautonomia: a report of genetic and
clinical studies, with a review of the
literature. Medicine 1970; 49:343.
178. Gitlow SE, Bertani LM, Wilk E, et al:
Excretion of catecholamine metabolites by
children with familial dysautonomia.
Pediatrics 1970; 46:513.
179. Eng CM, Slaugenhaupt SA, Axelrod FB,
et al: Prenatal diagnosis of familial
dysautonomia by analysis of linked
CA-repeat polymorphisms on
chromosome 9q 31-q 33. Am J Med
Genet 1995; 59:349.
180. Axelrod FB, Iyer K, Fish I, et al: Progressive
sensory loss in familial dysautonomia.
Pediatrics 1981; 67:517.
181. McKusick VA: Mendelian Inheritance in
Man. Baltimore: Johns Hopkins University
Press; 1990:1382.
182. Riley CM: Familial dysautonomia: clinical
and pathophysiological aspects. Ann NY
Acad Sci 1974; 228:283.
183. Mass E, Wolff A, Gadoth N: Increased
major salivary gland secretion in familial
dysautonomia. Dev Med Child Neurol 1996;
38:133.
184. Goldberg MF, Payne JW, Brunt PW:
Ophthalmologic studies of familial
dysautonomia: the Riley-Day
syndrome. Arch Ophthalmol 1968;
80:732.
185. Worobec-Victor SM, Bain MAB:
Oculocutaneous genetic diseases. In: Renie
WA, ed. Goldberg’s genetic and metabolic
eye disease. Boston: Little, Brown and
Company, 1988:515.
 CHAPTER
Immunologic Disorders of the Conjunctiva,
46 Cornea, and Sclera
C. Stephen Foster
Key Features
• The eye may be affected by immunologically driven
inflammation
• Understanding the mechanism of the immune reaction helps to
guide therapy
• The immune dysregulation may be from a systemic, potentially
lethal disorder
The eye can be affected by any of the immunologic hyper-
sensitivity reactions, and understanding the mechanism of a
particular patient’s inflammatory problem lays the groundwork
for correct treatment. The diagnostic pursuit of a mechanistic
understanding of a patient’s inflammatory problem is, at the
very least, sight saving and even may be life saving.
In this chapter, ocular diseases are grouped by the primary
Gell, Coombs, and Lackmann hypersensitivity reactions that
exist at the heart of the inflammatory mechanism. The four
types of hypersensitivity reactions rarely exist in pure form (i.e.,
in isolation from each other) in human pathologic states; it is
typical for hypersensitivity reactions to have more than one of
the classic Gell and Coombs responses to the inflammatory
problem. In cases in which it is known to occur, these com-
binations of types of mechanisms are pointed out in the various
ocular diseases presented and discussed in this chapter.
OCULAR ALLERGIC DISORDERS
SEASONAL ALLERGIC CONJUNCTIVITIS
Seasonal allergic rhinitis and seasonal allergic conjunctivitis
(SAC) are caused by a pure type 1 hypersensitivity mechanism.
Indeed, these are perhaps the only ocular inflammatory diseases
to satisfy all of Koch’s postulates, as re-phrased by Witebsky, for
proving that an inflammatory problem is immunologic: (1) one
or more causative antigens has been identified (e.g., ragweed
pollen), (2) the details of the immunologic response to the
antigen have been elucidated (e.g., immunoglobulin E
(IgE)–ragweed antibody production), (3) an animal model of the
disorder has been produced, and (4) adoptive transfer between
syngeneic animals has been accomplished.
People who develop SAC are atopic. Hay fever (either sea-
sonal or perennial), asthma, and eczema or atopic dermatitis
are considered the ‘major’ atopies, and idiopathic urticaria,
nonhereditary angioedema, and food allergies are classified as
‘minor’ atopies. Together, the atopies affect 10–20% of the
general population, and males are over-represented in that
number. SAC occurs in a large proportion of people afflicted by
seasonal rhinitis. Diagnosing the disorder usually is not
difficult. The patient complains of the typical symptoms of
ocular itch and watering, often in association with the sneezing
and nasal congestion symptoms of seasonal allergic rhinitis.
The seasonal influence on the appearance and disappearance of
the symptoms is obvious from the history, and a positive family
history of atopy is obtained in ~70% of patients with SAC.
Signs of ocular inflammation, even during the time of maximal
symptoms, usually are unimpressive. The globes usually are not
obviously inflamed. Indeed, the conjunctiva may appear totally
white and quiet. Further inspection by biomicroscopy, however,
often reveals mild edema of the bulbar conjunctiva and signs of
inflammation in the tarsal conjunctiva, both upper and lower.
Increased mucus is found in the preocular tear film and in the
inferior fornix.
Appropriate laboratory testing to establish the diagnosis
includes a quantitative serum IgE level, analysis of conjunctiva
(tarsal conjunctival scraping or conjunctival biopsy) with
specific attention to the presence of mast cells and eosinophils,
particularly in the epithelium, and skin testing for hyper-
sensitivity to ubiquitous environmental allergens. The latter is
most appropriately performed by skilled allergists.
Treatment of SAC should include the following steps: environ-
mental controls, mast cell-stabilizing agents throughout the
patient’s known allergy seasons, therapy for the nose with mast
cell stabilizers (e.g., azelastine) and aerosolized corticosteroid
(e.g., fluticasone propionate), systemic antihistamines when
environmental allergen exposure is unavoidable, topical com-
bination antihistamine and mast cell stabilizers (e.g., olopa-
tidine), and desensitization immunotherapy.
The value of the involvement of an expert allergist in the
care of a patient with SAC and other ocular allergic disorders
cannot be overemphasized. The allergist is a better environ-
mental detective than most ophthalmologists and can provide
the patient with specific instructions for environmental control
procedures, ranging from specific air-conditioning units,
furnace air-filtering devices, air purifiers, mattress and bedding
material alterations, and specific housecleaning techniques as
well as issues relating to existing carpeting and pets, the elimi-
nation of which is sometimes essential, particularly in the more
severe forms of allergy. The allergist also is the best resource for
expert skin testing and identification of the allergens responsible
for provoking episodes of SAC and can determine whether the
use of systemic antihistamines and the embarkation on the
lengthy road of desensitization immunotherapy are appropriate.
Topical antihistamines may be helpful, temporarily, in
patients with mild SAC, but because these agents competitively
inhibit only one mediator liberated by the mast cells, they are
not as effective a therapeutic strategy as are mast cell-stabilizing
agents during long-term therapy. The latter, used correctly,
stabilize mast cell membranes, and inhibit degranulation of all
the mast cell mediators, thereby preventing major SAC attacks, 585
 CORNEA AND CONJUNCTIVA

The diagnosis of GPC usually is not difficult to make.

FIGURE 46.1. Giant papillary conjunctivitis, stage 2. By slit-lamp
biomicroscopy with white light in the absence of fluorescein dye, the
large (greater than 1 mm in diameter) papillae in the upper (lower
portion of the lid) tarsal conjunctiva are evident, but just barely.
Compare this with the ease of viewing of these papillae after the
instillation of 2% fluorescein dye (see Fig. 46.2).
Symptoms of decreasing contact lens tolerance and increasing
mucus production in a previously successful contact lens wearer
are the primary features that should stimulate suspicion of
GPC. Some degree of ocular itch may be present, and exam-
ination of the upper tarsal conjunctiva discloses conjunctival
hyperemia and tarsal papillae greater than 1 mm in diameter
(Fig. 46.1). The geographic extent of the papillary response and
the size of the papillae, as well as the presence or absence of
epithelial erosions on the apices of the papillae, are important
features guiding therapy. Two percent fluorescein dye instilled
into the preocular tear film, with subsequent eversion of the
upper eyelid and examination of the tarsal conjunctiva with
cobalt blue-filtered light, facilitates the recognition of low-
profile papillae because the fluorescein dye outlines the
macropapillae as it lies in the valleys at their bases (Fig. 46.2).
The dye also shows stained epithelial defects at the apices of
macropapillae (Fig. 46.3).
Treatment of GPC may be difficult in the patient who is
determined to continue with contact lens wear. In most cases,

and limiting the amount of each of the hundreds of mediators

typically released by mast cells when they degranulate. Thus,
4% disodium cromoglycate, 2% nedocromil sodium, and
lodoxamide have been shown to be safe and effective in the
treatment of patients with SAC.1,2 Additionally, the dual acting
(H1 inhibition plus mast cell stabilization) agents, olopatidine,
azelastine, and epinastine 0.05% are even more user friendly in
the case of patients with ocular allergy because of twice daily
dosing effectiveness.

GIANT PAPILLARY CONJUNCTIVITIS

Spring3 was the first to describe the condition now known as
giant papillary conjunctivitis (GPC). In 1974, he reported that
78 of 170 soft contact lens wearers developed an allergic reaction
on the upper tarsal conjunctiva, presenting with complaints FIGURE 46.2. Same patient, same date, same flipped upper lid as
of contact lens intolerance and excessive mucus production. shown in Figure 46.1, after instillation of 2% fluorescein dye,
Allensmith and co-workers4 more definitively described this photographed using light filtered with a cobalt blue filter. Note the
collection of the fluorescein dye in the valleys between the papillae,
disorder and called it GPC because of the appearance of papillae
which outlines the bases of the periphery, making the detection of the
in the upper tarsal conjunctiva; these papillae grew larger when geographic extent of the bumps much easier.
the condition was left untreated. Biopsy of the conjunctival
papillae discloses mast cells in the conjunctival epithelium and
substantia propria, eosinophils in the same sites, and occasionally
SECTION 6

basophils in the conjunctival epithelium or substantia propria.

Mast cell participation in GPC is substantially greater than
would first appear to be the case on the basis of light micro-
scopic observations; ultrastructural studies show many more mast
cells than can be observed by light microscopy, with ultrastruc-
tural evidence of mast cell degranulation, leaving mast cell
remnants that cannot be seen at the light microscopy level.5
GPC develops as a result of tarsal conjunctival sensitization
to allergenic material present on the surface of the contact lens,
coupled with the trauma to the upper tarsal conjunctiva asso-
ciated with the excursion of the eyelid over the soft lens at each
blink, an event that occurs 10 000 to 16 000 times each day.
Scanning electron microscopy studies show that within 8 h of
wear, the contact lens becomes coated with a material
composed of mucus, protein, bacteria, cells, cell debris, and air-
borne pollutants.6 Vigorous contact lens hygiene, with lens
cleaning and enzyme treatment, fails to remove this lens FIGURE 46.3. Same patient, same eye, same flipped upper lid as
coating completely, and successive days of wear results in a shown in Figures 46.1 and 46.2. Note the subtle staining of the apices
586 steady buildup of the lens coat. of some of the papillae.
 Immunologic Disorders of the Conjunctiva, Cornea, and Sclera
FIGURE 46.4. Vernal keratoconjunctivitis with the obvious giant
papillae, or ‘cobblestones’, in the upper tarsal conjunctiva.
FIGURE 46.5. Vernal keratoconjunctivitis. Note the bulbar
conjunctival injection, the milky edema of the conjunctiva, and the
characteristic ropy mucous thread on the cornea.
however, patient education about proper lens hygiene, environ-
mental controls, modification of contact lens material and
design, and limitation of contact lens use can keep patients in
their contact lenses. Proper contact lens hygiene involves
vigorous daily cleaning with a soft contact lens cleaning agent,
hydrogen peroxide sterilization, lens storage in preserved saline
solution, and protein enzyme treatment at least twice a week;
some patients require enzymatic treatment of the lens every
other day. Conjunctival irrigation with the contact lens in place
four to eight times a day with unpreserved saline solution may
be of some benefit, and contact lens wearing time should be
kept to 50 h/week or less if the patient expects to remain a
successful contact lens wearer for many years. Finally, the
patient must be educated that the contact lens should be
considered a disposable device and encouraged to replace the
contact lens frequently. Low-cost disposable lenses may be ideal
for this reason, but even for those patients who cannot achieve
satisfactory wear with one of the available disposable lenses,
contact lens replacement every 4–8 weeks is recommended in
patients with GPC.
Modifying the contact lens edge-design or using a different
polymer material often is helpful in the care of patients with
GPC of moderate severity, in whom the aforementioned steps
have not been successful in achieving lens comfort. Prescribing
a new lens of different polymer and edge-design modifies the
conjunctival trauma and allergen adherence profile in com-
parison with the original lens that resulted in the GPC. This,
combined with a vigorous lens hygiene program, may be all that
is required for a contact lens-intolerant GPC patient.
If all these steps result in improved patient comfort and lens
tolerance, but the patient still has distracting symptoms, then a
mast cell stabilizing agent may be added.
VERNAL KERATOCONJUNCTIVITIS
Vernal keratoconjunctivitis (VKC) is an allergic conjunctival
inflammatory disorder with (in most cases) an associated
secondary keratopathy. VKC is characterized by the classic
hallmark of giant papillae, usually in the upper tarsal con-
junctiva but in some cases in the conjunctiva at the corneo-
scleral limbus. It is a disease predominantly of young men,
with pronounced seasonal (spring) influence, probably sec-
ondary to vernal allergens, but perennial forms exist as well. It
also can affect women, and some patients do not ‘outgrow’ their
VKC. A personal or family history of atopy usually is uncovered,
and in many cases, specific allergens to which the patient is
sensitive can be determined by history and by scratch and prick
allergen skin testing. One particularly notorious provocative
allergen in patients with VKC is the house dust mite and its
feces.
The predominant symptom of VKC is itching. As a rule, the
patient spontaneously complains of profound itching. Excessive
tearing, mucus production, photophobia, and burning or foreign
body sensation are common symptoms.
The classic sign of palpebral VKC is the giant papillae or
cobblestone in the upper tarsal conjunctiva (Fig. 46.4). These
papillae markedly increase the mass of the upper lid, and hence
ptosis is an additional typical sign. Inflammation of the bulbar
conjunctiva is variable, but a ropy, lardaceous thread almost
invariably can be found in the inferior fornix (Fig. 46.5).
Patients with VKC also may develop the ‘mucus fishing
syndrome’ because of this elastoid, irritating mucous thread,
with the result that an especially ocularly pernicious conspiracy
between these two problems is established.
The keratopathy of VKC typically begins as a diffuse super-
ficial punctate keratitis. If the inflammation continues with an
outpouring of inflammatory mediators into the tear film and
with associated epithelial toxicity and possibly conspiracy from
CHAPTER 46
the mechanical effects of the large papillae, a frank epithelial
defect appears next. These defects have been termed ‘shield
ulcers’ because of their position and morphology (Fig. 46.6).
Epithelial defects are trophic, defying the therapeutic strategies
that usually are successful in healing corneal abrasions or
epithelial defects (i.e., lubrication, patching, wearing a soft
contact lens). The longer such trophic defects persist, the higher
is the likelihood of eventual stromal ulceration and permanent
corneal scarring. Secondary microbial infestation also may
complicate this condition (Fig. 46.7). Successful treatment of
such defects invariably requires control of the ocular
inflammatory problem (discussed later).
The limbal form of VKC was first described by Arlt in 1846,7
pre-dating the description by von Graefe of the palpebral
form by 25 years.8 This form, common in highly pigmented
people, is characterized by the presence of large papillae in
the conjunctiva at the corneoscleral limbus, with associated
collections of inflammatory cells rich in eosinophils at the
apices of the limbal papillae, the so-called Horner–Trantas 587
 CORNEA AND CONJUNCTIVA
FIGURE 46.6. Shield ulcer in a patient with vernal
keratoconjunctivitis. This ulcer had persisted for 4 months before
referral.
FIGURE 46.7. Vernal keratoconjunctivitis with a persistent shield
corneal ulcer treated with topical corticosteroids. Note the suppurative
keratitis in the central cornea caused by Candida albicans infection.
SECTION 6
FIGURE 46.8. Limbal vernal keratoconjunctivitis. Note the white
588 Horner-Trantas dots on the apices of the limbal papillae.
FIGURE 46.9. Atopic keratoconjunctivitis with massive inflammatory
mound formation at the limbus.
dots (Fig. 46.8). In especially severe forms of limbal VKC,
the steady accumulation of inflammatory cells may result
in formation of a frank mound on the peripheral cornea
(Fig. 46.9).
The histopathology of the conjunctival papillae discloses
not only the cells typically associated with allergic reactions
(mast cells and eosinophils) but also large collections of
mononuclear cells, fibroblasts, and newly secreted collagen.
This tremendous influx of cells and collagen formation
increases the mass of the upper lid in palpebral VKC. The
histopathologic and immunopathologic characteristics of the
tissues has led some authorities9,10 to conclude that VKC is
not a pure type 1 Gell and Coombs hypersensitivity reaction,
but rather a combination of both type 1 and type 4 reactions.
Immunohistochemical studies show that the mononuclear
cells are rich in helper (CD4) T cells and that the cytokines
produced by the inflammatory cells are, among other things,
inducing abnormal expression of class 2 HLA glycoproteins
on conjunctival epithelium and stromal cells.9
Epidemiology
VKC has a worldwide distribution, with pronounced regional
variations in prevalence. It is most common in the
Mediterranean region and Central and South America. It is
relatively rare in North America and Northern Europe. It may
represent as much as 3% of serious ophthalmic disease in some
regions,11 whereas in Northern Europe and North America, the
prevalence is ~1 in 5000 cases of eye disease.12 VKC has been
reported to affect patients from 1 month to more than 70 years
of age, but at least 50% of the patients in most reported series
are between 5 and 25 years of age. In most patients, the disease
resolves spontaneously within 10 years of onset. It has been
associated with keratoconus, atopy, and atopic cataract. In a
study by Dart,13 78% of 120 patients with VKC developed the
disorder before the age of 16 years. Dart found that the corneal
complications of VKC in this population occurred almost
exclusively in patients with palpebral or mixed palpebral and
limbal VKC. He found no differences in serum or tear IgE levels
among VKC patients with the various forms of the disease; the
VKC patients did have higher than normal levels of IgE, and
specific IgE to cat dander and to house dust mites was detected.
Twenty-seven percent of the study population lost vision as
a result of VKC, and Dart commented that ‘therapeutic
complications are also common and may lead to blindness’.
 Immunologic Disorders of the Conjunctiva, Cornea, and Sclera
Therapy
Allergen avoidance
Although it usually is an unpleasant, expensive, time-
consuming exercise, policing the patient’s environment, and
scrupulously cleaning it of all potential allergen provocateurs
is critical to the long-term stability of patients with VKC.
Involvement by an expert allergist is essential; this allergist
should perform not only the appropriate patch, scratch, and
prick tests as well as the serum radioallergosorbent test (RAST)
but also the environmental detective work and motivational
and educational work necessary for a successful environmental
control program. Obviously, the family must be convinced of
the long-term benefits, not only to the patient but also to the
family as a whole, before they will seriously embark on a
complex program that sometimes involves removal of expensive
carpeting, installation of air conditioning, installation of air-
filtering systems in the home heating system, removal of
beloved pets, and other measures. The wisdom, importance,
and usefulness of this component of the patient’s care cannot
be overemphasized.
Systemic medication
Systemic antihistamine therapy is superior to topical ocular
antihistamine therapy in patients with the more complicated
allergic eye diseases, primarily because these diseases last so
long but also because these allergic patients sometimes become
sensitized to the preservatives present in the commercially
available ocular antihistamines. The use of cetirizine, fexo-
fenadine, or loratidine usually is sufficient. In the patient with
a significant ‘neuroconjunctivitis’ component of itch-scratch-
itch, the use of slowly escalating doses of hydroxyzine
(beginning with 50 mg at bedtime and slowly increasing as
needed) may interrupt this pernicious cycle.
Systemic desensitization immunotherapy may be indicated
in the patient who has striking sensitivity to a limited number
of allergens. Performing desensitization immunotherapy on a
patient with ocular allergy is not easy, however, and some
features of this practice are different from the typical practice of
desensitization immunotherapy in the patient with allergies not
affecting the eyes (see Atopic keratoconjunctivitis).
Ocular therapy
Mast cell-stabilizing agents are the mainstay of successful
treatment of patients with VKC. They have been shown,
unequivocally, in randomized, placebo-controlled clinical
trials,14,15 to be both safe and effective. Mast cell-stabilizing
agents available for ocular use in the United States include 4%
disodium cromoglycate, 0.1% lodoxamide, olopatidine, epinastine,
and azelastine have H-1 receptor blocking (antihistamine)
activity plus mast cell stabilization properties and are effective
with twice daily application. Nedocromil sodium, 2% eye drops,
is available in some of the Western European countries.
Topical corticosteroid therapy is required for breakthrough
attacks of highly active VKC inflammation after the patient has
encountered a stimulating allergen. The so-called pulse therapy
strategy involves administration of 1% prednisolone sodium
phosphate, 1% prednisolone acetate, or 1% rimexolone four
times daily for 2 days, with subsequent tapering to three times
daily for the succeeding 2 days, twice daily for 2 days after that,
once daily for an additional 3 days, and subsequent discon-
tinuation thereafter. Long-term low-dose maintenance topical
steroid therapy is inappropriate.
Adjunctive ocular therapy may be required for secondary
infection, for extreme mucus production and mucous plaque
formation on shield ulcers (e.g., with 10–20% N-acetylcysteine
drops, four times daily), and for persistent epithelial defect (e.g.,
prolonged bandage soft contact lens, fibronectin, epidermal
growth factor). The key to healing a persistent epithelial defect
is control of the associated inflammation.
ATOPIC KERATOCONJUNCTIVITIS
‘Atopic keratoconjunctivitis’ was defined by Hogan16 in 1952 as
allergic keratoconjunctivitis occurring in association with
atopic dermatitis (eczema). This definition, although imprecise
(patients with SAC, after all, are atopic), is in common usage
and is used here to connote the patients with the most severe
form of atopic ocular disease seen in association with eczema.
The argument by some physicians that, other types of atopic
conjunctivitis, such as chronic allergic conjunctivitis or
perennial atopic conjunctivitis, also are atopic ocular diseases
and therefore can be confused with atopic keratoconjunctivitis
is not a constructive one, particularly in view of the fact that in
those latter disorders, keratitis or significant keratopathy is not
part of the clinical picture. Corneal disease is, however, typical
of patients with atopic keratoconjunctivitis.
The term ‘atopy’ originally was coined by Coca and Cooke in
1923.17 It is derived from the Greek atopos, meaning out of
place, and it is defined by a group of findings occurring in
patients with a family history of allergic disease. These findings
include hay fever, bronchial asthma, and atopic dermatitis as
the ‘major’ atopies; and food allergies, urticaria, and non-
hereditary angioedema as ‘minor’ atopies. About 10–20% of the
general population is affected by one or another of the atopic
disorders.18 The reported incidence of ocular involvement in
atopic dermatitis is between 25% and 42%.19,20 This represents
a substantial number of people who are at risk of bilateral
blinding corneal complications from this complex inflam-
matory disorder. Atopic keratoconjunctivitis is always bilateral.
The symptoms include ocular itch, burning and foreign body
sensation, excessive tearing, and an abundant mucoid
discharge. Cicatrizing conjunctivitis may develop with chronic
conjunctival inflammation, and lid dermatitis and chronic
blepharitis with lid thickening and meibomian gland dys-
function are typical. Loss of vision occurs as a result of corneal
scarring and neovascularization.21 The disorder has been
neglected somewhat in the ophthalmic literature, and this is
especially regrettable because successful care of the atopic
patient is complex and commonly fragmented, with a failure to
provide long-term care by the physicians involved.
Atopic patients may have multiple immunologic derange-
ments, but one of the most notable is a defect in the T cells
responsible for regulating IgE production to one or more
allergens (ubiquitous environmental materials usually not
associated with allergy).
CHAPTER 46
Therapy for AKC must occur in concert with an allergist, and
allergen avoidance is critical. Systemic signal transduction
inhibitor therapy (cyclosporine) can be life transforming, and
similar treatment of the surface (tacrolimus for the skin and
cyclosporin for the eye), combined with long-term mast cell-
stabilizing therapy (e.g., olopatidine), can result in a very
limited need for the use of topical corticosteroid eye drop
therapy for breakthrough episodes of AKC.
TYPE 2 HYPERSENSITIVITY DISEASES OF
THE EYE
Although no disease affecting the eye has been definitively
proved to occur on the basis of a Gell and Coombs type 2
hypersensitivity reaction, the ocular consequences of cicatricial
pemphigoid, dermatitis herpetiformis, and pemphigus vulgaris
are believed to occur on this basis. This belief is based on the
nonocular findings of autoantibody deposition at the site of
disease activity22 and on the in vitro findings that such 589
 CORNEA AND CONJUNCTIVA
antibodies are pathogenic.23 Furthermore, the immuno-
pathologic characteristics of biopsy sampled ocular tissue
affected by these disorders is essentially the same as those
characteristics seen in skin affected by these blistering and
scarring autoimmune diseases. Hence, although circumstantial,
the evidence is strong that these diseases represent type 2
inflammatory disorders affecting conjunctiva.
OCULAR CICATRICIAL PEMPHIGOID
Ocular cicatricial pemphigoid (OCP) is a systemic autoimmune
disease with both ocular and non-ocular manifestations. Unlike
its blistering cousin, bullous pemphigoid, cicatricial pemphigoid
produces scarring of the affected skin. The so-called Brusting–
Perry dermatitis of cicatricial pemphigoid typically is confined
to the scalp and thorax. Scarring also is the typical consequence
of the inflammation that affects conjunctiva and other mucous
membranes. Indeed, the disease can be fatal when it produces
strictures from scarring in the esophagus or in the trachea.
Epidemiology
Although the estimated prevalence of this disease is only 1 in
20 000 ophthalmic cases,24 in fact, the disease is probably more
common than is recognized, because, the earliest stages of the
disease are underrecognized, and patients sometimes are
treated for ‘chronic conjunctivitis’ for many years before the
latter stages of the disease become grossly apparent. OCP has a
worldwide distribution, affects all races, and affects females to
a slightly greater extent than males. Although, it is said to be a
disease of old age (60s and 70s at disease onset), again, because
of the subtle nature of the subepithelial fibrosis in the earliest
stages of the disease, many cases probably begin when patients
are in their 40s and 50s.
Pathogenesis
At least two forms of OCP exist: idiopathic and drug induced.
Studies strongly suggest that, just as in the case of so many
other autoimmune diseases, a ‘two-hit hypothesis’ best fits the
available evidence regarding the pathogenesis. The first hit is
a genetic predisposition; in the case of OCP, the susceptibility
gene is at or closely linked to the HLA-DQw7 gene, and
people carrying this gene are at about a 9.6% relative risk of
developing OCP.25
The second hit in the development of pemphigoid most
probably is contact with a triggering or exciting agent in the
genetically susceptible person. Such an agent might be a virus,
as yet undefined; contact with a specific drug also may provoke
the onset of the autoimmunity in the genetically susceptible
SECTION 6
person. One notable example of this occurred in Great Britain
in the 1970s when the new b-blocking agent for treatment of
systemic hypertension, practolol, was introduced. Shortly after
its introduction, an epidemic of cicatrizing conjunctivitis with
features indistinguishable from idiopathic cicatricial pem-
phigoid appeared in British ophthalmic clinics.26 Similarly, so-
called pseudopemphigoid or drug-induced pemphigoid is seen
most typically in patients who have been receiving topical
medication for the treatment of glaucoma. Such cicatrizing
conjunctivitis cases have been seen after the topical use of
pilocarpine, epinephrine, timolol, and echothiophate iodide.27
Autoantibodies to a component in the conjunctiva, in the
vicinity of the lamina lucida of the basement membrane zone
are produced by people with OCP, and although the traditional
techniques for detecting autoantibodies fail to detect circulating
antibasement membrane zone antibodies in many OCP
patients, more sensitive techniques have disclosed the presence
of such antibodies in 100% of the patients tested thus far.28 Our
590 research aimed at defining the OCP antigen has disclosed that
the OCP antigen is different from the relevant antigen for
bullous pemphigoid. The antigen is a 205-kDa protein, the b4
peptide of a6 b4 integrin.29 The precise location of the antibody
binding site is in the intracytoplasmic portion of the b4 peptide
in the basal epithelial cell. 30
Just as in bullous pemphigoid, the autoantibody in OCP is
pathogenic in cicatricial pemphigoid. Antibody deposition, with
subsequent complement activation, probably results in a
cascade of events, including signal transduction disturbance
across the BMZ and basal epithelial cell, mast cell degranu-
lation from anaphylatoxin, with subsequent effects on
conjunctival vasculature from the vasoactive amines liberated
from the mast cells, recruitment of macrophages and
lymphocytes, liberation of cytokines from these inflammatory
cells, vascular damage and conjunctival epithelial damage from
these cytokines, upregulation of class 2 glycoprotein expression
on conjunctival epithelium and fibroblasts, with possible sub-
sequent contribution by these class 2 glycoprotein-bearing cells
to the inflammatory process, and fibroblast activation with
abnormal type 3 collagen secretion and with subsequent
cicatrization.31 Enormous numbers of immunologically active
cells are present in the substantia propria of patients with OCP,
and the predominant cells present are helper T lymphocytes
and macrophages.32 Systemic immunologic derangements are
present as well, including slightly abnormal proportions of
circulating helper T cells and evidence of systemic immuno-
reactivity with elevated levels of soluble interleukin-2 receptors,
elevated levels of soluble CD8 glycoprotein, and elevated levels
of tumor necrosis factor-a in the serum (unpublished
observations). These findings emphasize the unequivocal fact
that OCP is a systemic autoimmune disease. Even today, some
ophthalmologists still harbor the mistaken belief that OCP is a
local ocular problem that can be treated with local ocular
measures. This is not correct, and attempts to treat this disease
through local measures invariably result eventually in loss of
vision for the patient. The disease is systemic and must be
treated systemically.
Clinical Features
We initially described four clinical stages of OCP,33 and
subsequently described a more refined, precise staging system
that can enhance detection of even subtle disease progression.34
Stage 1 of the disease is characterized by chronic conjunctivitis
with mild conjunctival or corneal epitheliopathy, or both, and
subtle subepithelial fibrosis of the conjunctiva (Fig. 46.10).
FIGURE 46.10. Ocular cicatricial pemphigoid. Flipped upper lid
demonstrating the subepithelial fibrosis of the superior tarsal
conjunctiva.
 Immunologic Disorders of the Conjunctiva, Cornea, and Sclera
FIGURE 46.11. Ocular cicatricial pemphigoid, stage 1. Note the
fibronic striae under the inferior tarsal conjunctiva.
FIGURE 46.12. Ocular cicatricial pemphigoid, stage 2. Note the
subepithelial fibrosis under the tarsal conjunctival epithelium, with
formation of an extensive ‘feltwork’ of the new collagen, and the loss
of the normal depth of the inferior fornix (i.e., fornix foreshortening).
The latter is easily overlooked. It is best seen under the
epithelium of the upper or lower tarsal conjunctiva and presents
as fine white striae (Fig. 46.11) that, as they accumulate, may
coalesce to form a fine ‘feltwork’. Stage 2 is characterized by
progression of the cicatrizing process, with contraction of
the newly formed collagen, which foreshortens the inferior
fornix. The subepithelial fibrotic striae still may be seen
under the epithelium of the tarsal conjunctiva (Fig. 46.12).
Stage 3 is characterized by formation of the first obvious
symblepharon (Fig. 46.13). Stage 4, or end-stage disease, is
virtually untreatable and consists of a totally dry eye with
ankyloblepharon and keratinization of the cornea (Fig. 46.14).
Associated keratopathy, beginning with epitheliopathy and
progressing to corneal scarring and neovascularization, may
begin in stage 2.
A common myth about OCP is that it is a dry-eye syndrome
and, more specifically, that it is a mucin-deficient dry-eye
syndrome. This is not true until the later stages of the disease.
Indeed, in the earlier stages of the disease, patients with OCP
actually have an overproduction of mucus. This mucin, mixed
with proteins and nucleic acids from damaged cells, adheres to
the ocular surface epithelium and produces areas over which
FIGURE 46.13. Ocular cicatricial pemphigoid, stage 3. The cicatrizing
process has progressed to the point of formation of symblephara.
FIGURE 46.14. Ocular cicatricial pemphigoid, stage 4. Note the
leatherization of the ocular surface and the adhesion of the lids to the
globe.
the tear film breaks up more rapidly than it would normally
(Fig. 46.15). The findings of a relative decrease in the numbers
of goblet cells in conjunctival biopsy specimens from patients
CHAPTER 46
with OCP and of a more rapid tear film breakup time were
features that led Lemp35 to conclude, erroneously, that this
disease is a mucin-deficient dry-eye syndrome. It is true that as
the cicatrizing process progresses, with deformation of lash
follicles and compromise of lacrimal ductules and meibomian
gland ductules, aqueous and oil (and eventually mucin)
deficiencies begin to appear. With the appearance of the sec-
ondary phenomena of trichiasis, distichiasis, and tear film
abnormalities, the blinding consequences are corneal epithe-
liopathy, epithelial defect formation, secondary infection with
corneal ulceration, and corneal neovascularization (Fig. 46.16).
Non-ocular manifestations of cicatricial pemphigoid include
mucosal lesions in the nose, mouth, pharynx, trachea, eso-
phagus, anus, urethra, and vagina. Any patient in whom OCP
is suspected must be questioned carefully about the presence of
dysphasia or difficulty breathing. If the physician believes that
even the remote possibility exists of esophageal or tracheal
involvement, careful endoscopic evaluation for this possibility
is essential. 591
 CORNEA AND CONJUNCTIVA
FIGURE 46.15. Conjunctival biopsy of a patient with ocular cicatricial
pemphigoid. Note the normal surface specializations of the epithelium
(inferior half of the photograph) with microplicae and microvillae. The
superior half of the photograph, however, shows a thick mucous mat
adherent to the ocular surface epithelium.
TABLE 46.1. Differential Diagnosis of Cicatrizing Conjunctivitis
Cicatrizing pemphigoid
Atopic keratoconjunctivitis
Ocular rosacea
Scleroderma
Corynebacterium diphtheriae conjunctivitis
Chemical burn
Squamous cell carcinoma
Intraepithelial epithelioma
Stevens–Johnson syndrome
Lyell’s syndrome
Sarcoidosis
Trachoma
Adenovirus conjunctivitis
Trauma
Sebaceous carcinoma
SECTION 6
Diagnosis
A variety of disorders can cause subepithelial fibrosis of the
conjunctiva with or without inflammation (Table 46.1), many
of which can be excluded on the basis of history. The definitive
establishment of a diagnosis of OCP requires the demon-
stration, by immunopathologic technique, of immunoreactant
(immunoglobulin or complement component) deposition at
the epithelial basement membrane zone of biopsy sampled
affected inflamed conjunctiva (Fig. 46.17). Performing
immunopathologic studies on sampled conjunctiva is difficult
because of differing requirements from immunopathology of
skin, kidney, liver, and other organs and because few centers
with skilled ocular immunopathologists exist. In addition to
the usual requirements of extremely careful, expert handling
and processing, the availability of a full panel of antibody
reagents, including antibodies to serve as positive and negative
controls, is essential. A further advantage is the ability to
592 analyze the tissue by enhanced immunoperoxidase and even
FIGURE 46.16. Ocular cicatricial pemphigoid with extensive
keratopathy, corneal scarring, and neovascularization.
FIGURE 46.17. Conjunctival biopsy of a patient with active ocular
cicatricial pemphigoid, shown with immunofluorescence microscopy.
The primary antibody used on this specimen was antiimmunoglobulin
A. The bright apple-green, linear, continuous line of fluorescence of
the epithelial basement membrane zone shows that this patient has
large amounts of immunoglobulin-A deposited at the epithelial
basement membrane zone, an abnormal finding.
immunoelectron microscopic techniques in cases in which
the traditional immunofluorescence analysis is negative.
Treatment
Systemic therapy
Caring for patients with cicatricial pemphigoid is difficult and
multidisciplinary, requiring the close involvement of both the
ophthalmologist and a chemotherapist. The ophthalmologist’s
role is to apprise the chemotherapist of the state of inflam-
matory activity in the conjunctiva. The chemotherapist’s role is
to modify treatment based on therapeutic response and
systemic drug tolerance. A hand-in-glove relationship between
the ophthalmologist and an oncologist or hematologist can be
an effective collaboration. Randomized, controlled clinical trials
have shown that the systemic chemotherapeutic agents are both
safe and effective, when used properly, in ~90% of patients with
OCP.33 The current therapeutic recommendations remain the
same as previously described36: if the definitively diagnosed
pemphigoid clearly is active (inflamed) and progressive, the use
of dapsone is recommended, provided the patient is not allergic
to sulfa-containing drugs and is not deficient in glucose-6-
 Immunologic Disorders of the Conjunctiva, Cornea, and Sclera
TABLE 46.2. Therapy for Ocular Cicatricial Pemphigoid
Agent Initial Dose
Dapsone 25 mg b.i.d.
Methotrexate 7.5 mg once weekly
Azathioprine 2 mg/kg/day
Cyclophosphamide 2 mg/kg/day
Prednisone (adjunctive) 1 mg/kg/day
Cytosine arabinoside 0.2 mg/kg; 5 days/month
phosphate dehydrogenase. If the response to dapsone is
incomplete, once-weekly methotrexate or daily azathioprine or
mycophenolate mofetil is added to the therapeutic program. If
there is no response to the dapsone, it is discontinued, and
either azathioprine or mycophenolate mofetil or once-weekly
methotrexate is used. Patients who fail these regimens and
those who have extremely active, rapidly progressive OCP are
treated with high-dose systemic prednisone and either daily oral
cyclophosphamide or intravenous-pulse cyclophosphamide.
After the inflammation is controlled, the daily prednisone is
tapered, changed to alternate-day therapy, tapered further, and
eventually discontinued within 3 months. Selected patients
who also failed to respond to these treatment regimens adequately
have responded to subcutaneous cytosine arabinoside. This
approach to OCP therapy has been reserved for otherwise resist-
ant cases because of its logistically cumbersome administration
requirements. Cyclosporine is remarkably ineffective in treating
OCP. The chemotherapeutic drugs used to treat this disease
and their starting dosages and dose ranges are listed in
Table 46.2. Finally, intravenous biologic therapy with intravenous
immunoglobulin or with intravenous daclizumab.
Adjunctive ocular therapy
Removal of lashes that are abrading the cornea is an essential
part of the care of patients with OCP. Epilation is temporarily
effective, but the lash that has re-grown after epilation in fact
may produce more damage than the original lash that was
epilated; the re-grown lash produces a stabbing-type injury to
the cornea, and the re-growth is short and stiff, whereas the
original lash may have been a long, supple lash that was
brushing over the surface of the cornea. Permanent destruction
of the lens follicles is the ideal treatment, although accom-
plishing this can be difficult. Electrolysis, cryoablation, and
marginal lid rotation and follicle extirpation surgeries all have
been employed with varying degrees of success. Keratinized
posterior lid margin conjunctiva may respond to topical retinoid
therapy provided the immunologically driven conjunctival
inflammation has been brought completely under control with
immunosuppressive therapy. The effect of topical retinoids is
variable and often unimpressive, however, and lid margin
mucous membrane grafting procedures may be required to treat
extensive keratinization of tarsal conjunctiva effectively.37
Aqueous deficiency should be treated with punctal occlusion
and attention to the lids and meibomian glands (warm
compresses and meibomian gland massage with lid hygiene,
with or without systemic tetracycline therapy), and ocular
lubricants, preferably without preservative, may be indicated.
Corneal hypoesthesia and lagophthalmos should be treated
with tarsorrhaphy.
Corneal surgery is highly ill-advised in patients with
advanced OCP. The outcome almost invariably is the formation
of an epithelial defect, with subsequent stromal ulceration and
Maximal Dose
150 mg/day
15 mg once weekly
3 mg/kg/day
3 mg/kg/day
1 mg/kg/day
0.3 mg/kg; 5 days/month
perforation. Penetrating keratoplasty may be used to treat a select
few patients with corneal scarring but with good lid function
and reasonably good tear production. In most patients with
pemphigoid and significant corneal pathology, however, corneal
sensibility is profoundly impaired, the eye is very dry, and normal
lid function is impaired. For these patients, keratoprosthesis is
the only realistic surgical alternative that holds any hope for
visual rehabilitation.
PEMPHIGUS VULGARIS
Pemphigus vulgaris is a blistering autoimmune skin disease
that in the past was universally fatal. It affects people of all ages
and has widespread geographic distribution, but Ashkenazi Jews
are markedly overrepresented in groups of patients with
pemphigus vulgaris. This link led to the discovery of the HLA
association with this disease; susceptibility to the disease is
carried by the HLA-DQ3 gene DQw8. The relevant antigen of
pemphigus vulgaris is the intercellular cement of epithelial cells
(an adhesion molecule or cadherin previously designated PVA
for pemphigus vulgaris antigen) and now known to be des-
moglein 3, a member of the cadherin transmembrane adhesion
molecule family which interacts with plakoglobin in the
desmosome. Additional targets of the autoimmune auto-
antibody response are probably operative as well, most
particularly anticholinergic receptor antibodies.38
The circulating autoantibody (IgG, IgA, or IgM) deposits on
this 130-kDa glycoprotein, activating complement, releasing
plasminogen activator, forming plasmin, and dissolving the
bonds between adjacent epithelial cells. The epithelial cells then
separate from one another (acantholysis) forming blisters under
the surface epithelium. These blisters rupture, leaving an intact
layer of basal epithelium attached to a normal basement
CHAPTER 46
membrane at the base of the blister.
Ocular Manifestations
Lid skin manifestations of pemphigus vulgaris are not rare, but
frank conjunctival manifestations are. Conjunctival bullae with
subsequent rupture and erosions can occur, however; but these
heal with no evidence of the subepithelial fibrosis, fornix fore-
shortening, and symblepharon formation typical of OCP.39 Rare
cases of the latter have been reported, perhaps because of
secondary infection or because the patient had concomitant
pemphigus vulgaris and OCP.40
Treatment
In the past, patients with pemphigus vulgaris died of sepsis and
electrolyte imbalance from the extensive fluid loss from open
skin lesions. High-dose systemic corticosteroid therapy changed
that, and the introduction of combined cytotoxic immuno-
suppressive therapy with corticosteroid therapy appears to have
induced cases of long-term remission. The cytotoxic agents 593
 CORNEA AND CONJUNCTIVA
employed in the care of patients with pemphigus vulgaris have
included azathioprine, cyclophosphamide, methotrexate and
intravenous immunoglobulin.
DERMATITIS HERPETIFORMIS
Ocular Manifestations
Dermatitis herpetiformis is an autoimmune blistering derma-
tosis characterized by a pruritic eruption, usually of the scalp,
buttocks, lower back, and extensor surfaces of the arms.
Mucous membrane involvement, including ocular involvement,
is rare but can occur. The disease is chronic, with remissions
and exacerbations. The autoantibody generally is of the IgA
class, and immunochemical studies of biopsy sampled affected
skin show deposition of IgA and complement in a granular (or
in rare cases, linear) pattern at the dermal–epidermal junction.
The target autoantigen is tissue transglutaminase 2. Gluten-
sensitive enteropathy is associated strikingly with dermatitis
herpetiformis, and there is an HLA-DR3 and HLA-B8 genetic
association. The ocular manifestations reported are those of a
chronic or recurrent cicatrizing conjunctivitis producing
subepithelial fibrosis and symblepharon.41
Treatment
Dapsone is an extraordinarily effective therapy for dermatitis
herpetiformis. The usual caveats apply regarding sulfa allergy and
glucose-6-phosphate dehydrogenase deficiency. Starting with doses
of 50 mg/day and escalating to, in most cases, 150–200 mg/day
is typical. Concomitant systemic corticosteroid therapy and
antihistamine therapy also may be used, and dietary analysis
with gluten restriction is employed by many dermatologists in
the care of patients with dermatitis herpetiformis.
TYPE 3 HYPERSENSITIVITY DISEASES OF
THE EYE
MOOREN’S ULCER
The evidence that Mooren’s ulcer has a type 3 hypersensitivity
mechanism as at least part of its etiopathogenesis is not vast.
This disease is included here, however, because some
circumstantial evidence for this type of mechanism exists and
because the disease has some striking similarities to the
peripheral ulcerative keratitis (PUK) associated with some of the
classic type 3 circulating immune complex systemic disorders,
such as rheumatoid arthritis and polyarteritis nodosa (PAN).
Mooren’s ulcer, in fact, was first described by Bowman in
1849,42 and McKenzie43 later remarked on ‘chronic serpiginous
SECTION 6
ulcer of the cornea’ in 1854. Mooren’s name, however, became
attached to this curious disorder because of his publication of
cases in 1867.44 The disease is rare, with fewer than 200 cases
described in the world’s ophthalmic literature; some of the
cases included in reports of ‘Mooren’s ulcer’ are in fact not true
Mooren’s ulcer but rather cases in which PUK was the
presenting manifestation of an occult systemic disease.
Mooren’s ulcer is, by definition, idiopathic, occurring in the
absence of any diagnosable systemic disorder that could be
responsible for the progressive destruction of the cornea. It also
is strictly a PUK, with no associated scleritis. This latter point
is of substantial importance because so many of the mis-
diagnosed cases were in patients who had PUK in association
with adjacent scleritis, necrotizing or otherwise. Wood and
Kaufman45 emphasized the distinction between limited
Mooren’s ulcer, unilateral, usually occurring in older people,
and bilateral ‘malignant’ Mooren’s ulcer, which typically was
relentlessly progressive despite all previously described
594 treatments.
Interestingly, my experience has been just the opposite; most
of my patients with progressive, bilateral Mooren’s ulcer have
been older than 40 years,46 and a careful review of all reported
cases suggests that the bilateral form of the disease is
overrepresented in the older patients in the world’s reported
cases of Mooren’s ulcer.47
This latter form of the disease was observed to occur most
often in younger males. An especially interesting group of young
African males has been described with bilateral Mooren’s ulcer,
and some evidence was collected suggesting that helminthiasis
was associated with the disease and that progression of PUK
was halted by local ocular therapeutic measures combined with
systemic therapy for the helminthiasis.48 Although Schazlin49
and others have conjectured that the Ascaris and Ancylostoma
species parasites caused the Mooren’s ulcer, possibly through
antigen–antibody reactions to helminth toxins deposited in the
peripheral cornea, helminth infestation is epidemic in the
countries where these cases of Mooren’s ulcer in young African
males have occurred, and yet the disease, even in these endemic
areas of ascariasis, is rare. Other putative causes of some cases
of Mooren’s ulcer have included trauma, herpes, hepatitis C
virus, and ocular surgery. Again, with the exception of the cases
that follow trauma, it is difficult to indict the other agents as
true causes of PUK associated with Mooren’s ulcer simply
because herpes simplex virus, varicella-zoster virus, and
cataract surgery themselves can be associated with subsequent
PUK, often with associated necrotizing scleritis. This is not the
entity that Mooren described.
Clinical Features
Mooren’s ulcer is a PUK that begins in clear cornea at the
corneoscleral limbus and progresses centrally, circumferentially,
and posteriorly through the cornea, leaving a thinned, vascu-
larized corneal residua in its wake (Fig. 46.18a). The edge of the
progressive ulcer is undermined, and the extent of dissolution
of the cornea may be surprising as one gently probes the
overhanging edge of the ulcer with a thin probe (Fig. 46.18b).
White blood cell infiltrates in the corneal stroma in advance of
the edge of the ulcer are characteristic. The condition is painful,
and the pain is usually well out of proportion to obvious signs
of ocular (conjunctival) inflammation. A low-grade iritis may be
present, and spontaneous or traumatic perforation may occur.
The epithelium of the central edge of the ulcer remains intact;
conjunctival epithelium covers the thinned, vascularized cornea
left in the wake of the advancing ulcer. This may give the
impression that there is a progressive corneal thinning with
associated keratitis but without an epithelial defect. This is not
true, and the presence of the narrow, crescent-shaped epithelial
defect can be seen after instillation of 2% fluorescein eye drops,
with subsequent forced lid closure for 30 s and with evaluation
of the eye using cobalt blue light. The destructive process
progresses slowly, with eventual destruction of the entire extent
of the cornea, leaving thinned, vascularized residua. The
sometimes unbearable pain the patient has experienced
throughout this saga, suddenly vanishes when the process has
finally swept over the entire geographic extent of the cornea, but
this process may take 4–18 months.
Pathogenesis
Immunoglobulins and complement are found in the peripheral
cornea of patients with PUK typical of Mooren’s ulcer, and this
has suggested the possibility that Mooren’s ulcer represents a
type 3 immune complex deposition hypersensitivity reaction.
As with all type 3 diseases, the predominant cell attracted to the
site of immune complex deposition is the neutrophil, which is
found in great abundance in the area of corneal destruction.
The corneal destruction results from liberation of proteases and
 Immunologic Disorders of the Conjunctiva, Cornea, and Sclera

a b

FIGURE 46.18. (a) Mooren’s ulcer. Note the peripheral ulcerative keratitis that has begun at the 3 o’clock position and progressed clockwise to
the 6:30 position, counterclockwise to the 1 o’clock position, and centrally to involve (apparently) ~2.5 mm of the cornea. Note also the
inflammatory infiltrates in advance of the edge of the ulcer. (b) Probing of the edge of this ulcer demonstrates that the extent of the undermining
and hence of destruction of the corneal stroma is astonishingly much greater than was apparent on inspection by slit-lamp biomicroscopy.

collagenase from the neutrophil granules. The substantia

TABLE 46.3. Differential Diagnosis of Peripheral Ulcerative
propria of the conjunctiva adjacent to the ulcerating cornea is Keratitis
filled with plasma cells. Immunoglobulins found in the peri-
pheral cornea may be manufactured locally by these plasma Ocular, Infectious
cells in the conjunctiva. Schaap and associates50 found Any microbe
circulating autoantibodies to cornea in patients with Mooren’s
ulcer, and Berkowitz and colleagues51 found circulating immune Ocular, Noninfectious
complexes in patients with PUK associated with Mooren’s ulcer. Mooren’s ulcer
Interestingly, removal of conjunctiva adjacent to the ulcerating
Systemic, Infectious
cornea, combined with resection of the necrotic, ulcerating
cornea and application of tissue adhesive to exclude neutrophils Gonococcus
from access to the region, results in an instantaneous cessation Bacillary infection
of corneal destruction. This surgical procedure is curative in
most patients with the limited, unilateral form of Mooren’s Acquired immunodeficiency syndrome
ulcer.52 The destructive process resumes, however, in patients Tuberculosis
with the bilateral form once the conjunctiva has re-grown to the
Syphilis
limbus and has been re-populated with the immunocompetent
cells responsible for antibody and cytokine production. These Systemic, Noninfectious
patients usually require systemic cytotoxic chemotherapy to Rheumatoid arthritis
stop the progressive destruction.
Evidence of autoimmunity directed against an antigen or Systemic lupus erythematosus
antigens from corneal stroma in a patient with bilateral Polyarteritis nodosa
Mooren’s ulcer in 197953 have been found, and Gottsch and
Wegener’s granulomatosis
associates have identified a specific corneal protein as the
probable target autoantigen in this autoimmune disease.54 Relapsing polychondritis

Diagnosis
The differential diagnosis of PUK is shown in Table 46.3.
Scrapings for culture generally establish an infectious cause in
infectious PUK, although I have experience with three patients
whose PUK was caused by a limbal vasculitis secondary to
herpes simplex virus; the diagnosis was established only after
conjunctival resection and analysis, by immunohistologic tech-
niques, for the presence of herpes antigens. The vasculitic
collagen diseases typically cause an associated scleritis with the
PUK that may accompany them. But because each of these may
produce isolated PUK, and because PUK may be the presenting
manifestation of one of these potentially lethal systemic
vasculitic disorders and may precede obvious nonocular clinical
manifestations by many months, any patient with presumed
Mooren’s ulcer should be evaluated for the possibility of an
occult systemic vasculitic disease. Mooren’s ulcer can then be
appropriately diagnosed after these other disorders are excluded
(see Table 46.3).
CHAPTER 46
Behçet’s disease
Sarcoidosis
Inflammatory bowel disease
Rosacea
Treatment
The recommended initial treatment of progressive Mooren’s
ulcer (Fig. 46.19) is wide conjunctival resection to bare sclera,
extending at least to the 2 o’clock position on either side of the
peripheral ulcer and posteriorly for 4 mm. This is followed by
resection of the overhanging lip of ulcerating cornea and
application of tissue adhesive, with soft contact lens application
and with subsequent topical 1% prednisolone sodium
phosphate application four times daily. Others have advocated
aggressive (every 15–30 min) topical steroid therapy, describing 595
 CORNEA AND CONJUNCTIVA
FIGURE 46.19. Mooren’s corneal ulcer with perforation, after
conjunctival resection, ulcer débridement, and application of
cyanoacrylate tissue adhesive at the perforation site at the 2 o’clock
position on the limbus.
good results with this approach in patients with the limited
form of Mooren’s ulcer.52 Systemic tetracycline may be used
for its anticollagenolytic properties, as may topical 1%
medroxyprogesterone, eight times daily.
Bilateral progressive Mooren’s ulcer almost always requires
systemic cytotoxic chemotherapy to stop the progressive
corneal destruction. I typically treat bilateral cases that have not
progressed too extensively with the aforementioned approach
(i.e., conjunctival resection with tissue adhesive, soft contact
lens, and topical steroids) first. With the first appearance of
recurrence of keratitis, however, I will institute therapy with
systemic methotrexate (7.5–15 mg once weekly), azathioprine
(2 mg–1 kg–1 day–1), or cyclophosphamide (2 mg–1 kg–1 day–1).
This approach has been highly successful.53 A small number of
reports have been made of successful therapy of unilateral
Mooren’s ulcer treated with topical cyclosporine.55 Systemic
therapy for the bilateral cases typically is continued for 6
months, with subsequent attempts at tapering and discon-
tinuation of the medication.
The anamnesis and the ocularly pernicious nature of this
disease are documented amply in the ophthalmic literature.
Attempts at reparative corneal surgery almost always are
unsuccessful unless the underlying Mooren’s disease activity
has been controlled. Even when the disease has ‘burned itself
out’, attempts at corneal grafting usually are associated with
SECTION 6
recurrence of Mooren’s ulcer and destruction of the graft.56 An
example of this latter circumstance was seen in a patient57 who
underwent grafting 15 years after Mooren’s ulcer had destroyed
his cornea. This patient was not immunosuppressed before
grafting, and Mooren’s ulcer recurred in the edge of the
transplant. The graft perforated, the patient developed
endophthalmitis, and the eye was enucleated. The immune
system has a remarkable memory, and this is a vivid illustration
of that. Patients with ‘burned-out’ Mooren’s ulcer should be
immunosuppressed before cataract surgery or corneal grafting
procedures. Furthermore, I disagree vigorously with the
statement by Schazlin47 that ‘since the value of immuno-
suppressive therapy is less clear than other treatments, they are
recommended only in the severest and most resistant cases’.
On the contrary, it is believed that the evidence for the efficacy
of systemic immunosuppressive chemotherapy for progressive
bilateral Mooren’s ulcer is strong and that the evidence suggests
that such treatment should be employed sooner rather than
596 later in the care of such patients, before the corneal destruction
has become so extensive that surgical efforts at rehabilitation
are required.
Rehabilitative surgical therapy for this disease generally
requires two procedures: (1) lamellar tectonic grafting, followed
by (2) definitive central penetrating grafting. The lamellar graft
is required because of lack of sufficient peripheral cornea into
which a penetrating graft might be secured.
COLLAGEN VASCULAR DISEASES
Rheumatoid Arthritis
Rheumatoid arthritis is a crippling, potentially lethal collagen
vascular disease that can affect the eye in many ways, the most
common of which is through autoimmune damage to the
lacrimal gland with resultant dry-eye syndrome. It is possible
(even likely) that some of the ocular devastation and morbidity
associated with rheumatoid arthritis could be prevented if
patients with the earliest ocular manifestations of this disease
were treated more aggressively. For example, since rheuma-
tologists have turned to high-dose intravenous-pulse steroid
therapy and to once-weekly methotrexate therapy earlier in the
care of patients with the nonocular manifestations of
rheumatoid arthritis, fewer of the serious, late ocular
manifestations of this disease have been seen. I have cared
for one patient with rheumatoid arthritis who developed
pronounced, acute dacryoadenitis with associated profound
keratoconjunctivitis sicca (KCS); this patient’s lacrimal gland
inflammation resolved with systemic corticosteroid therapy,
with resultant resolution of the KCS. Two additional
recurrences of this phenomenon were similarly treated with like
outcomes.
Keratoconjunctivitis Sicca
The KCS associated with rheumatoid arthritis can be highly
destructive. It usually manifests with the typical dry-eye
symptoms of foreign body sensation, blepharitis, excessive
mucus production, and a sensation of dryness or paradoxical
tearing, all of which progress the longer the patient is awake.
The typical signs observable on evaluation include a diminished
marginal tear strip, superficial punctate keratitis, and ocular
surface epitheliopathy with rose bengal staining in the typical
horizontally oriented diamond pattern in the intrapalpebral
fissure (Fig. 46.20). In especially severe or neglected cases, the
superficial punctate keratitis may progress to filamentary
FIGURE 46.20. Keratoconjunctivitis sicca after instillation of 1% rose
bengal solution. The photograph has been taken with a red-free light
source. Note the punctate staining of the corneal epithelium in the
interpalpebral fissure.
 Immunologic Disorders of the Conjunctiva, Cornea, and Sclera
FIGURE 46.21. Keratoconjunctivitis sicca with filament formation in
the supernasal sector of the peripheral and midperipheral corneal
epithelium.
keratopathy, epithelial defect formation, stromal ulceration, and
corneal perforation (Fig. 46.21).
Treatment for KCS always should include vigorous attention
to the meibomian glands and lid margins. In KCS, meibomian
gland dysfunction nearly always is present, and a deficient oil
layer component to the preocular tear film is at least as
important (if not more so) than a deficient aqueous layer in the
abnormal function of the tear film. Lid hygiene with vigorous
warm compresses and lid massage, with or without the use of a
systemic tetracycline derivative, therefore is indicated in the
care of patients with KCS. Additionally, I am relatively
aggressive about puncta occlusion in these patients. If Schirmer
values (after the use of topical anesthetic and subsequent
inferior fornix blotting with tissue) are consistently 3 mm or
less, he occludes the puncta permanently with a hyphrecator.
For Schirmer values of 3–10 mm, I insert punctum plugs into
the inferior puncta. Artificial tears are still used, but better
insights gained through recent studies may indicate that use of
commercially available artificial tears may be more harmful
than helpful.58 In any case, my current practice is to be cautious
with the use of artificial tears and to have the patient
experiment with one commercial product in one eye and
another in the other eye, trying various preparations until the
best preparation is found. The deleterious effects of preser-
vatives in some of the commercial preparations, as compared
with the lack of convenience and risk associated with the
preparations not containing preservatives, should be weighed
by both the physician and the patient. Topical cyclosporine
(Restasis ®) twice daily can result in decreased lacrimal gland
lymphocytic infiltration and increased tear production in
patients with active dacryoadenitis but residual glandular acini
capable of function after resolution of imflammation.
Additional tear conservation methods, including specially
prepared glasses with side shields, the use of room humidifiers
in dry climates and during the winter, and even marginal
tarsorrhaphy, are other appropriate therapeutic measures that
may be employed, depending on the severity of the case.
Episcleritis and Scleritis
Between 1981 and 1996, 172 patients with scleritis and 94
patients with episcleritis were cared for by me, and these
patients formed the basis of our text on the sclera.59 Three of
the 94 patients with episcleritis had rheumatoid arthritis, and
32 of the 172 patients with scleritis had rheumatoid arthritis.
The episcleritis in the patients with rheumatoid arthritis was
FIGURE 46.22. Diffuse scleritis. Note the slightly purple contribution
to the red appearance of the eye.
relatively trivial in all cases, although it generally required the
use of a systemic nonsteroidal antiinflammatory drug (NSAID)
for resolution, unlike typical idiopathic episcleritis. Topical
corticosteroids are never indicated in the care of a patient with
episcleritis because the evidence is abundant that, although
they may be effective temporarily, their use prolongs the natural
history of resolution of episcleritis and makes each recurrence
more difficult to treat through a rebound phenomenon.59
Topical NSAIDs are also ineffective in treating episcleritis.60
Scleritis, on the other hand, is an extremely serious problem
that threatens the patient’s vision. It always requires systemic
treatment, and topical steroids may be indicated as adjunctive
therapy. Additionally, it is critical that the ophthalmologist
distinguish between episcleritis and true scleritis because the
latter carries serious nonocular implications. Patients with
rheumatoid arthritis who develop true scleritis must be watched
carefully because they are at higher risk of developing necro-
tizing scleritis and subsequent potentially lethal systemic
vasculitis.61–63 Distinguishing between episcleritis and scleritis
can be difficult. I agree with Watson and Hazelman64 that the
presence of scleral edema is the sine qua non for establishing
that a patient has scleritis. All of the other discriminatory signs
and symptoms are important, but the critical issue is whether
one can determine that scleral edema is present. Patients with
scleritis commonly complain of severe pain; those with
episcleritis rarely do. Patients with scleritis have an intensely
red eye with a violaceous or purple hue (Fig. 46.22), whereas
CHAPTER 46
patients with episcleritis have pink or bright-red conjunctival
and episcleral inflammation from dilation of the vessels in the
superficial episcleral vascular plexus (Fig. 46.23). These dilated
vessels typically blanche with the use of topical phenylephrine
(5% drops), whereas the dilated vessels in the deep episcleral
vascular plexus associated with scleritis often remain dilated
after the use of such drops. Palpating the globe through the
closed lids or through the lids while the patient is looking in one
gaze or another generally elicits tenderness to the touch in the
patient with true scleritis and little discomfort in the patient
with episcleritis. Slit-lamp biomicroscopy with a thin slit beam
is the only way to determine whether underlying scleral edema
exists. If it does, the beam is bowed forward as it makes its
excursion across the scleral surface (focusing through the con-
junctiva and the blanched conjunctival and episcleral vessels
after the use of topical phenylephrine).
In 32 patients with true scleritis associated with rheumatoid
arthritis, 11 had diffuse scleritis, five had nodular scleritis, 11
had necrotizing scleritis, four had scleromalacia, and one had 597
 CORNEA AND CONJUNCTIVA

FIGURE 46.23. Diffuse episcleritis. Note the brilliant red characteristic FIGURE 46.24. Necrotizing scleritis. Note the loss of sclera
of the conjunctival inflammation. superiorly, down to choroid, and the associated extensive avascular
area temporal to this area of near perforation in the right eye.

posterior scleritis. The patients with necrotizing scleritis
presented the most difficulty in management and experienced
the most extensive nonocular systemic vasculitic problems
(Fig. 46.24). Associated uveitis carried an especially poor visual
prognosis.65
Systemic therapy always was necessary for the successful
treatment of patients with scleritis. Systemic NSAIDs with or
without short-term high-dose systemic corticosteroid therapy is
the appropriate initial treatment in most patients with scleritis,
with the notable exception of those with necrotizing scleritis.
Patients with rheumatoid arthritis who develop necrotizing
scleritis are at high risk of dying from a vasculitic event within
2–5 years of onset of the necrotizing scleritis61,62 unless they are
treated with systemic immunosuppressive chemotherapy.63
Cyclophosphamide (2 mg–1 kg–1 day–1) is probably most effective
for the ocular and nonocular consequences of rheumatoid
vasculitis, although methotrexate (7.5–15 mg once weekly),
azathioprine (2 mg–1 kg–1 day–1), and cyclosporine (5 mg–1 kg–1
day–1) may also work. The choice of drug and management of
the patient should involve close collaboration between
chemotherapist and ophthalmologist. High-dose short-term
systemic prednisone may be indicated for temporary control
while chemotherapy induction is in progress, with subsequent
(within 4 weeks) prednisone taper, a switch to alternate-day
dosing, and eventual (within 3 months) discontinuation of the
prednisone.
Oral NSAIDs are usually sufficient for treatment of diffuse or
nodular scleritis. Just as in the case of NSAID response to
arthritis, however, the response of the patient with scleritis to
the NSAID initially chosen is unpredictable. I typically try at
least three NSAIDs in succession before concluding that a
patient’s scleritis is NSAID resistant. A list of the NSAIDs I
have used, with dosages, is given in Table 46.4. Eight of 11
rheumatoid arthritis patients with diffuse scleritis responded to
NSAID therapy alone.
Peripheral Ulcerative Keratitis
Peripheral corneal ulceration may be seen in patients with
rheumatoid arthritis in association with adjacent severe scler-
itis, usually necrotizing, but PUK may develop in the absence
of clinically obvious scleritis (Fig. 46.25). The pathogenesis of
these lesions has a vasculitic basis, with immune complex
localization in peripheral cornea and limbal vessels, chemotaxis
of inflammatory cells (particularly neutrophils and histiocytes),
and inflammatory cell enzyme liberation with resultant
collagen and proteoglycan destruction. The PUK typically

TABLE 46.4. Nonsteroidal Antiinflammatory Drugs

SECTION 6

Trade Name Generic Name Dose

Dolobid Diflunisal 500 mg b.i.d.

Naprosyn Naproxen 250–500 mg b.i.d.
Indocin Indomethacin 75 mg SR b.i.d.
Motrin Ibuprofen 800 mg t.i.d.
Feldene Piroxicam 20 mg q.d.
Butazolidin Phenylbutazone 100 mg t.i.d.
Nalfon Fenoprofen 600 mg t.i.d.
Voltaren Diclofenac 75 mg b.i.d.
Tolectin Tolmetin 400 mg t.i.d.
Meclomen Meclofenamate 100 mg q.i.d.
Ansaid Flurbiprofen 100 mg t.i.d.
Orudis Ketoprofen 100 mg t.i.d.
598
 Immunologic Disorders of the Conjunctiva, Cornea, and Sclera
a b
FIGURE 46.25. (a) Peripheral ulcerative keratitis in a patient with rheumatoid arthritis. Note the 360-degree ring infiltrate of inflammatory cells
with associated destruction of the peripheral cornea. (b) Same eye as shown in (2), after conjunctival resection, ulcer débridement, application of
cyanoacrylate adhesive, and application of a soft contact lens.
progresses both centrally and circumferentially, relentlessly,
unresponsive to commonly used topical forms of therapy for
corneal ulceration. The extraocular significance of PUK is
underrecognized; its significance is the same as that of necro-
tizing scleritis (see earlier) even if evidence of vasculitis at the
time of PUK development is lacking. Treatment for these
lesions is the same as that for necrotizing scleritis, that is, with
systemic immunosuppressive therapy after wide conjunctival
resection, ulcer débridement, application of tissue adhesive, and
the application of a soft contact lens. Topical (medroxypro-
gesterone, 1%, every 2 h while awake) and systemic (tetracyc-
line, 250 mg by mouth four times daily) collagenase inhibitors
or collagenase synthetase inhibitors may be used to some effect,
although it is negligent not to immunosuppress patients with
rheumatoid arthritis who develop PUK or necrotizing scleritis.
The most effective drug, as stated previously in the discussion
of necrotizing scleritis, is cyclophosphamide, but depending on
whether one or both eyes is involved and depending on the
speed with which the process is progressing, once-weekly
methotrexate or daily azathioprine or cyclosporine may be used
instead.
Marginal Furrow
Marginal corneal thinning without obvious inflammatory cell
infiltration into the cornea and without an overlying epithelial
defect may occur in patients with rheumatoid arthritis.66 The
cause of these marginal furrows, which often are in the inferior
aspect of the cornea, is unknown. The corneal epithelium is
intact over the progressively thinning cornea, and changing
degrees of corneal astigmatism may result. The lesions rarely
progress to the point of threatened perforation, do not
vascularize, and have no known effective treatment. Systemic
collagenase inhibitors, such as tetracycline and doxycycline,
may slow progression of these lesions.
Corneoscleral Ulceration after Cataract Surgery
Peripheral and, more rarely, central corneal ulceration and
necrotizing scleritis have been reported in patients with
rheumatoid arthritis (usually associated with KCS).67–71
Although Smith and Schanzlin67 impugn unrecognized dry eye
and epithelial damage at the time of cataract surgery, with
delayed treatment of postoperative epithelial defects as the
primary culprit in these cases, in many cases (particularly those
in which the pathology is strictly peripheral), the pathogenesis
appears to come from a slow accumulation of immune com-
plexes and immune complex-mediated vasculitic damage after
surgical trauma. The evidence for this mechanism of the
destructive lesions is particularly compelling in cases in which
the onset of PUK and necrotizing scleritis was delayed by weeks
or months after the cataract operation. Conjunctival resection
and removal of ulcerating scleral tissue at the time of scleral
transplantation in some of these patients has disclosed classic
vasculitis identical to that seen in scleral biopsy specimens from
patients with rheumatoid arthritis who developed idiopathic
necrotizing scleritis.71 Corneal ulcers in patients with
rheumatoid arthritis after cataract surgery may progress to
descemetocele formation, perforation, and loss of the eye.67–70
In cases of central corneal ulceration, aggressive treatment of
dry eye and exposure, through tarsorrhaphy and the use of
tissue adhesive if the progression of the central ulcer is rapid, is
essential if the globe is to be preserved. In patients with peri-
pheral ulceration with or without necrotizing scleritis, systemic
immunosuppression after resection of conjunctiva and
demonstration of unequivocal vasculitis is indicated if the globe
is to be salvaged.
Systemic Lupus Erythematosus
Episcleritis and Scleritis
Episcleritis or scleritis may occur in patients with systemic
lupus erythematosus (SLE) and may be the initial manifestation
of the disease. True scleritis is a reasonably accurate guide to the
CHAPTER 46
presence of significant systemic activity in patients with SLE
and resolves only with adequate control of systemic disease
activity; it does not respond to topical therapy. In my practice,
one of 94 patients with episcleritis had SLE, and seven of 172
patients with scleritis had SLE.59 The episcleritis was more
persistent than idiopathic episcleritis, and the patient insisted,
eventually, on systemic therapy. Systemic NSAIDs usually
eliminate the episcleritis completely, with the side benefit of
eliminating or decreasing the arthralgias experienced in a
significant proportion of patients with SLE. Systemic
hydroxychloroquine (200 mg twice daily by mouth) also was
highly effective in eliminating the episcleritis from one SLE
patient who was placed on this drug primarily for the skin and
constitutional symptoms associated with the SLE. Five of seven
SLE patients with scleritis responded completely to oral
NSAIDs, whereas two patients required the addition of
prednisone to the treatment regimen. No patient with SLE-
associated scleritis required the institution of immuno-
suppressive therapy for control of the scleritis. 599
 CORNEA AND CONJUNCTIVA
Keratitis
Corneal manifestations of SLE are confined primarily to ocular
surface epitheliopathy, although PUK has been described, and
keratitis with neovascularization has been described in patients
with discoid SLE. KCS, however, is extremely common in SLE
patients with inadequately controlled systemic disease.
Pillat72 reported superficial keratitis in one of 16 SLE
patients, and Gold and co-workers73 found a 6.5% incidence of
keratitis in an SLE outpatient population. Spaeth74 found that
88% of SLE patients hospitalized at the National Institutes of
Health had superficial punctate keratitis with fluorescein cor-
neal staining, even though their Schirmer values were normal.
Reeves75 reported the occurrence of deep bilateral segmental
interstitial keratitis and subsequent recurrent iritis in a patient
who, 1 year later, developed cutaneous, articular, and hema-
tologic manifestations of SLE. Halmay and Ludwig76 had des-
cribed similar findings in SLE patients.
Analysis of the entire population of SLE patients presenting
to me between 1981 and 1996 found that 16 of 47 patients had
corneal complications from the disease and that 62.5% of these
patients had associated KCS.
Therapy for the corneal disease associated with SLE includes
control of the underlying SLE disease activity as well as
adjunctive therapy for the eye. The well-known approach to
KCS (see the previous discussion of KCS associated with
rheumatoid arthritis), as well as the brief, judicious use of
topical corticosteroids for associated true inflammation of the
cornea, is the appropriate approach.
Polyarteritis Nodosa
Cornea External ocular manifestations of PAN include ptosis,
exophthalmos, extraocular muscular paresis, episcleritis,
conjunctival chemosis, scleritis, and PUK. Corneal and scleral
lesions in this disease are highly destructive and progressive
unless the correct diagnosis is made and control of the under-
lying systemic disease is achieved.
PUK may occur in PAN and may be its presenting
manifestation.77 A clinical characteristic of PUK in these cases
is corneal ulceration at the corneoscleral limbus that is
progressive centrally and circumferentially. This ulceration is
associated with ocular pain and inflammation and with
undermining of the central edge of the ulcer, resulting in an
overhanging lip of cornea producing a peripheral ulcer that
morphologically resembles Mooren’s ulcer. Adjacent sclera has
been involved in all of the reported cases, and this involvement
is a distinguishing characteristic from idiopathic localized PUK
of Mooren’s corneal ulcer.
Local therapy for these ulcers has failed routinely. Diagnosis
SECTION 6
of the underlying systemic condition and institution of
adequate systemic therapy to control the disease and the
destructive ocular lesions are essential. It is possible, however,
to retard progressive corneal destruction in cases of PUK
associated with PAN using local therapy while the underlying
systemic disease is being controlled. Conjunctival resection,
ulcer débridement, application of cyanoacrylate tissue adhesive
to the ulcer bed and to a small rim of surrounding normal
cornea and sclera, and application of a continuous-wear
bandage soft contact lens can be used to delay the degradation
process while the patient is being immunosuppressed. The use
of topical corticosteroids to inhibit inflammatory cell activity
and migration does not appear particularly effective and may be
harmful because it inhibits new collagen formation. Inhibitors
of collagenase synthesis, such as 1% medroxyprogesterone
drops, and competitive inhibitors of collagenase, such as
N-acetylcysteine (20% drops) and systemic tetracycline deri-
vatives, are adjunctive forms of therapy that may retard ulcer
600 progression while the systemic disease is being controlled.
The histopathology of the corneal lesions of PAN shows
almost exclusively a neutrophil infiltrate of the cornea, with
large numbers of plasma cells, histiocytes, and lymphocytes
and with various numbers of eosinophils in the adjacent
conjunctiva. Microvasculopathy typically is demonstrable in the
conjunctiva and in the sclera that is resected from areas of
associated necrotizing scleritis. The mechanisms involved in
the production of corneal and scleral lesions in PAN appear the
same as those associated with the vasculitis lesions extra-
ocularly: immune complex deposition and inflammatory cell
chemotaxis into the area of deposition of immune complexes,
with vessel and adjacent tissue damage from the digestive
enzymes liberated from the phagocytes attracted to the area of
immune complex deposition.
Sclera
The scleritis of PAN may be diffuse, nodular, or necrotizing. It
is always painful and never responds to local therapy or to oral
NSAIDs. Successful resolution of PAN always requires the
use of systemic cytotoxic immunosuppressive agents, and
cyclophosphamide is the most effective agent. PAN is a lethal
disease that, if left untreated, has a 5 year mortality rate of
87%.78 Treatment with systemic corticosteroids improves the
5 year survival rate to only 50%,79 whereas the use of oral
cyclophosphamide results in induction of permanent remission
in most cases and in a 5 year survival rate of 80%.79 The
ophthalmologist who diagnoses the patient with scleritis with
occult PAN not only has provided the opportunity to save
the patient’s eye but also has set the stage for saving the
patient’s life.
In my practice, PAN was diagnosed in two of 172 patients
with scleritis.59 One patient had diffuse scleritis, and the other
had necrotizing scleritis; both cases were unilateral. PUK
accompanied the necrotizing scleritis.
Wegener’s Granulomatosis
Ocular involvement commonly occurs in Wegener’s granu-
lomatosis (50–60% of cases), and the ocular lesion may be the
presenting symptom. Focal ocular manifestations of Wegener’s
granulomatosis include conjunctivitis, episcleritis, scleritis, and
PUK. Conjunctivitis and episcleritis are the most common, and
if the underlying Wegener’s granulomatosis is not diagnosed,
these relatively benign ocular manifestations become chronic
and eventually may be accompanied by the more ominous PUK
and necrotizing scleritis.
Cornea
PUK is reported with increasing frequency as the initial
significant manifestation of Wegener’s granulomatosis.80 It
commonly is preceded by localized conjunctivitis or episcleritis,
followed by the onset of true scleritis and the development of
intrastromal peripheral corneal inflammatory infiltrates. Pain
may be mild or severe. The corneal ulceration develops with
breakdown of the peripheral corneal epithelium, and the
crescentic peripheral corneal ulcer progresses centrally and
circumferentially, just as do that of Mooren’s ulcer and that of
PUK associated with PAN (Fig. 46.26). The ocular lesion is
relentlessly progressive despite local medical and surgical
therapy; control of the ocular lesion depends entirely on control
of the underlying systemic disease. Strategies to delay the
disease process, as mentioned previously in the discussion of
PUK associated with PAN, are appropriate while systemic
immunosuppressive chemotherapy is being instituted. In my
practice, three of seven patients with Wegener’s granulomatosis
presented with PUK as the initial manifestation of their occult
Wegener’s granulomatosis. Careful review of systems and
laboratory pursuit of the diagnosis (before the advent of the
 Immunologic Disorders of the Conjunctiva, Cornea, and Sclera
FIGURE 46.26. Necrotizing scleritis and peripheral ulcerative keratitis
in a patient with Wegener’s granulomatosis.
antineutrophil cytoplasmic antibody (ANCA) testing) resulted
in a definitive establishment of the diagnosis in all cases,
through established histopathologic criteria. With the advent of
ANCA testing, ophthalmologists should be able to establish the
diagnosis of Wegener’s granulomatosis more quickly, and less
ocular damage and less permanent renal or pulmonary damage
should result.81
Multiple studies have confirmed that the presence of ANCA
is highly sensitive and specific for Wegener ’s granu-
lomatosis.82–84 The specificity is ~99%, and the sensitivity
depends on the extent of disease and is 96% for active
generalized disease, 67% for local regional disease, and 32% for
patients in remission after initial loco-regional symptoms.
Experience with ANCA testing in a large population of patients
with scleritis demonstrates the extraordinary utility of this test
in diagnosing occult manifestations.81 Indeed, every patient
with scleritis should undergo ANCA testing as part of the
diagnostic laboratory evaluation. Microscopic polyangiitis and
crescentic glomerulonephritis also can produce a positive
ANCA test, and patients with the so-called perinuclear ANCA
(P-ANCA) staining pattern usually have these other two
diseases. Patients with Wegener’s granulomatosis can also show
this pattern, although they usually show the diffuse granular
cytoplasmic pattern of staining (C-ANCA). Specific auto-
antibody analysis by ELISA discloses that autoantibodies
directed against myeloperoxidase (MPO) accounts for the
perinuclear pattern of stain on immunofluorescence (IF)
analysis, while antibodies directed against proteinase 3 (PR3)
account for the cytoplasmic pattern of IF staining.
ANCA testing is important not only diagnostically but also
therapeutically. Patients with Wegener’s granulomatosis are
treated to bring about a total resolution of ocular and nonocular
manifestations of the disease and, if at all possible (short of
producing systemic toxicity), to normalize the serum ANCA.
Failure of the ANCA to disappear from the patient’s serum is
associated with a significant risk of reactivation of Wegener’s
disease after discontinuation of cyclophosphamide or other
immunosuppressant agents. In several patients with the limited
form of Wegener’s granulomatosis and with ocular manifes-
tations, all ocular and sinus inflammation was abolished, and
remission of disease was maintained for more than 1 year, with
subsequent discontinuation of systemic cyclophosphamide; but
in the patients in whom the ANCA did not normalize,
Wegener’s disease activity recurred, not only in the eye but also
in the lungs or kidneys. Thus, extreme vigilance is essential in
treating patients with Wegener’s granulomatosis, limited or
otherwise, who fail to exhibit ANCA normalization while on
systemic chemotherapy.
Histopathologic analysis of resected conjunctiva from the
area adjacent to PUK rarely is diagnostic, but biopsy of sclera in
patients with necrotizing scleritis commonly is diagnostic. In
the presence of a typical granulomatous inflammatory reaction
with epithelioid cells and multinucleated giant cells,85
eosinophil infiltration may have special diagnostic significance,
especially if these cells are ‘activated’.
Sclera
The scleritis associated with Wegener’s granulomatosis may be
diffuse, nodular, or necrotizing. In my practice, three of seven
patients with Wegener’s granulomatosis and scleritis exhibited
diffuse scleritis, one had nodular scleritis, and three developed
necrotizing scleritis.81 As mentioned previously, specimens of
ulcerating sclera commonly show neutrophils in the area of
active scleral degradation, fibrinoid necrosis, and surrounding
granulomatous inflammation, with epithelioid cells and
multinucleated giant cells (Fig. 46.27). If intrascleral vessels are
obtained in the biopsy specimen, true necrotizing vasculitis
with inflammatory cell infiltration into the vascular wall and
fibrinoid necrosis of the vessel may be seen. Even if true
vasculitis cannot be seen, a constellation of histopathologic
features should make the ophthalmologist and pathologist
particularly suspicious of Wegener’s granulomatosis.85
I have never seen a patient with Wegener’s granulomatosis
with associated PUK who did not have involvement of the
adjacent sclera. As in patients with Wegener’s disease who have
PUK, systemic therapy is the key to salvage of the globe in those
with scleritis. Systemic cyclophosphamide has been shown,
unequivocally, to be the treatment of choice for patients with
Wegener’s granulomatosis; the 5 year mortality rate for patients
with this disease who are not treated or who are treated with
only systemic prednisone is 95%,86 whereas the 5 year mortality
rate for patients treated with systemic cyclophosphamide is
10%.86 Cyclophosphamide is started at a dose of 2 mg–1 kg–1
day–1, with dosing restricted to breakfast and with encourage-
ment of high levels of fluid intake in the afternoon and evening.
Monitoring of the ANCA levels, the peripheral hemogram, liver
enzymes, blood urea nitrogen, chest radiograph, sinus films,
and ocular lesions longitudinally is recommended. The drug
dosage is adjusted according to clinical disease activity, ANCA
levels, and systemic tolerance of the medication. A particular
cause of concern is when the patient appears clinically to be in
CHAPTER 46
FIGURE 46.27. Hematoxylin and eosin stain showing the
histopathology of a scleral biopsy in a patient with Wegener’s
granulomatosis. Note the extensive array of multinucleated giant cells. 601
 CORNEA AND CONJUNCTIVA
remission but the ANCA levels remain high, breakthrough
relapse attacks or relapse of potentially lethal Wegener ’s
granulomatosis activity after drug cessation is common in these
patients. Ordinarily, however, cyclophosphamide should be
tapered and withdrawn after a 6 month–1 year period of total
clinical quiescence and replaced by longterm once a week
methotrexate maintenance therapy. Azathioprine maintenance
is also acceptable. Patients who relapse are retreated with
cyclophosphamide. This disease does not respond well to
trimethoprim and sulfa combinations. Adjunctive prednisone is
used as needed for disease control when bone marrow tolerance
of cyclophosphamide is at its limit and clinical remission has
not been achieved. Azathioprine, methotrexate, chlorambucil,
and cyclosporine are alternative therapies; scattered anecdotal
reports have been made of successful induction of remission
using these drugs in patients intolerant to cyclophosphamide.
Pneumocystis carinii prophylaxis is generally accomplished with
twice weekly trimethoprim–sulfa preparations.
Relapsing Polychondritis
Relapsing polychondritis can be fatal when it affects the trachea
or the kidney. The most obvious manifestations of this disease
usually are in the external ear and in the cartilage of the nose
(Fig. 46.28), but the eye can be involved, and when it is,
necrotizing scleritis with or without PUK is the most ominous
manifestation. Because the disease is rare and the multisystem
involvement may not be simultaneous, the definitive diagnosis
of relapsing polychondritis often is delayed; in this case, the
prognosis is poor.
In 11 of my cases of relapsing polychondritis with scleritis,87
achieving complete control of necrotizing scleritis associated
with relapsing polychondritis was extremely difficult. Three of
these patients had necrotizing scleritis, and two had PUK.
Scleritis was bilateral in four patients, and the ocular inflam-
mation was the presenting manifestation of the relapsing
polychondritis in three patients. Seven developed auricular
chondritis, six developed nasal chondritis, six developed arthritis,
two developed tracheal chondritis, four developed damage to the
cochleovestibular system, one developed renal involvement,
and one developed central nervous system vasculitic
manifestations of the disease. Cytotoxic immunosuppressive
chemotherapy was required to bring about total resolution of
the destructive inflammation in seven of the 11 patients.
Although dapsone is often effective in the care of patients with
relapsing polychondritis in whom auricular or nasal chondritis
is the primary manifestation, it was ineffective in all of the
patients studied who had necrotizing or nodular scleritis and
was effective in only two of the four patients with diffuse
SECTION 6
scleritis. Cyclophosphamide was the only effective drug in the
FIGURE 46.28. Facial profile of a patient with relapsing
polychondritis. Note the destruction of the nasal cartilage from
602 episodes of nasal chondritis.
three patients with necrotizing scleritis, in whom high-dose
systemic steroids, penicillamine, methotrexate, and azathioprine
had failed to control the progressively destructive inflammation.
The need for immunosuppressive therapy to control some
cases of relapsing polychondritis is not surprising; it is an
autoimmune disease in which autoantibodies to type 2 collagen
(which exists in both sclera and cartilage) and cell-mediated
immunity to cartilage components have been found. It is
interesting in this regard that sclera and cartilage share a
common phylogenetic origin.
Scleritis is a marker of severity of the underlying disease in
patients with connective tissue and vasculitic syndromes, and
as in other vasculitides, such as rheumatoid arthritis, Wegener’s
granulomatosis, PAN, and Behçet’s disease, the onset of necro-
tizing scleritis is a reliable sign of potentially lethal systemic
vasculitis and a clear indication for immunosuppressive
chemotherapy.
Progressive Systemic Sclerosis
Progressive systemic sclerosis, or scleroderma, is associated
with subepithelial fibrosis of the conjunctiva, KCS, and
blepharophimosis. The tear insufficiency has been well docu-
mented, and it is treated using the usual techniques, mentioned
previously in the discussion of KCS in rheumatoid arthritis.
The fornix foreshortening requires no treatment, but simply the
recognition that progressive systemic sclerosis is associated
with subepithelial fibrosis of the conjunctiva, which should not
make the ophthalmologist consider the associated diagnosis of
cicatricial pemphigoid.88–90 Chapter 330 illustrates both the
ocular and the non-ocular salient clinical findings in patients
with progressive systemic sclerosis.
Stevens–Johnson Syndrome
Stevens–Johnson syndrome (SJS), or erythema multiforme
major, is a life-threatening systemic illness most commonly
precipitated by a type 3 hypersensitivity reaction to a microbe.91
Mycoplasma pneumoniae is a common cause of this disease in
children, and children affected by SJS caused by M. pneumoniae
may die of the consequences of the SJS or may die of the
undiagnosed pneumonia. Herpes simplex virus is a common
and underrecognized cause of precipitation of SJS, and other
viruses, including polio, vaccinia, variola, and mumps, have
been associated with fulminant SJS. Mycobacterium
tuberculosis and various other microbiologic agents also have
been implicated. In truth, many cases of SJS that are blamed
on medication probably have occurred as a result of a
hypersensitivity reaction to the microbe for which the patient
was taking the medication and not from the medication itself,
which typically is blamed. Some drugs, however, are clearly
implicated in cases of SJS. Notable examples include
sulfonamides, tetracycline, penicillin, NSAIDs, allopurinol,
barbiturates, and various immunizing vaccines.
The disease is characterized by the systemic manifestations
of fever, malaise, headache, loss of appetite, nausea, and
vomiting. The dermatologic manifestation is a generalized
erythematous papular eruption, including involvement of the
soles of the feet and the palms of the hands, with eventual
emergence of the so-called target or iris or bull’s-eye lesion with
an erythematous center surrounded by a zone of relatively
normal-appearing skin and then by an erythematous ring
outside of that (Fig. 46.29). Mucous membranes typically are
involved in SJS, with nose and mouth the most common sites
affected, but vagina, anus, and conjunctiva are affected in a high
percentage of cases. The mucosal involvement is one of bullae
formation and rapid rupture of these bullae, with subsequent
scarring in the area of the epithelial erosions (Fig. 46.30). The
nails are also affected by the disease (Fig. 46.31).
 Immunologic Disorders of the Conjunctiva, Cornea, and Sclera
FIGURE 46.29. Erythema multiforme major. Note the classic target
lesions affecting the palms of the hands.
FIGURE 46.30. Erythema multiforme major with oral mucosal
involvement and mild conjunctival involvement.
The best evidence suggests that the immunopathology of SJS
is a combined mechanism involving circulating IgA-containing
immune complexes and a lymphocytic vasculitis in areas where
these IgA complexes lodge in vessel walls.92 A lymphocytic
microvasculitis is an immunopathologic characteristic of the
lesions of SJS.
Ocular manifestations
Conjunctivitis, conjunctival bullae formation, keratopathy, and
secondary infection are the typical acute ocular manifestations
of SJS. These manifestations may clear without chronic
sequelae, but in severe cases of SJS, the chronic consequences of
the acute exanthem are the features with which the
ophthalmologist and the patient must struggle for the rest of
the patient’s life. The subepithelial fibrosis of the conjunctiva
produces these chronic consequences, with many features
similar to those of stage 3 or 4 cicatricial pemphigoid. Speci-
fically, the subepithelial fibrosis produces conjunctival fornix
foreshortening, symblepharon formation (Fig. 46.32), meibo-
FIGURE 46.31. Fingernail involvement in erythema multiforme major.
Note also the typical skin lesions.
FIGURE 46.32. Ocular involvement in Stevens–Johnson syndrome,
with subepithelial fibrosis, fornix foreshortening, and dense
symblepharon formation.
mian gland duct compromise, misdirection of lash follicles with
resultant trichiasis and distichiasis, and chronic keratopathy
secondary to KCS, meibomian gland dysfunction, the abrading
action of the misdirected lashes, and the abrading action of
CHAPTER 46
the keratinization of tarsal conjunctiva posterior to the grayline
of both the upper and the lower lids (Fig. 46.33). Corneal
epithelial defect formation, neovascularization, and stromal
ulceration with stromal scarring or perforation are the blinding
consequences of this cruel disease.
Treatment
General supportive topical antibiotic and corticosteroid therapy
during the acute phases of SJS is the therapy most typically
employed for the eyes. However, increasing interest in acute
intervention with brief high dose corticosteroid therapy93
(provided the patient is not in sepsis) and/or with IV-Ig therapy
(provided the patient is not dehydrated) is gaining in
popularity.94 Acute care of the ocular manifestations of SJS may
appropriately include careful ocular hygiene with regard to
crusts and mucus, judicious use of topical corticosteroids,
vigilance for the formation of adhesions between raw surfaces,
gentle separation of such adhesions when observed, and use of
topical antibiotics for prophylaxis of infection. 603
 CORNEA AND CONJUNCTIVA
FIGURE 46.33. Typical keratinization of the tarsal conjunctiva for
~2 mm posterior to the mucocutaneous junction, lower lid, in a
patient with Stevens-Johnson syndrome.
Treatment of the chronic ocular consequences appropriately
includes control or correction of the trichiasis and distichiasis,
preferably through permanent destruction of the follicles of the
aberrant lashes, treatment of KCS if it exists (SJS patients are
often not tear deficient), treatment to the extent possible of
meibomian gland dysfunction and meibomian duct obstruction
through the use of warm compresses and lid massage, and
treatment of keratopathy secondary to the keratinized posterior
lid margins. Topical retinoids may or may not be helpful in the
latter regard; lubrication with ointments may help in some
cases. When the problem is severe, however, removal of the
keratinized tissue and replacement by mucosal membrane
grafting is the most definitive and effective therapy for this
problem (Fig. 46.34).
Scleral lens therapy, for protection from abrading lashes and
keratinized lid margins and for retention of fluid in the space
formed between the cornea and the posterior surface of the
scleral lens, can be extremely helpful, not only for chronic
management but also for postoperative protection of limbal
stem cell and corneal grafts. After all, placement of delicate
limbal stem cell allografts or the epithelium of a corneal
transplant into the same hostile environment that has resulted
in recurrent or persistent corneal epithelial defects is unlikely to
succeed in repopulating the patient’s ocular surface with
healthy cells that can resist the assault of offending lashes, KCS,
and keratinized posterior lid margins unless some protection is
SECTION 6
provided against these assailants.
Immunosuppressive therapy has no role in the care of
patients with the chronic consequences of SJS. Whether such
treatment might be of benefit during the acute phases of the
disease is unknown and probably cannot be answered unless or
until a proper prospective study of the immunopathogenesis of
the disease is performed. In many cases (e.g., in patients in
whom a microbe-like herpesvirus or M. pneumoniae has
stimulated the SJS), acute intervention with immuno-
suppressive agents probably would be contraindicated. There is
one extremely rare case, however, in which immunosuppressive
chemotherapy is effective in the care of patients with SJS:
immunologically driven, truly recurrent SJS. This rare
phenomenon, well described in skin and in oral mucosa,90 also
has been described in nine of my patients with recurrent
conjunctival inflammation for many years after the acute
exanthem of SJS.95 Immunohistochemical studies of the
affected conjunctiva that continued to exhibit pronounced
604 inflammation after appropriate control of all the potentially
FIGURE 46.34. A patient with Stevens–Johnson syndrome who had
extensive keratinization of the tarsal conjunctiva is shown after
resection of that keratinization and performance of buccal mucosal
membrane grafting to both upper and lower lids.
confounding variables, such as lid margin keratinization, sicca
syndrome, meibomian gland dysfunction, trichiasis, and
distichiasis, disclosed IgA deposition in vessel walls and
lymphocytic microvasculitis. Immunosuppressive chemo-
therapy in these nine patients resulted in abolition of the
recurrent attacks of chronic inflammation that was producing
progressive scarring of the conjunctiva.
Lyell’s Syndrome
Lyell’s syndrome, or toxic epidermal necrolysis (TEN), can
occur as a result of Staphylococcus aureus infection in infants
and young children. The staphylococcal toxin production of
TEN produces the so-called scalded skin syndrome, with
generalized peeling of the epidermis in large geographic areas
of the skin and of the mucous membranes.96 Ocular
manifestations in this group are not extremely common but
can occur and include mucopurulent conjunctivitis with
conjunctival scarring and keratopathy. The more common form
of TEN, however, seen in older people, usually is not
staphylococcal but may be microbe related nonetheless. The
medication used often is blamed for the exanthem, however,
and such medications have included penicillin, allopurinol,
NSAIDs, sulfonamides, and phenytoin. As is the case in SJS,
TEN is systemic and the patient may die. Indeed, the mortality
rate for both SJS and TEN approaches 30%,97 and this is
probably underappreciated by most ophthalmologists.
The immunopathogenesis of TEN probably begins with drug-
skin binding with an aberrant immune response to this bound
form of the drug and with resultant attack on skin and mucous
membrane. Complement and immunoglobulin deposition
within the epidermis and mucosa occurs, with resultant
inflammatory cell infiltration and damage secondary to the
inflammatory cells.
Headache and malaise precede the appearance of bullae on
the skin. Unlike the tense subepidermal bullae of SJS, the bullae
of TEN are intraepidermal and hence are flaccid. The bullae
rupture, large expanses of epidermis are lost, and mucous
membrane lesions often develop in the nose, mouth, trachea,
esophagus, and conjunctivae.
Ocular manifestations
The ocular manifestations of TEN essentially are identical to
those of SJS. Conjunctivitis with epithelial erosions and
subepithelial fibrosis and fornix foreshortening, symblepharon
formation, trichiasis, distichiasis, and meibomian duct and, in
 Immunologic Disorders of the Conjunctiva, Cornea, and Sclera
FIGURE 46.35. Blinding keratopathy consequences include scarring
and neovascularization in a patient with toxic epidermal necrolysis.
some cases, lacrimal duct obstruction occur. Keratopathy with
corneal ulceration, neovascularization, perforation is typical, as
in the case of SJS (Fig. 46.35).
Treatment
The treatment of TEN is the same as that of SJS, with acute
supportive care, antibiotic prophylaxis, ocular hygiene,
attention to adhesions, and judicious use of topical corti-
costeroids. Care of the chronic consequences are as described
previously for SJS.
TYPE IV HYPERSENSITIVITY REACTIONS
PHLYCTENULOSIS
Phlyctenulosis, which was relatively common when tuber-
culosis was prevalent, is relatively rare in developed countries
today. The lesions, which are granulomatous, typically appear
at the corneoscleral limbus (Fig. 46.36), but in severe disease,
they may occur in conjunctiva posterior to the limbus, in the
cornea, or both. Organisms cannot be demonstrated in these
lesions, which are believed to be secondary to a classic Gell
and Coombs type IV delayed-type hypersensitivity reaction to
FIGURE 46.36. Phlyctenulosis. Note the bowing forward of the thin
slit beam as it sweeps across the phlyctenular conjunctival lesion near
the corneoscleral limbus.
FIGURE 46.37. Limbal scarring at the 3 o’clock and 5 o’clock
positions in a patient who previously had phlyctenulosis. Note also
that this patient now has Saltzmann’s nodular degeneration with
corneal lesions in the periphery and midperiphery of the cornea, in the
7 o’clock sector.
bacterial proteins. Although the disease is still seen today in
American Eskimos and in disadvantaged cultures in which
tuberculosis is still prevalent, the occasional case seen in more
developed societies is found generally in association with
staphylococcal proteins. Candida and Coccidioides species and
lymphogranuloma venereum have also been associated with
phlyctenulosis.98
The epithelium overlying this inflammatory lesion generally
develops a defect at the apex during the course of ~1 week, with
subsequent ‘ulceration’ of the lesion and the residua of a limbal
scar (Fig. 46.37). When the cornea has been involved to a
significant degree, the patient’s clinical picture may evolve to
that of Saltzmann’s nodular corneal degeneration (Fig. 46.38).
Topical corticosteroids are the mainstay of treatment for
phlyctenulosis. The lesions are exquisitely sensitive to topical
steroids, and the equivalent of 1 drop of 1% prednisolone
sodium phosphate twice daily is generally effective within 48
to 96 h. Concomitant topical antibiotic therapy and lid hygiene
to control the almost invariably associated staphylococcal
blepharitis and meibomianitis is mandatory. The application of
CHAPTER 46
FIGURE 46.38. Saltzmann’s nodular degeneration with a solitary
lesion at the 6 o’clock periphery. 605
 CORNEA AND CONJUNCTIVA
FIGURE 46.39. Drug allergy contact dermatitis affecting the lids of
the right eye.
warm compresses with lid massage and lid scrubs twice daily,
along with an antibiotic such as bacitracin ointment instilled
into the cul-de-sac twice daily, typically is sufficient.
DRUG ALLERGY (CONTACT
HYPERSENSITIVITY)
Type 4 contact hypersensitivity reactions to ocular medications
are not rare, and the most culpable medications in provoking
such reactions in the susceptible patient’s conjunctiva and lid
skin are neomycin, atropine, penicillin, and antazoline. The
diagnosis must first be suspected, based on a history of ocular
medication use and the clinical appearance (Fig. 46.39), which
typically includes erythematous, scaly dermatitis, affecting
upper and lower eyelids, with the lower lid skin (and even down
onto the cheek) being more affected than the upper. Resolution
of this problem, including the erythematous conjunctiva,
within 4 days of cessation of medication use offers strong
circumstantial evidence of the accuracy of the diagnosis.
Definitive diagnosis requires patch testing, with the allergen
applied to the skin under an occlusive patch for 48 h. The site
of application is unremarkable 24 h later but shows a classic
delayed-type hypersensitivity response with erythema and
induration 48–72 h after application.
Cool compresses usually are indicated, along with with-
drawal of the medication, in the treatment of patients with
contact dermatitis. Occasionally, 0.5% hydrocortisone skin
cream applied to the affected skin area is indicated, depending
on the severity of the allergic reaction and on the social
circumstances of the patient.
SECTION 6
a b
FIGURE 46.40. (a) Corneal transplant endothelial rejection with dramatic stromal swelling in the inferior half of the graft. (b) Same eye as shown
606 in (a), higher magnification, illustrating the classic endothelial rejection line.
CORNEAL TRANSPLANT REJECTION
Corneal transplantation, like any other solid organ trans-
plantation, provokes a systemic immune response in the
recipient, contrary to the mistaken beliefs of Sir Peter Medawar
dating from his important observations in the early 1950s.99
The cornea and the anterior chamber are immunologically
privileged by virtue of the avascularity of the cornea and by
virtue of the anterior chamber route of class 2 HLA glycoprotein
presentation of alloantigens from the transplanted cornea and
not by virtue of antigen ‘invisibility’ from the systemic immune
system. Rapid, potent systemic immune responses occur after
foreign antigen presentation into the cornea or anterior
chamber, but curiously, the systemic immune response results
predominantly in a tolerating sequence of events that actively
produces the immunologic privilege of these sites.100–104 Such
immunologic privilege includes the tolerance usually enjoyed by
the antigens on corneal allografts.
As with transplant rejection of other solid organs, however,
transplant rejection of transplanted corneas represents pri-
marily a type 4 hypersensitivity reaction. Such a reaction is
mediated primarily by T cells, although lymphokines liberated
by these T cells may recruit other cell types that can participate
in the damage to the graft. Such surrogate effector cells include
natural killer cells and macrophages. In extremely rare cases,
hyperacute rejection of corneal grafts may occur in the
previously sensitized recipient by virtue of the presence of
preexisting antibodies in the blood of the recipient that react
with donor tissue. In this so-called antibody-dependent cellular
cytotoxicity reaction, neutrophils, macrophages, and killer K
cells participate with the preformed antibodies in destroying the
graft cells.
Treatment of corneal graft rejections appropriately includes
the administration of corticosteroids through all routes and
with sufficient dosage to reverse the rejection reaction.
Aggressive topical corticosteroid administration (e.g., 1%
prednisolone sodium phosphate hourly while awake and
dexamethasone phosphate ointment at bedtime) generally is
sufficient to reverse corneal transplant rejection reactions in
patients with reactions of mild to moderate severity (Figs 46.40
and 46.41), whereas this treatment combined with sub-
conjunctival corticosteroid (dexamethasone sodium phosphate,
4 mg) and systemic corticosteroid (prednisone, 60 mg by
mouth each morning for 5 days with subsequent tapering
and discontinuation) often is required to treat a severe rejection
reaction effectively. In a small number of desperate cases
 Immunologic Disorders of the Conjunctiva, Cornea, and Sclera
involving one-eyed patients who had undergone multiple
corneal transplantations, I have employed intraocular
dexamethasone sodium phosphate (400 µg) and systemic
therapy with immunosuppressive regimens (azathioprine,
2 mg–1 kg–1 day–1, combined with cyclosporine, 5 mg–1 kg–1
day–1) and in one case have salvaged a patient’s corneal graft
with the use of intravenous anti T-cell antibodies.
In the high-risk corneal graft recipient, HLA typing with
subsequent screening of the recipient’s serum for preformed
antibodies against candidate corneal grafts is appropriate, as is
obtaining a cornea from a donor who is as closely HLA matched
as possible. Preemptive treatment with solid organ immuno-
suppressive regimens, including combination prednisone,
cyclosporine, and azathioprine therapy, coupled with topical
corticosteroid and cyclosporine therapy, is employed in such high-
risk recipients. The value of HLA matching for such patients
has been shown in a well-designed Dutch study.105 A multi-
centric trial to answer this question in the American population
concluded that HLA matching provided no significant
advantage for the high-risk corneal graft recipient but that
ABO matching did.106 A randomized study comparing topical
cyclosporine with placebo for such patients was discontinued
when interim data analysis disclosed that there was no
statistically significant benefit derived from topical cyclosporine
and therapy. The sponsoring pharmaceutical company,
regrettably, has declined to publish these negative results.
GRAFT-VERSUS-HOST DISEASE
Graft-versus-host disease (GVHD), after bone marrow
transplantation, occurs because the donor T lymphocytes recog-
nize the multiple differences (even in closely matched HLA
donors and recipients) among the various polymorphic minor
histocompatibility antigens on recipient tissues, and these
donor lymphocytes then attack the recipients’ cells. It is not
clear why, but the primary targets of this immunologic attack
are skin (Fig. 46.42), liver, intestine, oral mucosa, conjunctiva,
lacrimal gland, vaginal mucosa, and esophageal mucosa. Acute
GVHD develops in 35–45% of bone marrow recipients; the
occurrence of GVHD has a profound influence on patients’
survival rates; ~90% of patients who have little to no acute
GVHD survive, and only 45% of patients with moderate to
severe acute GVHD survive.107 The primary effector cells in the
affected tissue of patients with GVHD are T-lymphocytes,
specifically CD8 lymphocytes.
FIGURE 46.41. Same eye as shown in Figure 46.40, after 1 week of
intensive (hourly) topical 1% prednisolone sodium phosphate therapy
with partial resolution of the transplant rejection.
Multiple immunosuppressive chemotherapeutic regimens
have been tested for their efficacy in prevention of acute GVHD.
Combination preemptive therapy with methotrexate, cyclo-
phosphamide, and cyclosporine has been the most thoroughly
studied regimen. Methotrexate typically is given immediately
after the bone marrow transplantation, and cyclosporine is
sometimes administered subsequently. At least 3 months of
therapy is required for a therapeutic effect.
Chronic GVHD develops 3–15 months after bone marrow
transplantation in ~45% of bone marrow recipients.108 In
addition to T-cell infiltration into the target tissues, com-
plement and autoantibody deposition is found at the
dermal–epidermal junction and in conjunctiva. Chronic GVHD
patients experience recurrent and sometimes fatal bacterial
infections. The disease may be preventable through the
aggressive use of prednisone and other immunosuppressants
after bone marrow transplantation. The most effective pre-
vention of chronic GVHD is prevention of acute GVHD
because patients who do not experience acute GVHD have only
a 25% risk of developing chronic GVHD, whereas those who
experience acute GVHD have a 60–80% probability of
developing chronic GVHD.104 Older bone marrow recipients
are at higher risk of developing late-onset, chronic GVHD, but
the form of the disease seen in these recipients generally is
less severe and more amenable to immunosuppressive
chemotherapy.
Treatment of chronic GVHD is with aggressive immuno-
suppressive chemotherapy, typically employing a polyphar-
macologic approach with systemic corticosteroids and one
or more immunosuppressants. The most commonly used
immunosuppressants with good effect have been azathioprine
and cyclosporine.108
The external immunologic ocular manifestations of GVHD
are extraordinary and ocularly devastating, producing profound
morbidity for patients suffering from this problem. The most
impressive ocular manifestation is KCS. The severity of the
KCS usually is extreme, and treating it can present a major
challenge (Fig. 46.43). The usual treatment modalities are
described in the section on KCS (see earlier), but increase in
aggressiveness of therapy generally occurs much more rapidly in
patients with GVHD. It is not uncommon for these patients to
have profound corneal epitheliopathy and to develop epithelial
erosions, persistence of these erosions, and subsequent stromal
ulceration. Permanent puncta occlusion and tarsorrhaphy
CHAPTER 46
FIGURE 46.42. Typical skin manifestations of graft-versus-host
disease. 607
 CORNEA AND CONJUNCTIVA

particularly the retina, nearly impossible at times. As with the

immunologic attack on the conjunctiva, the only effective
treatment of these manifestations is systemic treatment that
brings the other systemic manifestations of GVHD under
control.

FIGURE 46.43. Profound corneal scarring and neovascularization as
a consequence of the severe keratoconjunctivitis sicca associated
with graft-versus-host disease.
should be performed very early in the course of the physician’s
care of the GVHD patient who develops KCS.
An underrecognized ocular consequence of GVHD is the
immunologically mediated T-lymphocyte attack on the
conjunctiva, with resultant chronic conjunctivitis.9 This
immunologically driven inflammation responds only to
systemic therapeutic techniques that bring the systemic
manifestations of the GVHD under control (see previous
discussion). Adjunctive therapy that has had some ameliorating
effect has been the use of topical 2% cyclosporine drops four
times daily.
Other underrecognized consequences of GVHD are cataract,
uveitis, and retinitis. The basis of these ocular manifestations
is not clear, and they are typically unrecognized because of the
profound KCS that is blamed for the patient’s symptoms and
that makes adequate examination of the intraocular structures,
SUMMARY
The eye can be affected by any of the immunologic
hypersensitivity reactions, and this chapter attempts to describe
the current state of knowledge regarding the immunologic
inflammatory diseases of the conjunctiva, cornea, and sclera.
Some of the diseases, such as Mooren’s ulcer and
phlyctenulosis, are strictly ocular. Others, such as scleritis
associated with long-standing rheumatoid arthritis, are ocular
manifestations of preexisting, previously diagnosed and treated
systemic disorders. A major point of emphasis in this chapter,
however, is the idea that some potentially lethal systemic
diseases may be silent or occult systemically but may produce
an ocular inflammatory lesion that is the first obvious clinical
manifestation of the disease. A second point of emphasis in this
chapter has been the idea that the onset of necrotizing scleritis
or of PUK in a patient with previously diagnosed systemic
disease indicates a distinct change in the character of the
underlying systemic disease: the vasculitic component to the
disease now should be foremost in the minds of the physicians
caring for such patients because failure to recognize this
important harbinger of necrotizing scleritis and PUK for
subsequent potentially fatal systemic lesions may place the
patient in jeopardy. Finally, a third major point of emphasis
is the notion that the physician who investigates a patient
thoroughly in an effort to understand the immunologic
mechanisms central to the ocular inflammatory lesion is the
physician who is best prepared to care for the patient. Similar
remarks can be made for most of the uveitic syndromes and the
diseases associated with vasculitis, but the material
encompassed in this chapter is restricted to the anterior
segment.

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31. Dutt JE, Ledoux D, Baer H, Foster CS:
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33. Foster CS: Cicatricial pemphigoid [Thesis
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34. Tauber J, Jabbur N, Foster CS: Improved
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35. Lemp MA: The mucin-deficient dry eye. Int
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37. Shore JW, Foster CS, Westfall CT, Rubin
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40. Buhac J, Bhol K, Padilla T, et al:
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42. Bowman W: The parts concerned in the
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45. Wood TO, Kaufman HE: Mooren’s ulcer. Am
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46. Soukiasian SH, Foster CS: Mooren’s ulcer:
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47. Chow CYC, Foster CS: Mooren’s ulcer. Int
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48. Keitzman B: Mooren’s ulcer in Nigeria. Am J
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49. Schazlin D: Mooren’s ulceration. In: Smolin
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50. Schaap OL, Feltkamp TEW, Breebaart AC:
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51. Berkowitz PJ, Arentsen JJ, Felberg NT,
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52. Brown SI, Mondino BJ: Therapy of
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53. Foster CS: Systemic immunosuppressive
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54. Gottsch J, Liu S, Minkovitz J, Goodman D,
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55. Wakefield D, Robinson LP: Cyclosporine
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56. King JH: Destructive marginal ulceration: a
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57. Foster CS, Kenyon KR, Griner J, et al: The
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58. Gilbard J, Rossi SR, Heyda KG: Ophthalmic
solutions, the ocular surface, and a unique
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59. Foster CS, Sainz de la Maza M: Sclera.
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60. Lyons CJ, Hakin KN, Watson PG: Topical
flurbiprophen: an effective treatment for
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61. Watson PG, Hayreh SS: Scleritis and
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62. McGavin DDM, Williamson J, Forrester JV,
et al: Episcleritis and scleritis: a study of
their clinical manifestations and association
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63. Foster CS, Forstot SL, Wilson LA: Mortality
rate in rheumatoid arthritis patients
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ulcerative keratitis: effects of systemic
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91:1253–1263.
64. Watson PG, Hazelman BL: The sclera and
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65. Sainz de la Maza M, Foster CS, Jabbur NS:
Scleritis-associated uveitis. Ophthalmology
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66. Grayson M: Marginal furrows: a
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67. Smith RE, Schanzlin DJ: Rheumatoid
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68. Cohen KL: Sterile corneal perforation after
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69. Gelender H: Descemetocele after
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70. Insler MS, Boutros G, Boulware DW:
Corneal ulceration following cataract
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71. Sainz de la Maza M, Foster CS:
Necrotizing scleritis after ocular surgery:
a clinicopathologic study. Ophthalmology
1991; 98:1720–1726.
72. Pillat A: Über das Vorkommen von Choroiditis
bei Lupus Erythematodes. Graefes Arch Clin
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73. Gold DH, Morris DA, Henkind P: Ocular
findings in systemic lupus erythematosus.
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74. Spaeth GL: Corneal staining in systemic
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75. Reeves JA: Keratopathy associated with
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76. Halmay O, Ludwig K: Bilateral band-shaped
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77. Wise GN: Ocular periarteritis nodosa. Arch
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78. Fronert PP, Scheps FG: Long-term follow-
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79. Leib ES, Restivo C, Paulus HE:
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80. Fauci AS, Wolff SM: Wegener’s
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81. Soukasian SH, Foster CS, Niles JL,
Raizman MB: Diagnostic value of
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82. Ludemann G, Gross WL: Autoantibodies
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83. Savage CS, Winearls CG, Jones S, et al:
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84. Niles JL, McCloskay RT, Ahmed MF, et al:
CHAPTER 46
Wegener’s granulomatosis autoantibody is
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Blood 1989; 74:1888.
85. Ahmed M. Niffenegger JH, Jakobiec FA,
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86. Fauci AS, Haynes BF, Katz P, Wolff SM:
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87. Hoang Xuan T, Foster CS, Rice BA: Scleritis
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94. Hynes AY, Kafkala C, Daoud YJ, Foster CS:
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95. Foster CS, Fong LP, Azar D, Kenyon KR:
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SECTION 6
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98. Thygeson P: Observations on non-
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99. Medawar PB: Immunity to homologous
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100. Wetzig RP, Foster CS, Greene MI: Ocular
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101. Foster CS, Wetzig RP: Immune reactions
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102. Foster CS, Monroe JG, Campbell R, et al:
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mice in the vitreous induces either
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103. Streilein JW, Niederkorn JY, Shadduck JA:
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105. Koch-van-Alphen CC, Volker-Dieben HJ,
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107. Storb R, Prentice RL, Sullivan KM, et al:
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 CHAPTER
Allergic and Toxic Reactions: The Immune
47 Response
Mark B. Abelson, Gail L. Torkildsen, and Ira J. Udell
Key Features
• Ocular allergies affect an estimated 20% of the population in
industrialized countries
• Types of ocular allergies include:
• Allergic conjunctivitis (seasonal and perennial) – most
common, mild, caused by environmental allergens such as
ragweed, tree pollen, animal dander, and dust mites.
Characterized by ocular itching, hyperemia, chemosis,
eyelid edema, and tearing. Treatments include allergen
avoidance, cold compresses, antihistamines, mast cell
stabilizers, combination antihistamine/mast cell stabilizers,
NSAIDs, and corticosteroids.
• Atopic keratoconjunctivitis – rare, serious, chronic
condition seen in 24–40% of patients with atopic
dermatitis. Characterized by itching, redness, photophobia,
keratopathy, corneal ulcers, keratoconus, anterior polar
cataracts, mucous discharge, atopic blepharitis.
Treatments include topical corticosteroids and mast cell
stabilizers.
• Vernal keratoconjunctivitis – rare, serious, usually seen in
warm climates in males ages 3–20 years with family history
of atopy. Characterized by ptosis, ropy mucous discharge,
photophobia, large, nonuniform cobblestone papillae,
Horner–Trantas dots, limbal nodules, neovascularization,
corneal shield ulcers, and itching. Treatments include
allergen avoidance, cold compresses, antihistamines,
corticosteroids, and mast cell stabilizers.
• Giant Papillary Conjunctivitis is not a true allergic reaction but
an inflammatory reaction of the upper tarsal conjunctiva
associated with the presence of contact lenses, surgical suture
barbs, and ocular prostheses.
• Toxic keratoconjunctivitis is an ocular toxic reaction due to use
of certain drugs, vehicles, and preservatives.
THE ALLERGIC RESPONSE
Ocular allergies can range from mild (as in seasonal and
perennial allergic conjunctivitis (SAC and PAC)) to severe and
vision threatening, as in atopic and vernal keratoconjunctivitis
(AKC and VKC). Allergic diseases affect an estimated 20% of the
population in developed countries worldwide, including an
estimated 22 million people in the United States.1 Data from
epidemiological studies in a number of countries suggest that
the prevalence of allergic diseases has increased substantially
since the 1940s.2 This rising prevalence of asthma and allergic
diseases has not been definitively explained, but researchers
have proposed causes including increasing air pollution and
diesel exhaust, genetics, and the so-called hygiene hypothesis,
which proposes that atopic diseases are prevented by infections
in early childhood.
T-cells play an important role in the allergic response. Upon
activation, naive T-helper (Th0) cells differentiate into Th1 or
Th2 cells. Th1 cells secrete interferon (IFN)-g, tumor necrosis
factor (TNF), and lymphotoxin, and are associated with cell-
mediated immunity. Th2 cells secrete interleukin (IL)-4, IL-5,
and IL-13. Th2 cells are important for immunity and resistance
to parasitic infection and are associated with allergy and asthma.
The Th1/Th2 balance, which determines the type of immune
response the body will mount in response to a given allergen,
forms the basis of the hygiene hypothesis. As allergen
sensitivity develops, the balance in this specialization shifts
toward higher Th2 and lower Th1 levels. According to the
hygiene hypothesis, a lack of triggers for Th1-type immune
response, such as exposure to infections, endotoxins, and dirt
in childhood, would result in a preponderance of Th2-type
immune responses responsible for allergic disease.
The primary factor that influences this differentiation is the
presence of certain cytokines at the time of T cell activation.
Cells exposed to IL-12 produce IFN-g and become Th1 cells,
whereas T cells exposed to IL-4, a product of other CD4+ T
cells and mast cells, tend to become Th2 cells.3 In vitro studies
show that Th1- and Th2-type immune responses downregulate
each other when activated. For example, typical Th2-type
cytokines like IL-4 and IL-10 inhibit the production of Th1-
derived cytokines such as IFN-g, and vice versa.4
Genetics also play a role in the predisposition to allergic
diseases. The risk for atopic disease is doubled in children who
have one parent with a history of atopy, and it is more than 50%
if both parents have such history. Although the specific type of
allergy expressed by individuals may differ within a family, the
incidence of allergic disorders is approximately three times
higher in atopic families than in nonatopic families.5 Further-
more, children who have both a maternal and a paternal family
history of atopy generally manifest allergy before puberty.6
Several genes are suspected to be associated with atopy, such as
5q31–33. It appears that children do not inherit the allergic
disorder per se, but instead an ‘allergic predisposition’ that can
be contributed equally by both parents, suggesting that this is
an autosomally carried trait.6 Nongenetic risk factors include
small family size, higher socioeconomic status, use of
antibiotics, and residing in an urban environment.
THE ROLE OF MAST CELLS IN THE ALLERGIC
RESPONSE
The mast cell mediates type I (immediate) hypersensitivity
reactions. Mast cells are characterized as containing either
tryptase (T) or tryptase/chymase (TC) based on immuno-
histochemical staining of these endoproteases. Both types
of mast cells develop from the same CD34+ mononuclear 611
 CORNEA AND CONJUNCTIVA
precursor. Normally, the conjunctival mast cells are mostly the
TC subtype.7 Mast cells of the T subtype are increased in
epithelial and subepithelial layers in SAC and PAC and much
more in VKC. In AKC, the TC subtype predominates which
may be responsible for fibrosis.8 Mast cell heterogeneity can be
seen across species, which means that drawing conclusions
based on animal data must be done cautiously.9 In normal
human patients, their distribution is limited to the substantia
propria of the conjunctiva, whereas in patients with VKC,
mast cells are also found in the conjunctival epithelium.10
Approximately 50 million mast cells can be found in the ocular
and adnexal tissues of the human eye.
The allergic cascade begins when antigen binds and cross-
links with two immunoglobulin (Ig)-E receptors located on the
surface of conjunctival mast cells. The cell membrane surface of
a mast cell has as many as 500 000 IgE receptors, 10% of which
are occupied in vivo.11 In GPC, 30% of mast cells are degranu-
lated, and in VKC up to 80% appear to be degranulated.12 IgE
molecules, the major homocytotropic antibodies, may adhere to
these surface receptors. The allergen–IgE antibody–mast cell
union results in the activation of a serine esterase, initiating a
change in the Fc portion of the IgE molecule, which is attached
to the mast cell membrane.13 This event leads to an intra-
cellular biochemical cascade resulting in mast cell degranu-
lation and the release of histamine, eosinophil cationic protein
(ECP), high molecular weight neutrophil chemotactic factor, and
platelet activating factor (PAF). These agents attract eosinophils
and neutrophils (cells that contain secondary mediators), that
then restore homeostasis or produce tissue alterations in
chronic allergic disease. The signs and symptoms of an acute
allergic reaction are the result of this intricate network of
mediator interaction (Fig. 47.1).
MAST CELL AND EOSINOPHIL MEDIATORS
Mediators have been categorized into three different groups
based on their mode of action. The first group includes sub-
stances such as histamine and the prostaglandins that mediate
their actions by binding to a specific cell membrane receptor.
The second group comprises substances of cell or plasma origin
Histamine
Endothelial cells
APC
SECTION 6
B cell Th2 cell
E-selectin ICAM IgE
VCAM
IL-3, IL-4,
IL-6, IL-8,
IL-13, TNFa,
Blood vessel Histamine
612 Adapted from McGill, J I et al. Br J Ophthalmol 1998;82:1203-1214
that directly damage tissues, including eosinophil major basic
protein (EMBP) and complement. The third group consists of
chemotactic factors that attract cells such as eosinophils and
macrophages to the inflammatory site and includes the
arachidonic acid metabolites.14
Histamine
Histamine, stored in granules of mast cells and basophils, is the
central mediator of ocular allergy and inflammation. The
conjunctiva has at least two histamine receptors, H1 and H2.
Selective H1-receptor activation results mainly in itching,15
while selective H2-receptor activation primarily elicits redness.16
Thus, it appears that H1 and H2 receptors may be associated
with neuronal tissue and vascular tissue, respectively. Instillation
of histamine into the eye reproduces the exact clinical picture
of acute allergic conjunctivitis in a dose-dependent fashion:
itching, redness, chemosis, tearing, and eyelid swelling.17
Histamine levels were not found to be consistently elevated
in patients with allergic conjunctivitis; however, they were
found to be elevated in tear samples from patients with VKC
(16 ng/mL) (normal, 5ng/mL).18 One study examined the
presence of histaminase activity in human tears after in vivo
conjunctival allergen challenge. Histaminase inactivation
resulted in a 15-fold elevation of histamine recovery.19 These
results demonstrate the presence of histaminase activity in
human tears and suggest that histaminase activity may have
confounded the role of histamine in ocular allergic disorders
other than VKC. The finding of insufficient histaminase
activity in tears of patients with VKC may play some as yet
unknown role in the cause of the disease.20
Eosinophils
Eosinophils have a dual role in allergic disease: either spurring
inflammation or acting to quiet it. EMBP causes mast cell
degranulation and corneal damage. ECP and eosinophil derived
neurotoxin (EDN) can also cause corneal damage. Other
eosinophil mediators modulate the mast cell response through
negative feedback: histaminase inactivates histamine, phos-
pholipase inactivates PAF, and aryl sulfatase inactivates certain
leukotrienes.13 PAF is a potent eosinophil and neutrophil
FIGURE 47.1. Illustration of the allergic
response.
Fibroblasts
Basophil
IL-3
IL-4
IL-4
IL-5 Eosinophil
IL-3, IL-4
IL-10
Mast cell
Histamine
Nerve
 Allergic and Toxic Reactions: The Immune Response
chemotactic factor21 and an inflammatory mediator that modu-
lates vascular permeability. PAF has been found in basophils,
mast cells, eosinophils, monocytes, polymorphonuclear leuko-
cytes, and macrophages. Arachidonic acid is broken down by
cyclooxygenase into prostaglandins and thromboxanes, which
produce itching and conjunctival redness. Leukotrienes are pro-
duced from the breakdown of arachidonic acid by lipoxygenase,
which act by recruiting macrophages.
Eosinophil infiltration is observed in both VKC and contact
lens-associated giant papillary conjunctivitis (GPC), suggesting
that the tissue damage seen in these diseases may be, in part,
the result of eosinophilic mediators.10 Patients with VKC have
increased tear levels of both major basic protein and Charcot–
Leyden crystal protein, with the magnitude of the increase
correlating with the severity of the disease.22 EMBP, the highly
toxic substance released by eosinophils, is thought to be linked
to the corneal damage in VKC, that is, keratitis and shield ulcer
formation.22
Significant major basic protein deposition in conjunctival
specimens from patients with VKC, corneal vernal plaque
specimens, and GPC has been seen with no correlation between
the intensity of major basic protein deposition and the severity
of disease.23 Thus, in both VKC and GPC, the release of major
basic protein from eosinophils can contribute to the sustained
mast cell degranulation that occurs, leading to a more severe and
long-lasting process than that found in acute allergic conjunctivitis.
ALLERGIC CONJUNCTIVITIS
It is estimated that 90% of cases of ocular allergies are in the
form of seasonal and perennial allergic conjunctivitis (SAC and
PAC).24 While allergic conjunctivitis is a fairly mild disease, it
can result in considerable costs in terms of lost productivity and
decreases in quality of life. Many allergic patients report sleep
disruption, daytime fatigue, learning impairment, decreased
cognitive function, and decreased productivity. The economic
burden is substantial as well, with an estimated 3.5 million lost
workdays, 2 million missed school days, and 28 million days of
restricted activity or productivity loss per year in the US due to
allergies.25
SAC is a recurrent condition caused by airborne allergens, such
as pollens from ragweed, grasses, or trees. There are regional
variations throughout the United States and most fluctuate
seasonally. PAC results from allergens that exist year round,
including dust mites, animal dander, or mold. Whether seasonal
or perennial, allergens come into contact with the tear film and
then traverse the conjunctiva to join IgE antibodies attached to
conjunctival mast cells. This allergen–IgE antibody–mast cell
union results in mast cell degranulation, release of chemical
mediators, and the manifestation of ocular allergic signs and symp-
toms: itching, redness, chemosis, tearing, and eyelid swelling.
In the mild-to-moderate forms, allergic conjunctivitis can be
described as a series of acute type I hypersensitivity reactions
including mast cell degranulation and the release of preformed
mediators, which are responsible for the signs and symptoms
presented by the patient. The mast cells also releases enzymes,
such as histaminase, that act as control mechanisms to ‘shut
off ’ the release of mediators so that the overall effect is discrete
and self-limiting. If the allergic stimulation continues, additional
mast cell degranulation, cellular infiltrates, and inflammation
results. These are the late-phase allergic reactions, as opposed to
the early-phase response observed with the milder forms.
CLINICAL FEATURES AND DIAGNOSIS
Itching is the hallmark symptom of ocular allergy. Its absence
makes the diagnosis suspect. Symptoms of redness, tearing,
burning, and sinus pressure behind the eyes and ears can also
be present. Allergic conjunctivitis is an intermittent condition
that may not be manifested at the time of the ophthalmic
examination. It is important to inquire specifically about envi-
ronmental triggers such as cats, trees, grasses, ragweed, dust
and molds, as well as provoking factors like lawn mowing, pet
exposure, or camping. Patients who take anti-allergic medica-
tions systemically or nasally need to be asked specifically about
eye symptoms, since it is estimated that 90% of patients with
allergic rhinitis have ocular symptoms as well.26
Sometimes, a chart review will reveal that office visits occur
during the same month every year. The month or season that
the patient experiences symptoms is important to diagnosing
their allergic trigger. The patient is usually an excellent source
for identifying the allergen to which she or he is sensitive.
For example, in the north-eastern United States, March is
commonly tree pollen season, May is grass season, and mid-
August through mid-September is ragweed season. Additionally,
a personal or family history, either current or in childhood, of
eczema, asthma, rhinitis, or other atopic history may be
indicative of ocular allergy. When questioned, patients may
deny asthma but respond positively to questions related to
wheezing in cold air or upon exertion.
Perennial ocular allergens such as dust, pet hair and dander,
mites or mold can present a constant aggravation. If a patient
experiences intense itching, redness, and discomfort while
indoors, and during times of the year inconsistent with seasonal
allergens, then perennial ocular allergies may be involved. In
one investigation, the dust collected from nearly all of the
homes sampled contained pet allergens, even though less than
half of the homes had pets.27
Since clinical signs may be absent at the time of an office
visit, gathering a medication history of allergy medications,
inhalers, nasal sprays, and over-the-counter eye drops is
important. These medications may only be used seasonally or
as needed. A patient may be symptomatic, but the adnexal
tissue may appear normal. Most patients, however, have some
microchemosis and dilatation of conjunctival vessels, or
swelling of the lids (Fig. 47.2). Microchemosis is evident only
with careful slit-lamp examination. With the slit beam
narrowed, a slight elevation of the conjunctiva can be seen. An
additional clue is to notice the conjunctival redness prior to
dilation and compare it to the conjunctiva after dilation. Venous
CHAPTER 47
FIGURE 47.2. Classic acute allergic conjunctivitis. Conjunctival
hyperemia is evident as is chemosis. Hyperemia and swelling of the
lids may or may not be present on examination, but patients will often
associate their exacerbation with knuckle rubbing. The characteristic
symptom reported by patients is itching. 613
 CORNEA AND CONJUNCTIVA
congestion can lead to the allergic ‘shiners’ often seen in
children. Nasal symptoms are so common that sometimes just
listening to the sniffles points a clinician in the right direction.
Watching for the ‘allergic salute’ (nasal rubbing) while taking a
history is also important.
An acute reaction may yield a clear or white exudate, whereas
a chronic reaction is characterized by a mucopurulent, thicker,
stringier exudate. Pallor of the palpebral conjunctiva may occur
as a result of edema. A papillary vasodilatory reaction may
occur with the absence of giant papillae. Excessive conjunctival
chemosis can lead to corneal dellen; however, the limbus
and cornea are usually normal. The presence of purulent
discharge, follicles, cobbles, or keratitis is inconsistent with a
sole diagnosis of allergic conjunctivitis. A complaint of ocular
itching that, with further questioning, specifically involves the
lid and lid margin may suggest a meibomian gland dysfunction
or allergic blepharodermatitis.
The ocular allergic reactions elicited by animal dander, dust
mites, and molds are similar to those seen in seasonal allergic
conjunctivitis. The conjunctival and periorbital lid swelling can
be impressive and the conjunctiva may actually balloon beyond
the lids.
The differentiation of allergic conjunctivitis from dry eye
syndrome hinges on the presence or absence of the distin-
guishing characteristic of allergy, which is ocular itching. Both
conditions may produce mild conjunctival vasodilatation and
a burning sensation with intermittent exacerbations. Not
infrequently, these diseases occur concurrently. Patients with
dry eye may be more susceptible to allergic conjunctivitis owing
to decreased tear film production and a decreased ability to
wash away and dilute airborne allergens, thus acting as a barrier
to the adherence of allergens to the conjunctival surface.
Corneal and conjunctival staining, tear meniscus level assess-
ment, Schirmer’s testing, and tear break-up time evaluations
may aid in properly categorizing dry eye. Ocular allergic
symptoms rarely include foreign body sensation, although this
is common in dry eye. Another distinguishing factor may be the
type of mucus present. In allergy, it tends to be thin and clear,
rather than the stringy white mucus associated with dry eye.
If the diagnosis is in doubt, a conjunctival scraping positive
for eosinophils is certainly indicative of allergy. However,
because eosinophils may exist more deeply in the conjunctiva,
a negative scraping is inconclusive.28 High levels of total and
specific serum and tear levels of specific IgE have a strong corre-
lation with ocular allergy. Tear cytology positive for eosinophils
would suggest that an IgE-mediated response is present.
Neutrophils may also be seen. A positive skin test for specific
allergens has been shown to be between 71 and 87% predictive for
SECTION 6
positive ocular reactivity.29,30 Finally, a positive ocular challenge
to the allergen is most decisive for a positive diagnosis,30,31 but
may be impractical unless all other tests are inconclusive.
HISTOPATHOLOGY AND PATHOGENESIS
Most animal work using models of ocular anaphylaxis
corroborate the clinical picture of mild to moderate ocular
allergic reactions, with 70–100% of mast cells degranulated,32
no effect on goblet cells,33 and significant early-phase increase
in neutrophils.34 Mast cells have been shown to regranulate
within 24 h.35 Macroscopically, eyes can also appear normal,
despite degranulated mast cells and an increase in microscopic
interstitial edema.36
The conjunctiva is capable of mounting immediate immune
responses to external insult. There is strong evidence of a late-
phase reaction as well.37 In an allergen challenge study, the
numbers of neutrophils, eosinophils, lymphocytes, and mono-
614 cytes found in conjunctival scrapings were quantified and
correlated with the clinical profile, total serum IgE, and serum
IgE to rye I antigen. Significant increases in neutrophils of
patients occurred after 20 min (P <.001), and in eosinophils at
6 h (P <.005), compared with values of control subjects. Thus,
significant inflammatory changes in conjunctival scrapings are
present long after allergen exposure has ended.
The late phase of allergic conjunctivitis is manifested by
either a sustained or discrete second peak of allergic response
4–24 h after allergen exposure.38 Tear cytology has also shown
increases in eosinophils or neutrophils in allergen-challenged
human eyes long after the immediate reaction had disappeared.39
A recent study40 using a modified Conjunctival Allergen
Challenge (CAC) design30 showed that some individuals who
were sensitive to low doses of allergen had a late-phase reaction.
When subjects were given an allergen challenge and then
rechallenged 24 h later, these individuals showed heightened
ocular itching and redness. An anti-inflammatory agent
(corticosteroid) was shown to inhibit signs and symptoms of the
late phase component. This modified CAC model will further
elucidate the late phase phenomenon and may develop into a
tool for assessing other antiinflammatory agents.
SACs and PACs are type 1 hypersensitivity reactions. Allergens
penetrate the conjunctival epithelium and bind to IgE receptors
on mast cells. This leads to mast cell degranulation and release
of chemical mediators including histamine. Histamine causes
itching, increases in vascular permeability and recruitment of
immune cells. Preformed mediators include histamine, tryptase,
and heparin. Mediators formed in response to allergen binding
include prostaglandins (from membrane-bound arachidonic acid),
thromboxanes, leukotrienes, PAF, cytokines, chemokines, and
growth factors. Histamine, bradykinin, and prostaglandins
stimulate pain and itching.
TREATMENT
No patient should suffer unnecessarily, and treating ocular
allergies represents a tremendous opportunity to improve the
quality of patient’s lives. The first and foremost step in the treat-
ment of allergic conjunctivitis is the removal of the offending
allergen, if possible. The severity of the allergic condition is
directly proportional to the level and duration of exposure to the
allergen. Depending on the allergen to which the patient is
sensitized, limiting time spent outdoors, using air conditioners,
or avoiding animal dander will all dramatically improve the
condition. Tear substitutes can be used to dilute the allergen
and wash some allergen away.
In the short term, using over-the-counter H1 antihistamine–
vasoconstrictor combinations (as needed) can successfully
alleviate the primary symptom, itching, and the primary signs,
redness and swelling. A study of the effects of antazoline
phosphate 0.5% in combination with naphazoline hydrochloride
0.05% in the allergen challenge model of allergic conjunctivitis
showed significant relief of itching, redness, chemosis, and lid
swelling immediately and 2 h after administration of the drug.31
However, these agents are short-acting and may lead to rebound
vasodilation.
A more effective therapeutic option for allergic conjunctivitis
is a combination antihistamine plus mast cell stabilizer, such as
olopatadine, epinastine, ketotifen, or azelastine. Olopatadine
0.1% is the most frequently prescribed combination antihista-
minic agent with mast cell stabilizing properties. It attaches to
the H1 receptor site to prevent histamine from binding and
therefore provides initial relief of itching. Owing to its mast cell
stabilization, IgE cross-linking is prevented, histamine release is
halted, and further mast cell degranulation is inhibited. In this
way, olopatadine provides both quick and long-lasting relief
from the signs and symptoms of allergic conjunctivitis. The
 Allergic and Toxic Reactions: The Immune Response
olopatadine molecule is the most researched of all topical ocular
anti-allergic agents, and has been shown to be the most effective
treatment for ocular allergy.41 A new formulation, olopatadine
0.2%, has been introduced as the first and only available
antiallergic agent indicated for once-daily dosing. This product
has demonstrated enhanced efficacy and extended duration of
action (up to 24 h) (Vogelson, Abelson), while still remaining
safe and well-tolerated in both adults and children (Lichtenstein).
The once-a-day formulation will provide increased convenience
and compliance for patients.
The classic H1 antihistamines, levocabastine and emedastine,
have been shown to significantly inhibit ocular itching, but these
are combined with a-adrenergic agents in order to maximize the
alleviation of conjunctival redness. The topical antihistamine
levocabastine has been shown to be effective in the relief of
itching.42,43
Topical nonsteroidal antiinflammatory drugs such as ketorolac
and flurbiprofen inhibit the activity of the cyclooxygenase
pathway and the production of prostaglandins. They inhibit
vasodilation and edema and are useful in reducing itching and
conjunctival injection, but not very helpful in ridding the eye of
excess immune cells.
Single-action mast cell stabilizers include cromolyn sodium
4%, pemirolast potassium 0.1%, nedocromil, and lodoxamide
0.1%. Cromolyn and lodoxamide are indicated for the treatment
of VKC and AKC, but not allergic conjunctivitis. Cromolyn in
vitro inhibited activation of neutrophils, eosinophils, and mono-
cytes, thus blocking tissue destruction.44 This may explain why
this drug is more effective in chronic cell-mediated disorders
such as VKC and atopic conjunctivitis and not as effective in
the type 1 hypersensitivity allergic conjunctivitis. Cromolyn has
also been shown to inhibit the release of substance P and other
neuropeptides from nerve endings.45
Pemirolast has been demonstrated to be upto 100 times more
potent than cromolyn in vitro and in animal studies, and in
clinical trials, has been shown to be effective at completely
resolving itching in some patients.46
Nedocromil has been shown to be more effective than
cromolyn in stabilizing mucosal mast cells, and this difference
may account for its effectiveness in a disease in which cromolyn
has not shown efficacy.47 Nedocromil has also been shown to
block eosinophils, neutrophils, macrophages, monocytes, and
platelets.48 In clinical trials, nedocromil was more effective than
cromolyn in resolution of itching, hyperemia, epithelial keratitis,
corneal pannus, and other symptoms in patients with ocular
allergy.49
The clinical efficacy of lodoxamide may be superior to that
seen with cromolyn, as has been reported in the literature for
the treatment of AKC, VKC, and GPC.50,51 It is unclear how
effective lodoxamide is for seasonal allergic conjunctivitis. One
clinical study of lodoxamide showed it to be more effective than
the placebo in treating seasonal allergic conjunctivitis during
peak pollen exposure.51
Steroids, such as loteprednol etabonate, are an option for
allergic conjunctivitis but are usually reserved for AKC and
VKC. Corticosteroids inhibit the production of arachidonic
acid itself, reducing the production of all three eicosanoids
(prostaglandins, thromboxanes and leukotrienes) reducing
redness, edema, and inflammation. However, their long term
use can be associated with side effects including delayed wound
healing, increased intraocular pressure, local immunosuppression,
and resultant superinfection and induction of cataractogenesis.
ATOPIC KERATOCONJUNCTIVITIS
Atopic keratoconjunctivitis (AKC) represents the ocular
manifestation of atopy, a hereditary condition characterized by
FIGURE 47.3. Atopic keratoconjunctivitis. Note the slight corneal
haze, the conjunctival changes, and the accompanying blepharitis
marked by structural changes at the lid margin and loss of lashes.
eczema, rhinitis, asthma, and atopic dermatitis. The term
atopy was originally paraphrased by Coca and Cooke52 in 1923
from its Greek equivalent meaning ‘out of the ordinary’.52 In
1953, Hogan was credited for first describing AKC as a distinct
entity, characterized by severe, chronic external ocular inflam-
mation associated with atopic dermatitis.53 Although atopy is
common, AKC is rare. Atopic dermatitis is present in ~3%
of the general population.54 Ocular involvement in atopic
dermatitis is estimated to occur in 25–40% of patients.55,56 AKC
is characterized by atopic dermatitis of the eyelids as well as by
papillary conjunctivitis, disruption of the corneal epithelium,
and in severe cases, conjunctival and corneal scarring (Fig. 47.3).
Treatment may need to be administered for years, and patients
commonly develop complications from both the disease and the
therapeutic interventions.
CLINICAL FEATURES AND DIAGNOSIS
AKC can appear in childhood and continue for 4–5 decades, at
which time the disease can spontaneously resolve.54 The peak
incidence is reported to occur between the ages of 30 and
50 years57 with an age range between 9 and 76 years.58 The
symptoms of AKC last throughout the year and are almost
always bilateral.54 Patients regularly complain of moderate to
severe itching, and may also describe a burning sensation,
photophobia, and blurring of vision. Tearing is frequently noted
CHAPTER 47
and can be accompanied by a distinct mucous discharge that is
mucopurulent, thick, ropy, and white.54
Gross examination of the ocular adnexae may reveal
indurated lid margins that are often thickened and scaly, and
there may be secondary blepharitis. Exudative, vesicular, or
crusted lesions may be observed elsewhere from atopic
dermatitis. Maceration of the inner or outer canthi may be
observed,59 and punctal stenosis can occur.57 An additional lid
fold (Dennie’s line) may be present.
The conjunctiva may be pale in comparison with that seen in
the other allergic disorders;54,58 however, limbal hyperemia and
chemosis can be seen with exacerbations of the disease.
Papillary hypertrophy is prominent in the inferior palpebral
conjunctiva, but not exclusively so. In contrast, VKC and GPC
tend to have papillae in the superior tarsus.54,58 Conjunctival
scarring is frequently a serious consequence of AKC, and
shrinkage of the inferior fornix may result. The characteristic
increase in tearing associated with AKC may be a result of the
loss of the inferior cul-de-sac. Subepithelial fibrosis, forniceal 615
 CORNEA AND CONJUNCTIVA
shortening, and symblepharon are seen in patients with AKC.58
In this stage of the disease, both the bulbar and the tarsal
conjunctiva appear hyperemic.
Corneal scarring, suppurative keratitis, and keratoconus are
the major reasons for loss of vision in patients with AKC and
may require immediate attention. Corneal involvement can begin
as superficial peripheral keratitis, with or without infiltrates.
Gelatinous infiltration, opacification, Horner–Trantas dots, and
true cysts may be seen.53 Generally, patients with AKC display
punctate epithelial keratopathy and intraepithelial microcysts.59
Ulcers resulting from AKC are typically ovoid, horizontally
oriented, and have irregular borders. Peripheral corneal neovas-
cularization is a prominent sign.59 Severe cases are marked by a
hazy vascularized cornea, interfering with normal visual
function.60 Keratoconus is estimated to be found in 25% of patients
with atopic dermatitis61 and in 16% of patients with AKC.59
Cataractogenesis is estimated to occur in ~8–10% of patients
with atopic dermatitis54,61 and is unique to AKC. Patients
treated with or without steroids may develop cataracts. Almost
90% of the AKC-associated cataracts are bilateral and are either
anterior subcapsular (shield-like) or posterior polar. They have
been observed as early as the teenage years, and the rate of
progression can vary from several months to many years. The
fundus may show degenerative changes and retinal detachment
may occur.
Contact lenses are often not tolerated by patients with atopy,
but if contact lenses are worn, signs of GPC may be super-
imposed on those of AKC. A study of the clinical features
of AKC revealed that 95% of the patients surveyed had
concomitant eczema and 87% had concomitant asthma.59 Hay
fever, migraine headaches, and rhinitis have also been reported
as part of this symptom complex.58
The ocular inflammation is perennial, although exacerbations
related to airborne allergens are common. Animal dander was
suspected as the precipitative factor in 51%, dust-type allergens
were thought to be causative in 43%, and food allergies were
suspected of being responsible in 35% of patients questioned
in one study.59 Patients diagnosed with the condition are
encouraged to control their environment as much as is practical
and to identify and avoid offending allergens if possible.
Patients should be questioned extensively about other atopic
conditions and about a family history of atopy. Patches of dry,
erythematous, pruritic skin may be ignored by patients who fail
to recognize it as eczema. Secondary staphylococcal infection of
the lids may complicate the AKC-associated blepharitis.
Laboratory tests measuring serum and tear IgE levels may
confirm the diagnosis of AKC if the levels are elevated. In the
serum, IgE is normally less than 100 IU/mL.58 Conjunctival
SECTION 6
scrapings that contain eosinophils and mononuclear cells are
also indicative of AKC, although this procedure is not encour-
aged because it may promote additional scarring.
AKC may not always be the sole allergic disorder presenting;
some investigators feel that it can occur in conjunction with
acute allergic conjunctivitis and even VKC.58 The presence of
severe itching and induration of the lids is indicative of AKC
and can be used to differentiate it from chronic blepharitis.57
Moreover, herpes simplex and staphylococcal infections are not
uncommon in patients with AKC and must also be considered.57,62
HISTOPATHOLOGY AND PATHOGENESIS
The underlying mechanism of AKC is thought to be both a type
1 hypersensitivity and a type 4 delayed hypersensitivity
response.57 Histological evaluation of conjunctival biopsies from
patients with AKC reveals an elevated number of mast cells and
infiltration by basophils, eosinophils, and lymphocytes. Mast
616 cells are found in much higher densities in the conjunctival
epithelium of patients with AKC and tryptase and heparin may
contribute to papillae formation and conjunctival scarring.63
Pronounced degranulation of eosinophils and neutrophils has
been reported, contributing to the clinical picture of the disease.62
The epithelium of patients with AKC appears convoluted, and
small fibrovascular stalks have been described.63 Goblet cell
proliferation and epithelial pseudotubular formation have also
been noted in conjunctival biopsies of patients with AKC.64
Differences have been shown among the conjunctival T-cell
subpopulations characteristic of patients with AKC, normal
individuals, and patients with ocular cicatricial pemphigoid
with respect to the antigen-recognizing receptor TCR. TCR
a- or b-containing T cells are found in the normal conjunctiva
and in the conjunctiva of patients with ocular cicatricial
pemphigoid; TCR g or d predominated, suggesting that TCR
g or d may play a role in the autoimmune diseases but not the
allergic disorders. TCR g or d cells were not found in patients
with AKC, but an increase in TCR a- or b-containing cells was
noted in the substantia propria when compared with that of
normal individuals. This could prove to be a method of
differentiating between severe AKC and the other cicatrizing
disorders. In addition, a statistically significant increase in
Langerhans’ cells (CD1+) was found in the epithelium and in
the substantia propria of patients with AKC.65
IgE levels are elevated in both the serum and the tears of
patients with AKC. Although these levels generally do not
correlate with the severity of the disease,59serum IgE has been
observed to decrease when the patient goes into remission.
Although exposure to allergens is associated with exacerbation
of the disease, allergen-specific (pollen, mite, and cat) IgE levels
were not elevated in the tears of patients with AKC compared
with those of nonatopic controls. Allergen-specific IgE levels
were elevated in the serum, however. Despite the increase in
circulating IgE antibodies, the number of IgE-bearing lympho-
cytes in circulation appears to remain within normal limits. By
contrast, the number of complement-bearing lymphocytes has
been reported to increase.
The cellular immune response appears to be dysfunctional in
patients with AKC, as evidenced by the absence of type 4 delayed
hypersensitivity responses to Candida and streptokinase-
streptodornase antigens and the inability of some patients to
become sensitized to dinitrochlorobenzene,66 depressed mito-
genic T-cell responses to phytohemagglutinin,67 and an increased
susceptibility to fungal and viral diseases.68 The circulating
immune cell profile includes fewer peripheral T cells but increased
B cells and eosinophil counts. The classic theories attempting
to explain the pathogenesis of AKC have been based on the
dysfunction of the cellular aspect of the immune system,
postulating that regulation of IgE synthesis is not properly
maintained by the T-cell population. Excessive binding by the
overabundant IgE molecules to resident mast cells may induce
a somewhat continual release of histamine and other mediators,
producing the clinical picture observed with this disease. Indeed,
the dense numbers of mast cells in AKC biopsies have been
observed to be in various states of degranulation.64
Early experiments in animals identified immune response
genes, called Ir genes, which were linked to the major histo-
compatibility complex of these animals.69–71 The Ir gene was
found subsequently to control the function of T-cells and related
intercellular interactions.71 Later research on IgE has revealed
similar relationships in humans and the role of the T cell on IgE
expression.72 Combined with the strong family history asso-
ciated with the disease and the observation that, under histo-
logical evaluation, the human conjunctival epithelium stains
intensely for HLA-DR glycoproteins,64 these findings suggest
that the actual site of the aberration in AKC lies at the
chromosomal level.
 Allergic and Toxic Reactions: The Immune Response
TREATMENT
The mast cell stabilizer cromolyn sodium has been reported to
be effective in the treatment of AKC.60 One study reported a
reduction in itching, watering, and photophobia in 66% of
patients taking one to two drops qid.72 Another trial revealed a
statistically significant improvement in photophobia, discharge,
papillary hypertrophy, limbal changes, and corneal changes
compared with placebo treatment.74 In contrast, a clinical trial
concurrently treating one eye with 2% cromolyn sodium and
the fellow eye with 1% medrysone every 6 h showed cromolyn
to be ineffective, whereas medrysone was highly effective in
improving both objective and subjective parameters.75 Package
labeling advises that the efficacy of cromolyn sodium is
dependent on its instillation at regular intervals. Furthermore,
a loading period of up to 2 weeks may be necessary before the
complete drug effect is observed. Other mast cell stabilizer
treatment possibilities include lodoxamide, pemirolast, or
nedocromil. For some patients, mast cell stabilizer/
antihistamines are often effective treatments for signs and
symptoms of AKC.
Typically, topical vasoconstrictor–antihistamine combinations
are beneficial for the relief of symptoms in the less severe forms
of the disease. Symptomatic relief has also been achieved to
some extent by lowering tear pH with saline irrigation and the
use of mucolytic agents54 and cold compresses.60 Systemically
administered antihistamines may alleviate the symptoms, but
may also induce dry eye symptoms, which could complicate the
ocular condition.76,77 The use of systemic antihistamines is
usually reserved for those with atopic conditions affecting
other organ systems. Terfenadine (60–120 mg bid), astemizole
(10 mg/day), and hydroxyzine hydrochloride (50 mg at bedtime
with gradual introduction into daytime with dosage escalation)
were found to be effective.58
Corticosteroids bring about the most dramatic improvement
in symptoms, but these agents must be used with caution
because of the increased risks. Pulse-type therapy is an alter-
native available to the practitioner. Topical steroids in strong
concentrations (1%) are used up to eight times per day for
periods not exceeding 7 days, followed by a rapid tapering.
Continuous wear soft contact bandage lenses used in com-
bination with 0.125% prednisolone acetate instilled qid have
been successful in the treatment of AKC-related epithelial
defect.58 In severe recalcitrant cases of AKC, systemic steroids
(such as a medrol pack) may be used.
The treatment of corneal ulceration with antibiotics and the
treatment of blepharitis, resulting from opportunistic infection
of the compromised lid epithelium, should not be ignored. The
prevalence of such conditions was demonstrated by the isolation
of Staphylococcus aureus from 67.6% of the lids of patients with
AKC in a study by Tuft and colleagues.59
Application of topical tacrolimus on eyelid skin may be
effective for treatment of severe atopic dermatitis of the eyelids,
and may have secondary benefits for AKC.78 Because a defi-
ciency in the suppressor T-cell population has been implicated
in the failure to arrest IgE responses, drugs geared toward
modulating the numbers, maturity, and function of this immune
cell subpopulation were evaluated with some success in the
1970s and 1980s. Indeed, the numbers of CD8 or cytotoxic
suppressor T-cells do not differ between AKC and normal
patients in conjunctival biopsies, whereas CD3, CD4, and CD5
T-cell subpopulations are greatly increased in AKC.64 Further
understanding of the roles of the various control mechanisms of
the immune system with respect to atopy may warrant
additional investigation into therapeutic agents of this type.
Topical cyclosporine A 0.05% seems to be safe and have some
effect in alleviating signs and symptoms of severe AKC
refractory to topical steroid treatment.79 Oral cyclosporine at
a dosage of 400 mg/day has been used in combination with
topical treatments in patients whose serum IgE levels were
exceedingly high.58 The use of lymphokines is also being explored
in the treatment of immune disorders. The identification of a
subset of the T-cell population that produces IgE-binding factors
when stimulated by glycosylation-enhancing factor but
produces IgE-suppressive factors when exposed to glycosylation-
inhibiting factor suggests that recombinant human IgE-
suppressive factors can be developed for therapeutic purposes.72
Regardless of the therapeutic choice made to treat the ocular
symptoms, the systemic nature of the disease warrants a
multidisciplinary approach to ensure complete and efficacious
control of AKC. Other targets for therapeutic intervention
include inhibition of tryptase, cyclooxygenase, leukotrienes,
bradykinins, platelet activating factor and IgE. Cytokine
antagonism and agonism, T-cell or eosinophil inhibition and
adhesion molecule antagonism might provide potent new
modes of treatment.
VERNAL KERATOCONJUNCTIVITIS
Vernal, derived from the Greek meaning ‘occurring in the
spring’, is a rare, serious form of ocular allergy. The disease
characteristics include a predilection for warm rather than cold
climates, a frequent family and personal history of atopic
disease, a higher than 2:1 frequency in males over females, and
an early onset, with remission by the late teens, very frequently
at the onset of puberty.80,81
Vernal keratoconjunctivitis (VKC) has a hereditary predis-
position with exogenous factors, such as climate, season, and
allergen exposure, determining the likelihood and severity of
this disease. Arid areas with the potential for wind and desert
storms, such as the Middle East and North Africa, have the
highest incidence of VKC. Dahan82 noted a high frequency of
VKC in black patients in South Africa, almost all of whom had
the limbal form. Patient histories in Israel and in Egypt had
shown VKC to be year-round and rarely associated with atopy
in these areas.83,84
CLINICAL FEATURES AND DIAGNOSIS
The clinical manifestations of VKC include raised conjunctival
cobblestones over the upper tarsal plate, and almost never over
the lower plate, with no significant conjunctival hyperemia
(Fig. 47.4). Bulbar conjunctival cobblestones, papules, or
follicles are almost never observed. The cobbles are large and
pleomorphic, and rarely are they evenly distributed. Bulbar
CHAPTER 47
conjunctival vasodilatation is diffuse and presents as pink,
rather than red. Horner–Trantas dots, first described in the
1880s,85 are chalk-white, raised superficial infiltrates straddling
the limbus with no specific meridional predilection (Fig. 47.5).
Gelatinous, translucent, globular deposits at the limbus vary
greatly in size and shape, from a 2 mm circle to an arc to a 360o
ring. Diffuse keratitis is present in more severe cases. The
shield ulcer, a central ovoid epithelial defect with a white fibrin
coating, is well delineated with no surrounding haze (Fig. 47.6).
These ulcers are almost never associated with iritis. Copious,
tenacious cordlike mucus with highly elastic properties is
always present in VKC. The common and often debilitating
symptoms of VKC are itching, photophobia, and pain.
The full manifestation of VKC provides few diagnostic
difficulties. The large cobblestones of the upper tarsal plate are
pathognomonic for this disease, but they do require lid eversion
to be identified. Thus, this is an indispensable component of
the external ophthalmic examination. These cobbles differ from
those in GPC by being dramatically larger in height and breadth 617
 CORNEA AND CONJUNCTIVA
FIGURE 47.4. Vernal conjunctivitis. The palpebral form is
characterized by enlarged papillae, referred to as cobblestone papillae
because of their shape and size, that are almost always confined to
the upper tarsus. The conjunctiva is often pink.
FIGURE 47.5. Vernal conjunctivitis. The limbal form exhibits
Horner–Trantas dots, which are white infiltrates found at the limbus
that can vary greatly in size and specific location or pattern. In more
severe cases, diffuse keratitis may be observed.
and varying in shape in a different pattern than the homo-
geneous cobbles seen in GPC or in the follicles in viral
SECTION 6
conjunctivitis. Scarring is not present, regardless of the number
of years that VKC has been present. If scarring exists, it is more
suggestive of the Arlt lines found in trachoma. The develop-
ment of ptosis is related to the presence of keratitis and
photophobia, producing a protective response. Additionally, it
can be caused by the increased bulk of upper tarsal conjunctiva
or myositis of the levator muscle. Ptosis and conjunctivitis in
combination can also be found in trachoma, chlamydia, GPC,
herpes zoster, and follicular conjunctivitis.
The pattern of vasodilatation seen in VKC is nondescript but
gives the conjunctiva a pink color rather than the red observed
in severe corneal ulcers and infectious conjunctivitis. Mild to
moderate chemosis, sometimes visible only with a slit-lamp as
pinkish fluid slightly separating the conjunctiva from the
underlying episclera, is commonly seen in VKC, rather than the
ballooning chemosis of acute conjunctivitis. However, other
forms of conjunctivitis also present with this mild chemosis.
The gelatinous nodules of limbal VKC are vascular and rapid
618 in onset and respond promptly to topical steroids, factors that
FIGURE 47.6. Vernal conjunctivitis. The shield ulcer is the most
serious consequence of vernal conjunctivitis. It is a centrally located,
white, fibrinous defect in the corneal epithelium. It lacks the
surrounding haze often seen with other ulcer types and is rarely
accompanied by iritis.
differentiate them from other limbal tumors. The chalk-white
Horner–Trantas dots are elevated and straddle the limbus,
unlike immune marginal infiltrates, which have a surrounding
lucid area, involve the corneal stroma, and are separated from
the limbus by ~1 mm. The shield shape of the central corneal
ulcer with its overlying white plaque differs from that of other
corneal ulcers in the absence of a surrounding haze, iritis, and
purulent discharge. However, the keratitis of VKC is diffuse and
provides no help with differential diagnosis.
Itching is pathognomonic for all allergic disease. Yet, the
intensity of the itching seen in patients with VKC requires
vigorous knuckle rubbing, and this observation is very typical of
this disease. The lids, other than manifesting mild edema, are
not involved. There is usually no excoriation of the lateral
canthal area or associated allergic blepharitis.
A careful and complete ocular examination, including upper
and lower lid eversion, careful limbal evaluation, attention to
the nature of the mucus, observation by slit-lamp of chemosis,
absence of conjunctival scarring, lid involvement, follicles, and
pretragal adenopathy, will inevitably lead to a correct diagnosis
of VKC.
HISTOPATHOLOGY AND PATHOGENESIS
The cobblestones in VKC represent dramatic collagen
proliferation and ground substance and cellular accumulation.
Mast cells are found in increased numbers, 80% degranulated,
located more superficially in the conjunctiva, and more likely
to be found in 1-m light microscopic or electron microscopic
sections.10,86 Eosinophils are also found in increased numbers
and located more superficially; thus, they are frequently avail-
able for recovery in scrapings. VKC is the only ocular surface
disorder in which greater than two eosinophils can be found
per 25-power objective field.28 Mast cells, lymphocytes,
macrophages, basophils, and rarely, polymorphonuclear cells
are also present.
The first stage in the development of VKC is heralded by a
prehypertrophic phase of hyperemia and a thin, milky-white
pseudomembrane. Subsequently, hypertrophic changes occur
that are related to a stromal infiltration with large papillae
covered by an epithelial monolayer with mucoid degeneration
in the crypts between papillae. The early cellular and vascular
phase is replaced by collagen deposition, hyaluronization,
 Allergic and Toxic Reactions: The Immune Response
decreased vascularity, and an overall decrease in inflammatory
cells. The limbal papillae, although differing in clinical appear-
ance, undergo the same pathologic transformation.87 Horner–
Trantas dots consist mainly of eosinophils and degenerated
cellular debris, but they may also contain polymorphonuclear
cells and lymphocytes.85
VKC represents a cell mediated hypersensitivity reaction by
T-cells. The increased number of degranulated mast cells and
their more superficial location was an important finding by
Allansmith and colleagues in implicating the central role of
these cells in VKC. The epithelium of normal subjects
contained neutrophils and lymphocytes but not plasma cells,
eosinophils, mast cells, or basophils. All these cells are found
in the conjunctival epithelium of the vernal patient. The
substantia propria of patients with VKC has substantially fewer
lymphocytes and more eosinophils, basophils, and mast cells.10
Leonardi and colleagues observed the role of mast cells in VKC
by observing the increase of mast cells in the stroma and
epithelium and the increase of thick, abnormal collagen fibers
in seven eyes of patients with VKC.88
A comparison of the percentage of degranulated mast cells
in VKC and GPC showed little difference.10 The fact that
histamine levels are dramatically elevated in VKC and not in
GPC suggests that there is a higher level of histamine release
from the mast cells of patients with VKC.18,89
There is increased mucus production in allergic con-
junctivitis, but more so in VKC. The mucus in VKC differs in
consistency from the thin strands seen in allergic conjunctivitis.
The thick, tenacious, ropy strands have an elastic quality
described as the Maxwell Lyon sign. This chewing gum-like
mucus contains inflammatory cells, specifically, dramatic
numbers of eosinophils and their Charcot–Leyden granules.90
Mucopolysaccharides, possibly hyaluronic acid, have been
noted by Neumann and Blumenkrantz.91 In certain patients,
the pH of the secretions has been noted to be highly alkaline.92
Such tear pH elevations have been found otherwise only in
severe rosacea keratitis.93
The central role of the mast cell and its mediators in VKC is
well established. The increased number and superficial location
of mast cells, dramatically degranulated, with increased tear
histamine and IgE levels94 leave little doubt that these factors
contribute to the pathogenesis of this disease. It remains
unknown why certain individuals respond to allergens with
such severity, and undoubtedly many other factors are involved.
Tear assays have shown 10-fold increases in tear histamine
levels from patients with VKC. Histaminase inactivation in
acute allergic conjunctivitis led to 10-fold increase in recovery
of histamine from tears (levels similar to the highest levels
found in patients with VKC).19 This suggests the possibility of
a histaminase dysfunction contributing to the high histamine
levels seen in VKC. In support of this theory, blood histaminase
levels were found to be reduced in patients with VKC.95,96
In addition to histamine, allergen-specific IgE antibodies
in tears and serum, tear IgG,97 and tear tryptase levels98 are
elevated in patients with VKC. Tear EMBP has been eluted from
shield ulcers.22 The mucoid plaque overlying the shield ulcer
has been shown to contain eosinophils and their granules.99
Table 47.1 summarizes the mediator effects presently eluci-
dated in VKC.
TREATMENT
Therapy for both AKC and VKC, as for all allergic disease,
should be aimed primarily at the identification of the allergen
and, when possible, its elimination or avoidance. Although
these patients generally have multiple sensitivities to allergens
such as grasses, dust, and mites, avoidance can be extremely
TABLE 47.1. Mediators Identified in Vernal Conjunctivitis and
Their Effects
Mediator Levels Origin Effect
Histamine Ø Mast cell Itch/vasodilatation/chemosis
EMBP Ø Eosinophil Keratitis/ulcer
Tryptase Ø Mast cell Effect on ground substance?
PGF Ø Mast cell Vasoactivity; chemotaxis
ECF Ø Eosinophils Mast cell Eosinophil
chemotaxis
ECF, eosinophil chemotactic factor; EMBP, eosinophil granule major basic
protein; PGF, prostaglandin F.
helpful in acute exacerbations. The use of air conditioning with
the appropriate filters can also be helpful. Avoidance of wind,
which is usually pollen bearing, and use of glasses or goggles to
decrease airborne allergen contact should also be suggested. As
mentioned previously, in North Africa, the wind coming off the
desert has long been associated with increased incidence and
exacerbations of VKC. A final suggestion to the patient is to
limit digital manipulation and knuckle rubbing of the eye as
much as possible, as this has been shown to cause mechanical
release of mast cell mediators.
Cold compresses are helpful in treating AKC and VKC,
perhaps related to a vasoconstricting effect or to some minor
role in mast cell stabilization. Elimination of the allergen will
yield dramatic benefits, particularly in the presence of shield
ulcers and keratitis. The use of bandage lenses should be
avoided since they can trap allergen in the precorneal tear films
and worsen keratitis.
Tear substitutes are helpful because of their barrier function,
their allergen dilutional function, and their irrigating ability.
The use of vasoconstrictors can inhibit vascular transudation,
thus decreasing chemosis.100 Although rebound vasodilatation
does not occur with the ocular use of vasoconstrictors, overuse
must be avoided to prevent tachyphylaxis and medicamentosa.101
Topical antihistamines can provide short term itching relief
for VKC patients by virtue of their H1 activity.15 In combination
with vasoconstrictors,31 these may provide adequate control in
mild conditions or allow for a decrease in the dosage of, or a
delay in the use of, topical steroids. A topical antihistamine–
mast cell stabilizer, such as olopatadine, can also be applied
owing to its H1 activity and inhibition of mast cell
degranulation. Olopatadine 0.1% has also been shown to reduce
the number of goblet cells in brush cytologic specimens of VKC
CHAPTER 47
patients after 2 months of treatment, which, in turn, decreased
the amount of mucus discharge.102
The double-edged sword of steroids is acutely evident in VKC
therapy. Therapeutic response to topical steroids can be
dramatic. However, the potential for superinfection and delayed
wound healing as well as cataract and glaucoma development
must be taken into account. For these reasons, pulse therapy of
a topical steroid such as prednisolone phosphate 1%, six to eight
times per day for up to 1 week, followed by rapid tapering to the
lowest levels needed for patient functioning, should be
prescribed. Steroids should not be used to eliminate the last
vestige of vasodilatation or itching, nor should the clinician
expect immediate resolution of the cobbles. Cobbles can remain
for many months without creating clinical problems. Surgical
removal of these cobbles with cryotherapy should be avoided,
because the resultant scarring of the conjunctiva can lead to lid
and tear film abnormalities that will persist as a life-long
problem after the VKC has spontaneously resolved. Patching
with antibiotic–steroid combinations is highly effective in 619
 CORNEA AND CONJUNCTIVA
treating shield ulcers, and in recalcitrant cases of shield ulcer
plaque, debridement is highly effective with or without
amniotic membrane.
Studies have shown the ability of cromolyn to often decrease,
and occasionally eliminate, the amount of or need for steroid
use in certain patients with VKC.84,103,104 Other agents used
in VKC treatment include topical lodoxamide,50 nedocromil,47
levocabastine,42 ketotifen,105 and cyclosporine.106,107 Topical
cyclosporine 2% provides a marked reduction in the symptoms
and signs of VKC, and is helpful as a steroid-sparing agent. It is
an immunomodulator inhibiting the clonal expansion of the
helper T subset of lymphocytes and the release of interleukins.
GIANT PAPILLARY CONJUNCTIVITIS
Giant Papillary Conjunctivitis (GPC) is not a true allergic
reaction, but is instead an inflammatory reaction of the upper
tarsal conjunctiva associated with the presence of contact
lenses, surgical suture barbs, and ocular prostheses.108 Initially,
GPC was mistakenly classified as an allergic reaction because of
its vague resemblance to VKC. Both diseases are characterized
by a papillary reaction on the upper tarsus, but the similarity
ends there. The papillae of GPC are small and even, between
0.3 and 1 mm in diameter. In VKC they are large (greater than
1mm in diameter), irregular, and often have mucus between
them. VKC patients report severe itching, whereas there is
limited itching seen with GPC. The epidemiology of the two
diseases is also quite different. While VKC is commonly seen in
young males and generally resolves after puberty, GPC, though
it is often seen in both female and male children and teenagers,
may affect patients at any age. In addition, histopathological
examinations show no increase in histamine or eosinophil
levels in GPC, which are two hallmark signs of chronic ocular
allergy.109
GPC is characterized by similar copious tearing, foreign body
sensation, copious production of mucus, and the proliferation of
subepithelial collagen, leading to the eruption of giant papillae.110
Patients may have blurred vision and excessive contact lens
movement serious enough to cause intolerance to lens wear.
Both VKC and GPC arise from similar underlying patho-
physiologic mechanisms. VKC and GPC are best explained
by a hyperactivity of resident mast cells, lymphocytes, and
fibroblasts, and the proliferation of collagen and formation of
conjunctival papillae. Advances in theory have been translated
to rational treatment.
GPC and similar conjunctival papillary disorders are the
result of: (1) genetics, (2) the appropriate triggering agent, (3)
sufficient duration of exposure, (4) sufficient area of exposure
SECTION 6
on the conjunctival surface, and (5) the particular geometry of
the exposure. Thus, the onset of disease is the result of two
broadly defined factors-genetics and physical trauma.
Retrospective epidemiologic studies of GPC have revealed a
strong association of GPC and wearing contact lenses
, especially hydrogel lenses.111 Younger patients were shown to
have a higher risk of developing GPC. Gender and tear film
breakup time were not found to be associated with the con-
dition. GPC is almost exclusively bilateral with a mean onset
time of 31.4 months after commencing lens wear. A retro-
spective study of the personal histories of patients with GPC
disclosed a higher incidence of atopy. Like PAC, GPC showed a
bimodal distribution, with peaks in the spring and late summer
and autumn. Patients with GPC reported a higher incidence of
allergy to pollen as well as to drugs and medications, but the
only statistically significant discriminator between patients
with GPC and comparison patients was sensitivity to
thimerosal. The seasonal onset of GPC diagnoses in 1987 and
620 1988, and the increase in reported allergies within the GPC
group, suggests a strong association between atopy and the
development of GPC.112
GPC can result from an exposed suture end that abrades the
upper palpebral conjunctiva.113 This syndrome consists of a
mucoid ocular discharge with blurred vision, a foreign body
sensation, upper lid edema, and blepharoptosis concomitant
with giant papillae of the upper palpebral conjunctiva. Removal
of the offending suture(s) results in resolution of the papillae
and symptoms.
CLINICAL FEATURES AND DIAGNOSIS
Contact lens-associated GPC is observed in wearers of both
rigid gas-permeable and soft lenses, although the incidence is
greater in wearers of soft contact lenses. Superficial neovascu-
larization, contact lens-associated superior limbic keratocon-
junctivitis, and GPC are all associated with wearing soft contact
lenses.114 Furthermore, mechanical irritation from the large-
diameter soft contact lenses and a tendency for these lenses to
be coated with mucoprotein deposits increase the potential
problems.115
Signs and symptoms of GPC range from minimal discomfort
upon inserting or removing contact lenses to complete
intolerance of the contact lenses.108 The earliest symptoms of
GPC (which precede signs) are irritated eyes when the lenses are
removed, accumulations of mucus in the nasal corner of the
eye, and slight blurring of vision due to coatings on the surface
of the contact lens. Symptoms of more advanced GPC may
include foreign body sensation and reports of mucus gluing the
eyes shut during sleep.
Signs of GPC may range from mild hyperemia of the upper
tarsal conjunctiva, with strands of mucus streaking the other-
wise smooth conjunctival surface, to the presence of milky-white
discharge covering broad areas of giant papillae. Early in the
progress of GPC, the conjunctiva remains translucent, but careful
observation reveals that the conjunctiva is somewhat thickened.
As GPC progresses further and infiltration by inflammatory cells
continues, the conjunctiva acquires a more opaque appearance.
Conjunctival papillae larger than 0.3 mm in diameter are
abnormal.110 In GPC, as these abnormally large papillae
emerge, they push aside the normal smaller papillae, their
apexes flatten, and there may also be conjunctival ulceration.
The precise location of papillae varies with the type of contact
lens worn by the patient.116 Wearing soft contact lenses tends to
induce papillae that appear first in the upper zone of the tarsal
area and then progress toward the lid margin. Enlarged papillae
may be seen throughout the upper tarsal conjunctiva in
advanced cases of GPC. Wearing hard contact lenses tends to
induce fewer and smaller papillae, with crater-like flattened
(rather than rounded) tops. Those papillae are likely to be found
first along the lid margins.
HISTOPATHOLOGY AND PATHOGENESIS
Disruptions of normal host defenses are present in both VKC
and GPC. Although patients with VKC and GPC are found to
have normal tear concentrations of tear lysozyme, for example,
tear concentrations of lactoferrin are reduced in VKC and
GPC.117 Lactoferrin, an essential component of the nonspecific
immune protection of the external eye, is reduced in patients
with active VKC and GPC, although patients with inactive GPC
had normal tear levels of lactoferrin.118 The contribution of
reduced lactoferrin to the onset and course of VKC or GPC
remains unclear. It is possible, however, that decreased
lactoferrin in the tears of patients with VKC and GPC somehow
contributes to the increased ocular inflammation and to the
troublesome bacterial contamination of worn contact lenses.
 Allergic and Toxic Reactions: The Immune Response
Eosinophil degranulation commonly occurs in VKC and GPC.
EMBP is elevated in conjunctival tissues of patients with VKC
and GPC.119 The cytotoxic effects of these cationic proteins are
almost certain to play a major role in the conjunctival inflam-
matory reaction and the subsequent deposition of collagen in
the pathogenesis of VKC and GPC.
The significance of atopy in GPC is emphasized by the
finding of elevated tear concentrations of IgG and IgE in GPC,
perhaps owing to the presence of antigenic coatings on the
surface of the worn contact lens. Tear IgE levels in patients with
GPC were significantly increased, especially in the more
symptomatic eye (geometric mean of 6.9 IU/mL, P <.01), com-
pared with those in a control group who also wore contact
lenses (2.1 IU/mL). Increased tear IgG levels (50.7 g/mL, P <.01)
were found in the more symptomatic eyes of patients with
GPC.120 In eight of the 18 patients, tear IgM was measurable
(> 4.7 g/mL), whereas none of the patients in the control groups
had detectable amounts of IgM in their tears. Studies with
transferrin as a marker for the vascular leakage of serum proteins
into the tears showed that local production was responsible for
the increased tear immunoglobulin levels.
TREATMENT
Successful treatment depends on early recognition of the
condition, although signs and symptoms will resolve if the
patient refrains from wearing the contact lens. The first concern
is the prevention of GPC. Prevention depends on: (1)
encouraging strict lens hygiene, and (2) prescribing the
appropriate lens type and edge design. Treatment of GPC
likewise depends on finding the appropriate lens material and
design and encouraging proper lens hygiene. Treatment also
requires proper therapy to control the conjunctival
inflammatory response.
Regarding hygiene, lens cleaning agents and saline solution
for rinsing and storing lenses should be thimerosal free.
Enzymatic cleaning of the lens with papain preparations is
essential to minimize the accumulation of lens coatings and to
remove build-up of environmental antigens that may adhere to
the lens coating. Finally, lenses should be replaced frequently.
Fluorescein staining of the apices of enlarged papillae, heavy
mucus, significant conjunctival hyperemia, and movement of
the lens on blinking (decentering) are all indications that the
patient should discontinue wearing contact lenses until the
signs and symptoms of GPC resolve. A marked increase in
immunoglobulin deposition and enhanced IgG:IgA (P <.001) is
common to high-water content lenses (especially those of
nonionic composition) used on an extended wear basis, when
compared with low-water content lenses used on a daily wear
basis. It is thus hypothesized that use of high-water content
lenses on an extended wear basis leads to a greater degree of
inflammatory or immune stress.121 However, frequent changing
of the lens material or design allows patients to continue
wearing contact lenses. Because GPC seems to occur less
frequently with hard than with soft contact lens wear, if GPC
develops with soft contact lens wear, changing to the rigid gas-
permeable lenses may resolve the problem.
Hydrogel contact lenses appear to result in an overall
prevalence of GPC of ~20%. The stiffness of the material,
rather than thicker edges, is presumed to be the principal factor
behind the higher incidence of GPC in silicone hydrogel lens
wearers. Changes to lens design and the introduction of a
steeper base curve may reduce the incidence levels previously
reported.
There is no need to wait until the enlarged papillae regress
before reintroducing contact lenses. In fact, it may take several
months or even years for the enlarged papillae to disappear. In
the absence of signs indicating the need for withdrawing lenses,
contact lenses may be reintroduced 3–5 days after symptoms
such as hyperemia and excessive mucus production have been
resolved.
Unlike in the treatment of VKC, topical corticosteroids have
not proved particularly effective in the treatment of GPC. A
short course of corticosteroids may quiet the inflammation
before long-term management of the disease is begun.
TOXIC KERATOCONJUNCTIVITIS
Toxic keratoconjunctivitis (keratoconjunctivitis medicamen-
tosa) is one of the most frequently encountered problems in
the subspecialty of cornea and external disease. Taking a careful
history is critical to establishing correct diagnoses in
ophthalmology. Potent medications used inappropriately can
result in toxic or hypersensitivity reactions. For example, a
patient with an underlying dry eye problem may be
misdiagnosed as having viral conjunctivitis. Various antibiotics
or antiviral agents may be prescribed without subsequent
improvement or with worsening of the initial problem. Owing
to a deteriorating clinical condition, the practitioner may alter
therapy using other agents possibly aggravating the clinical
picture. As a general rule, if a patient has been treated with
multiple medications over the course of weeks to months, with
no apparent improvement or worsening of the original
complaint, all topical medications should be discontinued for at
least a few weeks. This allows the practitioner to re-establish a
baseline clinical status and may result in clinical resolution.
Toxic symptoms may range from mild irritations to
ulcerative keratitis with potentially sight-threatening visual
consequences. By definition, a toxic substance is poisonous and
‘may cause a disturbance of structure or function’.122 Although
irritation implies inflammation, generally speaking, toxicity
and irritation are terms that may be used interchangeably.
Differentiating between an allergic and a toxic reaction may be
difficult because some drugs may elicit both reactions with
similar biochemical mechanisms. In general, allergic reactions
require repeated exposure to the sensitizing agent and a
sufficient amount of time to elapse for sensitization of the
immune system. This time period may range from 5–10 days to
years, depending on the potency of the sensitizing agent and the
susceptibility of the exposed individual. Patch testing may help
differentiate allergy from toxicity; however, false-positive and
false-negative test results may frustrate the clinician in a
situation in which a careful history and examination are more
likely to establish the etiology.
Factitious (self-induced) disease results from mechanical
CHAPTER 47
trauma or toxicity from eye drop abuse. The incidence of
iatrogenic keratoconjunctivitis was found to be 13% at one
tertiary center.123 Healing was prolonged, taking 7–93 (median
28.5) days. From a research standpoint, toxicities from drop
abuse are often not documented in the medical chart and not
reported. Factitious disease should only be considered after
iatrogenic causes have been investigated.
In addition to redness, the conjunctival tissue response to
toxic agents may result in follicular or papillary excrescences.
Typically, the reaction is more prominent in the inferior bulb,
fornix, and tarsal conjunctiva. Conjunctival scrapings may reveal
mononuclear cells, a few neutrophils, and mucus. Eosinophils,
the hallmark of an allergic reaction, are generally absent unless
a combined allergic and toxic mechanism is present. Epithelial
cells, mononuclear cells, and polymorphonuclear cells may show
toxic large basophilic cytoplasmic granules.122
The most common manifestation of corneal toxicity is a coarse
punctate epithelial keratopathy. Heaped-up opaque epithelium,
swirl patterns, and pseudodendrites may occasionally develop.122 621
 CORNEA AND CONJUNCTIVA

These morphologic patterns have been reported most com-
monly in cases of idoxuridine toxicity, an antiviral now used
infrequently. Possibly the leading cause of pseudodendritic ker-
atitis is timolol, a b-blocker used in the treatment of glaucoma.
Toxic ulcerative keratitis is the most severe form of corneal
toxicity. Schwab and Abbott have reported on 19 such cases, of
which five were factitious and 14 iatrogenic.124 In this series,
the corneal epithelial defects were typically oval, occurred
inferonasally, and had gray rolled edges with surrounding
intense superficial keratitis. The lusterless conjunctiva and
cornea stained well with both rose bengal and fluorescein dyes.
An additional corneal abnormality, punctate marginal keratitis,
may be a result of allergic hypersensitivity or, on occasion, a
hypersensitivity reaction to topical drugs such as gentamicin
(the most common offending agent), atropine, anesthetics, and
epinephrine.125 Multiple small perilimbal infiltrates are evident
circumferentially with a characteristically clear zone noted
between the infiltrates and the limbus.
Cicatrizing conjunctival and keratinizing changes may
develop, particularly in patients using glaucoma medications
and antiviral agents. These changes, which may completely
mimic ocular cicatricial pemphigoid, include punctal occlusion,
canalicular obstruction, fornix and tarsal conjunctiva scarring,
corneal vascularization, and keratinization.122,126–129
Conjunctival and Tenon’s capsule specimens from glaucoma
patients have shown a significant increase in the number of
macrophages, lymphocytes, mast cells, and fibroblasts, whereas
goblet cells were decreased in patients who took at least two
glaucoma medications for 1 year.130 This data suggest that
glaucoma therapy may induce inflammation and may worsen
the prognosis for future glaucoma surgery.
Drug-induced ocular cicatricial pemphigoid or drug-induced
pseudopemphigoid has been reported by a number of
investigators.126–129 Topical drugs implicated in this reaction
include miotics (echothiophate iodide, pilocarpine), sympa-
thetic agents (dipivefrin hydrochloride), b-blockers (timolol
maleate), and antivirals (idoxuridine, trifluorothymidine).
Fortunately, this side effect occurs rarely.
Numerous studies have to date been unable to differentiate
between cicatricial pemphigoid and drug-induced cicatricial
pemphigoid on the basis of histopathologic, ultrastructural, or
immunofluorescent criteria.126,127,131–133 Tauber reported a 26%
incidence of glaucoma in their patients with cicatricial
pemphigoid (29 of 111 patients).134 In this study, 27 of 29
patients had a history of glaucoma medication use, suggesting
that long-standing glaucoma therapy may induce or increase
the susceptibility of certain individuals to the development of
ocular cicatricial pemphigoid.

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107. Secchi AG, Tognon MS, Leonardi A: Topical
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 CHAPTER
48 Lid Inflammations
Audrey S. Chan and Kathryn A. Colby
Lid inflammation or blepharitis, a common problem in
ophthalmic practice,1 is also a frequent cause for visits to
physicians. In a general practice, 2.3% of visits were for ocular
problems, 70% of which were diagnosed as bacterial con-
junctivitis, allergic conjunctivitis, meibomian cyst, or
blepharitis.2 Blepharitis is a condition that can often be
controlled but never permanently cured, because the underlying
factors of sebaceous gland dysfunction and skin flora cannot be
permanently and irrevocably altered. Chronic blepharitis can
lead to severe dry eye, trichiasis, lid notching, reduced corneal
sensation, corneal scarring with neovascularization and
marginal keratitis. There is considerable amount of overlap of
symptoms among blepharitis and other inflammatory disorders
affecting the lids.
ANATOMY OF SEBACEOUS GLANDS IN
THE EYELID
Sebaceous glands are present in the eyelids as both meibomian
glands and the glands of Zeis. The sebaceous glands in the lid
are embryologically derived from a common pilosebaceous unit
that differentiates during the second month of gestation. Unlike
the glands of Zeis, which are associated with cilia, meibomian
glands are modified sebaceous units that lack hair follicles.
They are vertically oriented in parallel rows through the tarsus.
There are ~20–22 meibomian glands on the lower lids and
22–24 on the upper lids. The glands of Zeis are located on the
lid margin anterior to the opening of the meibomian glands.
Absence of meibomian glands may be a marker for ectodermal
dysplasia syndrome.3
A disruption in normal meibomian gland function leads to a
variety of disorders that affect the ocular surface, the most
common of which is blepharitis, which causes secondary
evaporative dry eye. The meibomian glands play a critical role
in maintaining tear film homeostasis and stability. The
meibomian secretions produce the oily outer layer of the tear
film, which prevents evaporation of the aqueous tear layer and
creates an optically smooth surface. The structural and
refractive integrity of the ocular surface depends on the quality
of meibomian secretions. The symptoms of ocular burning,
stinging, and irritation are the result of tear film instability
causing evaporative dry eye. In this chapter the treatment and
diagnosis of the many disorders stemming from lid
inflammation will be discussed.
COMPOSITION OF MEIBOMIAN GLAND
SECRETIONS
Sebaceous glands are holocrine glands. Each meibomian gland
consists of several acini connected by a long central duct that
opens at the lid margin. Each acinus is lined by cuboidal
epithelium that houses storage granules containing lipid
material. When the cell becomes engorged with lipid, the
nucleus of the cell becomes pyknotic and the apex of the cell
membrane ruptures into the lumen of the gland opening; the
cell spills forth its lipid and cellular contents into the duct.
The lipid material flows to the orifice of the gland and onto the
skin or into the tear film. The formation of sebum is dependent
on cellular proliferation. There is also increasing evidence
that meibomian gland secretion is modulated via neuronal,
hormonal and vascular influences.4,5 Vasoactive intestinal
peptide (VIP) innervation has been shown to be present in acinar
cells and also provides innervation to the lacrimal glands.6
Studies of meibomian gland growth and differentiation in cell
culture may provide better understanding of meibomian gland
function.7,8
Stagnation of meibomian gland secretion results in several
histopathologic features. Features of meibomian gland dysfunc-
tion include signs of obstruction and dilatation of the ducts,
enlargement of acini with cystic degeneration and squamous
metaplasia, foreign-body reaction and granuloma formation,
a mild increase in inflammatory cells, and abnormal
keratinization.9 Obstruction of the meibomian gland ducts with
stagnation of secretions may cause increased pressure within
the ducts, thus inhibiting cellular differentiation and causing
squamous metaplasia. Keratinization of the meibomian gland
ductal epithelium may be the initiating event.9–12
The chemical composition of meibum has been extensively
studied. Meibomian gland secretion is distinct from other forms
of sebum in that it has a relatively low melting point of
19–32°C, which allows the secretion to remain fluid at lid
temperature.13 Approximately 84% of meibum is composed of
nonpolar lipid wax esters and sterol esters. Cholesterol is the
main esterified ester with relatively longer carbon chains of 20
carbons or more in comparison to wax esters.14 Triglycerides
and free fatty acids are found in smaller amounts in meibum.
Unsaturated fatty acids are particularly important to the
maintenance of normal meibum properties. Solidified paste-like
meibum such as the type often found in blepharitis patients
contains relatively low concentrations of unsaturated fatty
acids.15 These lipid abnormalities may account for many of the
signs and symptoms of chronic blepharitis, such as tear-film
instability and inspissation of secretions.
One study conducted by Shine and McCulley investigated the
composition of polar lipids from patients with meibomianitis.
They found that patients with meibomianitis had higher
concentrations of an unknown type of polar lipid and polar
lipids with unsaturated fatty acids and amide acyl chains when
compared with normal patients or patients with other forms of
chronic blepharitis. From these results they hypothesized that 625
 CORNEA & CONJUNCTIVA

a b c

FIGURE 48.1. Hordeolum. There is focal inflammation and abscess formation around the mouth of a meibomian gland (a) and around a lash
follicle/gland of Zeis (b). The abscesses may be single or multiple (c), involving more than one lid.

changes in the polar lipid phase of the tear film could lead to
tear-film instability and thus dry eye symptoms.16
The lipid abnormalities may be physiologic or induced by
bacterial lipases.17 Work has also been directed toward analysis
of meibomian secretions and interactions with bacterial
lipases.18–21 S. epidermidis and S. aureus produce triglyceride
lipase and cholesterol and wax esterase. These exoenzymes
hydrolyze wax and sterol into free fatty acids thereby altering
the composition of meibum. Increased levels of free cholesterol
from esterase activity have been found to stimulate proliferation
of S. aureus in vitro.22 Low levels of tetracyclines used for the
treatment of blepharitis have been found to inhibit bacterial
lipases reducing free fatty acid production thereby changing the
composition of meibum and inhibiting S. aureus proliferation.23

INFLAMMATIONS OF THE SEBACEOUS

GLANDS

HORDEOLUM
An acute focal inflammation of the eyelid may occur when a
meibomian or a Zeis gland becomes infected (Fig. 48.1). This is
called a hordeolum, or in the vernacular, a stye. It represents
an acute pyoderma. The most common bacterial cause is
Staphylococcus aureus. The process is usually self-limited,
with spontaneous drainage of the abscess and resolution within
5–7 days. Warm compresses are helpful in localizing the FIGURE 48.2. A hordeolum can be drained by nicking the pustule at
inflammation. More rapid drainage of a hordeolum can be the gland opening and expressing the purulent contents.
promoted by nicking the pustule at the mouth of the occluded
orifice using the sharp tip of a needle or blade and then applying
focal pressure to express the pus (Fig. 48.2). No anesthesia is by the absence of tense erythema, pain, or leukocytosis. The
required. Systemic antibiotics are unnecessary unless there is focal inflammation around the involved gland may cause
SECTION 6

significant cellulitis, in which case a semisynthetic penicillin,
erythromycin, or clindamycin should be administered.
Preseptal and orbital cellulitis are discussed in Chapter 239.
CHALAZION
A chalazion is a granuloma that develops around a sebaceous
gland in the lids as a foreign-body reaction to sebum released
into the surrounding tissue. It may evolve from a hordeolum
or may occur secondary to inspissation of sebum at the open-
ing of a gland with engorgement and rupture of the gland
contents into the surrounding tissue (Fig. 48.3). Histo-
pathologic evaluation of chalazion contents reveals histiocytes,
multinucleated giant cells, lymphocytes, plasma cells,
polymorphonuclear leukocytes, and eosinophils.24 The acute
inflammatory process may be intense, creating enormous lid
edema that may spread to the opposite lid and sometimes
across the bridge of the nose to the lids of the other eye. The
626 local lymphatic congestion can be differentiated from cellulitis
pointing of the lesion through the skin anteriorly or into the
subconjunctival–tarsal space, where it may eventually drain
spontaneously or persist as a chronically inflamed granuloma.
In the chronic phase, a chalazion may appear as a quiet but
large swelling of the lid. Large lesions of the upper lid may cause
distortion of vision from induced astigmatism. As with
treatment of a hordeolum, warm compresses are useful in
trying to localize the inflammation and cause spontaneous
drainage. Topical antibiotics cannot directly affect the inflam-
mation inside the gland but are an adjunctive therapy in trying
to decrease the local bacterial flora. Many chalazions, especially
if they are small and of short duration, will be cured or
improved within a month of medical treatment (Table 48.1).25
Rosacea is common in patients with chalazions.26
Treatment of the chalazion in the chronic phase consists of
surgical drainage or intralesional steroids. When the transcon-
junctival surgical approach is used, it is important to make the
incision(s) perpendicular to the lid margin, parallel to the orien-
tation of the meibomian glands. Horizontal conjunctival–tarsal

a b
c d
incisions will create scar tissue across the ducts of the
meibomian glands, resulting in blockage of sebum in the
proximal ducts. The sebum then backs up, causing rupture of
the ducts and more granuloma formation, which perpetuates
rather than cures the problem. Incisions can be made in the
skin, especially if the chalazion has already ruptured through
the skin. The incision in the skin should be parallel to the lid
margin to minimize visible scarring. Excision of a chalazion
using a carbon dioxide laser and curettage has been reported.27
Triamcinolone acetonide, a soluble aqueous steroid sus-
pension, in a concentration of 5 mg/mL, can be injected directly
into the center of a chalazion. The total volume varies from
0.05 to 0.2 mL, which is injected transdermally or directly
perpendicularly through the conjunctiva and tarsus.24 The
advantage to the latter approach is that it decreases the risk
of dermal depigmentation and atrophy, which sometimes
accompany intradermal use of fluorinated corticosteroids.28,29
The injections can be repeated anywhere from 2 days to
1 month apart if the initial injection does not result in com-
plete resolution of the lesion. Success rates vary from 76% to
93%.24,30–32 A recent retrospective case series from Jules Stein
Eye Institute reported an 83% success rate after triamcinolone
acetonide injection of primary or recurrent chalazia.33 Hard
lesions present for more than 6 months are less likely to
respond.31 Accidental intravitreal injection of a steroid,
resulting in macular pucker and optic atrophy, has been
reported.34
Care must be taken not to mistake sebaceous cell carcinoma
of the eyelid for recurrent chalazions. If any doubt exists,
especially in an older patient, the excised material should be
sent for histopathologic examination.35–37
TABLE 48.1. Treatment Options for Chalazions
Treatment Duration
Compresses Less than 6 wk
Intralesional
steroids/incision 2 wk–6 mo
Incision/drainage Longer than 6 mo
Lid Inflammations
FIGURE 48.3. A chalazion is a granulomatous
reaction to the inspissated secretions of
meibomian gland. It appears as a swollen
tumor mass involving the eyelid. It may be
associated with local inflammation around the
mouth of a single gland (a). It may show no
external signs of inflammation (b), with only a
slight engorgement of vessels on the tarsal
conjunctiva (c). Chalazions may be very small,
presenting as only small granulomas at the
posterior lid margin (d). They may involve
multiple glands and different lids (b). They may
cause blurring of vision owing to induced
astigmatism from the pressure of the mass on
the cornea. The patient in b and c complained
of visual acuity that was reduced to 20/25; this
returned to 20/15 when the chalazion was
excised.
BLEPHARITIS
Key Features
• Blepharitis is a common chronic eye disease.
• The goal of treatment is management of symptoms; the
condition cannot be cured.
• New treatment for dry eye address the inflammation of the
ocular surface now known to play a role in this condition.
Classification of Blepharitis
Blepharitis can be broadly classified anatomically as either
anterior or posterior, anterior blepharitis comprising staphy-
lococcal and seborrheic forms and posterior blepharitis
primarily involving the meibomian glands. Many other
classification schemes have been proposed based on clinical
symptoms and findings.38
Mathers and Choi evaluated 513 patients to create a clas-
sification tree to separate blepharitis and dry eye conditions into
clinically relevant groups based on objective physiologic
measurements.39 According to their classification scheme, nine
categories were established based on meibomian gland drop
out, lipid viscosity, lipid volume, Schirmer testing, and tear
CHAPTER 48
evaporation. Interestingly, the presence or absence of bacteria
was not included as one of their objective measurements,
arguing that the mere presence of bacteria does not necessarily
indicate infection. Although Staphylococcus aureus can be
found more frequently in blepharitis,40 they contend that
bacterial infection alone may not be causative, but instead may
secondarily exert some effect on meibomian glands resulting in
blepharitis.
Infection Size Hardness
Yes Small–medium Soft
No/yes Small–large Rubbery
Yes Small–large Hard
627
 CORNEA & CONJUNCTIVA
TABLE 48.2. Clinical Spectrum of Blepharitis
Type Major Feature Common Associations
Staphylococcal Acute inflammation 80% female
Seborrheic blepharitis Oily, greasy scales Spotty glandular
around lashes involvement
Meibomian seborrhea Excess secretions
from glands
Seborrhea Solidified with plugged
secretions
Rosacea Facial dermal involvement Keratitis
Seborrhea/rosacea Chalazions
Using cluster analysis Mathers and Choi were able to devise
a decision tree to place patients into distinct categories based on
the results of the objective tests. Meibomian gland drop out was
found in only two groups, which they identified as (1) rosacea
and (2) obstructive meibomian gland dysfunction (MGD)
dry-eye patients. Both of these groups had obstructive MGD but
differed by lipid volume and viscosity. According to the cluster
analysis, seborrheic MGD was divided into three groups, with
only one group having a low tear-evaporation rate. It was
previously thought that seborrheic patients have low tear-
evaporation rates owing to the presence of excess lipid
secretion. However, in this study the authors found two
subgroups of seborrheic patients with high lipid volumes with
high tear evaporation and dry eye. Evaporation was an
important variable in the study that helped to classify patients
into relevant groups. Through the use of physiologic
parameters, the authors hope to shed insight into the
underlying pathologic mechanisms of blepharitis and dry eye.
McCulley and colleagues suggest categorizing blepharitis
into six groups to facilitate a rational approach to investigation
and therapy17 (Table 48.2). The group that they called
‘staphylococcal’ tended to have more acute lid inflammation of
shorter duration. Eighty percent of the group consisted of
women. Keratitis sicca affected 50% of the group. The results
of lid cultures were positive for S. aureus in 46% of patients
versus 15% in the control group. Interestingly, fully 90% of
both control and ‘staphylococcal’ patients had positive
S. epidermidis cultures.
McCulley and colleagues’ seborrheic blepharitis group had
SECTION 6
oily, greasy scales and crusting around the cilia with spotty
involvement of clusters of glands. A third group had combined
seborrheic and staphylococcal involvement. A fourth group
had seborrheic blepharitis with excess secretions from the
meibomian glands (meibomian seborrhea), and a fifth group
had seborrheic blepharitis with secondary inflammation of the
meibomian glands with solidified secretions within the ductules
that were difficult or impossible to express. There was no
increase in recovery of bacteria in these patients compared with
normal controls. McCulley and colleagues’ final group was
described as having primary meibomitis with associated
generalized dermal involvement in the form of acne rosacea or
seborrheic dermatitis. These patients, with what McCulley and
colleagues called ‘meibomian keratoconjunctivitis’, had a
marked instability of the tear film. They tended to have the
most severe dry eye signs and symptoms.41
In clinical practice, patients do not tend to fall neatly into a
pigeonhole category (Fig. 48.4) but present on a continuum
628 between categories. As their blepharitis waxes and wanes over
Dry Eye Microbiology
50% keratitis sicca Staphylococcus aureus
common
Bacterial flora within
normal limits
Bacterial flora within
normal limits
Yes
years, they may sometimes fit into each and every category.
Nevertheless, the concept of multiple types of blepharitis is
useful in sorting out the various components and approaches to
therapy (Table 48.3).
Role of Staphylococcus Aureus
Traditionally, the cause of blepharitis has been attributed to
S. aureus,42–44 despite the fact that S. aureus can not always be
recovered on culture.45 However, despite decades of study, its
contribution to blepharitis remains poorly defined. Cultures of
normal lids and those with blepharitis reveal similar, very
frequent colonization with coagulase-negative staphylococcal
species, Propionibacterium acnes, and Corynebacterium
species.46–49 S. aureus is not disproportionately represented in
patients with blepharitis,49 unless the group with S. aureus has
been deliberately subdivided from the larger group.17 In one
large study, however, quantitative growth of S. aureus was
significantly heavier in patients with blepharitis.49
The role of staphylococcal toxins in blepharitis is still not
well established despite extensive research in toxin production.
a-Lysin from bacterial strains isolated from patients with
blepharitis produced dermal necrosis when tested in rabbits,50
but a-lysin was also found to be produced by all isolates of
S. aureus-colonizing lids of normal controls as well as by
patients with blepharitis.51 Enhanced cell-mediated immunity
to S. aureus was demonstrated in 40% of patients with chronic
blepharitis but not among normal controls.52 The same group
of patients was tested with intradermal thiomersal and only an
expected 6% showed cell-mediated immunity to thiomersal,
suggesting that they were not hyperimmune.53
Mondino and coworkers developed a rabbit model of
staphylococcal blepharitis and postulated that hypersensitivity
to the S. aureus cell wall, particularly to ribitol teichoic acid,
plays a role in the pathogenesis of staphylococcal blepharitis.54
Rabbits immunized with cell wall or ribitol teichoic acid
also developed 4–5 mm of peripheral corneal vascularization,
and several developed corneal phlyctenules or catarrhal
infiltrates.54 Subcutaneous vaccination with S. aureus phage
lysate did not prevent development of phlyctenules or
blepharitis in rabbits given topical applications of viable
S. aureus in both eyes. In fact, the control group was less
affected than the vaccinated group.55
DEMODEX
Demodectic mites can be found inhabiting hair follicles
(Demodex folliculorum) and sebaceous glands (D. brevis). Their
role in causing blepharitis has not been well established,12,56–59
 Lid Inflammations

a b c

d e

FIGURE 48.4. (a) This patient with chronic blepharitis shows the typical heavy crusting and scales along the bases of the eyelashes. There is a
fairly uniform swelling to the lids with a chronic spotty redness to the lid margins. (b) Patients with long-standing blepharitis will demonstrate
patchy loss of lashes (madarosis) and whitening or loss of pigmentation in lashes (poliosis), as well as chronic crusting and scale formation along
the bases of the lashes. There may be patchy focal involvement, with some portions of the lids affected more than others. a and b show the
right and left eyes, respectively, of the same patient. (c) Focal pouting of the individual meibomian glands may be seen, accompanied by
telangiectasia of the lid margin. (d) The upper and lower lids may be asymmetrically involved. (e) Patients with chronic ‘staphylococcal’
blepharitis frequently have a dry eye, as demonstrated here by rose Bengal staining. They are also susceptible to peripheral marginal ulcers of
the cornea (b and c). It is postulated that hypersensitivity to components of the Staphylococcus aureus cell wall plays a role in the pathogenesis
of staphylococcal blepharitis and peripheral corneal infiltrates.

TABLE 48.3. Treatment of Blepharitis

Major Findings Treatment

Scales and crusts around lashes
Ocular irritation
Focal swelling
Recurrent hordeola and rosacea
Meibomian gland dysfunction
Lid hygiene: warm compresses, lid scrubs, dilute shampoo; topical antibiotics with efficacy against
Staphylococcus species
Test for dry eye (Schirmer with anesthesia); artificial tears; punctal occlusion; Restasis
Hot compresses; manual expression of glands
Oral tetracycline and derivatives; topical metronidazole
Warm compresses, oral omega-3 fatty acids, Restasis

but it is tempting to postulate that in heavy infestations they translucent cylinders resembling clear plastic insulation or
could cause mechanical plugging of the gland orifices and cuffs enclosing the base of a lash for a distance of ~1 mm is

secondary blepharitis.
D. folliculorum and D. brevis, the hair follicle mites, are the
most common ectoparasites of humans. They are colorless,
spindle-shaped, and only 0.3–0.4 mm long. The anterior third
of their bodies has four pairs of very short legs, which limits
their mobility. They are almost always found with their
posterior down in the hair follicles, especially in the nasolabial
folds, the nose, and the eyelids. They feed on the cells of the
follicular or sebaceous glandular epithelium by piercing the cell
wall with their convex U-shaped chelicerae. Their complete life
cycle is ~15 days. The female mite lays her eggs deep in the
gland. The larvae are conveyed passively with secreted sebum
into the pilosebaceous canal. Nymphs move in the dark into
another follicle.57,60
Demodex can be identified in normal-appearing eyelids by
epilating lashes and observing the mites clinging to the lashes
under the microscope (Fig. 48.5). They cannot be seen by slit
lamp. They have been noted by electron microscopy beside an
eyelash at the lid margin (Fig. 48.6).61 The presence of
CHAPTER 48
suggestive of the presence of Demodex in the follicle. Such
cylinders were demonstrated in 26% of patients with blepharitis
without Demodex and in 44% of patients with Demodex. They
were found in almost 66% of patients with a heavy infestation
of Demodex. There was no correlation with the finding of
scales, hyperemia of the lid margin, clubbed hairs, or itching.57
The incidence of Demodex infestation increases with age. It is
rarely seen in children62 but involves virtually everyone older
than 70 years of age.57,58,62,63 Patients with rosacea and perioral
dermatitis frequently have significant mite infestation of the
face.64 Some authors attribute the lesions of rosacea to a cell-
mediated immune response to D. folliculorum because
inflammatory infiltrates, including helper–inducer T cells, can
be found around the mites.65
Treatment for Demodex of the eyelids is problematic and its
necessity is questionable in the first place.57,66 Substances
effective in killing the organisms are simply unusable in oph-
thalmic practice because they are highly toxic, irritating, and
malodorous. Absolute and ethyl alcohol, ether, xylol, acetone, 629
 CORNEA & CONJUNCTIVA

FIGURE 48.5. A Demodex mite is colorless and spindle-shaped. The
anterior section of the body has four pairs of very short legs, which
limits the mite’s mobility.
From Smolin GR, Tabbara K, Whitcher J: Lids. In: Infectious diseases of the eye.
Baltimore: The Williams & Wilkins Company; 1984.
FIGURE 48.7. Early rosacea may be easily overlooked as being a
ruddy complexion. The fine, blotchy inflammation of the skin over the
malar areas and the nose is typical of early rosacea. There may be
little lid inflammation with slightly increased prominence of
conjunctival veins.

and benzene kill Demodex within minutes.57 Danish ointment,

which contains 14% sulfur as potassium polysulfides, also kills
the organisms within minutes.57 In a recent study,67 treatment
with 2% mercury oxide ointment was reported to be successful
in reducing Demodex concentrations. The mites can survive in
concentrations of metronidazole that are unachievable in
serum.68

ROSACEA
Rosacea is a very common chronic inflammatory disorder of
the midline facial skin and blush area of the chest, with an
onset mainly between the ages of 30 and 50 years, although it
can also occur during childhood. In ocular rosacea, women are
affected slightly more often than men, but the disease is often
more severe in men. The early stages of rosacea consist of facial
erythema. This may be overlooked as ‘high coloring’ or a ‘ruddy’
complexion (Fig. 48.7). The next stage includes the develop-
ment of fine telangiectasias, especially around the nose, and FIGURE 48.8. More pronounced rosacea gives rise to inflammatory
recurrent episodes of inflammatory papules and pustules papules and pustules.
(Fig. 48.8). Severe involvement results in facial disfigurement Courtesy of Curatek Pharmaceuticals and Arthur Sober, MD.
from rhinophyma and markedly dilated superficial telang-
iectatic blood vessels on the nose, cheeks, and chin (Fig. 48.9).69 rosacea have a high incidence of chalazion formation. In a series
SECTION 6

Ocular involvement is common, affecting up to 58%.66 Ocular
signs and symptoms may precede significant skin changes in up
to 20% of cases.70 Keratoconjunctivitis sicca is much more
common in patients with rosacea26 (36.6%) compared to age-
matched and sex-matched controls (4.1%).71 Patients with
of patients older than 19 years of age who were scheduled for
chalazion excision, 57% had rosacea.26
The ocular signs of rosacea are similar to those of chronic
blepharitis with chronic low-grade conjunctivitis and tear-film
instability giving rise to ocular surface irritation and

FIGURE 48.6. (a) Scanning electron

micrograph of the lower eyelid of a 35-year-old
woman who underwent a full-thickness lid-
shortening procedure reveals the dome-shaped
tail of a Demodex folliculorum mite contiguous
with an eyelash. (b) Higher magnification
reveals the characteristic annular bands of the
abdomen.
(a and b) From English FP, Zhang GW, McManus DP,
Campbell P: Electron microscopic evidence of acarine
infestation of the eyelid margin. Am J Ophthalmol
a b 1990; 109:239–240.
630

FIGURE 48.9. Severe
rosacea results in
facial disfigurement
from rhinophyma and
markedly dilated
superficial
telangiectatic blood
vessels. Ocular
involvement is
common.
From Browning DJ, Proia
AD: Ocular rosacea. Surv
Ophthalmol 1986;
31:145–158.
irregularity. Patients with rosacea have a tendency toward
disproportionate conjunctival hyperemia. They frequently
complain of foreign body sensation, burning, tearing or redness
that may be worse toward the end of the day, and contact lens
intolerance. The most common ocular manifestations include
meibomian gland dysfunction, lid telangiectasis, conjunctival
hyperemia, and blepharoconjunctivitis. Corneal changes result
from involvement of the lid and conjunctiva. Initially there
is marginal vascular infiltration,72 followed by the formation of
superficial peripheral corneal neovascularization. As the disease
progresses, patients will develop subepithelial infiltrates that
appear near the limbus as round, oval or linear in shape. In
severe cases of rosacea, there may be peripheral corneal
vascularization (Figs 48.10a and b), thinning, ulceration, and
even perforation (see Fig. 48.10c and d) with serious visual and
ocular morbidity. Vitritis, which was not explained by other
causes, has been reported in two cases.66
The pathogenesis of rosacea remains unclear although it is
commonly thought of as inflammatory in nature. There is a
genetic predilection, and the disorder is common in people of
Celtic and Northern European ancestry.73 It has been reported
in blacks74,75 and Japanese.76 One hypothesis is that rosacea
a b
c d
Lid Inflammations
represents a dermal dystrophy in which there are degenerative
changes in perivascular collagen that lead to small vessel dila-
tation and eventually to incompetence of the vessels. Sub-
sequent leakage of potentially inflammatory substances into the
perivascular space leads to lymphedema and to the formation of
papules, pustules, and lupoid nodules.77,78 Recent studies have
documented increased levels of proinflammatory mediators
such as interleukin 1-alpha and matrix metalloproteinase-9
in the tears of rosacea patients compared with age-matched
controls, which seem to support the theory that rosacea is
inflammatory in nature.79–81
Patients with rosacea are also thought by some to have
lability of vascular regulatory mechanisms, accounting for
their tendency to flush. Patients with rosacea are twice as likely
to have a migraine compared with a control group.82,83
Histopathologic study of conjunctivae in subjects with rosacea
shows attenuation and infiltration by inflammatory cells,
mainly helper–inducer T (CD4) cells, phagocytic cells, and
antigen-presenting (CD14, Mac-1) cells. The substantia propria
corneae contains large subepithelial infiltrates of inflammatory
cells and sometimes granulomas. The mechanism involved
resembles a type IV hypersensitivity reaction.84 Other studies
linking rosacea to Helicobacter pylori are inconclusive.
TREATMENT OF ROSACEA AND CHRONIC
BLEPHARITIS
LID HYGIENE
Treatment of rosacea and chronic blepharitis is multifaceted.
Since there is no cure for rosacea, patient education is the key
to controlling symptoms. Lid hygiene is important in reducing
the oil and blepharitic scales around the cilia. This can be
accomplished in various ways. Simple bathing with warm to
hot water held against the lids with a washcloth will hydrate
and loosen fibrinous scales and mucus and heat the meibomian
gland contents to a more liquid state. The patient should be
instructed to brush the bases of the lashes with the cloth to
mechanically débride each lash. The patient may also be
instructed to gently press against the meibomian glands, rolling
a finger toward the lid margin trying to express the glandular
secretions. Using one or two cotton-tipped applicators against
the tarsal plates, the physician can gently but forcibly massage
FIGURE 48.10. Ocular rosacea may have
ocular involvement more severe than facial and
CHAPTER 48
lid involvement. Therapy tends to be chronic.
(a) A patient with rosacea after diagnosis and
treatment with oral tetracycline and low-dose
topical steroids shows quiet peripheral
neovascularization. The patient had systemic
hyperlipidemia with secondary deposition of
lipid in his peripheral cornea. (b) After cessation
of therapy, the patient had a flare-up of his
condition that responded to resumption of
therapy with retention of excellent vision.
(c) Peripheral corneal ulceration may occur,
leading to perforation (d). Corneal
transplantation may be necessary and has a
good prognosis if the underlying disease can
be controlled with topical and oral medication.
631
 CORNEA & CONJUNCTIVA
a b
TABLE 48.4. Comparison of Pharmacologic Aspects of Tetracyclines Used in the Treatment of Rosacea and Blepharitis
Tetracycline
Daily dosage 250 mg–2 g
Serum half-life 8h
Absorption from small bowel Fair; better on an empty stomach
Excretion Urine
Use in renal failure Avoid
Unusual side effects Pancreatitis; colitis
sebum and debris from the glands (Fig. 48.11). The patient may
use baby shampoo diluted with water to scrub the lid margins
and lashes with a washcloth, cotton-tipped applicators, or even
their fingertips. The patient should understand that the goal is
to clean the bases of the lashes and the lid margins, not just the
skin of the eyelids. In a study of eyelid-cleaning regimens in
contact lens wearers with chronic blepharitis, hypoallergenic
bar soap, baby shampoo, and commercial lid scrubs were all
shown to be effective in improving the slit-lamp findings.
Patients preferred the commercial lid scrub because of
convenience and ease of use.85 Antibiotic ointments with
efficacy against staphylococci, such as bacitracin and erythromycin,
are useful in controlling the more acute bacterial overgrowth
component of the disease but are not effective in eradicating
the severe forms of the disease.46,48 If one subscribes to the
Demodex gland-blocking theory of blepharitis, then one can
postulate that nightly application of ointment to the lid margins
mechanically impedes migration of nymphs from one follicle
to another, thus reducing the infestation. Patients who wear
mascara and eye make-up should replace their products,
SECTION 6
especially mascara, on a regular basis to reduce the likelihood of
reinoculating their lids with contaminated cosmetics.86
TREATMENT OF THE DRY EYE
Aggressive treatment of evaporative dry eye associated with
blepharitis is important to relieve the major symptoms of
stinging and burning. Artificial tears and ointments should be
used as needed. If they fail to control symptoms, a Schirmer
test with anesthetic should be performed and temporary
punctal occlusion tried if basal tear secretion is low. If there is
success with temporary punctal occlusion and no overflow
tearing, then punctal cautery or silicone plugs should be
seriously considered for the highly symptomatic patient.
Topical cyclosporine A 0.05%, Restasis, an immuno-
modulator that inhibits activation of T-lymphocytes, has been
studied for use in dry-eye patients and patients with meibomian
gland dysfunction. The suppression of T-cell activation reduces
632 cytokine production and release of inflammatory mediators.
FIGURE 48.11. (a and b) Expression of
inspissated meibomian glandular secretions is
helpful in controlling chronic blepharitis. A
bimanual technique, placing cotton-tipped
applicators on both sides of the lids, may also
be used.
Minocycline Doxycycline
50–200 mg 50–200 mg
16 h 18 h
Excellent Excellent
Urine; metabolized Feces
Avoid with caution None
Vertigo, tinnitus, skin, nail, and scleral
pigmentation
Although its exact mechanism of action in promoting tear-film
stability is not fully understood, it has been hypothesized that
Restasis decreases meibomian gland inflammation, thus
reducing the duct obstruction that predisposes to bacterial
colonization. In clinical studies, 15% of Restasis-treated
patients had an increase in Schirmer scores of 10 mm or more
compared with controls.87 Topical cyclosporine for the
treatment of meibomian gland dysfunction was evaluated in a
randomized prospective study.88 The investigators found a 50%
reduction in the number of meibomian gland inclusions at
3 months compared with placebo, suggesting that topical
cyclosporine A may be useful in the treatment of posterior
blepharitis.
Oral omega-3 fatty acid supplementation has been
investigated as an adjunctive treatment for dry eye based on
large epidemiologic studies showing a decrease in dry eye
symptoms among women with a higher intake of foods rich in
omega-3 fatty acids.89 Meibomian glands require essential fatty
acids to produce meibum. Increased dietary intake of omega-3s
as found in certain fish and flax seed oil, has recently been
shown to affect the polar lipid profiles of meibum as observed
by high performance liquid chromatography (HPLC).90
Eicosapentaenoic acid (EPA), a long chain omega-3 fatty acid,
blocks the gene expression of proinflammatory cytokines such
as tumor necrosis factor-alpha (TNF-a) and interleukin 1-alpha
that may play a role in meibomitis. Further investigation is
needed to clarify the relevance of dietary omega-3s on the
treatment of blepharitis and dry eye.
TETRACYCLINE
Tetracycline and its derivatives are very useful in treating
rosacea (Table 48.4). Tetracycline is an antibiotic that is
bacteriostatic in usual doses. It inhibits protein synthesis by
binding on the 30S ribosomes. This is similar to the action of
aminoglycosides. It has a broad spectrum of activity against
gram-positive, gram-negative, aerobic, and anaerobic bacteria;
spirochetes; mycoplasmas; rickettsiae; chlamydiae; and some
protozoa.91 Tetracycline, when administered orally, is absorbed

in the proximal small bowel and reaches peak levels in 1–3 h
after administration. There are three groups of tetracyclines,
differentiated by their pharmacology and duration of action.
Tetracycline hydrochloride is typical of the group of short-acting
compounds. It is inexpensive, most commonly used, and most
poorly absorbed. Milk and milk products as well as polyvalent
cations such as calcium, iron, aluminum, and magnesium
inhibit its absorption. The drug is concentrated in the liver
and excreted in the bile. Minocycline and doxycycline are long-
acting analogues with half-lives of 16 and 18 h, respectively,
versus 8 h for tetracycline. They are absorbed almost com-
pletely. Except for doxycycline, they are excreted mainly in the
urine.92
The side effects of tetracycline are chiefly gastrointestinal
and are dose-related. Diarrhea is related to changes in bowel
flora, which are least pronounced with doxycycline because it
is well absorbed. The diarrhea usually subsides when the
antibiotic is stopped; however, pseudomembranous colitis has
been reported.93 Tetracycline-induced pancreatitis has also been
reported.94 Tetracyclines should not be given to patients with
renal failure. These agents should be used cautiously in patients
with hepatic disease because they have been associated with
hepatic toxicity.92 Tetracycline crosses the placenta and
accumulates in fetal bones and teeth.93 It causes a permanent
gray-brown or yellowish discoloration of growing teeth, which
appears to be dose-related.94 Doxycycline does not bind with
calcium to the same degree as other tetracyclines and may cause
less dental discoloration.95 Tetracyclines should be avoided by
pregnant or lactating women and by children younger than 8
years of age.
Allergic reactions that occur with tetracyclines include
urticaria, fixed-drug eruptions, periorbital edema, and mor-
billiform rashes. An allergy to one analogue implies an allergy
to all. Photosensitivity is not an allergic reaction, rather a toxic
one. Superinfection occurs; the most common is oral or vaginal
moniliasis, which can be treated with specific topical
medication. Minocycline can cause vertigo and tinnitus.92
It may cause fatty infiltration of the liver, intrahepatic
cholestasis, and acute hepatitis.96 Prolonged administration of
minocycline can cause nail, skin, and scleral pigmentation
that is usually reversible.97 Minocycline has been associated
with a reaction of fever, arthritis/arthralgia, and livedo
reticularis.98 The syndrome was associated with a high titer of
serum perinuclear antineutrophil cytoplasmic antibodies
(p-ANCA) and antimyeloperoxidase (anti-MPO) antibody.
Symptoms resolved after stopping the drug but returned
when minocycline was restarted.92 Benign intracranial hyper-
tension has been reported.99 The use of outdated tetracycline
can cause damage to the renal tubules, resulting in Fanconi’s
syndrome.100 The formulations producing this syndrome have
been modified, so that it is unlikely to occur in the future.101
It has been reported that women on oral contraceptives have
become pregnant while taking tetracycline.102
Oral tetracycline has become the treatment of choice for
rosacea blepharitis or meibomian keratoconjunctivitis.76,92,103
Its mechanism of action cannot be fully explained by its
antibacterial effects because 75% of S. epidermidis strains are
resistant to tetracycline.19 Tetracycline was found to cause
significant decreases in the production of bacterial lipase in
sensitive and resistant strains of S. epidermidis without
decreases in bacterial growth. S. aureus showed parallel
decreases in lipase production and growth. McCulley showed
that low doses of tetracycline inhibited bacterial lipase
production by ~30%.23 Lipases act on wax and sterol esters to
release free fatty acids that can affect the solubility of other
lipids in the tear film or contribute to ocular inflammation.104
Tetracyclines can inhibit collagenase, decreasing the activity
Lid Inflammations
a b
FIGURE 48.12. A patient with rosacea before (a) and after 6 months
of metronidazole topical therapy (b).
(a and b) Courtesy of Curatek Pharmaceuticals and Arthur Sober, MD.
of matrix metalloproteinase-9, which has been implicated in
delayed corneal wound healing. Doxycycline is the most potent
inhibitor, followed by minocycline and tetracycline, which
corresponds to their ability to bind Zn2+. It is postulated that
the inhibitory mechanism is through tetracycline binding of
essential Zn2+ in corneal collagenase.105 This is compatible
with the findings that low oral dosage levels could cause
complete cessation of lipase production in sensitive strains of
S. epidermidis.103
Tetracycline may be administered in various dosage sched-
ules. Because rosacea is a chronic condition and because gastro-
intestinal side effects are dose-related, patients are typically
started at low doses such as doxycycline 100 mg twice a day or
tetracycline hydrochloride 250–500 mg once or twice a day for
several months. It takes ~6 weeks for symptomatic improve-
ment. In a study comparing doxycycline and tetracycline
therapy, greater symptomatic relief was seen in the tetracycline-
treated group after 6 weeks, but there was no difference between
the groups after 3 months.106 The drug should not be taken
at bedtime, because it may cause reflux esophagitis or stomach
irritation. The drugs are slowly tapered as clinical findings
warrant.
OTHER THERAPIES
Strongly fluorinated topical steroids should not be used on the
face, especially in rosacea, because the steroids themselves may
cause a confusing picture of steroid-induced rosacea-like
dermatitis.107,108
Topical metronidazole is highly effective in treating rosacea
dermatitis, showing significant improvements in over 70% of
CHAPTER 48
patients (Fig. 48.12).109–111 Metronidazole is a broad-spectrum
antibiotic and antiparasitic agent that has antiinflammatory
and perhaps immunosuppressive effects.112 It probably has little
effect against Demodex.68 It is not currently available in an
ophthalmic preparation, but careful application of the gel to the
lid margins has been shown to significantly improve the
adnexal changes in ocular rosacea. It did not significantly alter
the surface disease.113 Metronidazole reduces potent inflam-
matory mediators in skin in which palmitoleic acid is
present.114 Studies need to be done to show whether controlling
the facial dermal aspects of the condition has any effect on the
ocular manifestations.
CONCLUSION
Lid inflammation is a problem that is commonly encountered
in a general ophthalmic practice. The severity of symptoms falls
into a range of mild ocular irritation to severe discomfort and
reduced vision. It is therefore important to understand the 633
 CORNEA & CONJUNCTIVA
alterations in the normal physiology of the meibomian glands
that result in lid inflammation in order to target therapies
effectively. Along with patient education, encouragement and
proper treatment, blepharitis can be successfully controlled in
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CHAPTER 48
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635
 CHAPTER
49 Viral Disease of the Cornea and External Eye
Deborah Pavan-Langston
Viral infections of the external eye range from the benign to
malignant, from a transient keratitis of mononucleosis to the
progressive sarcoma associated with AIDS. Transient acute
follicular conjunctivitis, with or without keratitis, may be seen
with any of the DNA or RNA agents. The former include the
herpes viruses, adenoviruses, poxviruses, and papillomaviruses.
The latter include the paramyxoviruses (measles, mumps, and
Newcastle disease), the retrovirus (HIV), the picornavirus of
acute hemorrhagic conjunctivitis, the togaviruses (rubella and
arbovirus), and the orthomyxovirus (influenza). With the excep-
tion of congenital rubella, the RNA viruses tend to be the more
benign, and the DNA viruses more associated with a notable
ocular morbidity and loss of vision. Many, but not all, of the
organisms that most seriously affect the anterior segment are
amenable to therapy; however, some progress despite our best
efforts, and others need only palliative treatment. Diagnostic
tests and currently available antiviral and antiinflammatory
agents are briefly reviewed, followed by a discussion of the
clinical disease and management of the major anterior ocular
viral infections. Antiviral drugs are discussed in detail in
Chapter 20.
DIAGNOSTIC TESTS
Diagnosis of ocular viral disease is usually made based on
clinical impression only. When objective data are needed,
however, the four most commonly used approaches are: (1)
examination of skin, conjunctival, or corneal scrapings
(herpetic, adenoviral, and pox infections); (2) molecular and
immunologic assays; (3) viral culture; and (4) measurement of
circulating antibodies. A fifth approach is histopathologic study
of tissue obtained at keratoplasty. This is done almost
exclusively in herpetic disease as other forms of viral disease are
not sufficiently severe to warrant biopsy or are obtained only as
postmortem specimens.1–5
External ocular scrapings may be taken with a sterile platinum
spatula or the edge of a curved surgical blade, then these are
smeared on a slide and stained for light microscopic examina-
tion. The cellular inflammatory reaction indicative of viral
infection is predominantly a monocytic white cell infiltrate.
The simplest cytopathologic examination uses heat fixation of
the slide followed by Giemsa’s staining. Herpetic infections
caused by herpes simplex virus (HSV) and herpes varicella-
zoster virus (VZV) are characterized by some multinucleated
epithelial cells with ballooning degeneration and a mixed mono-
nuclear and polymorphonuclear leukocyte (PMN) reaction. As
Giemsa’s stain obscures nuclear detail, the eosinophilic viral
inclusion bodies of Lipschutz, also called Cowdry-A inclusions,
are best seen in the epithelial cells after the slide is fixed in
Bouin’s solution or 95% ethanol and stained with the
Papanicolaou method. The inclusion bodies appear as an
eosinophilic intranuclear mass within a clear halo and are
associated with clumping or margination of the basophilic
chromatin on the nuclear membrane. Fluorescent antibody
staining may also reveal herpetic antigen in the nucleus or
cytoplasm. This technique is rapid and as reliable as tissue
culture recovery but is not generally available outside of major
medical centers because an ultraviolet microscope is required.
The stained preparations are also unstable, which prevents
storage for later evaluation.1–4,6
Polymerase chain reaction (PCR) has also become a useful
and sophisticated tool for diagnosis of numerous herpetic and
other diseases. PCR has been used to detect both HSV and VZV
in the tear film and corneas of patients.2,7–9 PCR has further
been used to identify HSV DNA in irido-corneal-endothelial
and in Posner–Schlossman syndromes.10,11 While Kaye has
reported finding HSV DNA by PCR in corneal lesions unrelated
to HSV, the first two reports raise the specter of HSV as an
etiologic agent in syndromes not previously associated with this
infectious agent.12,13
Multiplex PCRs (mPCRs) have been successfully developed
for detection of DNA and RNA agents in the investigation of
congenital infection and an mPCR for the viruses most
commonly requested in a diagnostic virology laboratory (CMV,
Epstein–Barr virus (EBV), enterovirus, HSV-1, HSV-2, and
varicella-zoster virus).14 Nested PCR was performed as the most
sensitive assay currently available, and detection of the
amplicons using hybridization to labeled probes and enzyme-
linked immunosorbent assay detection was incorporated into
three of the four assays. In a number of cases this technique
reveals an agent not diagnosed clinically which affected sub-
sequent treatment and course.
A new, easy to use, multipotential derivative of PCR is the
Smartcycler II (Cepheid, Sunnyvale, CA) real-time PCR system
for detecting HSV-1, VZV, adenovirus, and Chlamydia
trachomatis in ocular infections.15 This test may be performed
quickly in a small conventional laboratory or office with results
comparable to those of a central molecular laboratory.
Sensitivity for adenovirus, HSV-1, VZV, and Chlamydia
trachomatis were 85%, 91%, 100%, and 95% respectively while
specificity was 98%, 100%, 100%, and 100%.15
Enzyme-linked immunosorbent assay (ELISA) tests are used
not only for viral antigen detection but are particularly useful
for detection of IgM in the presence of IgG. Only IgM anti-
bodies, if present in the serum, are bound to the solid phase and
are therefore easily detectable. This is important diagnostically
as IgM antibodies appear early during infection and last only a
few weeks whereas IgG comes after 1 or 2 weeks but lasts for
years. Quantitative documentation of a fourfold rise in either
IgM or IgG strongly supports a diagnosis. Serum should be 637
 CORNEA AND CONJUNCTIVA
drawn as soon as possible in the acute illness and again 2–3
weeks later for comparative titers. Finding a positive IgM in a
single specimen may also be diagnostic in a very ill patient, e.g.,
disseminated varicella, or a patient with ongoing infection, e.g.,
HIV. Elevation of IgM may also indicate re-activation of a latent
infection. IgM detection is most useful in diagnosis of VZV,
EBV, CMV, measles, rubella, coxsackie viruses, and hepatitis. As
IgM does not cross the placental barrier, finding IgM in a
newborn is diagnostic of intrauterine infection.1,16–19
Immunologic ELISA diagnostic kits are available for specific
diagnosis of HSV infections in ocular scrapings: the Herpchek
kit (Dupont) and the Virogen kit may be purchased
commercially.20 The Virogen test relies on the cross-linking
of latex particles to produce an agglutination reaction visible
to the human eye without magnification; Herpchek uses an
immunostaining system that relies on a color change for
antigen detection. The Herpchek is 100% specific and has a
sensitivity of 99% compared with the shell vial tissue culture
method but because of the equipment needed require the
facilities of medical centers. The Virogen is easy to set up in an
office but has about 26% sensitivity.20,21
Serologic testing is also the principal method of diagnosis in
EBV infection. Rapid diagnosis of acute EBV infectious mono-
nucleosis can usually be made on clinical grounds, atypical
lymphocytosis, and a positive rapid heterophile.22–24 The
Monospot test has numerous pitfalls but tests for viral capsid
antigen (VCA), EB nuclear antigen (EBNA) (see ‘EBV’ ahead)
also of use particularly in late diagnostic testing where only IgG
VCA and EBNA are high for several years but early antigen (EA)
and IgM VCA are negative. In HIV testing, the conventional
ELISA is used to screen for anti-HIV antibodies and the Western
blot test, a more sophisticated and complex antibody assay,
used for confirmation of diagnosis in those screening positive by
ELISA.25 The HIV Oraquick rapid HIV-1 testing (OraSure
Technologies, Inc., Bethlehem, PA) (blood) has been found to be
a highly reliable rapid test preferred by patients in screening
programs and enhancing the effectiveness of screening
programs.26
In adenoviral infections, Giemsa-stained smears of con-
junctival exudates reveal lymphocytes and degenerated epithelial
cells with a few polymorphonuclear leukocytes (PMNs). If the
reaction is so acute as to induce pseudomembrane formation, a
PMN response will predominate over the mononuclear. No
light-microscope-visible inclusion bodies are formed. As with
herpetic infections, fluorescent antibody testing or ELISA may
be used for rapid definitive diagnostic testing on scrapings taken
during the first week of infection, but the drawbacks noted with
the use of these techniques in herpetic disease also apply here
SECTION 6
thus favoring the Smartcycler II.15,1,27
Smallpox and vaccinia have only recently become infectious
agents of concern again, the former having been declared extinct
by 1980. Now, however, it is a potential bioterrorism agent and
its preventative, vaccinia vaccination, make both these agents
of importance in ocular disease such as cellulitis, conjuctivitis,
and acute or chronic keratitis or iritis. In pox infections, acute
disease is characterized by an outpouring of PMNs followed
by a mononuclear reaction days later. Giemsa-stained smears
may reveal diagnostic eosinophilic bodies of Guarnieri in the
cytoplasm of epithelial cells. Diagnostic tests of use are not
routine serologic testing but ELISA, radioimmunoassay, or
monoclonal antibody assays.1,28–31
Viral recovery on tissue culture is the most definitive method
of diagnostic testing but, unfortunately, may take several days
to become positive and is not widely available outside of major
medical centers.1 Ocular or periocular lesions are swabbed with
calcium-alginate-tipped applicators and eluted either into viral
638 carrier medium or into viral monolayer tubes and sent directly
to the laboratory, where the carrier medium is inoculated into
cultures and the inoculated cell monolayers incubated at 37ºC.
Once a virus is recovered in culture, its precise identity is
confirmed by serologic testing using antibody specifically
directed against the suspected agent.
HSV and the poxviruses grow on almost any cell monolayer
such as human embryonic or rabbit kidney or chick embryo.
The recovery rate from acutely infected ulcers is about 70% if
the specimen is taken within 2–3 days of the appearance of the
lesion, as the viral titer is highest just before, and as the corneal
or skin lesions appear and then decreases as the clinical findings
become more prominent.30 The use of antiviral agents prior to
culture will drop recovery rate to 4% even in early disease.32
VZV and adenovirus are more fastidious; therefore, cultures
must be done early in disease, and these require cells of human
origin. They may be isolated directly from ocular cultures, but
because of their fastidious nature diagnosis is usually by
immunofluorescence, viral neutralization or PCR.1–3,15,37
CMV has been isolated from virtually all forms of body fluid
from tears to blood, or breast milk. Transmission is via the
congenital, oral, and sexual routes, blood transfusion and tissue
transplantation. Diagnosis, however, is usually made by tests
such as immunoassays, and PCR testing as discussed above.
EBV and human immunodeficiency virus (HIV) are generally
not grown in culture for diagnostic purposes.1
ANTIVIRAL DRUGS
Twenty antiviral drugs are currently FDA-approved for clinical
use. Half of those are for the treatment of HIV infections
(acquired immune deficiency syndrome). The others are used
for herpes virus (e.g., herpes simplex virus, varicella zoster
virus, and cytomegalovirus), hepatitis B virus, hepatitis C
virus, or influenza virus infections. Recent studies have focused
on antiviral therapies for virus infections that appear amenable
to antiviral drug treatment, as well as for virus infections
for which, to date, no antiviral drugs have been approved, e.g.,
adenoviruses, human herpes virus type 6, poxviruses, corona
virus, severe acute respiratory syndrome, and hemorrhagic fever
viruses.38 A vaccine has been approved for human papilloma
viruses related to cervical carcinoma.
There are nine antiviral drugs with proven efficacy in ocular
viral disease: idoxuridine (IDU, Herplex), vidarabine (ara-A, Vira
A), trifluridine (TFT, F3T, Viroptic), acyclovir (ACV, Zovirax),
famciclovir (FCV, Famvir), and valacyclovir (VCV, Valtrex) and
bromovinyldeoxyuridine (BVDU, Brivudine). Ganciclovir (DHPG,
Cytovene), foscarnet (PFA, Foscavir), and HPMPC (Cidovir)
have specialized roles. All but BVDU are approved by United
States Food and Drug Administration (FDA) in one or more
forms: drops, ointments, pills, or for injection.39–42 Because
of greater convenience and overlapping efficacies two antiherpes
drugs are no longer commercially available: IDU and
vidarabine. BVDU is licensed throughout Europe. This chapter
discusses these various antiviral agents where pertinent in
various clinical therapy sections5,43 (see Chapter 20).
CORTICOSTEROIDS: PROS, CONS,
INITIATION, AND WITHDRAWAL
TECHNIQUE
Corticosteroids (steroids), specifically the glucocorticoids, are of
use in those viral diseases characterized by vision-threatening
immunologic keratitis or keratouveitis seen in certain cases of
stromal HSV, VZV, or adenoviral disease. These drugs interfere
with the distribution and function of immunologically
competent lymphocytes, amoeboid white cell migration, and
release of white cell digestive enzymes. Topical steroids inhibit

antibody-forming cells and cell-mediated immunity (CMI) in
the cornea and iris. As there is little effect on the number of
immunocompetent cells in the draining lymph nodes, the host
is still capable of immune reaction upon reduction or cessation
of steroid therapy.44–46
Steroids interfere not only with corneal neovascularization but
also with mucopolysaccharide and collagen formation, substances
critical to the integrity of the corneal structure. Medroxyprog-
esterone acetate (Provera) is a mild steroid that suppresses both
latent and active collagenases, thus differing from other steroids
by not interfering with collagen synthesis yet still suppressing
CMI and neovascularization.47 At 1.0% concentration, medroxy-
progesterone has an antiinflammatory efficacy roughly
equivalent to 0.12% prednisolone. It appears to be a safer topical
steroid for use if these drugs must be used to suppress immune
reaction in the presence of corneal thinning or melting.
The factors in support of steroid use in the eye are inhibition
of (1) white cell infiltration, (2) release of toxic hydrolytic
enzymes, (3) scar-tissue formation, and (4) neovascularization.
Because these drugs induce vascular constriction, they keep the
eye ‘white and quiet’, thus enhancing patient comfort. On the
negative side of steroid administration are (1) suppression of the
normal immune-inflammatory response, allowing spread of
potentially superficial viral infection, inhibition of collagen
synthesis in corneal ulceration, (2) opening the eye to oppor-
tunistic bacterial or fungal infection through suppression of the
immune defense system, and (3) steroid-induced glaucoma and
cataract.
It may be difficult to withdraw treatment once a patient is
committed to topical steroid therapy. Too abrupt cessation often
results in rebound inflammation. A useful rule of thumb in
tapering a patient from topical steroid therapy is ‘never reduce
dosage more than 50 percent of the current level of therapy.’
The higher the dosage the more rapid the taper and, conversely,
the lower the dosage the more prolonged the withdrawal period.
If a patient is at risk of scarring involving the visual axis, it is
preferable to initiate steroid therapy to inhibit structural
damage and then slowing wean the dose down to whatever level
is necessary to keep the disease quiescent. Periodic attempts at
further tapering should be made over time. As an example, a
patient may require 1% prednisolone qid to bring an immune
keratitis under control over a 1–2-week period. Dosage may
then drop to tid or bid (but no lower) over the next 3–4 weeks,
then down to everyday over several weeks before switching to
0.12% prednisolone qid so that the decrease is not more than
50% of the total dose. The physician is then positioned to take
the patient down through a more prolonged withdrawal using a
much weaker drug concentration. Excellent alternative medium
to weak strength steroids with less tendency to cause glaucoma
and cataract are rimexolone 1% (Vexol), lotoprednol 0.5%
(Lotemax), and the weaker lotoprednol 0.2% (Alrex) and
fluoromethalone 0.1% or 0.25% or 0.1% ointment. Not
infrequently, a rebound may begin during withdrawal, signaling
a need to go back up to the next higher dosage for a longer
period of time. It is not uncommon, nor necessarily worrisome,
if a patient is unable to discontinue topical steroids altogether.
Many patients do very well on one drop a day, every other day,
or even once-weekly using 0.12% prednisolone to hold an
otherwise scarring immune reaction in check.
It should be noted that patients with a history of previous
HSV epithelial keratitis and nonwhite patients are more likely
to develop HSV epithelial keratitis during treatment of stromal
keratouveitis with steroids.48 Such patients should be put on
prophylactic oral antiviral therapy during the period of steroid
use or at least until the dose is down to once daily or less.
The issue of band keratopathy being so frequent in HSV
patients chronically on topical steroids has raised the question
Viral Disease of the Cornea and External Eye
as to the etiology: chronic HSV or the steroid. Abel et al have
reported that the normal tear concentration of calcium and
phosphate are near spontaneous precipitation levels.49,50 While
the added burden of phosphate when delivered as a steroid salt
has never been proved to cause band keratopathy, the index of
suspicion is raised such that acetate forms of topical steroids
might be advisable in patients showing tendencies toward
calcium precipitation.
Oral steroids given short-term may be necessary in situations
such as iritis in the presence of a melting ulcer with topical
steroids not being initiated until some control over the ulcer
has been achieved. Common dosing schedule is 20 mg of
prednisone po with meals tid for 5 days, then bid for 5 days, and
finally q AM (to minimize adrenal suppression) for 4 days.
Medical history to ascertain any contraindications to oral
steroid should be taken before starting any such regimen.
Cyclosporin A (Cy A), FDA-approved for dry inflammatory
disease, may be useful in controlling herpetic inflammation
without the risk of elevating intraocular pressure.51 It has been
reported, however, that herpetic epithelial keratitis persisted in
a corneal graft until Cy A was discontinued suggesting that the
drug has the same potential to enhance the infectious
component of herpetic disease as steroids.52 Cy A should,
therefore, be covered with antiviral agent prophylactically. In
another study, 10 patients with HSK using 2% Cy A drops qid
and ACV 3% ointment five times per day for 2 months had
complete resolution of the stromal disease, vision increased by
at least two Snellen lines in eight of the 10 patients and there
were no episodes of epithelial infection.51 Heilingenhaus’ study
of 18 patients with stromal HSK treated with Cy A drops and
acyclovir ointment reported that the condition can be treated
successfully with CsA eyedrops, especially in nonnecrotizing
disease. Some cases of necrotizing keratitis required a
combination of CyA and steroids, but the latter at lower dose
than without Cy A. Cy A, then, may be particularly helpful in
the presence of steroid glaucoma, herpetic corneal ulcers, and to
taper off topical corticosteroids. Additional use of oral acyclovir
may be judicious to prevent recurrence of epithelial HSV
keratitis.53
HERPETIC DISEASE OF THE ANTERIOR
SEGMENT
The herpes viruses of interest in the context of anterior segment
disease are HSV, VZV, EBV, and CMV.
HERPES SIMPLEX VIRUS (HSV)
CHAPTER 49
Ocular HSV is a multifaceted disease capable of inducing the
most difficult complications through both infectious and
immune pathogenetic mechanisms.
Epidemiology
HSV is the most common infectious cause of corneal blindness
in the developed world, with up to 500 000 cases diagnosed
annually in the United States alone.5 There are a number of
excellent ocular epidemiologic studies which emphasize the
importance and morbidity of this disease.30,54,55 A study out
of Moorfields Eye Hospital in London sampled their clinic
population to ascertain the incidence with herpetic eye disease
of the anterior segment. Age, gender predominance, and inci-
dence of bilateral disease among these patients had not changed
in 20 years. The most common disease form was stromal
keratitis, with significant morbidity and visual loss. They also
noted that HSV disease of the anterior segment utilizes only 1%
of out-patient clinic resources but 17% of external disease
specialists’ clinic time. There was a disturbing and statistically 639
 CORNEA AND CONJUNCTIVA
significant correlation between total length of follow-up and
reduced visual acuity. While the prevalence of anterior segment
herpetic eye disease appears not to have increased in incidence
the visual prognosis had worsened.29
In a more recent study in France, the overall incidence of
HSV keratitis was 31.5/100 000 person-years (p = 0.05). The
incidence was 13.2/100 000 person-years for new cases (p = 0.05)
and 18.3/100 000 person-years for recurrences (p = 0.05).56 The
most frequent types were dendritic keratitis (56.3%, n = 153),
stromal keratitis (29.5%, n = 81), and geographic keratitis
(9.8%, n = 27). Associated with 35% of the keratitis cases were
conjunctivitis (18.8%, n = 67), uveitis (11.8%, n = 42), and/or
lid involvement (8.6%, n = 31).56
Humans are the only natural reservoir of herpes. Sources of
infection are by direct contact with infected lesions, by salivary
droplets from children and adults with active disease (cold
sores), and via the saliva or fomites of asymptomatic, virus-
shedding carriers.57,58 The marked frequency of trigeminal HSV
and VZV was demonstrated in ganglia removed at autopsy from
immunocompetent individuals with no history of HSV
infections who were screened by PCR for latent HSV-1 and
(VZV) DNA. HSV-1 DNA was found in the vast majority of
samples (> 90%) and VZV DNA in ~50% of samples. Both
DNA types were distributed throughout each latently infected
ganglion.55 Pepose’s recent report on the changing epidemiology
and new emerging disease patterns reveals an epidemic increase
in genital HSV-2 (30% increase in type 2 antibodies in the
United States since 1976).59 Approximately, one in four people
in the United States over age 30 is infected with HSV-2. In
contrast to developing nations where HSV is acquired early in
life and is ubiquitous, primary acquisition of herpes simplex
type 1 is becoming progressively delayed in many industrialized
countries. Changes in sexual behavior among young adults have
been associated with a recent increase in genital HSV-1
infection, resulting from oral–genital rather than genital–genital
contact which strongly suggests that we will or may already be
seeing an increase in the incidence of type 2 HSV keratitis.
Multiple recurrences are far more common with genital or
oral HSV in comparison with the recurrence of ocular disease.
The reported rates of HSV-2 genital recurrence are 0.33/month
(89% in 24 of 27 patients), for orolabial HSV 0.12/month (42%
in five of 12 patients) in one study, and only 40% over a 5 year
period for another study on ocular HSV epithelial recurrence
rates.59–61 The elderly (> 60 years) appear particularly
susceptible to microbial keratitis with HSV being the most
common cause (8% out of 62% positive cultures or PCR in 190
patients) and causing greater damage than that seen in younger
populations.62 Further, it has been found that recurrent
SECTION 6
epithelial herpes is frequently associated with corneal re-
infection with a different HSV-1 strain with PCR typing of
strains from successive recurrences revealing 37% were of a
strain different from that causing the previous recurrence.63
Iatrogenic sources of patient infection are the physician’s
unwashed hands and the contaminated Schiotz or applanation
tonometer head. HSV is viable for up to 2 h on a dry tonometer
head and up to 8 h on the one kept moist. Dry wiping and a
variety of ophthalmic solutions such as anesthetics and dilating
agents have minimal antiviral effect. Swabbing the tonometer
with 70% isopropyl alcohol is 100% effective in killing virus,
and this should be done between patients along with hand
washing with a soapy solution. Dipping the tip into Dakins
solution followed by careful rinsing and wiping dry with a sterile
pad is also 100% effective. By the age of 5 years, at least 60% of
all children have been infected with type 1 (oral) HSV, usually
through the mouth or nares, with only about 6% developing
clinically apparent primary disease. Less than 4% of primary
640 HSV presents as overt ocular disease.58 The oral and nasal portals
of entry allow the virus access to the trigeminal ganglion, which
also innervates the eye. The vast majority of first ocular (not
primary) or recurrent ocular herpes infections are due to re-
activation of latent trigeminal ganglion virus with subsequent
appearance of the virus in the eye alone, or associated with an
eruption of cold sores around the mouth or nose.
The spectrum and recurrence of (HSV) keratitis in children
and adolescents has been reported in a retrospective cohort
study of 23 patients under age 16 years and diagnosed with
HSV keratitis. Dendritic or punctate epithelial keratitis and
stromal keratitis occurring concurrently with epithelial keratitis
was seen in 14 patients (61%). Six patients (26%) had HSV
keratitis OU. Eleven of these 23 children (48%) developed
recurrent HSV keratitis at a median of 15 months after the first
documented episode and in three patients, amblyopia occurred.
The study concluded that children with herpetic keratitis may
frequently have bilateral ocular involvement, like adults are at
risk for recurrent keratitis, and in addition have the added risk
of amblyopia.64
HSV tear shedding studies have revealed that people with no
history of ocular herpes may have HSV in the tear film and
patients with known history of HSV keratitis show no greater
risk than the rest of the population of shedding asympto-
matically.8,65 Tear antibodies to HSV-1 may be detected in
the absence of detectable serum or parotid saliva antibodies.
The former finding suggests that the ocular surface may be the
initial infection site for this virus in some healthy, clinically
normal subjects, and the latter suggests that there is a
preferential homing of committed B lymphocytes to different
mucosal surfaces.66 The significance of these findings in terms
of latency and recurrent disease is yet to be determined.
Other recent studies on the tears and saliva in 50
asymptomatic patients determined DNA copy number and
frequency of shedding by real-time PCR sampling bid over 30
days.67 While only 74% of the patients were sero-positive for
HSV-1 (by IgG ELISA and neutralizing antibodies), 98% shed
virus at least once from either or both saliva and tears during
the test period. This indicated that the percentage of
asymptomatic subjects who intermittently shed HSV-1 DNA in
tears or saliva was higher than the percentage of subjects with
positive ELISA or neutralization antibodies to HSV. As most
HSV transmission occurs during asymptomatic shedding, it is
easy to see why the frequency of this infection is so widespread.
Other epidemiologic characteristics of ocular herpes have
been noted. Liesegang and co-workers reported an incidence of
8.4 new cases of first ocular herpes per 100 000 person-years
during the period from 1950 to 1982.68 In 1980, the overall
prevalence of a history of ocular herpes was 149 cases per
100 000 population. Initial ocular HSV episodes included
incidences of 54% blepharitis or conjunctivitis, 63% epithelial
keratitis, 6% stromal keratitis, and 4% uveitis. Age-adjusted
rates by sex were comparable and there were no seasonal trends
in incidence, although rates increased with time. In some
contrast, a study by Bell et al of 141 patients with documented
infectious dendritic/geographic keratitis revealed that there was
a significant predominance of men in the population of patients
over 40 years of age.69 Of the 65 patients who suffered more
than one episode, 34% had a mean recurrence rate of one or
more episodes per year and 68% had more than one episode
within 2 years of each other. The cold weather months of fall
and winter correlated with increased herpetic recurrences and
flu-like viral respiratory infections. The epidemiologic report by
Shuster et al on ocular HSV in 119 patients revealed that 24%
had a recurrence within 1 year of the first ocular occurrence
and 33% had them within 2 years.70 There was a positive
correlation between short intervals between past attacks and
short intervals between future recurrences.

In contrast to the foregoing studies, the HEDS report on
recurrences as a risk factor noted that a history of epithelial
keratitis within the past year was not a risk factor for recurrent
epithelial keratitis, but that previous, especially multiple,
episodes of stromal keratitis markedly increased the probability
of subsequent stromal keratitis.71 Similarly, another HEDS study
differed in its interpretation of the role of factors stimulating
re-activation.72 Psychological stress, systemic infection, sun-
light exposure, menstrual period, contact lens wear, and eye
injury were recorded on a weekly log. The exposure period was
considered to be the week before symptomatic onset of a
recurrence. No association was found between any of the other
exposure variables and recurrence. Psychological stress did not
appear to be a trigger of recurrences of ocular HSV disease. It
was concluded that recall bias substantially overestimated the
importance of factors such as systemic illness or stress that did
not have a causal association with ocular HSV.
HSV Vaccine
Although much interest and work is now being conducted with
an aim to developing either a vaccine to prevent a primary
infection or recurrent disease, at present, neither passive immu-
nization nor existing circulating antibodies have significant
influence on the development of disease.73–75 Neutralizing anti-
body titers in the serum remain constant during and between
recurrences or may fluctuate to high levels in the absence of an
episode of infectious disease.76–79 Approaches to the induction
of protective responses by altering innate and adaptive immu-
nity using novel vaccines such as recombinant viral vaccine
vectors and DNA vaccines specifically tested in models of HSV
infections of the eye may offer some true efficacy.73 As Nesburn
et al have noted, however, most vaccines fail to induce local
ocular immune responses and, without adjuvant, may induce a
state of immunological tolerance. Using epitope-based vaccines
delivered via the ocular mucosal (OM) route.74 The generation
of local and systemic peptide- and virus-specific T cells
confirmed the potent immunogenicity of peptides-CpG2007
formulation when applied via this route and suggest the clinical
feasibility of developing an OM delivery system using epitope-
based vaccine.
Clinical Disease
Ocular herpetic disease may be primary neonatal, primary, or
recurrent. Primary disease is infectious disease of the non-
immune host, and recurrent disease occurs in the immune (or
previously immune) host and may be either infectious or
immune, or both (Table 49.1).
TABLE 49.1. Classification of Anterior Herpes Simplex Disease
I. Primary Infection
A. Neonatal
B. Primary (children, adults)
II. Recurrent Infection
A. Blepharitis
B. Conjunctivitis
C. Infectious dendritic or geographic epithelial keratitis
D. Sterile corneal neurotrophic ulceration
E. Stromal keratitis – primarily immune
1. Interstitial keratitis
2. Immune rings
3. Limbal vasculitis
4. Disciform keratitis
F. Endotheliitis/trabeculitis – primarily immune
G. Iridocyclitis – primarily immune
Viral Disease of the Cornea and External Eye
Neonatal HSV infection
Approximately 1 in 10 000 infants is born with neonatal HSV,
20% type 1 and 80% type 2. The clinical manifestations of this
rare but usually devastating disease include local skin, eye, or
oral infection, central nervous system (CNS) disease, and dis-
seminated HSV in which the visceral organs are also affected.
The local forms may accompany either of the other two forms.
Despite antiviral therapy earlier studies have reported a mor-
tality rate of ~57% in disseminated disease and 10% in CNS
disease, with a very high rate of CNS damage in survivors.80,81
More recent studies by Freij et al and by Kimberlin et al
reported that while most exposure to the virus occurs in an
infected birth canal, 5% of infants acquire the infection in
utero.82,83 After an incubation period which can last as long as
2–4 weeks, neonatal HSV disease then manifests in one of three
ways: (1) disseminated disease, with visceral organ involvement
(including infection of the brain in two-thirds to three-quarters
of patients), (2) central nervous system disease (with no other
visceral organ involvement, but with skin lesions in two-thirds
of patients), or (3) disease limited to the skin, eyes, and/or mouth
(i.e., SEM disease).The mortality rate is 31% for disseminated
infection and 6% for localized central nervous system disease
with long-term neurologic sequelae seen in 17% and 70% of
survivors, respectively. Diagnosis is made by isolating the virus
from skin lesions or other involved sites. PCR detection
of viral DNA in cerebrospinal fluid (CSF) or serum is now the
diagnostic test of choice for central nervous system or
disseminated neonatal herpes. Supportive measures and
neuroimaging studies are often required.
Treatment is with high-dose intravenous acyclovir (60 mg–1
kg day–1 in three divided doses) for 3 weeks, with adjustments
–1
made for infants with renal or hepatic insufficiency. Infants
with disease localized to the skin, eyes, and mucous mem-
branes can be treated for 2 weeks if the CSF PCR reaction assay
is negative for HSV DNA. Cesarean delivery will prevent
infection in infants when women have active lesions at the
onset of labor. Suppressive acyclovir therapy beginning at
36 weeks gestation is often prescribed for women with frequent
recurrences of genital herpes. Neonates delivered through an
infected birth canal should be screened between 24 and 48 h
of age with viral cultures of eyes, nasopharynx, mouth, and
rectum. If positive, they should be treated with acyclovir even
if asymptomatic.
Kimberlin et al’s study on the natural history of neonatal
HSV in the era of acyclovir was somewhat discouraging. The
investigators found that comparisons between patients treated
between 1981–1988 and 1989–1997 revealed that the mean
time between the onset of disease symptoms and initiation of
CHAPTER 49
therapy has not changed significantly from the early 1980s to
the late 1990s. It is this delay in thinking of HSV in any acutely
ill infant, not so much the lack of excellent treatment, that
causes the unchanged incidence of morbidity and mortality in
neonatal HSV.
Acute neonatal ocular HSV is most frequently a con-
junctivitis often associated with ulcerative keratitis, which may
be diffuse microdendrites or serpiginous epithelial defects or
simply a punctate keratitis. Stromal involvement at or within
days of birth may occur but is extremely rare and usually
indicates intrauterine infection rather than infection during
passage through an infected birth canal.84,85 Other ocular com-
plications that may occur acutely but more commonly after the
acute phase has passed include necrotizing chorioretinitis,
cataracts, optic neuritis, variable forms of comitant and non-
comitant strabismus due to CNS damage and phthisis. The
appearance of opsoclonus may also be an early sign of HSV CNS
infection and warrants full radiologic and infectious disease
workup.86 641
 CORNEA AND CONJUNCTIVA
The diagnosis of ocular HSV must be considered in any
infant with nonpurulent conjunctivitis or keratitis, particularly
those in whom there has been fetal monitoring with a scalp
electrode, as this group appears to be at particular risk for all
forms of neonatal HSV infections.81,87 Evidence of focal ulcer-
ative dermatitis should be sought by thorough examination of
the entire infant, as this may assist in the ocular diagnosis and
save the child’s life through institution of earlier therapy.
Diagnostic scrapings and cultures and radiologic imaging as
described earlier are useful; serial serologic tests will confirm
diagnosis but only belatedly.
Therapy of neonatal ocular herpes
An emergency pediatric or infectious disease consultation
should be obtained. Although there is only a 6% incidence
of corneal scarring, about 37% of these children will have
visual acuity of less than 20/200 due to the long-term sequelae
of other ocular forms of HSV, which are best treated
systemically.80,88 This is quite independent of the life-saving
aspects of such therapy.
Therapy of focal ocular disease is with oral antivirals such as
acyclovir (ACV) and topical antivirals regardless of whether the
etiologic agent is type 1 or type 2 HSV. Both types are equally
sensitive to the commercially available agents. In a study to
evaluate the use of oral acyclovir in pediatric patients with HSV
keratitis, Schwartz and Holland reported seven pediatric
patients with ages ranging from 6 weeks to 5 years at the time
of presentation (mean 1.9 years).89 All patients received oral
ACV; six of seven patients also received topical antiviral medi-
cations. Three of seven patients had topical antiviral therapy
fail before being placed on oral acyclovir and the remaining four
patients were placed on oral acyclovir primarily. All patients
resolved their keratitis. Three patients were maintained on
prophylactic dosage of oral ACV because of recurrent disease or
because they had been chronically treated with topical corti-
costeroids for immune stromal keratitis. There were no adverse
drug reactions. The authors concluded that oral acyclovir is
effective and safe in treating HSV infectious epithelial keratitis
in pediatric patients. It is beneficial in treating infectious epithe-
lial keratitis and as prophylaxis while treating with topical
corticosteroids for immune stromal keratitis or for preventing
recurrent infectious epithelial keratitis.
Kimberlin et al reported use of intravenous ACV, 60 mg/kg
for 21 days in 72 infants under the age of 28 days.90 Six patients
dropped their absolute neutrophil count (ANC) to 500–1000/mm3.
In all cases, the ANC recovered during continuation of acyclovir
at the same dosage or after completion of acyclovir therapy,
and there were no apparent adverse sequelae of the transient
SECTION 6
neutropenia. However, decreasing the acyclovir dosage or
administering granulocyte colony-stimulating factor should be
considered if the ANC remains below 500/mm3 for a prolonged
period.
1. Intravenous acyclovir (60 mg–1 kg–1 day–1 in three divided
doses) for 2 weeks if disease localized to eyes, skin, and
mucous membranes and CSF PCR reaction assay is
negative for HSV DNA.
2. Decreasing the acyclovir dosage or administering granulocyte
colony-stimulating factor should be considered if the ANC
remains below 500/mm3 for a prolonged period.90
3. Topical trifluridine 9x/day µ 14 days.
4. Topical antibiotic drops such as Polytrim qid.
5. Periocular skin lesions should be kept clean with warm
sterile compresses two to three times daily and the lesions
should be kept dry between compresses. Some physicians
advocate topical antibiotic ointments such as ophthalmic
bacitracin. This is effective in minimizing secondary
642 infection.85
Primary ocular HSV (POHSV)
Primary ocular HSV (POHSV) is an acute first HSV infection of
the nonimmune host. It is to be differentiated from a first
ocular occurrence in a patient previously infected with HSV
elsewhere, e.g., orally, as the latter patients exhibit disease
similar to recurrent forms. POHSV may present as a ble-
pharitis, conjunctivitis, keratoconjunctivitis without, but more
commonly with, significant periorbital skin involvement, and,
rarely, iridocyclitis.5,91–93 As noted, < 4% of POHSV presents
as overt disease. But whether overt or not, it would appear that
all patients, once infected with HSV at any site, become viral
carriers with the agent residing in a latent state in the trigemi-
nal ganglia and, if there has been an infectious keratitis, the
cornea.5,94–99
Clinically, overt disease begins 3–9 days after exposure to an
infected carrier and typically manifests itself as an intense,
occasionally hemorrhagic vesicular (blistering) periocular
dermatitis or blepharitis, follicular conjunctivitis that may be
pseudomembranous and/or have geographic ulceration, corneal
ulceration, iritis, and a nonsuppurative preauricular adenopa-
thy. The skin eruption remains fairly localized to the periocular
area in the immunocompetent host and is a self-limited disease
that resolves entirely without scarring and often without
specific therapy (Fig. 49.1).5,92,100–102
A keratitis will develop in more than 60% of patients and,
due to the lack of immune inhibition, is usually atypical. There
may initially be diffuse punctate staining that converts within
24 h to multiple diffuse microdendritic epithelial defects, or
there may be serpiginous ulcers without clear-cut branching
effect covering much of the corneal surface (Fig. 49.2).
Iritis is uncommon in POHSV but, if it occurs, may produce
extensive atrophic damage.91
Therapy of POHSV Specific therapy is highly successful and
often results in healing with little to no scarring owing to
absence of a preprogrammed immune response. The most
commonly used drug is acyclovir because of ease of use and
compliance as well as efficacy. For ambulatory patients, therapy
is tailored according to age. For children less than 8 years of age,
oral acyclovir is administered at a dosage of 20 mg/kg every 8 h.
A pediatric suspension of 200 mg/5 mL is available. For children
FIGURE 49.1. Acute primary herpes simplex virus (HSV) blepharitis
with extensive vesicular eruption of the lids and the periorbital area
but no ocular involvement. Skin lesions healed within 3 weeks without
scarring.

a ophthalmic bacitracin may be used on badly ulcerated
b cephalic nucleus of the brain stem, and in some cases, to the
FIGURE 49.2. (a) Acute primary HSV epithelial dendritic keratitis with
diffuse serpiginous ulcers. (b) Same eye 3 weeks later with complete
epithelial healing and a clear stroma.
60 lb or over dosage can be the standard 400 mg po tid to 5id
either by pills or pediatric suspension, with the higher doses
being used in patients with altered immune systems, e.g., excema,
asthma, or immunosuppression. Topical trifluridine (Viroptic)
five to nine times daily for 2–3 weeks may be added after about
5 days if response is slow or steroids are in use.43,103,104
Although no large controlled studies have been performed
with valaciclovir or famciclovir, these drugs have been shown to
be clinically equal to acyclovir systemically and, valciclovir,
superior topically. However, they are notably more expensive.
For difficult cases, however, it is noteworthy that famciclovir
has a longer intracellular half-life than acyclovir, and valciclovir,
which is hydrolyzed back to ACV results in five times the
bioavailability of the latter drug.105–107 Loutsch et al’s rabbit
study on oral FCV treatment showed that the drug in doses
comparable to 120 mg bid in humans significantly reduced
the severity of corneal lesions, reduced the number of HSV-1
genomes in the TG, improved survival, and therefore may
be beneficial in reducing the morbidity of HSV keratitis in
the clinic.
Dosage recommendations have not been established for
young children. For postpubertal children, dosage should mirror
that of adults. Valaciclovir is administered at 500 mg twice
daily. Famciclovir is administered at 125 mg three times daily.
Herpes simplex keratoconjunctivitis is treated with topical
triflurothymidine. Two drops are applied to the infected eye five
Viral Disease of the Cornea and External Eye
times daily until resolved. Recurrences are managed in a similar
manner. Some physicians administer oral acyclovir at the doses
noted above in order to prevent frequent recurrences. Ocular
and mucocutaneous HSV infections in the immunocompro-
mised host can be treated with either intravenous acyclovir
or one of the orally bioavailable antiviral therapies. For
hospitalized patients, therapy consists of IV acyclovir at 5 mg/kg
every 8 h for 7–4 days.104
1. Periocular dermatitis or blepharitis only:
A. Prophylactic acyclovir 400 mg po two to three times
daily (dependent on patient weight) for 10 days or until
lesions scabbed over. Use pediatric suspension
(200 mg/5 mL) at dose of 20 mg/kg in children less
than 60 lb (–28 kg.) (1 lb = 2.2 kg).
B. Warm wet soaks 5 min bid with drying of lesions
allowed between soaks. General good hygiene. Topical
skin.
2. Corneal ulceration (dendrites, geographic ulcers):
A. Oral antivirals for 10 days to 3 weeks as in 1.A above.
B. Add topical trifluridine five to nine times daily if
patient immunocompromised and therapeutic response
to oral antivirals not satisfactory by 5 days of oral
treatment.
C. Topical antibiotics bid for corneal ulceration.
D. Cycloplegics, cyclopentolate, or scopolamine bid if iritis
is present.
E. Fox shield or hand restraints in young children as
needed.
Recurrent ocular herpes
Latency Within the first 48 h of primary infection the virus
travels as an unenveloped particle by retrograde axoplasmic flow
to the sensory (trigeminal) ganglia, ciliary ganglia, mesen-
sympathetic ganglia where it enters a latent or dormant
state.48,94,95,101 The trigeminal ganglion (TG) is one of the main
sources of virus for recurrent disease particularly of the ocular
adnexa and cornea. Re-activation of HSV latent in the ciliary or
superior cervical ganlion of the Edinger–Westfall nucleus is
more likely to result in iritis. Virus in the suprachiasmatic or
paraventricular nuclei may cause retinitis or encephalitis.75,108
As noted, HSV may enter the TG via any of the three major
divisions of the trigeminal nerve, mandibular, maxillary, or
ophthalmic. Thus, an initial orofacial infection with HSV may
establish latent virus in the ophthalmic division of the
trigeminal ganglion and subsequently lead to ocular infection
after re-activation of virus with spread down the nerves to the
CHAPTER 49
eye rather than those to the nose or mouth, or down all three
(Fig. 49.3).29,109,110 Bilateral disease is rare, occurring in about
2% of patients.101
Further, several studies indicate that the cornea may also
serve as a site of viral latency capable of re-activation and that
serial recurrences are due to re-activation of the same latent
virus, not new exogenous infection.98,111–114 In a study by Rong
et al, corneal specimens from 18 patients with quiescent herpes
simplex keratitis (HSK) were obtained at keratoplasty. PCR
amplification followed by Southern blot hybridization detected
HSV-1 genome in these human corneal samples. The DNA
sequences from either the TK or the LAT gene were identified
in 15 of 18 HSK corneas (83%). These data indicate that the
HSV genome was retained, at least in part, in human corneas
during quiescent HSV infection, giving further support to the
concept of corneal extraneuronal latency which may, in turn, be
one source of herpetic disease in previously nonherpetic grafted
eyes. This has further been confirmed by Remeijer et al and
others.12,96,99,112–114 It has also been shown that viral infection 643
 CORNEA AND CONJUNCTIVA

HSV re-activating factors The mechanisms of viral re-

activation are not well understood. A common theory is that
the ganglion (and possibly corneal) cells harboring the HSV
genome are in a nonpermissive state for replication during
latency. A yet-to-be-established stimulus then allows the
ganglion cell to become permissive for active HSV replication.
On a cellular level, this may translate into a deficiency of
immune competence or intracellular messenger systems.