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ADVANCES IN EYE RESEARCH

VOLUME 2
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VOLUME 2
WILLIAM L. THOMSEN
EDITOR
Nova Science Publishers, Inc.

New York
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Contents
Preface vii
Chapter I Advances inPathogenesis of Pterygium 1
Louis Tong, Wanwen Lan,
Andrea Petznick and Aihua Hou
Chapter II Role of Prisms in the Management of Horizontal Deviations 37
Rehab Rashad Kassem
Chapter III MRSA (Methicillin-resistant Staphylococcus Aureus)
and Eye Infection 69
Theodora Tsirouki, Sofia Androudi
and Evangelia Tsironi
Chapter IV Proteomics and Metabonomics of the Vitreous Fluid as a New
Approach to Identify New Candidates in the Pathogenesis
of Diabetic Retinopathy 93
Rafael Simó and Cristina Hernández
Chapter V Measuring Cornea 115
Cristina M. Oliveira
and S. Franco
Chapter VI Retinal Control of the Refractive State of the Eye 137
Francisco J Carrillo-Salinas and Jaime Tejedor
Chapter VII Proliferative Vitreoretinopathy: Signaling Mechanism Involved
and Available Treatments 157
Bose Karthikeyan, Venkataraman Deepak and

Sangiliyandi Gurunathan
Chapter VIII Neuropathology of Cultured Retinas: Degenerative Events
and Rescue Paradigms 177
Camilla Mohlin, Ingela Liljekvist-Soltic, Jenny Olofsson
and Kjell Johansson
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Copyright © 2012. Nova Science Publishers, Inc. All rights reserved. May not be reproduced in any form without permission from the publisher, except fair uses permitted under vi Contents
Chapter IX Non-Invasive Imaging of Retinal Gliosis for Screening

Neurotoxicity and Neuroprotection in Mouse Models
of Human Diseases 191
Lang Zhuo and Saravana Kumar
Chapter X Proteomic Approach to Chronic Eye Diseases 205
Takashi Kanamoto
Chapter XI Lens Plasticity and Intralenticular Translocation of Crystallins 215
Jeffrey O. Henderson and Larry J. Takemoto
Index 223
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Preface
This series presents original leading edge results in the field of eye research. The focus in
this compilation is on the role of prisms in the management of horizontal deviations;
advances in the pathogenesis of pterygium; MRSA and eye infections; the pathogenesis of
diabetic retinopathy; retinal control of the refractive state of the eye; proliferative
vitreoretinopathy; the neuropathology of cultured retinas; non-invasive imaging of retinal
gliosis and lens plasticity and intralenticular translocation of crystallins.
Chapter I - Pterygium is a relatively common human ocular surface disease characterized
by fibrovascular proliferation of the conjunctiva, involving up to 200 million people globally.
It causes irritation, induces astigmatism and tear film disturbance, and may threaten vision as
it invades the cornea. The pathogenesis of this complex disease is unknown although
pterygium is known to be associated with overexposure to excessive ultraviolet (UV) light
and defects in matrix reorganization, cell migration and cell adhesion. Recent studies have
focused on the epidemiological, genetic and molecular factors related to pterygium.
Epidemiology studies have continued in different populations of the world, the prevalence
ranges from 3.8% in aged Beijing population to 12.3% in Singapore Malays. Notably,
pterygium is found to be more common at latitudes less than 30 degrees, which are generally
associated with longer sunlight hours. Genetic studies have found the association of single
nucleotide polymorphisms in genes such as CYP1A1, GSTM1, XRCC1 and G6PD with
pterygium, although controversy is present in the reports. The presence of some genetic
predisposition to free radical damage related to UV radiation may increase susceptibility to
pterygium formation. Molecular studies have implicated matrix metalloproteinases (MMPs)
and their natural tissue inhibitors, vascular endothelial growth factor (VEGF), E-cadherin and
S100 proteins in pterygium pathology. Inflammatory mediators that have been found to be
involved in pterygium include CD40 from the TNF-receptor superfamily, cyclo-oxygenase,
and interleukin-4. Free radicals-induced stress generated by UVB results in oxidative damage
to DNA, triggering ocular surface inflammatory responses. Basal cells of pterygium epithelial
tissues express higher levels of p53 and Bax, as well as the apoptosis-inhibiting protein Bcl-
2, suggesting cell cycle defects in pterygium pathogenesis. In addition, gene expression
studies have found structural and adhesion molecules to be involved in pterygium formation.
Experiments suggest that pterygium may be a limbal basal epithelial stem cell disorder, or a
process of epithelial mesenchymal transition. Abnormal fibroblasts derived from conjunctival
epithelium may have greater proliferative and invasive tendencies, and may express MMPs
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Copyright © 2012. Nova Science Publishers, Inc. All rights reserved. May not be reproduced in any form without permission from the publisher, except fair uses permitted under viii William L. Thomsen
that facilitate dissolution of the corneal Bowman‘s layer. VEGF has been found to be an
important factor in driving the abnormal vasculature in the stroma of some pterygia. The
S100 proteins are a group of calcium binding cornified envelope proteins that play diverse
roles in stress regulation, differentiation and cell proliferation. Interestingly, mRNA
transcripts and proteins of members such as S100A9 have been found to be elevated in the
pterygium tissue and the proteins elevated in patients‘ tear fluid as well. The definitive
treatment for pterygium is surgical resection with autologous conjunctival autografting or
recent modifications of the technique. This strategy is largely successful although post-
resection recurrences can still occur. Mitomycin C (MMC) has been used as adjunctive
therapy, however it is also associated with serious and potentially sight-threatening
complications. More recently, clinical research has been conducted to compare conjunctiva
grafting using fibrin-based adhesives with suturing the autograft via conventional sutures.
Currently, there is no definitive non-surgical method of treating primary pterygium, although
early studies in anti-VEGF treatment have been conducted in human trials. Recently,
researchers have published a murine pterygium model based on injection of human pterygium
epithelial cells. The availability of this model suggests that novel modalities of treatment can
now be evaluated for their effect in-vivo. This is particularly useful for methods of treatment
with unconfirmed efficacy and safety but have in-vitro evidence that is promising. This is a
critical step in the development of drugs for pterygium retardation in humans. With advances
in the knowledge on pathogenesis and formation of pterygium, one may expect important
new developments in pterygium treatment to follow in the next decade.
Chapter II - Prisms are important tools for the diagnosis and treatment of strabismus as
they are used to measure and neutralize ocular deviations. A prism is essentially two
angulated plane refracting surfaces. It displaces, as well as deviates, light rays. The total
angle of deviation is the sum of the deviations produced at each of the two surfaces.
Chapter III - Staphylococcus Aureus (SA) suggests one of the major human pathogens. It
is a common cause of disease, both in healthcare facilities, as well as in the community. SA
constitutes such a problem to physicians, throughout the world, due to its great virulence. It
causes an extensive variety of human diseases, many of which are life threatening. Actually,
SA is estimated to be responsible for 20-40% of mortality in bacteremias. It spreads from
human to human, and it is easily adapted in a variety of environmental conditions. However,
the most important problem in its management is its capability to develop resistance to almost
every antibiotic. Staphylococcus is a genus of gram positive bacteria. There have been
described 32 species and 8 sub-species of genus Staphylococcus. SA is the most virulent
species, and has been thoroughly investigated over the years. To date, the SA genome
databases have been completed for 7 strains; 8325, COL, MRSA, MSSA, N315, Mu50, and
MW2. SA normally colonizes human nose and skin, a fact that influences the pathogenesis of
staphylococcal infection. Less often, SA is found on the respiratory or urinary tract. Healthy
individuals carry the bacteria from weeks to years, without manifesting a disease. It is
estimated that 20% of individuals are persistent carriers, who carry almost always one type of
SA. Another 20% almost never carry it, whilst the rest of the human population carries SA
intermittently.
Chapter IV - Diabetic retinopathy remains a leading cause of blindness and visual
impairment among adults aged <40 years in the developed world. Investigation into the
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processes involved in DR and the testing of new therapies are limited because the
unavailability of human retina samples and the lack of diabetic animal models that faithfully
replicates the features of human DR. Vitreous fluid obtained from diabetic patients
undergoing vitreoretinal surgery is currently used as a surrogate for the retina in clinical
research. However, the volume of vitreous fluid obtained after vitrectomy is approximately 1
ml and, therefore, only a few peptides or metabolites can be analyzed simultaneously. The
recent development of high-throughput techniques such as proteomics and metabonomics has
made it feasible to analyze both the protein and metabolite profiles in a massive manner. In
recent years author have been able to demonstrate by proteomic analysis that several proteins
such as complement factors (C3, C4b, C9 and factor B), Apo A1 and Apo-H were higher in
the vitreous fluid from patients with proliferative DR (PDR) than non-diabetic patients,
whereas the interstitial retinol-binding protein (IRBP) was lower. In addition, mRNA levels
in the retina run in parallel with proteins, thus suggesting that the intraocular production of
these proteins participates in the physiopathology of DR. Furthermore author have recently
identified by proteomic analysis genuine proteins (ie. hemopexin and clusterin) differently
expressed in the vitreous fluid from patients with diabetic macular edema. Finally, by using
metabonomics author have been able to shown that, apart from a higher abundance of both
lactate and glucose, significant deficits of galactitiol and ascorbic acid also exist in the
vitreous fluid from PDR patients. The potential role of all these candidates in the
pathogenesis of DR will be discussed in this chapter. In summary, proteomics and
metabonomics of the vitreous fluid are useful not only for identifying potential candidates in
the development of DR, but also for designing new approaches leading to a more efficient
pharmacologic treatment of this devastating complication of diabetes.
Chapter V - By virtue of its remarkable mechanical strength and optical transparency, the
cornea serves as the major refractive element of the eye while protecting its contents. These
properties are directly attributable to the cornea‘s structural architecture of collagen fibrils
and surrounding hydrated matrix containing proteoglycans, glycoproteins, other soluble
proteins, inorganic salts and keratocytes. The maintenance of corneal shape and transparency
is crucial for its optical function. The optical properties of the cornea are mainly governed by
its collagen fibril organization, which ensures that the cornea maintains correct surface shape
under the action of intraocular pressure on its internal surface.
Chapter VI - In this chapter, author summarize advances in the understanding of factors
that control refractive development and refractive state of the eye, particularly by retinal
mechanisms. When the normal refractive development process towards emmetropia
(emmetropization) fails, it leads to refractive errors, such as myopia or hyperopia, which are
highly prevalent in the general population. Different studies have reported a regulatory effect
of the retina in the refractive state of the eye, through a cascade of neurotransmitters or
growth factors signals, in which the involvement of different systems, including muscarinic,
glucagonergic and dopaminergic, has been demonstrated. The main findings described in the
literature will be presented. Transcription factors of neuromodulators, whose expression is
known to change with variations in light conditions, in particular Egr-1, could be of vital
importance in regulating refractive error. Different genetically modified mouse strains
showing the potential role of neurotransmitters and transcription factors, and the
experimental studies showing this effect, will be described. Other studies stress the
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Copyright © 2012. Nova Science Publishers, Inc. All rights reserved. May not be reproduced in any form without permission from the publisher, except fair uses permitted under x William L. Thomsen
importance of the ionic and water balance and content of the outer retina and choroid as
controlled by the retinal pigment epithelium, and its role in the mechanisms of myopic and
hyperopic changes. The influence of peripheral versus central retina in the development of
myopia has been a matter of debate, with contradictory conclusions regarding the importance
of retinal periphery and peripheral vision. Author will finally deal with this controversy, as
well as introduce potential therapeutic implications of some involved described regulatory
elements.
Chapter VII - Proliferative vitreoretinopathy (PVR) is the most common cause of visual
loss after retinal detachment. It is clinicaly characterized by uncontrolled proliferation of
Retinal pigment epithelial (RPE) cells in the vitreous and the concomitant effect of
membrane formation on bothside of the retina. In normal adult eye, blood retinal barrier
(BRB) comprises of non-proliferating RPE cells which are also essential for the survival of
photoreceptors. Continuous damage to the BRB causes numerous growthfactor release into
the vitreous and often precedes clinically recognisable PVR. Both retinal injury and BRB
damage enhances RPE cell migration into vitreous and transforms fibroblast like cells. The
early cellular cascade includes various growth factors which involve in autocrine or paracrine
loop is a key factor in PVR. Although several growthfactors are involved in PVR condition,
vascular endothelial growth factor (VEGF) and interleukin1-β are the major key factors
which play important role in cellular proliferation. Significant evidence suggests that multiple
intracellular pathways regulate inflammatory phase of PVR. Better understanding of
pharmacological agents and elucidation of the exact signaling mechanism involved in the
PVR associated metabolic alterations may reveal novel therapeutic approaches. Several
inhibitors were tested for efficient treatment of PVR but many of them were unsuccessful.
Although some had satisfied a few of criteria, the place of a promising agent for the potential
treatment of PVR still remains void.
Chapter VIII - This review examines the potential of retinal explant cultures as a model
system for neuroprotective strategies aiming at the treatment of retinal diseases. Early studies
on explanted retinas were initially concentrated on developmental and differentiation events
of different cell subtypes. A growing number of studies have examined different
neuroprotective strategies including growth factor supplementation as well as coculture with
progenitor and stem cells. Most studies have focused on neuroprotection of photoreceptors,
but protection of axotomized ganglion cells have also been explored. Both cell types are
injured during the culture procedure, and show an injury-sequence that is morphologically
comparable to the ones described in other experimental models and human retinal
dystrophies. Injury-related responses include photoreceptor sprouting as well as neuronal
remodeling, glial cell activation, and neuroinflammatory responses. In general, the explant
models offer several advantages: 1) degenerative pathways and remodeling events are rapid,
2) different neuroprotective strategies are possible and can be, compared to in vivo
experiments, rapidly evaluated, 3) a controlled in vitro environment, and 4) an easy
experimental procedure that is cost-effective.
Chapter IX - Reactive gliosis, a hallmark of neurotoxicity, is triggered by injury or insult
to the central nervous system (CNS) due to various factors such as drugs, chemicals, trauma
and diseases. Reactive gliosis involves the transformation of glial cells, such as astrocytes in
the brain and Müller cells in the retina, from a quiescent to an activated state. Glial fibrillary
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acidic protein (GFAP), expressed in astrocytes, is widely regarded as the ideal biomarker for
astrogliosis due to its sensitivity and specificity. As such, a non-invasive detection and
quantification of GFAP transcriptional activity would not only facilitate the objective
assessment of neurotoxicity from compounds but also enable the timely detection of CNS
related diseases [Alzheimer‘s disease (AD), Parkinson‘s disease (PD), multiple sclerosis
(MS), diabetic retinopathy (DR), etc.] and development of effective treatment strategies
which intervene at an earlier stage.
Chapter X - Proteomic analyses have been used to study different aspects of various
systemic and ophthalmic diseases, and the number of proteomics publications on eye diseases
has been rapidly increasing. Proteomic has been used to investigate chronic eye diseases,
such as dry eye, cataracts, glaucoma, diabetic retinopathy, and aged-related macular
degeneration. It is expected that the results of this proteomic approach can be used to
investigate the mechanism and diagnosis of diseases, and that information will help in
developing new treatments. Author shall present the summary of proteomic research for
several chronic eye diseases and show the up-dating of them.
Chapter XI - Very high concentrations of ―crystallin‖ proteins in the human lens are
necessary for refractile properties of this tissue, and abrupt changes in refractive indices
within the lens due to aggregation of these proteins result in scattering of light and
subsequent loss in the ability of the lens to focus light upon the retina. Such a condition
called cataractogenesis is prevalent in patients greater than approximately 60-65 years of age,
and is currently one of the most common causes of blindness in the world. Author have
previously demonstrated weak, heterologous interactions of alpha and gamma crystallins.
Author now hypothesize that such weak interactions contribute towards the plasticity of
heterologous interactions, facilitating a uniform refractive environment that will minimize
light scattering in a tissue of very high protein concentration. Loss of this lens plasticity, due
to covalent interaction of lens crystallins and formation of strong heterologous interactions,
would result in changes in refractive indices and subsequent lens opacification.
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In: Advances in Eye Research, Volume 2 ISBN: 978-1-61324-605-4
Editor: William L. Thomsen ©2012 Nova Science Publishers, Inc.
Chapter I
Advances in Pathogenesis of Pterygium
Louis Tong* 1,2,3, Wanwen Lan 1, Andrea Petznick 1 and Aihua Hou 1
1
Ocular Wound Healing and Therapeutics Laboratory,
Singapore Eye Research Institute, Singapore
2
Singapore National Eye Center, Singapore
3
Duke-NUS Graduate Medical School, Singapore
Abstract
Pterygium is a relatively common human ocular surface disease characterized by
fibrovascular proliferation of the conjunctiva, involving up to 200 million people
globally. It causes irritation, induces astigmatism and tear film disturbance, and may
threaten vision as it invades the cornea. The pathogenesis of this complex disease is
unknown although pterygium is known to be associated with overexposure to excessive
ultraviolet (UV) light and defects in matrix reorganization, cell migration and cell
adhesion.
Recent studies have focused on the epidemiological, genetic and molecular factors
related to pterygium. Epidemiology studies have continued in different populations of the
world, the prevalence ranges from 3.8% in aged Beijing population to 12.3% in
Singapore Malays. Notably, pterygium is found to be more common at latitudes less than
30 degrees, which are generally associated with longer sunlight hours. Genetic studies
have found the association of single nucleotide polymorphisms in genes such as
CYP1A1, GSTM1, XRCC1 and G6PD with pterygium, although controversy is present
in the reports. The presence of some genetic predisposition to free radical damage related
to UV radiation may increase susceptibility to pterygium formation. Molecular studies
have implicated matrix metalloproteinases (MMPs) and their natural tissue inhibitors,
vascular endothelial growth factor (VEGF), E-cadherin and S100 proteins in pterygium
pathology. Inflammatory mediators that have been found to be involved in pterygium
include CD40 from the TNF-receptor superfamily, cyclo-oxygenase, and interleukin-4.
*
Correspondence for reprints: Dr Louis Tong, Address: Singapore National Eye Center, 11 Third Hospital Avenue,
Singapore 168751, Telephone: +65-62277255, Fax: +65-63224599 , Email address: Louis.tong.h.t@
snec.com.sg
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Copyright © 2012. Nova Science Publishers, Inc. All rights reserved. May not be reproduced in any form without permission from the publisher, except fair uses permitted under 2 Louis Tong, Wanwen Lan, Andrea Petznick et al.
Free radicals-induced stress generated by UVB results in oxidative damage to DNA,

triggering ocular surface inflammatory responses. Basal cells of pterygium epithelial
tissues express higher levels of p53 and Bax, as well as the apoptosis-inhibiting protein
Bcl-2, suggesting cell cycle defects in pterygium pathogenesis. In addition, gene
expression studies have found structural and adhesion molecules to be involved in
pterygium formation.
Experiments suggest that pterygium may be a limbal basal epithelial stem cell
disorder, or a process of epithelial mesenchymal transition. Abnormal fibroblasts derived
from conjunctival epithelium may have greater proliferative and invasive tendencies, and
may express MMPs that facilitate dissolution of the corneal Bowman‘s layer. VEGF has
been found to be an important factor in driving the abnormal vasculature in the stroma of
some pterygia. The S100 proteins are a group of calcium binding cornified envelope
proteins that play diverse roles in stress regulation, differentiation and cell proliferation.
Interestingly, mRNA transcripts and proteins of members such as S100A9 have been
found to be elevated in the pterygium tissue and the proteins elevated in patients‘ tear
fluid as well.
The definitive treatment for pterygium is surgical resection with autologous
conjunctival autografting or recent modifications of the technique. This strategy is
largely successful although post-resection recurrences can still occur. Mitomycin C
(MMC) has been used as adjunctive therapy, however it is also associated with serious
and potentially sight-threatening complications. More recently, clinical research has been
conducted to compare conjunctiva grafting using fibrin-based adhesives with suturing the
autograft via conventional sutures. Currently, there is no definitive non-surgical method
of treating primary pterygium, although early studies in anti-VEGF treatment have been
conducted in human trials.
Recently, researchers have published a murine pterygium model based on injection
of human pterygium epithelial cells. The availability of this model suggests that novel
modalities of treatment can now be evaluated for their effect in-vivo. This is particularly
useful for methods of treatment with unconfirmed efficacy and safety but have in-vitro
evidence that is promising. This is a critical step in the development of drugs for
pterygium retardation in humans.
With advances in the knowledge on pathogenesis and formation of pterygium, one
may expect important new developments in pterygium treatment to follow in the next
decade.
Keywords: Pterygium, review, pathogenesis, epidemiology, genetics, metalloproteinases,

tissue inhibitors of metalloproteinases, inflammation, ultraviolet radiation, cytokines, cell
transformation, matrix remodeling, cell cycling, cell proliferation, stress-related
signaling, S100, NFκB, Phospholipase D, polo-like kinase signaling, metabolic
1. Introduction
Pterygium is a fibrovascular tissue that grows centripetally from the conjunctiva to the
cornea. There have been recent advances in the mechanism, pathology and treatment of
pterygium. This review focuses on the latest findings on the epidemiology, genetics,
molecular biology, including molecular intervention, and etiological factors in pterygium
(Figure 1). Surgical excision of pterygium with conjunctival autografting is largely successful
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although post-resection recurrences can still occur. Some studies have examined the use of
adjunctive treatments intra- or peri-operatively. Mitomycin C (MMC) has been used as
adjunctive therapy. However it is also associated with serious and potentially sight-
threatening complications. More recently, clinical research has been conducted to compare
conjunctiva grafting using fibrin-based adhesives with suturing the autograft via conventional
sutures. Reviews of surgical and adjunctive treatments, [2, 51] including a meta-analysis of
different modalities of treatment, [116] have previously been published and will not be
covered here. However, these reviews do not include therapeutic trials against specific
molecules which we will describe below.
Figure 1. This schematic shows the molecular intermediates that may trigger pterygium formation. Typically,
there may be processes in angiogenesis mediated by production of vascular endothelial growth factor
(VEGF). Inflammatory processes may be mediated by interleukin (IL)-6, -8 and cyclo-oxygenase (COX) 2,
macrophage chemoattractant protein (MCP) 3, transforming growth factor beta (TGFβ) and insulin-like
growth factor binding protein (IGFBP) 3. Metabolics changes associated with pterygium include low density
lipoprotein receptor (LDL-R), hydroxyl methyl glutaryl coenzyme A reductase (HMG CoA R) and glucose 6
phosphate dehydrogenase (G6PD). MMPs – matrix metalloproteinases; TIMPs – tissue inhibitors of MMPs.
Recently, there have been advances in the assessment of pterygium such as using Raman
spectroscopy and in vivo confocal microscopy. In-vivo laser confocal microscopy has been
used to detect inflammatory cells and dendritic cells in pterygium. [41, 65, 95-96, 143] One
possible way to perform the imaging is using the Heidelberg Retina Tomograph II Rostock
Cornea Module. [41] Using this technique, densities of corneal epithelial cells in the basal
layer, keratocytes in the anterior and posterior stroma, and dendritic cells in the pterygium
and adjacent cornea were assessed. [143] The cell density findings using confocal
microscopy were similar to those from impression cytology, suggesting that the former
procedure is promising for quantification of cells. [65] Laser confocal microscopy is also able
to determine the tortuosity and number of vesicles in the sub-basal nerve fibres. In one study,
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these nerve endings were found to be increased in pterygium compared to conjunctival

tissues in non-pterygium subjects. [96]
The confocal Raman spectroscopy is a very powerful technique for non-invasive study of
human skin and ocular surface under in vivo conditions. It is possible to obtain information
regarding the molecular composition of pterygium tissue down to several hundred
micrometers below the surface of the tissue. The Raman spectra of pterygium and normal
conjunctiva have different intensities as pterygium tissues have additional components not
present in the conjunctiva. For example, pterygium tissue contains elastic fibers, mast cells
and lymphocytes, while it has less unsaturated fatty acids. [119] This allows users to
distinguish between normal and disease conditions.
Previously, there was no reliable animal model for investigations of pterygium available.
A murine model that develops pterygium lesions in 70% of the cases was established by Cox
et al. [18] Ten thousand human pterygium epithelial cells mixed with Matrigel were injected
into the nasal conjunctiva close to the limbus in nude mice. The authors observed that
histological features in the murine lesions resembled human pterygium, such as neovessels
and fusiform cells migrating towards the cornea. However, the mouse model was tested for
up to 15 days only, while human pterygium is likely to have developed over a much longer
period. Clinically, pterygium is known to grow slowly but this has not been longitudinally
documented. The authors of this unique study [18] described the murine lesions as not
containing areas of stromal collagen degradation unlike in human pterygium.
As an in vitro model cannot mimic ocular surface microenvironments, it is important to
develop an animal model that allows for investigation into risk factors, mechanisms of
development and possible therapies of pterygium. Degradation of the Bowman‘s membrane
is a characteristic feature of human pterygium. The mouse eye, unlike the human equivalent,
possesses a very thin Bowman‘s membrane, [48] and therefore would not be ideal for
observing its destruction. Nevertheless, for want of a better alternative, this model may be
very helpful in pterygium research.
2. Epidemiology
It is estimated that pterygium may affect up to 200 million people in the world. [80] At
least [10] epidemiology studies on the prevalence of pterygium have been published since the
2000. This has been reported in Indonesia, [40, 123] Singapore Malays, [15] Singapore
Chinese, [147] Doumen county China, [149] Tibet, [79] Mongolia, [78] Barbados, [81]
Australia [88] and a Saharan population. [13] In general, the prevalence rates are lower when
an urban or large metropolitan center is being studied, compared to special occupational

groups such as Croatian fisherman, [141] Indian brine workers [86] or Amazonian villagers.
[100] The common factor in these special occupational groups may be sun exposure. In the
largest study reported (n=7990) in Chinese people, the prevalence was 8%.75 In special
occupational groups, prevalence rises to 18-23%, [86, 100, 141] and even to 33% in rural
Chinese people more than 50 years old. [149]
In a study with very high participation rate (97%) conducted in rural Sumatra in
Indonesia (n=1210), it was noted that pterygium was associated with increasing age. [40] Age
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as a risk factor was also supported by the Barbados (n=2781), [81] Australian (n=5147), [88]
Tibetan (n=2632), [79] Mongolian (n=2112), [78] Singapore Malay [15] and Singapore
Chinese (n=1232) [147] studies, even though these studies involved participants of diverse
and different ethnicities.
In the Singapore Chinese [147], Singapore Malay [15] and Australian [88] studies, male
gender was a significant independent risk factor, whereas in the China [149] and Tibetan [79]
studies, female gender was more at risk. In Indonesia, [40] however, the amount of outdoor
work contributed to the risk of having pterygium in both men and women, but male gender
was not an independent risk factor. Similarly, having an outdoor occupation or increased
lifetime ocular sun exposure was an independent risk factor in the Barbados [81] and
Australian [88] studies. In Singapore, [147] people working in factories, production lines,
machine operations, and agriculture had higher odds of having pterygium compared to office
workers. Interestingly, the use of sunglasses outdoors and having darker skin pigmentation
were found to be protective factors for development of pterygium. [81] In Australia, rural
residence, compared to urban ones, was found to be a risk factor for pterygium. [88]
Consistent with clinical impression, most pterygium (93%) was found on the nasal side
of the cornea. [149] The mean basal width was 3.3 mm (SD 1.51, range 0.1-9.5) and the mean
extent from the limbus was 1.4 mm (SD 1.18, range 0.1-8.0). [40] As one expects, pterygium
was associated with the presence of astigmatism. [40] Increased extension of pterygium from
the limbus, but not its basal width, was associated with severity of astigmatism. The
morphology (atrophic, intermediate or fleshy) of the pterygium is a relevant consideration
clinically as fleshy or non-translucent lesions have higher risks of recurrence after surgical
excision. [124]
Population-based investigations were able to shed light on the effect of pterygium on tear
film parameters employed in dry eye assessment. In the Mongolian study, pterygium was
found to be associated with a reduced fluorescein tear break up time and reduced Schirmer‘s
test, [78] and in the Tibetan study, with dry eye symptoms. [79]
3. Genetics of Pterygium
Previous reviews on pterygium have included the discussion of inheritable defects in

DNA repair [17] and genetic susceptibility in pterygium development. [21] A report on a
Chinese family suggested an autosomal dominant mode of inheritance in 11 members of the
family descended from one individual. [161] In another family with 11 affected members (7
bilateral and 4 unilateral pterygia), including a monozygotic twin, the inheritance of
pterygium was also autosomal dominant. The onset was early in life in the teens or twenties.
Three members of a Saudi Arabian family, aged 4, 6 and 20 years were found to have
aggressive pterygium. [54] The relationship between the polymorphism of matrix-
metalloproteinase (MMP)-1 promoter and loss of heterozygosity (LOH) in chromosomes 9q,
17q and 11q has been published, where LOH in chromosome 11q was mapped to the MMP-1
promoter. [21]
The polymorphisms in DNA repair genes have been studied, such as in the human-
8-oxoguanine glycosylase I (HOGG1), glutathione S-transferase I (GSTM1) and Ku70
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promoters. [17] Figure 2 shows how LOH and polymorphisms can predispose to downstream
processes associated with pterygium.
Figure 2. This figure summarises the pathological events in the formation of pterygium. Under quiescent
conditions (white), the conjunctival epithelium does not migrate onto the cornea and there is no excessive
matrix production. In the presence of predisposing factors, inflammatory reactions and epithelial
mesenchymal transition (EMT) can cause downstream events (red) which may then result in pterygium.
LOH- loss of heterozygosity; SNP- single nucleotide polymorphisms.
Recently, a study in Taiwan has found benzo[a]pyrene 7,8-diol 9,10-epoxide (BPDE), a

metabolite of an aromatic environmental pollutant, to form an adduct with DNA.The
formation of adducts was associated with polymorphisms in the cytochrome P4501A1
(CYP1A1) but not the GSTM1 gene. [133] This study raises an interesting hypothesis:
deficiency in DNA repair capacity related to CYP1A1 polymorphisms may predispose to
accumulation of metabolites such as BPDE, consequently altering other DNA such as those
in tumor suppressor genes and eventually resulting in pterygium. BPDE-like compounds,
including cigarette smoke-derived benzo[a]pyrene can be found in the environment,
explaining the association of increased time spent outdoors and pterygium. [40] This study
involved 103 primary pterygium patients with an average age of 70 years old without any
non-disease controls. This is a major limitation of the study since aging changes in non-
pterygial tissue may possibly also be associated with BPDE.
4. Angiogenesis
Pterygium is a lesion with not only an epithelial and stromal component but also
increased vascularity. The role of blood vessels in the formation of pterygium is not entirely
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clear, but studies have suggested that molecular events in vessel formation may be linked to
pterygium.
Vascular Endothelial Growth Factor and Clinical Trials
The vascular endothelial growth factor (VEGF) is a pro-angiogenic growth factor that
has been found to play a key role in angiogenesis in many diseases, especially in the
vascularisation of tumors. [117] Polymorphisms of VEGF, especially VEGF-460, have been
associated with pterygium formation in a study conducted in Taiwan involving 127
pterygium patients and 102 volunteers without pterygium. [130] It was reported that female
participants who had at least 1 C allele (C/C and C/T genotypes) had about a 2.5-fold
increased risk of developing pterygium compared to those with the T/T genotype. This result
was however different from another study in Taiwan with 133 patients and 105 volunteer
controls, which found no difference in the VEGF-460 genotypes between the groups. [8]
The discovery that VEGF-A was upregulated in pterygium compared to conjunctiva
tissue [20] suggests that angiogenesis is an important component in the pathology of
pterygium. Since many pterygia appear clinically to be vascular, this observation may be
linked to a hyper-proliferation of vasculature within the stroma of the pterygium tissue, led
by the formation of vascular endothelial tubes. Furthermore, the transcript level of VEGF-A
was higher in recurrent pterygium compared to primary pterygium, suggesting that VEGF-A
may also play a role in post surgical recurrence. In another study, VEGF was found to be
upregulated not only locally in pterygium (28 patients with 13 controls) but also systemically.
[69]
In a study of 52 pterygia and 7 normal conjunctiva, VEGF immunopositivity was found
to be higher in the stromal and vascular endothelial cells compared to epithelial cells. The
density of vasculature was also higher in pterygium tissue than in normal conjunctiva. [3]
An amniotic membrane used as an adjunct modality during pterygium surgery is known
to lower recurrence rate compared to bare sclera surgery. [2] Amniotic membranes were
associated with a variety of growth factors including anti-angiogenic factors that may inhibit
VEGF. [74] Patients who had amniotic membranes placed during surgery were also found to
have lower levels of VEGF in the tears. [70] It is possible that the reduced VEGF may be
linked to reduced angiogenesis hence recurrence. In another study, immunohistochemical
methods were used to localize VEGF to pterygium tissue and normal conjunctiva. VEGF was
detected in all primary and recurrent pterygium tissue and localized to epithelial, fibroblast
and vascular endothelial cells, whereas staining was very weak or absent in normal
conjunctiva. [60] In another study using immunohistochemistry, VEGF detection was 57%-
69% in primary pterygium, 100% in recurrent pterygium and 13% in the normal conjunctiva.
[154]
One should however note that these studies did not show that VEGF initiated
neovascularisation. It was still possible that VEGF was produced only after the presence of
another triggering factor, or after the formation of the fibrous component of pterygium.
Alternatively, since not all pterygia are equally vascular, VEGF may play only a secondary
role in pterygium formation, affecting primarily the vascular events.
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Since anti-VEGF therapy is widely used for choroidal neovascularisation in age related
macular degeneration, [146] investigators have recently evaluated such treatment for
pterygium. [39] The first published report on the use of topical bevacizumab, an antibody
against VEGF, was in a patient who had pterygium excision and impending recurrence. [150]
In patients with impending recurrence after primary pterygium excision, topical bevacizumab
4 times a day for 3 weeks was noted to be more efficacious for reduction of
neovascularisation than a 1 week treatment twice a day.
In another case report [126] subconjunctival bevacizumab was used to treat a case of an
inflamed primary pterygium without surgery after unsuccessful management with artificial
tears and the vasoconstrictor naphazoline. Seven weeks after treatment, irritative symptoms
and the degree of vascularisation/ hyperemia improved. Since these were only case reports,
no conclusive evidence of the efficacy of the technique was obtainable. However, these
reports have spurred other researchers to evaluate this modality of treatment.
Mandalos et al. [82] performed subconjunctival ranibizumab (another antibody against
VEGF) injection therapy prior to primary pterygium excision in 10 patients, and followed the
patients for up to 1 week post-operatively. This did not result in regression of any pterygium
vasculature. However, the lack of controls and the short duration of follow up were
limitations of the study. Galor et al. [39] administered subconjunctival ranibizumab either 3
days preoperatively or intraoperatively in 10 patients, but did not compare results with a
standard care group which just had pterygium excision without anti-VEGF therapy. The
pterygium in 3 cases among those who had injections preoperatively recurred at 6 months,
whereas there was none in those who had intraoperative injections.
In a prospective interventional controlled study, [110] 30 eyes of 30 patients were given
either 1.25 mg (0.1 ml) of bevacizumab subconjunctivally (15 patients) or 0.1 ml of balanced
salt solution (15 patients). This was given as an adjunctive treatment to primary pterygium
excision and conjunctival autograft surgery. In this study, the recurrence rate was exactly the
same (13%) in each group. [110] This study, though controlled, presented some data which
may not be easily explained. For example, the rate of recurrence of 13% in the balanced salt
group was unexpected since this was much higher than that in other published studies using
conjunctival autograft in primary pterygium excision. [2] This unexpectedly high recurrence
may be related to the small sample size (15 subjects in each group).
In another prospective interventional controlled study, 54 eyes of 54 patients who had
bare sclera surgery were either given avastin eyedrops (5 mg/mL) twice a day and
betamethasone eyedrops 4 times a day for one week (26 eyes) or only betamethasone
eyedrops 4 times a day (28 patients). The follow up examinations were conducted at 1 week,
3 weeks and 3 months. In this study all pterygia recurred but it was noted that the onset of
invasion was slower and there was less fibrous tissue observed in eyes treated with avastin
compared to control with only steroids. [36]
In summary, although there were promising early clinical reports of the use of anti-VEGF
agents in pterygium, only two controlled studies were published. The results of the controlled
experiments were disappointing and suggested that avastin is definitely not a replacement for
conjunctival autograft surgery. More studies are required to examine if anti-VEGF treatment
is a useful adjunct to standard pterygium excision with conjunctival autograft surgery.
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Ephrin and Eph Receptors
Eph receptors represent the largest family of endothelial cell receptor tyrosine kinases
(RTKs) known to play a role in central nervous system as axon guidance molecules. [99] The
ephrin-eph system has also been found to be involved in tumour and ocular angiogenesis.
[85, 135]
In a recent study involving 9 males and 19 females (aged 41 to 72) for excised pterygium
tissues and 2 males and 9 females (aged 21 to 68) for normal conjunctival tissues, it was
found that ephrinB2 and its receptor EphB4 were overexpressed in pterygia.
Immunohistochemical staining found ephrinB2 and ephB4 only in the basal epithelial layer
of normal conjunctival tissue and in contrast, all epithelial layers of the head of pterygium
tissue. In combination with the known relationships between Ephrin and ocular angiogenesis,
[85, 135] the results provide some evidence for the involvement of ephrin and its receptors in
the development of pterygium and related vascular events.[151]
5. Metalloproteinases and Their Natural

Tissue Inhibitors
MMPs are a family of zinc- and calcium-dependent proteinases, and play an important
role in the development of pterygium and invasion of the cornea. These enzymes are key
effectors and regulators of a variety of physiological and pathological processes such as
tissue invasion, [94] cellular differentiation, [156] wound healing, [97] inflammation, [83]
cell-cell, cell-matrix signalling, [121] neovascularisation, [121] cell survival and programmed
cell death (apoptosis) [84, 91, 98]. Nagase et al. [91] have classified secreted and membrane-
associated MMPs as follows: collagenases (MMP-1, -8 and -13), gelatinases (MMP-2 and -
9), stromelysins (MMP-3 and -10), matrilysins (MMP-7, -11 and -26), membrane-type MMPs
(MT-MMP; MMP-14, -15, -16, -17, -24 and -25) and other MMPs (MMP-12, -19, -20, -21, -
23, -27 and -28).
The balance of proteolytic activity in tissues depends on the relative activities of MMPs
and their natural inhibitors. MMP inhibitors may be non-specific such as plasma
glycoprotein, α2-macroglobulin, or specific, ie., tissue inhibitors of MMP (TIMP) [12]. The
four different TIMPs (TIMP-1, -2, -3 and -4) are multifunctional molecules that not only
participate in MMP inhibition but also in cell proliferation [114], apoptosis [46],
differentiation [104] and angiogenesis [107] for example TIMP1 is principally a natural
inhibitor of MMPs, but it also participates in cell proliferation, [45, 114] and apoptosis. [71]
Most MMPs are secreted in an enzymatically inactive state, as pro-enzymes, which can
be activated in a proteolytic (for example by direct cleavage of the propeptide by another
MMP) or a non-proteolytic manner (for example organomercurials) via a cysteine switch
mechanism [44, 137, 140]. Imbalances in the spatial and temporal control of MMP
expression can cause MMPs to become destructive. Together, MMPs can degrade all extra-
cellular matrix (ECM) components and may contribute in matrix remodeling and Bowman‘s
layer destruction. The involvement of MMP s and tissues in pterygium development may be
similar to that established in tumor invasion and progression, which have been extensively
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reviewed previously. [52, 145] The role of MMPs in pterygia pathogenesis has also been
reviewed in detail previously. [17, 21]
Table 1. MMP and TIMP expression in normal conjunctival and pteryia tissue collected
from human
Expression of MMP
Normal Normal Pterygium Pterygia
MMP References Method
conjunctival conjunctival fibroblasts
epithelium fibroblasts
MMP-1 Di Girolamo 2000a IHC ND √
Di Girolamo 2000b IHC √ √
Li 2001 NB, NH, ELISA,
little √
WB
Dushku 2001 IHC ND ND √ √
Di Girolamo 2003 IHC little √
MMP-2 Di Girolamo 2000a IHC, GZ little/no √
Di Girolamo 2000b IHC √ little/no
Li 2001 NB, WB, GZ √ √
Dushku 2001 IHC ND ND
√ little/no
ISH ND ND
Yang 2009 RT-PCR little little √ √
GZ little little √ √
MMP-3 Di Girolamo 2000a IHC ND ND
Dushku 2001 IHC ND ND √ little/no
Li 2001 NB, NH, ELISA,
√ √
WB, CZ
MMP-7 Di Girolamo 2001 IHC little/no √
MMP-9 Di Girolamo 2000a IHC Only neutrophils √
Di Girolamo 2000b IHC √ √
Li 2001 ND ND
Duskhu 2001 IHC little/no little/no √ little/no
Chintala 2005 IHC, GZ, WB ND
Yang 2009 RT-PCR ND little/no
GZ ND little/no
Tsai 2010 IHC ND √
MMP-10 Tsai 2010 IHC ND √
MMP-13 Di Girolamo 2000b IHC √√
TIMP-1 Di Girolamo 2000a IHC ND √
Di Girolamo 2000b IHC
Li 2001
Tsai 2010 IHC, WB √ √

TIMP-2 Di Girolamo 2000a IHC ND ND
Di Girolamo 2000b IHC little/no √
Li 2001 NB, WB √ √
TIMP-3 Di Girolamo 2000a IHC little/no ND √ √
TIMP-4 Di Girolamo 2000a IHC ND ND ND ND
Methods: ELISA (enzyme-linked immunosorbent assay), CZ (casein zymography), GZ (gelatine
zymography), IHC (immunohistochemistry), ISH (in situ hybridization), NB (northern blotting), NH
(northern hybridization), RT-PCR (real time PCR), WB (western blotting), ND – not detected, √ -
detected, empty fields – not tested.
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Transcripts and proteins of MMPs and TIMPs were detected in pterygium, summarized
in Table 1. Production sites of MMPs and TIMPs include pterygium epithelial cells and
fibroblasts. [25, 27, 32, 160] Inflammatory cells, often found in pterygium, also contribute
significantly to MMP production. [25-26] For example, MMP-9 was mainly localized in
neutrophils [25] and an accumulation of inflammatory cells in advanced-stage pterygium
tissue may explain increased production of this enzyme. [155] The role of inflammation in
MMP expression will be discussed in section 6. Little MMP/TIMP expression could be
localized in normal conjunctival tissue by immunohistochemistry, whilst strong
immunoreactivity was noted in pterygium head and fibroblasts, particularly for MMP-1. [25,
27] Table 1 also shows the expression of specific TIMPs in pterygium. TIMP-1 and MMP-1
expression have been found to increase in parallel, [25] and their inherently antagonistic
actions may explain the slow growth and invasion of pterygia. Expression of MMPs in tears
obtained from patients with pterygium has also been described. [162 27] Compared to tears
collected from normal controls, such tears had an increase in MMP-9 and MMP-9/TIMP
complex. [27]
In a study investigating a range of proteolytic enzymes and inhibitors, an overexpression
of MMP-1 and MMP-3 has been described in cultured pterygium head fibroblasts. [72]
Expression of MMP-9 in pterygium was somewhat controversial and its levels may depend
on the severity of pterygium. MMP-9 mRNA and protein were not detected in early-stage
pterygium whereas advanced stages expressed MMP-9 in tissues and fibroblasts, as analyzed
by gelatin zymography and RT-PCR [155] It should be noted, however, that Yang et al. [155]
utilized early passage fibroblasts for analysis and culturing itself may have partly contributed
to MMP-9 production in corneal fibroblasts. [37] Although studies performed in vitro with
cultured pterygium epithelial cells or fibroblasts are useful to explore MMP and TIMP
production, it is important to remember that the ocular surface is an interactive system
consisting of different cell types creating a microenvironment. Ocular surface components,
such as the tear film bathing and lubricating the eye, glands producing aqueous and lipids for
the tear film or conjunctival goblet cells excreting mucus cannot be mimicked by tissue
culture. Therefore, animal models represent a better option, as mentioned in section 1 [18].
It was proposed that any imbalance in the MMP/TIMP ratio may facilitate pterygium
growth [23, 72] and this theory was corroborated in a recent study investigating pterygium
epithelial cells silenced for endogenous TIMP-1 expression. [131] Recently, Tsai et al. [131
]revealed an important contribution of TIMP-1 in the development of the disease. The team
detected TIMP-1 in normal conjunctival tissue using immunohistochemistry and showed that
silencing TIMP-1 expression in pterygium epithelial cells resulted in a greater ability of cells
to migrate and invade tissue. [131] This is an important paper because it shows that TIMP-1
has a direct impact in the migration function of cells as determined by the Transwell assay
and the invasion of cells through a Matrigel coated Transwell assay. In terms of therapeutic
implications, TIMP-1 inhibition may present an exciting alternative therapy in development
of early pterygium.
The active forms of MMPs may contribute to the pterygium pathogenesis more than the
inactive forms. Attempts to identify activity of MMPs in pterygium tissue have therefore
been performed using zymography. This technique is highly sensitive, has the potential to
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differentiate between inactive and active forms of MMPs, and may semi-quantitatively
determine the amount of substrate degradation.
Casein zymography identified inactive MMP-3 enzymes in cultured pterygium head
fibroblasts. [72] Di Girolamo et al. [22] reported that normal conjunctival tissue contained
mainly inactive and little activated forms of MMP-7. In contrast, pterygium tissue contained
high levels of both inactive and active forms of this enzyme. MMP-7 was also detected in
supernatants collected from organ-cultured pterygium tissue. [22]
Not all forms of active MMPs can be detected or quantified using zymography. For
example, active MMP-9 may be mistakened for as inactive forms on zymography because the
molecular weight may not have changed from inactive MMP. Bannikov et al. [7]
demonstrated that proMMP-9 may be activated by binding to gelatin or collagen IV coated
surfaces without further processing of the propeptide. [7] This activation is due to a
conformational change of the protein structure during binding, which destabilises the
interaction between the propeptide and the active centre of the enzyme.7 Because of this
limitation of gelatin zymography, one has to exercise caution when interpreting studies which
find little active MMP-2 and MMP-9 in various cell culture media. [27, 72, 155] Together,
these data substantiate the idea that mainly active forms of MMPs are involved in the
degradation of extracellular matrix, which explains the invasion of the pterygium into the
Bowman‘s layer of the cornea.
6. Inflammation
Since the last major review on molecular mechanisms of pterygium, [17] recent studies
have implicated the involvement of immune cell infiltration, E cadherin epigenetics, S100
proteins, pololike kinases, phospholipase D, Trefoil factors and the NFkappa B (NFκB )
signaling pathways in pterygium (Figure 1). These topics will be addressed in the sections to
follow.
Inflammatory Cells and Pterygium
Several studies have pointed to an immune response-inflammatory component in

pterygium pathology. Previous studies suggest involvement of human histocompatibility
complex human leucocyte receptor (HLA)-DR antigen in pterygium [9, 53, 132] and HLA-
DR is generally presumed to be a marker of inflammation in the conjunctiva. In addition, the
invasion of T lymphocyte populations in pterygium has also been observed. [9, 53] A great
difficulty in the assessment of the role of inflammation in pterygium formation is due to the
cross-sectional design of the studies. It may be that inflammatory infiltrates emerge in
pterygial tissues in some cases after the formation of the pterygium. It is well known that
pterygium and pinguecula, a yellowish white lesion found on bulbar conjunctiva, could
contribute to dry eye syndrome, since they affect the spreading and stability of the tears in the
vicinity of the lesions. As dry eye is an immune mediated condition93 with infiltration of
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immune cells in the conjunctiva, the presence of lymphocytes in pterygium may be a

secondary phenomenon.
A more recent paper5 has confirmed that there was increased infiltration of CD4 and CD8
positive lymphocytes (in equal proportions) in pterygium tissue compared to un-involved
conjunctival tissue. The immunostaining for lymphocytes was performed in 16 primary
pterygium specimens, compared to only one control conjunctival tissue. Based on the clinical
signs of vascularity, conjunctival redness, congestion, oedema and thickness the primary
lesions were classified into mild/moderate (2 tissues), moderate (12 tissues), moderate/severe
(1 tissue) and severe (1 tissue). There was no significant difference in the extent of
lymphocytic infiltration between pterygia tissues of different clinical severity or differences
in use of anti-inflammatory medication. However, the sample size was small and there were
differences in the type of anti-inflammatory medications (ranging from prednisolone to
naphazoline to olopatadine) as well as the duration of usage.
One prospective randomized study has previously found a decrease in the clinical signs
and symptoms after 14 days of treatment of pterygium with indomethacin (a non-steroid anti-
inflammatory drug) or dexamethasone (a glucocorticoid) topically. [38] The study was
performed in 17 patients with inflamed pterygia and 33 patients with pingueculae. However
the symptoms evaluated in that study were also commonly experienced in dry eye syndrome,
namely photophobia, pain, foreign-body sensation, discomfort, and tearing, and the signs
reported were also related to those present in dry eye: conjunctival congestion, redness and
edema, and staining of cornea. Therefore, whilst these treatments are able to address dry eye
and inflammation, there was no evidence that they are targeting the primary pathology in
pterygium. In other words, the data from this paper do not show that pterygium is an
inflammatory condition to begin with.
Ultraviolet Radiation and Inflammation
Given that ultraviolet (UV) radiation is a major clinical risk factor for pterygium (Figure
2), it is tempting to suggest that this initiates a cascade of events that lead to pterygium
formation. In the previous section, we discussed the importance of MMPs. [23] The effect of
UV on MMPs and other stress signaling pathways will be discussed in the sections below.
UV is also known to play a role in the formation of reactive oxygen species (ROS) and
consequently the increase in oxidative stress in pterygium. ROS accumulation can induce
further molecular changes such as that of cyclo-oxygenase (COX)-2. [87] The family of COX
enzymes can convert arachidonic acid into prostaglandins, [49] many of which are pro-
inflammatory. Hence, upregulation of COX-2 may, at least in theory, contribute to

inflammation in pterygium. In the same way, COX-2 is known to play a role in UV-induced
carcinomas in the skin. [112]
In one study COX-2 was detected by immunohistochemistry in 63/93 primary pterygium
samples but not in any of the normal conjunctiva samples. [87] The 10 normal conjunctival
samples were from the medial bulbar conjunctiva of patients who underwent cataract surgery.
The mean age of the control group (52.1 years) was older than the pterygium group (43.4
years), so there is a possibility that the effect of age on COX expression may confound the
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reported results. The COX expression was localized to the basal and mid suprabasal layers of
the pterygium epithelium and in the vascular endothelial cells. It is intriguing that the
expression of survivin, a member of the inhibitor of apoptosis protein family (IAPs) was
correlated to the expression of COX-2 in pterygium. This led the authors to propose that
COX-2 overexpression leads to a resistance to UV-induced apoptosis via expression of
survivin, a situation also found in basal cell carcinoma of the skin. [127] Reduced apoptosis
may then lead to uncontrolled epithelial cells to form a fibroblastic growth in pterygium. In
this hypothesis it is not clear if COX-2 plays a role in the progression of some types of
pterygium or whether it is involved in triggering the lesion in the first place. Since this study
[87] used only the pterygium head tissues, it is not known if COX-2 is expressed in cases of
pinguecula or very early cases of pterygium.
Excessive exposure to UV light has been suggested to cause an induction of MMPs.
[147] This concept has been confirmed in pterygium-derived epithelial cells by Di Girolamo
et al. [21, 23] Pterygium cells were treated with different levels of UV-B irradiation and
differing time periods of up to 3 days. Data obtained by gelatin zymography and ELISA
revealed expression of MMP-1 and MMP-3 in a dose- and time-dependent fashion while no
modulation was achieved for MMP-2 and MMP-9. TIMP-1, 2, 3 and 4 appeared to be
relatively unaltered after UV exposure as shown by RT-PCR and reverse zymography, a
method for detection of TIMP. Similarly, limbal epithelial cells, but not corneal epithelial
cells, responded to UV-B irradiation with enhanced expression of MMP-1. This increased
proteolytic enzyme activity may not be sufficiently inhibited by TIMPs resulting in
conditions that may promote invasion of the cornea by diseased cells. However, currently
published studies investigated single dosed UV-B exposure exclusively. It is necessary to
investigate multiple doses of UV exposure and spectra that may trigger expression of MMPs
and TIMPs.
UV-exposed pterygium epithelial cells were also found to generate proinflammatory
cytokines, such as interleukin (IL)-6 and IL-8, but not IL-1α and tumor necrosis factor
(TNF)-α.24 In skin, however, UV radiation induces IL-1 [21] which in turn can activate other
cytokines or MMPs. The expression of MMP and TIMP can be modulated by cytokine
treatment in cultured pterygium epithelial cells. Specifically, treatment with a combination of
IL- 1α and TNF-α, but not IL-6 and IL-8, increased production of MMP-1, MMP-2 and
TIMP-1, while TIMP-3 expression remained unchanged. [23, 25] The late induction of
MMP-1 by UV light implies that this enzyme is only a secondary messenger. [23]
Inflammation increases the calibre of the vasculature, with a consequent increase in
blood flow and influx of, for instance, plasma proteins and leukocytes. [63] Increased
expression of MMP-9 associated with inflammation has been measured in tears from patients
with dysfunctional tear syndrome [16] or dry eye symptoms. [113] Apart from the role of
MMPs in matrix degradation, a view is emerging that MMPs may also be an important
modulator for inflammation by regulating chemokines and cytokines. [83, 98] MMPs have
been shown to activate chemokines, such as monocyte chemoattractant protein-3, [89] growth
factors and cytokines, such as transforming growth factor (TGF)-ß [159] or IL-1 [118].
Conversely, cytokines also have the ability to modulate MMP expression. Studies found that
IL-1, TNF-α and TGF-ß could induce MMP expression in primary cultures of human
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epithelial cells, [43, 72] primary cultures of human keratocytes [42] or mouse models of
Pseudomonas aeruginosa. [152]
Figure 3. The epithelial markers may decrease and mesenchymal markers increase in the process of epithelial
mesenchymal transition. For example, the hallmark of conjunctival epithelial cells (left column) are the
desmoplakin, tight junction proteins such as occludins and specialized secretory proteins such as mucin-1.
These proteins may be disrupted or downregulated during pterygium formation. When there is disruption of
cell:cell contacts, the affected cells become more fibroblastic or mesenchymal (right column). This may be a
key process in pterygium formation.
7. Cell Transformation and Wound Remodeling
In a recent review of pterygium, the role of epithelial-mesenchymal transition (EMT) has

been proposed. [17] Abnormal stimulation of epithelial cells may result in the loss of
epithelial characteristics, resulting in a differential pattern of gene expression due to action of
specific transcription factors such as TCF and LEF, and the consequential development of a
fibroblastic phenotype (Figure 3). These abnormal fibroblasts can elaborate matrix which
result in the abnormal deposition of stroma in pterygium. As the pathology of pterygium
lesion progresses centripetally from the periphery, one would expect differences in the spatial
expression of phenotypic markers relevant to pterygium formation. For example, at the
pterygium head, there may be abnormal epithelial cells that possess some mesenchymal
markers, whilst not fully transformed into fibroblasts. The following section begins with a
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discussion on differentiation markers in different parts of pterygium and how this may be
related to a stem cell disorder.
Spatial Differences in the Phenotype of Pterygium Epithelial Cells
Recently, Bai et al. reported an interesting study concerning the spatial localization of
stem cell markers in different parts of pterygium tissue [6] Surgically excised pterygium
specimens were dissected into head, neck and body. Using immunofluorescence, the authors
reported that p63α, a molecule involved in regulation of epithelial maturation, was strongly
expressed in the basal epithelia of the head, neck and body of pterygium. However, a
differential pattern of expression was found in the para-basal (adjacent to basal) layers of the
pterygium epithelium, in that the expression of p63α was strong in the head and body but
very weak in the neck of the pterygium. Interestingly, when pterygium epithelial cells were
separately cultured from the 3 different parts of pterygium tissue, they exhibited colony-
forming efficiencies in a manner that mirrored the intensity of the para-basal epithelial p63α
expression. In other words, the percentage of colonies obtained per number of seeded cells
was high for epithelial cells from the head and body, and in particular from the head of the
pterygium, but low for those from the neck of the pterygium.
Although previous localization of p63 in primary and recurrent pterygium has been
reported, [109, 115] neither of these earlier studies has highlighted spatial differences in the
expression of p63. In this way, Bai‘s findings6 were particularly important, highlighting that
spatial differences in differentiation may shed light on the formation of pterygium.
The Pax6 molecule is a master control for ocular morphogenesis and required for proper
maturation of corneal epithelium. [30, 73, 108] Bai et al. [6] detected this protein in full
thickness of the epithelium in head of pterygium but only in the superficial layers of
epithelium in the pterygium neck and body. MMP-2 and MMP-9 were immunolabeled in the
basal lamina of the head of the pterygium but only very weakly over the pterygium epithelia
of the neck and body. [6] Western blots showed that the detection of MMP-9 protein was
increased in the pterygium head compared to the body. These findings further implied an
abnormal differentiation process in the head of the pterygium, and that the destruction or
dissolution of the basal lamina may occur preferentially at the head of the pterygium.
The authors [6] showed in scratch tests that cells growing in the presence of recombinant
active MMP-2 and MMP-9, migrated faster compared to those incubated in anti-MMP-2 and
MMP-9. Even though MMPs have previously been linked to invasion in tumors, [145] Bai‘s
[6] data cannot be easily interpreted. This is because data from critical controls were not
reported. The data shown included cells grown in 10% fetal bovine serum (FBS), and in 0.2%
FBS with anti-MMP but did not include cells grown in 0.2% FBS without the anti-MMP. In
addition, instead of comparing cells growing on active MMP to those exposed to anti-MMP,
it would be more useful to compare cells growing on active MMP to those growing on active
MMP but incubated with anti-MMP.
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Figure 4. Differentiation disorder in pterygium formation. Top: The sequence of events that may result in
differentiation defects. Bottom: The localization of differentiation defects to the different parts of the
pterygium (body, neck and head).
Despite the above shortcomings, Bai et al‘s findings [6] suggest that limbal stem cells
either were destroyed in ptergyium, or had migrated out from the limbus to a different
location, leaving non-proliferative cells at the limbus (Figure 4A). The authors emphasized
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that the stem cell failure is the critical factor in the pterygium formation, not the extensive
active proliferation of surrounding conjunctival epithelial cells. Had the active proliferation
and movement of conjunctiva be the primary pathology, they argued that a lower proliferative
tendency should not be observed in the epithelium of the pterygium neck. The data imply that
actively proliferating cells in the head of the pterygium may drive progression of the lesion
(Figure 4B). Supporting this hypothesis is the previous observation of altered vimentin-
expressing limbal stem cells in the pterygium head. [33]
In this hypothesis, the destruction or anomaly at the limbus will be likely due to UV-
induced damage. [19, 68] Apart from the inflammation induced by light-generated ROS,
inflammation can also be propagated by ECM degradation. Since MMPs degrade ECM, the
destruction of the latter may be associated with release of cytokines (e.g., VEGF and basic
fibroblast growth factor) which will increase inflammation and angiogenesis and the other
observed processes in pterygium. Many issues remain unexplored. For example, the role of
the MMPs expressed by the pterygium fibroblasts was not explained, and how the
proliferation and migration of pterygium epithelial cells are related to the fibroblastic changes
was not evaluated in this paper. [6]
Matrix Remodeling
Apart from the cellular (epithelial and fibroblast) component, pterygium tissue also has
prominent stromal tissue. Previous studies have shown the derangement of elastic fibers in
the subepithelial stroma of the body of the pterygium, in particular, abnormal maturation of
elastic fibers may eventually lead to degenerative change or elastodystrophy [4, 142] This
process of accumulation of abnormal elastic fibers has also been reported in sun-damaged
skin, [90] and so it is tempting to suggest that the role of UV light in pterygium may be
related to the pathology of elastic fiber in this disease.
A recent paper by Perez-Rico et al. [105] reported the increased presence of immature
collagen III in the subepithelial areas of pterygium compared to normal conjunctival
specimens. This was demonstrated by Sirius red staining and polarized light microscopy. In
addition, increased tropoelastin (monomeric elastin) mRNA and protein were reported in
pterygium tissue. Fibulin-2 and -3 were localized to the subepithelial stroma of pterygium, as
well as along the blood vessel walls and lymphatics. Fibulins were involved in the molecular
organization of the corneal stroma and they tend to be located around corneal fibroblasts.
[31] They may have a role in stabilizing the ECM, and play a role in the stromal changes in
pterygium. Several questions remain, especially concerning the role of elastin in the head of
the pterygium, which was not investigated in this study.
E-Cadherin Epigenetics
Epithelial type (E)-cadherins are important components of the cell-cell junctions or cell
adhesion molecules in epithelial tissue. [136] Dysregulation of E-cadherin may disrupt
epithelial phenotypes and induce abnormal cell signaling events. Major evidence for the
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involvement of epigenetics in pterygium is reported by Young et al in 2010. [157] This paper

shows the interesting finding that hypermethylation of the promoter of E-cadherin was
associated with pterygium. Repression of E-cadherin gene by promoter hypermethylation
may cause loss of contact inhibition or activation of Wnt pathway in conjunctival epithelial
cells. If this is the event that triggers the transformation of conjunctival epithelial cells to
fibroblasts, it may explain the subsequent events of EMT as previously reported by Kato et
al. [59]
The epigenetics study [157] was performed in 120 participants from Taiwan, which may
not be comparable to Caucasians, such as those in Australian studies [23, 25, 28] in genetic
background. Thirty control tissues from un-involved individuals without pterygium were
used for comparison. There were however, several limitations in the study [157] which will
be discussed below.
First, the participants are relatively old (pterygium: mean of 62.5 years, controls: mean of
62.8 years). From epidemiology studies, we are aware that a large proportion of subjects are
younger than 50 years old, with a mean of 36.6 years (SD 13.1). [40] In other words, we do
not know if the hypermethylation phenomenon applies to the more common pterygia in
younger people.
Second, there was no indication of whether the hypermethylation phenomenon is a cause
or effect of pterygium formation, as the study was performed entirely in a cross sectional
design. After the onset of EMT, lymphocyte-enhancer factor (LEF) and T-cell factor (TCF)
transcription factors may alter the function of DNA methyltransferases to affect methylation.
Therefore it is possible that the onset of hypermethylation is consequential to pterygium
formation. To resolve this issue, one may hypermethylate the E-cadherin promoter and
evaluate the subsequent profile of the EMT target genes or other downstream effects in
cultured epithelial cells.
Third, the results of the study [157] were not adjusted for total UV exposures which can
be calculated for each participant. It is possible that environmental factors such as UV may
affect epigenetic events. The authors did not consider that UV exposure may be directly
linked to the hypermethylation events. In fact, longer hours of UV exposure in people without
pterygium can possibly be associated with increased hypermethylation of certain promoters.
For example, the synthesis of vitamin D may depend on sun exposure, [29] and the level of
vitamin D3 may affect demethylation of promoters such as osteocalcin in osteosarcoma cells.
[47] Furthermore, the activity of the DNA methyltransferases (Dnmt)1, Dnmt3a and Dnmt3b
were elevated in sun-exposed skin and sun-induced tumors. [92] On the other hand,
hypermethylation of promoters is quite rare after UV exposure, compared to many types of
UV-induced genetic events. [134]
Interleukin 4
The role of many cytokines has already been mentioned in previous reviews. [17, 21]
Here, we focus on more recent developments after publication of these articles.
One such area of research involves IL-4, a Th2 type cytokine that can induce changes in
integrin adhesion molecules or matrix components in carcinoma of the colon. [50]
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Transcripts of this cytokine have been found to be elevated in recurrent pterygium. [64] In
this study, recombinant IL-4 stimulated fibroblasts derived from the head of pterygia were
investigated. Induction of periostin upregulation was observed in an IL-4 dose dependent
fashion. Periostin was noted to play a role in the adhesion of fibroblasts to fibronectin, since
treatment with anti-periostin reduced the fibronectin-mediated fibroblast adhesion. This assay
was performed using Accutase, a proprietary cell detachment solution containing
collagenases and proteases.
Gene Expression Studies in Epithelial-Mesenchymal Transition
Unlike the above studies, not all published studies support the phenomenon of EMT in
pterygium. A gene expression analysis [56] carried out to identify the transcriptional
repertoire of pterygium did not find abundant expression of genes associated with EMT.
Nevertheless, genes associated with EMT were found in a pterygium cDNA library, including
S100A4, bone morphogenetic protein 7, heterogenous nuclear ribonecleoprotein A/B and
alanine-glyoxylate aminotransferase 2-like 2 and LGALS3/galactin-3. In this study, 15
specimens of pterygium tissues were pooled before reverse transcription and cloning into the
pCMVSPORT6 vector to make a cDNA library. The cloning was performed into NotI and
SalI endorestriction sites of the vector. The authors also noted that 29 out of 51 genes in the
pterygium cDNA library were associated with cell migration, including spermidine/spermine
N1-acetyltransferase (SAT) 1, a regulator of polyamines, Annexin A2 and S100A9.
It is assumed that the frequency of clones containing a particular gene coding sequence
reflect the abundance of the transcript for that gene. However, longer genes and transcripts
may also have a higher probability of producing more clones because of the increased
likelihood of encountering NotI and SalI sites. Secondly, when 3000 clones were randomly
selected for sequencing, some of the more prevalent transcripts may be missed. Thirdly,
pooling of samples from 15 patients may cause loss of important information. The authors did
not provide clinical/demographic information of these patients and therefore we do not know
how representative they are.
An Affymetrix gene chip (HG_U95Av2) representing more than 10000 genes showed
that fibronectin, collagen type III and versican were upregulated 9.0, 4.1 and 3.3 times
respectively. [57] However, this study only utilized 2 primary pterygium tissues, 1 recurrent
pterygium tissue and 3 superior conjunctiva samples and the small number of samples may
not be sufficiently representative of pterygium in general.
8. Cell Cycling/Apoptosis
Cell Cycling
In the sections above, we mentioned the role of extracellular matrix and remodeling as
possible mediators in the development of pterygium. The alteration of the steady state of
proliferating cells may be related to intrinsic cell cycling progression defects or to a reduced
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proportion of cells exiting the cell cycle (such as apoptotic cells). A study conducted by
Karukonda et al. [58] compared pterygium and normal conjunctival tissues from different
latitudes using flow cytometry. Interestingly, the study showed greater proliferation rates in
the entire ocular surface in patients living closer to the equator, but no significant difference
in cellular proliferation rates between pterygium and conjunctiva tissues were identified. [58]
In contrast, Tan et al. [125] showed significantly increased proliferation rates in pterygium
subepithelial fibrovascular tissue compared to matched conjunctival fibrovascular tissue
using DNA flow cytometry. Highest fibroblast proliferation rates were identified in recurrent
pterygium. [125] Inconsistent results found in both studies may be explained by the use of
different controls. Karukonda et al. [58] utilized normal conjunctival tissue adjacent to
pterygium tissue whereas Tan et al. [125] collected conjunctival samples from the superior
aspect of the conjunctiva which is protected from UV exposure by the eyelid.
Insulin-Like Growth Factor Binding Protein 3
The insulin-like growth factor binding protein (IGFBP) family has been described as
regulators of cell division in tumors and various cell types. [158] IGFBP-3, a member of this
family, can induce apoptosis by the nucleomitochondrial translocation of nuclear receptor
subfamily 4, group A, member 1 (NR4A1). [71] Recently, our group reported that IGFBP-3
was significantly down-regulated in pterygium tissue compared to normal conjunctiva. In
addition, immunohistochemistry of IGFBP-3 in pterygium and paired conjunctiva, as well as
western blots in paired tissue showed that reduced IGFBP-3 protein occurs in pterygium.
[148] The factors that regulate the expression of IGFBP-3 in pterygium remain unknown but
the known functions of IGFBP family in cancers suggest that IGFBP-3 down-regulation
could lead to hyperproliferative changes or defective apoptosis in pterygium. [148] In
contrast, in a cell culture experiment involving pterygium fibroblasts, IGFBP-2 was found to
be up-regulated. [122] The difference in the results between the two studies could be due to
differential regulation of IGFBPs in different cell types within pterygium tissue. Our group
reported that down-regulation was found primarily in the pterygium epithelium. [148]
Trefoil Factor 1
Trefoil Factor (TFF) 1 is a protease-resistant protein commonly found in tumours and

known to inhibit apoptosis. They are also secreted by mucin-secreting epithelial cells and
help to stabilize mucin. [11, 128]

In the epidemiology section above, UV has been discussed as a strong risk factor for
pterygium. The inflammation section contained a discussion of UV and ROS production and
the formation of ROS could trigger cell death via oxidative DNA damage. [106] If there is
significant DNA damage in this process, there would not be sufficient cells to form a tumour
or pathological lesion such as pterygium. However, if certain cells are protected from UV-
induced death, then these cells may form the basis of an enlarging and proliferating lesion.
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It has been found that the expression of TFF1 protects against cell death. [11] In a
previous report, TFF1 was found to be up-regulated in pterygium. [66] Recently, this cell
protection phenomenon has been reported in the context of UV-activated caspase 8-mediated
cell death in Chang conjunctival cells, [14] which is an immortalized conjunctival epithelial
cell line. The mechanism of cell protection also involves TFF1 upregulating X-linked
inhibitor of apoptosis protein (XIAP). [14] The signaling molecule IκB- α is a key inhibitor in
the NFκB pathway (see ―stress pathways‖ below). The protective effect of TFF1 is dependent
on functional IκB-α, since TFF1 did not produce protective effects on Chang cells with
mutated IκB-α.
Such apoptosis-suppression pathways may also be coupled with regulation of cell
migration [62] thereby performing a dual role in pterygium formation. The main limitation of
these studies is the fact that Chang conjunctival cells may not be representative of native
conjunctival epithelial cells in their response to disease processes.
9. Stress Related Signaling
S100 Proteins
The S100 protein family is a group of low molecular weight, calcium binding proteins
that are encoded within the epidermal differentiation gene complex on chromosome 1q21 in
humans. The roles of various S100 proteins in skin diseases and tumors have been previously
discussed. [35] Our laboratory has found protein levels of S100A4, A6, A8, A9 and A11 to
be elevated in pterygium compared to un-involved conjunctiva. In addition, the transcript
levels of S100A6, A8 and A9 were elevated in pterygium compared to controls. [111] The
advantages of this study include the fact that the control tissues were obtained from the same
eye as the pterygium during the surgical excision. Interestingly, the S100 proteins were also
detectable in tears of patients who had undergone pterygium surgery, using nanoLC-nano-
ESI-MS/MS. [162]
The S100 proteins could be involved in the pathology of pterygium in several ways.
Firstly S100A4 and A6 may be involved with fibroblast function and abnormal wound
healing. Secondly, S100A4 may stimulate abnormal cell proliferation as in epithelial tumors,
and lastly, S100A8 and A9 may be proinflammatory due to their ability to induce leukocyte
chemotaxis. [35]
This group of proteins are known substrates of transglutaminase (TGM) enzymes which
are intra- and extracellular enzymes that can covalently cross-link peptides. [76] One member
of this family is the ubiquitously present TGM2, which has been reported to be strongly
expressed in pterygia, especially in the basal epithelium on the dissoluted Bowman‘s layer
and fibrous tissue. [61] Mechanistically, TGM2 could exert certain actions in the cellular
matrix which are relevant to pterygium pathology. For example, cell surface TGM2 can bind
to fibronectin, fibrin and type I collagen, or released from injured cells to interact with these
molecules.[1, 76, 138-139]
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NF Kappa B Signaling
The NFκB pathway is an important signaling pathway that controls proliferation,

differentiation and migration of cells. In un-stimulated cells, the cytosolic NFκB components
are bound to inhibitors such as the IκBα and remain inactive. When cells are stimulated by a
variety of stimuli, the inhibitors of NFκB became phosphorylated and degraded by the
proteasome system. This resulted in the cytosol to nuclear translocation of NFκB. In the
nucleus, the NFκB can bind to various target gene promoters and regulate the transcription of
various genes. Through these mechanisms, this pathway can regulate the expression of more
than 200 genes. [67]
We recently published evidence that the NFκB pathway may be differentially activated in
pterygium. [120] Phosphorylation of the inhibitor of the NFκB, the IκBα, was increased in
excised pterygia tissues compared to un-involved conjunctiva tissues. In addition the nuclear
binding activity of NFκB (RelA and p50) was increased in pterygium. In that study, the roles
of the classical and non-canonical NFκB pathways were also investigated. Fibroblasts
derived from pterygium showed increased phosphorylation of IκBα and nuclear translocation
of RelA and p50 in a short time after stimulation with TNF-α. This was consistent with the
activation of the classical pathway. Prolonged stimulation by TNF-α however, resulted in the
increased nuclear localization of RelB, p100 and p52, which indicated the activation of the
alternate NFκB pathway. We also reported that upregulation of Regulated on Activation
Normal T Cell Expressed and Secreted (RANTES), Monocyte Chemoattractant Protein
(MCP)-1, Epithelial neutrophil activating peptide (ENA)-78 and MMP-1, 2 and 3 were
greater in TNF-α treated pterygium fibroblasts than in Tenon fibroblasts. It is well known that
inflammatory signals typically activate the classical NFκB pathway. It is uncertain whether
the non-canonical pathway can play a role in the induction of EMT, given that the non-
canonical pathway is normally concerned with developmental signals. If pro-inflammatory
stimuli can also affect the non-canonical pathway and consequently EMT, this may be a
possible mechanism for formation of pterygium. More studies are required to investigate the
roles of other regulatory pathways in the induction of inflammatory genes such as RANTES.
Phospholipase D
The phospholipase (PL) D is a family of enzymes that catalyses the metabolism of

phosphatidylcholine to produce phosphatidic acid, a precursor of various inflammatory
molecules. PLD 2, 3 and 4 transcripts were upregulated in pterygium. [129] PLD1/2 was
localized in the nucleus of basal epithelial cells in un-involved conjunctiva, but in pterygium
epithelial cells, this was diffusely localized in the cell periphery and cytoplasm. Such findings
are interesting because the subcellular trafficking of PLD2 between the nuclei and cytosol has
previously been linked to regulation of cell motility. Increased epithelial cell motility may be
a contributing factor in the progression of pterygium in the centripetal direction.
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Polo-Like Kinase Signaling
The Polo-like kinase (PLK) 1 is a member of the serine/threonine kinase that is known to
be involved in mitotic control in cancerous cells, [34] including hepatoblastomas, [153]
papillary carcinomas, [55] and gastric carcinomas. [144] Recently a research group has
reported that PLK-1 mRNA was upregulated in pterygium compared to normal conjunctiva.
[77] This study utilized 16 cases of ptergyium (13 primary and 3 recurrent) with a mean age
of 54.2 years. Thirteen cases of normal conjunctiva were obtained from donor eyes for
corneal transplantation, who had similar mean age of 54.5 years. The authors did not recruit
patients younger than 35 years old for the expression of PLK in pterygium, and most
importantly, the level of PLK protein or activity were not evaluated. Since many of the
kinases are regulated by phosphorylation and not by de novo synthesis, the significance of
this study in the pathogenesis of pterygium is currently unknown.
10. Metabolism
The discussion has so far focused on stress signaling. There has been evidence that
metabolic pathways such as lipid pathways may also be deranged. In a big population-based
study in Singapore (n = 3280), total cholesterol was significantly associated with presence of
pterygium. In a multiple logistic regression model, after adjustments for age, sex, cigarette
smoking, systolic blood pressure, education and occupation, the fourth quartile of serum
cholesterol level versus first quartile was associated with pterygium (odds ratio=2.2; 95%
Confidence Interval 1.1, 4.5). [15]
It is unclear how cholesterol metabolism is related to pterygium formation. However,
abnormal intracellular cholesterol homeostasis has been observed in human pterygium.
Previous experiments showed that primary pterygium fibroblasts have dramatically increased
intracellular cholesterol metabolism compared to normal conjunctiva. [101] The low-density
lipoprotein receptor (LDL-R) and hydroxyl-methylglutaryl-coenzyme A-reductase (HMG-
CoA-R), two genes involved in cholesterol metabolism, are increased at the mRNA level in
pterygium tissues. [102] The HMG-CoA-R is a rate-controlling enzyme of the mevalonate
pathway, the metabolic pathway that produces cholesterol and other isoprenoids. Because of
the nature of this pathway, any substance that inhibits HMG-CoA-R will increase liver LDL-
receptors and plasma LDL. [10]
Peiretti et al. [101] showed that pterygial fibroblasts have accumulation of cholesterol
esters more than conjunctiva fibroblasts. The authors performed a series of experiments that
showed when cell proliferation inhibitors (pioglitazone and everolimus) were added to
pterygial fibroblasts, the presence of cholesterol esters were reduced intracellularly. These
inhibitors affect the handling of cholesterol by the cells, since they induced some lipid
enzymes to increase (acetyl-CoA acetyltransferase 1, ATP-binding cassette sub-family B
member 1) but suppress other lipid enzymes (ATP-binding cassette, sub-family A1 and
neutral cholesterol ester hydrolase 1). These findings were suggestive, but not conclusive,
that cholesterol esters mediate the proliferative function of pterygial fibroblasts. In the study,
the same inhibitors have not been added to normal conjunctiva cells to observe for
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cholesterol ester accumulation. Secondly the effects of these inhibitors on the lipid enzymes
have not been studied in conjunctival cells or other kinds of abnormal fibroblasts, for
example, renal fibroblasts. Therefore, the relevance of this mechanism, when applied to
pterygium pathogenesis, is uncertain.
Another metabolic enzyme found to be deranged in pterygium is the glucose-6-phosphate
dehydrogenase (G6PD) enzyme. This enzyme, active in the pentose phosphate pathway, is
critical in converting glucose-6-phosphate to 6-phosphoglucono-δ-lactone. It is important for
energy metabolism by maintaining supply of the coenzyme nicotinamide adenine
dinucleotide phosphate (NADPH), which in turn is involved in maintaining reduced
glutathione. In the context of insufficient reduced glutathione, cells may not be able to protect
themselves from free radicals, such as in UV-induced oxidative damage. Recently, the
Peiretti et al group [103] studied G6PD activity in 123 pterygium patients and in 112 age-
matched control patients and found that the G6PD enzymatic defect may be a predisposing
factor for pterygium.
Conclusion
The research in pterygium is a rapidly expanding field and the increase in number of
molecules and mechanisms of interest over the last few years suggest that more specific
treatments may be available soon. For example, a single molecular therapy, the anti-VEGF
therapy, has already progressed to clinical trials. These potential new developments may be
used in the prevention of progression of early pterygium or as adjunctive treatments for
surgical excision of pterygium in the future. Clinicians and ocular surface researchers should
be kept abreast of new knowledge in the pathology of pterygium. In the light of rapid
development in the field, we expect that this review may need to be extensively revised in 2 -
3 years.
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Chapter II
Role of Prisms in the Management

of Horizontal Deviations
Rehab Rashad Kassem

Cairo University, Cairo, Egypt
Optics of Ophthalmic Prisms and Fresnel Optics
Ophthalmic Prisms
Prisms are important tools for the diagnosis and treatment of strabismus as they are used
to measure and neutralize ocular deviations (1,2).
A prism is essentially two angulated plane refracting surfaces. It displaces, as well as
deviates, light rays (Figure 1). The total angle of deviation is the sum of the deviations
produced at each of the two surfaces (3,4).
Figure 1. Ophthalmic prism. A prism has an apex and a base. A prism bends light towards the base of the
prism.
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Copyright © 2012. Nova Science Publishers, Inc. All rights reserved. May not be reproduced in any form without permission from the publisher, except fair uses permitted under 38 Rehab Rashad Kassem
A prism bends light towards the base of the prism (Figure 1). This occurs because light
has both particle and wave characteristics. As light passes through the prism, the part of the
light wave closest to the prism base has more prism to traverse than the part of the wave
closest to the apex. Light as a particle travels slower through the plastic prism than it does
through air, so light towards the base of the prism takes longer to exit than light traversing the
apex. The exit time differential causes the light to bend towards the base of the prism (1,3).
The power of a prism to bend light is measured in ―prism diopters‖ (3,4). The use of the
unit ―prism diopter‖ was first proposed by Prentice [5] and is defined as the amount of
displacement in centimeters of a light ray passing through the prism, measured 100 cm from
the prism (4). One prism diopter will shift light one centimeter at one meter or a displacement
of approximately ½ degree (1-3) (Figure 2).
Figure 2. Definition of "prism diopter".

Figure 3. Real image displaced towards the prism base. Virtual image observed towards the prism apex.
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Copyright © 2012. Nova Science Publishers, Inc. All rights reserved. May not be reproduced in any form without permission from the publisher, except fair uses permitted under Role of Prisms in the Management of Horizontal Deviations 39
When a prism is placed in the path of converging light rays, the rays will be bent towards
the base of the prism. Real images formed by these converging light rays also will be
displaced towards the base of the prism. If one looks through a prism at an object, the object
will appear to be displaced towards the apex of the prism. This displaced image is a virtual
image [3,4] (Figure 3).
Similarly, when a prism is placed in front of one eye, it moves the image off the fovea
causing a perceived image ―jump‖. The retinal image will shift towards the base of the prism,
but the perceived image jump is in the opposite direction, towards the apex of the prism (1,3).
To refixate on the shifted image, the eye will move in the direction of the apex of the prism,
thus aligning the fovea with the new image location (Figure 4). This is the basis for using
prisms to neutralize an ocular deviation (1).
Figure 4. Diagram shows the effect of a prism over one eye. a. Patient fixates on the object (star). b. A prism
is introduced & the image is displaced towards the base of the prism & off the fovea. The patient will
perceive the image to jump in the opposite direction; thus, a patient will perceive the image to jump in the
direction of the apex of the prism. c. Patient refixates in order to place the image on the fovea by rotating the
eye towards the apex of the prism.
Prisms can be used to optically neutralize or correct strabismus. A prism acts to change
the direction of the incoming image so the retinal images in each eye fall directly on the
fovea. Neutralization occurs when enough prism is placed in front of the eye so the two
foveas are aligned on the same object of regard. Prisms optically neutralize the deviation
even though the eye remains anatomically deviated (1).

Esotropia (ET) is an inturning strabismus with the image in the deviated eye falling nasal
to the fovea (Figure 5). Exotropia (XT), on the other hand, is an outward deviation with the
image falling temporal to the fovea in the deviated eye (2) (Figure 6). As a prism placed in
front of one eye will shift the retinal image towards the base of the prism, the rule for
neutralizing a deviation is to orient the prism so the apex is in the direction of the deviation.
For esotropia, the apex is directed nasally (Figure 7); for exotropia, the apex is directed
temporally (1,2) (Figure 8).
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Figure 5. Esotropia. Image falls nasal to fovea in esotropic left eye.

Figure 6. Exotropia. Image falls temporal to fovea in exotropic left eye.
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Figure 7. Prism placed apex in to neutralize an esotropia.

Figure 8. Prism placed apex out to neutralize an exotropia.
For neutralizing a strabismus, the prism can be placed over the deviated eye, the fixing
eye or split over both eyes. If a base-out prism is placed in front of the deviated eye of a
patient with ET, the retinal image shifts temporally towards the fovea. If the correct amount
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of prism is used, the retinal image will fall directly on the fovea of the deviated eye. The
deviation is thus optically neutralized by the prism even though the eye is still anatomically
deviated (1,2) (Figure 7).
On the other hand, if the prism is placed base-out in front of the fixing eye of a patient
with ET, the retinal image will be displaced temporally off the fovea of the fixing eye. The
fixing eye will see the image shift and will immediately rotate nasally to re-establish foveal
fixation. As the fixing eye rotates nasally, the deviated eye rotates temporally (because of
Herring‘s law), causing a version movement to one side. The previously deviated eye will,
therefore, become straight, while the fixing eye turns in. Consequently, when a base-out
prism is placed in front of the fixing eye, both eyes move in the same direction as the apex of
the prism and both foveas shift into alignment (1,2) (Figure 9).
Figure 9. Neutralization of an esotropia by placing the prism in front of the fixing eye. a. Esotropia with right
eye fixing. b. Prism is placed in front of the fixing eye (right eye), which displaces the image off the fovea of
the right eye. The right eye rotates to refixate the image and, because of Herring‘s law, both eyes rotate in
the direction of the apex of the prism. c. Deviation is neutralized, as the images are on both foveas.
The prismatic deviation of a given ophthalmic prism is based on the angular position of
the prism as it is held before the patient‘s eye (5,6). Accordingly, the amount of ocular
deviation produced or neutralized by a prism varies according to the position of the prism as
it is held before the patient‘s eye. There are three commonly used positions for holding
ophthalmic prisms (Figure 10). The first position is the Prentice position. In this position the
posterior face of the prism is perpendicular to the line of sight of the deviating eye. All of the
prismatic deviation occurs at the anterior face of the prism. The second position is the
position of minimum deviation. In this position the same prismatic deviation occurs
symmetrically at each of the two faces. In other words, the visual axis inside the prism is
perpendicular to the line bisecting the apex angle of the prism. The third position is the
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frontal plane position, where the posterior face of the prism is held in the frontal plane of the
patient (7).
Figure 10. Three common positions for ophthalmic prisms. a. Prentice position. b. Minimum deviation
position. c. Frontal plane position (Thompson and Guyton, 1983).
Figure 11. Regular ophthalmic prisms. a. Prism bars. b. Separate prisms.
Two types of prisms are available for use in ophthalmic practice: glass prisms and the
more commonly available plastic ophthalmic prisms (Figure 11). Glass prisms are calibrated
for use in the Prentice position. On the other hand, plastic ophthalmic prisms are calibrated
for use in the position of minimum deviation, which is stated by the manufacturer. The
prismatic deviation in the position of minimal deviation is very close to the deviation in the
frontal plane position (the difference in these two positions is never greater than 2 Δ with
available prisms). As the frontal plane position appears to be a more practical way to hold a
plastic ophthalmic prism, for the position of minimum deviation may be quite difficult to
judge in clinical practice, plastic ophthalmic prisms are used in this position instead.
Horizontal and vertical prism bars are calibrated for use in the frontal plane position, that is,
with the flat side of the bar held posteriorly, parallel to the frontal plane of the patient (7).
The frontal plane position is appropriate for holding plastic prisms when measuring
distance deviations. When measuring deviations at near, the rear surface of the plastic prism
should still be held perpendicular to the line of sight anterior to the prism, but this
necessitates rotating the rear surface of the prism out of the frontal plane of the patient (7).
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Variable results of strabismus surgery may be due in part to errors in prism measurement.
A major source of error arises when prisms are held in a position for which they are not
calibrated. The deviation difference between the Prentice and minimum deviation or frontal
plane positions is small for prism values below 20 Δ but increases significantly for large
prisms (7).
Table 1. Deviation in prism diopters for the addition of two plastic prisms stacked
together, with the posterior prism in the frontal plane position (7)
Added prism (labeled value Initial prism (labeled value in prism diopters)
in Δ) 10 12 14 16 18 20 25 30 35 40 45 50
1 11 13 15 17 19 21 27 32 37 43 48 54
2 12 14 16 18 20 23 28 33 39 45 50 56
3 13 15 17 19 22 24 29 35 40 46 52 58
4 14 16 18 21 23 25 30 36 42 48 54 61
5 15 17 20 22 24 26 32 38 44 50 56 63
6 16 19 21 23 25 27 33 39 45 52 59 66
7 17 20 22 24 26 29 35 41 47 54 61 68
8 19 21 23 25 28 30 36 42 49 56 63 71
9 20 22 24 27 29 31 37 44 51 58 66 74
10 21 23 25 28 30 33 39 46 53 60 68 77
12 23 25 28 30 33 35 42 49 57 65 74 84
14 25 28 30 33 35 38 45 53 61 70 80 91
16 28 30 33 36 38 41 49 57 66 76 87 100
18 30 33 35 38 41 44 52 61 71 82 95 110
20 33 35 38 41 44 47 56 66 76 89 104 122
25 39 42 45 49 52 56 66 78 93 110 133 165
30 46 49 53 57 61 66 78 94 114 141 183 264
35 53 57 61 66 71 76 93 114 144 195 315 _
40 60 65 70 76 82 89 110 141 195 339 _ _
45 68 74 80 87 95 104 133 183 315 _ _ _
50 77 84 91 100 110 122 165 265 _ _ _ _
Table 2. Deviation in prism diopters for the addition of two prisms (glass or plastic)
with one prism held in front of each eye (7)
Right eye prism (labeled value)

Left eye prism (labeled value)
10 12 14 16 18 20 25 30 35 40 45 50
10 20 22 24 26 29 31 36 41 47 52 58 63
12 22 24 26 29 31 33 38 44 49 55 60 66
14 24 26 29 31 33 35 40 46 52 57 63 69
16 26 29 31 33 35 37 43 48 54 60 66 72
18 29 31 33 35 37 39 45 51 57 63 69 75
20 31 33 35 37 39 42 47 53 59 65 71 78
25 36 38 40 43 45 47 53 59 66 72 79 86
30 41 44 46 48 51 53 59 66 73 80 87 94
35 47 49 52 54 57 59 66 73 80 87 95 103
40 52 55 57 60 63 65 72 80 87 95 104 113
45 58 60 63 66 69 71 79 87 95 104 113 123
50 63 66 69 72 75 78 86 94 103 113 123 133
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Glass and plastic prisms are available to a maximum power of 40 and 50 Δ respectively.
Significant errors in measurement could, therefore, arise from stacking two prisms in the
same direction to measure a large deviation or a deviation that is between the calibrated
values of two prisms in a set. The additivity errors of prisms can be reduced but not
eliminated by splitting prisms between the two eyes. The error can also be calculated so that
appropriate correction can be made (Tables 1 and 2). There is, however, no practical way to
measure accurately large strabismic deviations with prisms (7).
Fresnel Optics
The Fresnel Principle
Figure 12. A Fresnel prism can be imagined to be a series of small plastic prisms lying adjacent to each other
on a thin platform of plastic (Flom and Adams, 1996).
Any thick lens or prism can cause practical problems because of its weight: the higher
the power and the larger the diameter, the greater the weight problem becomes. Since light is
refracted only at the surface of an optical element and travels in a straight line elsewhere, the
refracting power of a lens or prism is based primarily on the relative angle between the two
refracting surfaces. This angle remains unchanged across a prism. The basis of the Fresnel
principle is to remove most of the nonrefracting portions of a conventional lens or prism; this
process results in a relatively lightweight, large diameter optical element. A Fresnel prism
can be imagined to be a series of small prisms lying adjacent to each other on a platform
creating a thin membrane (8) (Figure 12).
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Types of Fresnel Prisms

Formerly, Fresnel prisms were manufactured as hard acrylic prisms that were designed to
clip onto existing glasses. The principal disadvantage of these prisms was the conspicuous
Fresnel grooves (8).
In 1970, thin flexible membrane Fresnel prisms were developed. The membranes are
made of clear polyvinyl chloride (PVC) and are designed to be applied without adhesive to
the back surface of a conventional ophthalmic lens. These membranes are cosmetically
superior to the hard acrylic wafer prisms, because they are extremely thin (0.8 mm) and the
grooves are very narrow. Their relatively large diameter (64 mm) allows them to be cut to
conform to the shape of most spectacle carrier lenses. The refractive index (1.525) is very
close to that of optical crown glass. A range of powers from 0.50 to 30 Δ exists. These prisms
may be used as a permanent correction and as a direct substitute for the conventional prism;
however, they are most commonly applied in temporary use (e.g., for diagnostic use or where
the prism requires frequent changing) (8).
Application of Fresnel Prisms

The Fresnel membrane prisms are designed for in-office application (8,9). For prism
application, Fresnel (press-on) prisms are positioned base-out to neutralize an esotropia, and
base-in to neutralize an exotropia. The base orientation of the press-on prism is indicated with
the word ―base‖ (Figure 13).
The Fresnel prism is fashioned to the spectacle lens in the following manner. First, the
Fresnel prism is placed over the spectacle lens with proper base orientation. A pen is then
used to trace the shape of the spectacle lens onto the press-on prism just inside the rim of the
frame. The press-on prism is removed and cut with scissors just inside the line.
Figure 13. Application of Fresnel press-on prism over spectacle lens. Fresnel press-on prism positioned base-
out over the spectacle lens. The base orientation of the press-on prism is indicated with the word ―base‖
(inset).
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Both the spectacle lens and the press-on prism are washed with water. The prism is placed
with its smooth surface towards the inside surface of the spectacle lens. The spectacles are
then removed from water, holding the press-on prism in place. Any fine adjustments are
made by sliding the press-on optic. Air bubbles with most of the water are gently squeezed
out with the ball of the thumb. The edges of the press-on prism are inspected to make sure
that no overlapping occurs on the spectacle lens bevel or frame (Figure 14).
Several hours later the residual layer of water completely evaporates through the PVC
leaving the membrane tightly adherent to the spectacle lens carrier. The membrane adheres to
the lens for the same reason that two flat pieces of glass bond together when the air layer
between them has been removed. This glueless application is not injurious to the spectacle
lens, and successive Fresnel membrane applications to the same carrier lens are possible. For
removing the Fresnel membrane, it can be peeled off starting at the edge (8).
Figure 14. Application of Fresnel press-on prism over spectacle lens. a. Tracing the shape of the spectacle
lens onto the press-on prism. b. Cutting the Fresnel press-on prism. c. Applying water drops to the inside
surface of the spectacle lens. d. Placing the press-on prism base-out with its smooth surface towards the
inside surface of the spectacle lens, then squeezing out air bubbles with most of the water. e. Press-on prism
in place. f. Patient wearing bilateral base-out press-on prisms to neutralize an esoptropia.
Visual Performance of Fresnel Prisms

The distortions of conventional prisms and Fresnel membrane prisms have been
compared by Adams et al. [10]. Five distortions described by Ogle [11] were considered:
horizontal magnification, vertical magnification, curvature of vertical lines, asymmetric
horizontal magnification and change in vertical magnification with horizontal angle. It was
noted that horizontal and vertical magnification (i.e., overall magnification) increases rapidly
with increasing prism power in case of conventional prisms. On the other hand, the
magnification remains small for all powers of Fresnel prisms. For example, a 15 Δ
conventional glass prism yields 6% and 4.5% horizontal and vertical magnification
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respectively whereas a Fresnel membrane prism of the same power produces only 1.5% and
0% horizontal and vertical magnification respectively [10].
The oblique astigmatism and power error found in ophthalmic prisms are also important
clinically. Oblique astigmatism and spherical power vary across the surface of both Fresnel
membrane prisms and conventional prisms. Although astigmatism is usually higher at any
given viewing angle for the Fresnel prism, it remains almost constant across the prism. The
spherical error for any viewing angle, however, is always less in a Fresnel prism than a
conventional prism (8).
Prisms, whether of the conventional or Fresnel type, cause some reduction and distortion
in visual acuity. Much of this acuity loss is due to the distortions and chromatic aberration
associated with all prisms. Additional acuity-reducing factors, however, appear to be
associated with Fresnel prisms. Reflections at the prism facets and increased chromatic
dispersion (when the optical material is PVC) produce a loss in contrast of objects viewed
through a Fresnel prism. A slight decrease in acuity may occur when the groove width in
prisms becomes less than approximately 2 mm, because of diffraction of light (8).
The amount of reduction and distortion in visual acuity is directly related to the prism
power. Prism powers of up to 10 Δ are generally well tolerated and reduce visual acuity by no
more than one line. Further distortion in vision occurs with prism soilage. Frequent cleaning
avoids this problem. Press-on prisms are a Fresnel-type prism and thus have multiple lines
across the surface. Initially these lines are troublesome to patients but they soon learn to
ignore them. As a rule, children have a better tolerance to prisms than adults do (9).
The greatest advantages of Fresnel prisms for clinical use are their thinness, lightweight,
localized use on spectacles and quick and easy in-office application and modification. They
are cosmetically acceptable and their expense is modest. Their disadvantages, however, are a
noticeable loss of contrast, a slight loss of acuity, reflections and scattered light from the
prism facets and visibility of the grooves (8,9).
Use of Prisms in Measuring Ocular Deviation
Krimsky Test
A prism is combined with the light reflex test to measure an ocular deviation. A prism is
placed over one eye, preferably the fixing eye. The prism is placed base-out to measure an
esodeviation, and base-in to measure an exodeviation. Light is shone on both eyes, while the
patient fixes on an accommodative target at 0.33 meters. Prism power is increased till the
light reflex is symmetric in both eyes. This prism power is the measure of the patient‘s ocular
deviation in prism diopters. This test is less accurate than the prism and cover tests. It is,
however, of value where the latter are impossible as in infants and in patients with eccentric
fixation [12] (Figure 15).
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Figure 15. Krimsky test. a. Right esotropia. b. Krimsky test to measure esotropia.
Cover-Uncover Test
The cover-uncover test is designed to detect the presence of a tropia, without dissociating
an existing phoria [12]. The test is performed by very briefly covering then uncovering one
eye while observing the fellow eye for a tropia shift as the eye picks up fixation. While
performing the test, the patient is asked to fixate on an accommodative target. Each eye is
covered for only 1 to 2 seconds, and the cover is removed for several seconds before covering
the fellow eye to allow re-establishment of fusion. The results of the cover-uncover test are
interpreted as follows:
 If there is no shift of either eye after covering and uncovering each eye, orthotropia
is present.
 If briefly covering one eye produces a refixation shift of the fellow eye, a manifest
tropia is present. If the uncovered eye moves nasal to temporal to fix, this is
considered an esotropia shift, while if it moves in a temporal to nasal direction, this
is an exotropia shift.
 If a tropia is detected by the cover-uncover test, the deviation is then measured by
the prism and cover tests.
Prism and Cover Tests
Motor alignment is measured by the simultaneous prism and cover test and the alternate
cover and prism test at 6 metres in straight, vertical and horizontal gazes and at 0.33 metres in
straight gaze. Measurements are recorded without and with correction (one month after
spectacle prescription) and are repeated twice at least one week apart. Stability of deviation is
established by requiring measured angles of ET or XT within 5 Δ or less on 2 consecutive
visits at least one week apart [13].
When measuring the ocular deviation, an accommodative fixation target is used to keep
the patient‘s attention and ensure that he/she is appropriately accommodating [12]. For that
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purpose, small pictures with fine details are used at near, while pictures projected by the chart
projector or posters are used in the distance.
The deviation is measured using either prism bars or separate prisms. The horizontal
prism bar is used to measure horizontal deviations. For neutralizing a deviation, prisms are
held in front of the patient‘s eye so that the apex is in the direction of the deviation: for
neutralizing esodeviations, the apex is directed nasally; for exodeviations, the apex is directed
temporally [12].
When measuring distance deviations, prisms are held in the frontal plane position. If the
horizontal prism bar is used, the flat side of the bar is held posteriorly, parallel to the frontal
plane of the patient. In case the separate prisms are used, these are oriented in a way so that
the posterior face of the prism is held in the frontal plane of the patient. When measuring
deviations at near, the rear surface of the prism is rotated out of the frontal plane of the
patient, in order to maintain the rear surface in a position that is perpendicular to the line of
sight anterior to the prism (7).
When measuring a large deviation, or a deviation that is between the calibrated values of
two prisms in the set, prisms are split between the two eyes to avoid underestimation of the
angle size (7). This is only possible when performing the alternate cover and prism test
(ACPT) but not the simultaneous prism and cover test (SPCT), as in the latter the prism has
to be placed in front of the deviated eye. Accordingly, deviations exceeding 50 Δ can be
measured only by the alternate cover and prism test.
Simultaneous Prism and Cover Test

The SPCT is used to measure the amount of tropia present. To perform the SPCT, a
prism is presented in front of the deviated eye to neutralize the tropia, as the fixing eye is
simultaneously covered by an occluder (Figure 16). If the tropia is neutralized by the prism,
the eye under the prism will show no refixation shift.
Figure 16. Simultaneous prism and cover test.
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If the deviated eye shows a refixation movement, a residual deviation is present. The prism
and occluder are withdrawn from the eyes and, after several seconds, a different prism is
presented to the deviated eye as the fixing eye is simultaneously covered. This process is
repeated until a reversal of the shift of the deviated eye, with the fixing eye covered, is noted.
The prism power just below that producing reversal is taken as a measure of the deviation
(12).
Alternate Cover and Prism Test

The ACPT is used to determine the amount of prism necessary to neutralize the full
deviation including any latent phoria. A prism is placed in front of one eye in an attempt to
neutralize the deviation. Alternate cover testing is then performed with the prism in place by
alternately occluding each eye, then observing for a refixation shift of the uncovered eye to
the midline. While performing the test, the occluder is held over one eye for several seconds
to dissociate fusion, then rapidly moved to the fellow eye, so that one eye is always occluded.
If there is a residual refixation shift with the prism in place, the prism is changed to neutralize
the deviation. During changing prisms, care is taken to always keep one eye covered in order
to maintain binocular dissociation. The process is repeated until a reversal of the shift in the
uncovered eye is observed. The prism power just below that producing reversal is taken as a
measure of the deviation (12).
An overshoot of the refixating eye is sometimes noted even when the deviation is
neutralized. This is termed ―physiologic redress fixation movements‖. The point of
neutralization in these cases is judged as the point when the redress movement is equal to the
refixation movement. Another way to determine the angle of deviation in such cases is to
bracket the deviation by intentionally overcorrecting with too much prism then reducing the
prism until the best neutralization is achieved (12).
Patients with variable measurements on prism cover tests should have one eye patched
for at least 45 minutes to dissociate binocular vision prior to measurement of the deviation
(12).
Measuring in the Cardinal Positions of Gaze

The deviation is measured in the primary position in the distance (6 meters) and at near
(0.33 meters). The positions of gaze are measured in the distance by moving the head up,
down, right and left to measure the deviation in down gaze, up gaze, left gaze and right gaze,
respectively. The deviation is measured in vertical gaze positions to detect the presence of an
A/V pattern. The deviation is measured in horizontal gaze positions to detect lateral
incomitance (12).
Use of Prisms in Horizontal Comitant Ocular

Deviations (Prism Adaptation)
Prism adaptation, the preoperative use of prisms to determine the maximum angle of
strabismus and to estimate fusional potential, has been suggested as a method of improving
the results of initial surgery and minimizing the rate of reoperation (Figure 17). The Prism
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Adaptation Study Research Group [13] defined prism adaptation as the preoperative wearing
of Fresnel prisms to offset the angle of ET, with adjustment of prism power over time to
accommodate build-up to larger angles of ET, until fusion is achieved or it is demonstrated
that fusion cannot be achieved. Prism-adapted surgery refers to surgery for the angle of
deviation at which the prism wearer achieves fusion (13) (Figure 17).
Figure 17. Prism adaptation determines the maximum angle of strabismus. a. Esotropia 25 ∆ before prism
adaptation. b. After prism adaptaion, esotropia built-up to 50 ∆. Patient wearing 50-∆ press-on prisms (25-∆
prism over each eye), neutralizing the built-up esotropia. c. Esotropia 50 ∆ at the end of prism adaptation. d.
Postoperative orthotropia 6 months after bilateral 6-mm medial rectus recession to correct 50-∆ esotropia.
The use of prisms as a preoperative testing technique was first described by European
clinicians in the 1960s [14,15]. Later, the use of prisms as a preoperative test to predict the
success of surgery in acquired ET was popularised by Jampolsky [16] and coined the ―Prism
Adaptation Test‖ (PAT). The author advocated using prism adaptation preoperatively to
predict what will occur with the image movement from a suppressed region (nasal retina) to a
nonsuppressed region (temporal retina).
Improved surgical results were reported by Aust and Welge-Lussen [17] when prisms
were used to determine the angle of deviation (the ―target angle‖) to be corrected surgically.
Subsequently, articles appeared in the American literature reporting improved surgical results
following preoperative prism adaptation [18-24]. Delisle and co-authors [25] reported that the
PAT is indispensable if adequate surgery is to be performed in certain well-selected cases
such as symptomatic large esophoria, intermittent esotropia and recent-onset esotropia.
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These studies differed in methods of prism application, patient selection, length of

follow-up and techniques used to evaluate sensory and motor status after surgery. Data from
these studies suggested that preoperative prism adaptation might have a beneficial effect on
postsurgical alignment and the likelihood of achieving sensory fusion, thus substantially
reduce the rate of reoperation. All of these studies, however, were retrospective and
nonrandomized [13]. On 1990, the Prism Adaptation Study Research Group [13] performed
the first prospective, randomized, multicenter clinical trial of the effectiveness of using
preoperative prism adaptation for determining the surgical target angle in patients with
acquired esotropia.
Prism prescription for patients included in the PAS, was based on the distance deviation
measured by the ACPT with the patient wearing the proper refractive correction. The amount
of prism needed to neutralize the distance deviation on ACPT was mounted on the spectacles
in the form of base-out Fresnel press-on prisms. The prism power was divided as equally as
possible between the two eyes. After wearing the prisms for at least 10 minutes, the SPCT at
6 and 0.33 meters was performed, with the patient still wearing the prisms. If the patient‘s
eyes converged at an angle greater than 8 Δ, the total angle was again measured with the
ACPT, and sufficient prism was mounted to render the patient 0 to 8 Δ esotropic at 0.33
meters. The patient left the office wearing the prisms, with 0 to 8 Δ esotropic shift on the
SPCT at 0.33 meters. An exotropic shift of 5 Δ or less at 6 meters was permissible (13).
Patients were re-examined at weekly intervals. The sensory status was tested with the
patient wearing the prisms using the Worth Four-Dot test, the Titmus Stereo Test and the
American Optical Vectograph circle stereotest at 6 meters. Alignment through the prisms was
assessed using the simultaneous prism and cover test. If the deviation was greater than 8 Δ at
6 and 0.33 meters and no fusion was elicited, more prism was added until stability was
achieved. The patient again left the office with 0 to 8 Δ of ET and was followed up at weekly
intervals, with readjustment of prisms if necessary. Eventually, prism response or non-
response was determined (13).
The patient was classified as a prism adaptation responder if, while wearing prisms, the
SPCT deviation was between 0 and 8 Δ of esotropia at distance and near, and one of the
following applied: the patient reported a fusion response on the Worth Four-Dot test at 0.33
meters; or the patient reported diplopia on the Worth Four-Dot test at 0.33 meters, but
identified at least the first two consecutive circles (of nine) and the first two consecutive
animals (of three) on the Titmus Stereo Test (13).
The patient was defined as a prism adaptation nonresponder under any of the following
conditions: the patient developed a stable ET between 0 and 8 Δ (on the SPCT at distance
and near) for 30 days without developing the sensory response described above; or an
exotropic shift was noted through the prisms on the SPCT at distance or near, with a
suppression response to the Worth Four-Dot test; or the patient‘s deviation built up to exceed
60 Δ through the course of the adaptation process (this is the upper limit that can be
neutralized by bilateral Fresnel press-on prisms) (13).
In the PAS, the surgical target angle differed in each group of patients. Patients who did
not undergo prism adaptation, PA nonresponders and half of the PA responders had surgery
performed based on the ACPT distance deviation measured at the time of entry into the study.
The second half of the PA responders had surgery performed based on the prism-adapted
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angle, which was equal to the amount of prism worn plus the deviation measured through the
prism immediately before surgery (13).
A successful motor outcome in the PAS was defined as 0 to 8 Δ of horizontal deviation
when measured by the SPCT at distance fixation. Binocular sensory function was measured
with the Worth 4-Dot test at near. A fusion response of four lights was considered a success
(13).
The PAS yielded an estimated overall motor success rate, 6 months after surgery, of 72%
in non-prism-adapted patients. Among the entire group of prism adaptation responders, 83%
experienced a successful motor outcome. The highest motor success rate was recorded in the
prism adaptation responders who underwent surgery for the prism-adapted angle (89%),
versus 79% in the prism adaptation responders who underwent surgery for their entry angle
of deviation. Among nonresponders the motor success rate was 73%. A total of 7
overcorrections were seen among the 333 patients involved in the PAS: 5 in the 127 non-
prism-adapted patients and only 1 in the 61 prism-adapted patients who underwent surgery
for the prism-adapted angle. These data suggest that the prism adaptation process identifies a
group of patients who can safely undergo a larger amount of surgery without increasing the
risk of overcorrection (13).
Percentages of patients with motor success plus fusion on the Worth Four-Dot test,
obtained in the PAS 6 months after surgery, were as follows: 54% for non-prism-adapted
patients, 69% for PA responders undergoing prism-adapted surgery, 61% for prism
adaptation responders undergoing entry-angle surgery and 34% for PA nonresponders (13).
Repka and co-authors [26] evaluated the one-year motor and sensory outcomes for 92%
of the patients previously included in the PAS. Non-prism-adapted patients had a motor
success rate of 74%. The successful motor outcome for all prism adaptation responders was
82%, where prism adaptation responders operated on for their prism-adapted angle
maintained an excellent 90% motor success rate, and those operated on for their entry angle
had a decline to 75% success. The nonresponders showed a decline in success from 73% at 6
months to 63% at 1 year. Overcorrections remained equally rare at 1 year, when only 5
patients were so classified: one patient each was in the prism-adapted angle group, prism
entry angle group and the nonresponder group; two patients were in the non-prism-adapted
group. The combined motor and sensory success rates at the one-year follow-up became 59%
for non-prism-adapted patients, 67% for all prism adaptation responders, 75% for PA
responders undergoing prism-adapted surgery, 60% for prism adaptation responders
undergoing entry-angle surgery and 39% for prism adaptation nonresponders.
The results of the PAS indicate that prisms better define the target angle of surgery (13).
The prism responders showed superior outcomes when compared with the non-prism-adapted
(control) group, while the prism nonresponders experienced the lowest success rates (26). A
plausible hypothesis is that surgery places the eyes within a range of alignment, but final
alignment is based on sensory factors (27). Accordingly, because of their apparently lower
potential for fusion, nonresponders are a subgroup with an inherently poorer prognosis for
long-term stability of their postoperative alignment [13,26-28]. Conversely, responders had
superior outcomes because of the presence of sensory fusion in these patients [26].
The highest success rates were achieved in the fusing group (responders) whose surgical
dose was based on the angle measured after building it up on prism adaptation testing
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(13,26). The responders whose surgery was based on the preadaptation angle (entry angle),
however, had the same success rates as the controls who did not undergo prism adaptation
testing (29). It was suggested that the therapeutic benefit comes not from the act of prism
adaptation, but rather from the surgeon performing the greater amount of medial rectus
recession surgery suggested by the prism build-up during the adaptation process (28). A
question that has been raised, therefore, was whether the nonresponders would have had a
better outcome if more surgery had been done [29].
The author performed a study to evaluate the effect of performing prism-adapted surgery
to prism adaptation nonresponders. Among twenty patients with acquired esotropia, who
underwent preoperative prism adaptation, six patients (30%) were prism responders, while 14
(70%) were nonresponders. Prism responders as well as nonresponders, underwent surgery
for the prism-adapted angle, defined as the power of press-on prisms worn plus the distance
deviation measured through worn prisms by the alternate cover and prism test at the time
prism response was determined. Motor success, at the sixth postoperative month, was
obtained in 100% of prism responders and 93% of nonresponders. The author, therefore,
concluded that to maximize the benefit of prism adaptation, it is recommended that all prism-
adapted patients (responders and nonresponders) undergo surgery for the prism-adapted angle
(30).
The Prism Adaptation Study Research Group [13] stated that although the observed
benefits of prism adaptation were concentrated mainly in the prism adaptation responders
who built up to larger angles with the prism adaptation process and who underwent surgery
for their prism-adapted angles, this group, in whom the process makes a substantial
difference in postoperative outcome, cannot be identified with certainty in advance. They,
therefore, recommended that prism adaptation be considered for all patients with acquired
esotropia to determine the appropriate target angle for surgery.
On 1991, Repka and co-authors [31] developed an article to determine predictors of
prism response during prism adaptation by analyzing the characteristics of those patients who
were and were not prism responders in the PAS. They stated that the beneficial effect of
prism adaptation was observed only in prism responders, who comprised only 66% of the
prism-adapted patients in the PAS. It was, thus, reasonable to try to identify those patients
who are likely to benefit from prism adaptation, sparing those who will not benefit from the
extra cost and inconvenience of pursuing prism adaptation.
Demographic, timing and sensory features of the preoperative examination were
evaluated to determine their value in predicting whether a patient would respond to
preoperative prisms. The demographic features that were studied included sex, race, Hispanic
background, refractive error and size of the initial esodeviation. Timing features included the
age of onset of the esotropic deviation and the duration of esodeviation. Sensory tests studied
included the presence of alternating fixation, a fusion, diplopia or suppression response on
the Worth Four-Dot test at 0.33 meters with and without neutralizing prisms, Bagolini lenses,
the distance vectograph and the red-filter test. All of these tests were performed prior to prism
adaptation. The effects of amblyopia and visual acuity were similarly assessed [31].
Significant factors predicting a prism response included: patients who were older at the
time of onset of their esodeviation, duration of deviation less than 1 year, alternating fixation,
fusion on the Worth Four-Dot test at near with prism neutralization and equal vision.
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Demographic characteristics were similar for both responders and nonresponders except that
non-Hispanic patients were significantly more likely to respond to prisms than Hispanic
patients (31).
Repka and co-workers (31), however, stated that although approximately one-third of
patients with acquired esodeviations will not respond to prism adaptation, no test or other
characteristic was found that allowed the physician to predict which patients will not respond.
Nevertheless, it was suggested that despite the additional time and cost required for prism
adaptation prior to surgery, the potential benefit of improved ocular alignment demonstrated
in the PAS outweighs the disadvantages of prism adaptation. In conclusion, it was
recommended that all patients with acquired esodeviations be considered candidates for
preoperative prism adaptation (26,31).
On 1993, another prospective study was performed in Japan by Ohtsuki and co-workers
[32] to compare the motor outcome of patients undergoing surgery for the prism-adapted
angle of esotropia versus those who undergo surgery for the original angle of esotropia.
Results obtained in three groups of patients were compared: prism adaptation responders
undergoing surgery based on the prism-adapted angle, prism adaptation responders
undergoing surgery for the original angle of ET and prism adaptation nonresponders who
underwent surgery for the original angle of esotropia. The postoperative success rates were
evaluated 1 year after surgery. A successful result was defined as a deviation of 10 Δ or less
of horizontal deviation with the ACPT at 5 meters. The motor success rate was highest in the
group of prism adaptation responders undergoing prism-adapted-angle surgery (84%) and
lowest in nonresponders (50%). Success rates of 78% were reported in the group of prism
adaptation responders undergoing surgery for their original angle of esotropia. 84% of the
prism adaptation responders who underwent surgery for their prism-adapted angle showed
motor success with binocular vision on Bagolini‘s lenses test, while only 21% of prism
adaptation nonresponders did. These results indicated that preoperative prism correction not
only allowed a relatively accurate determination of the target angle for surgery, but also
promoted the development of binocular co-ordination.
On 1993, Herzau and Schoser [33] performed a retrospective study to evaluate the value
of the PAT in determining the degree of strabismus surgery. They concluded that
preoperative prism adaptation improves predictability of the effect in strabismus surgery. In
patients with early-onset strabismus, however, the authors demonstrated that prism adaptation
does not improve predictability; nevertheless, higher dosage of surgery seemed to be useful
in cases with increased deviation under prisms and did not lead to overcorrections. In
addition, the authors suggested that the test represented an additional method for evaluation
of the binocular sensory status.
Several authors argued against the value of preoperative prism adaptation in the
management of acquired esotropia. Giangiacomo [34] argued that the reason why patients in
the PAS undergoing prism-adapted-angle surgery had a more successful outcome than
patients undergoing entry-angle surgery might have been only because the former underwent
more measurements of their ocular deviation than did the latter. The former thus had a more
accurate preoperative evaluation.
Lang and Heinrich [35] argued that the highest rates of orthotropia obtained in the PAS
in patients who underwent prism adaptation and had prism-adapted-angle surgery, should
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merely be attributed to the late age at onset of strabismus in these patients as compared to the
other groups in the study.
Tennant [36] argued that if Repka and co-authors [26] claimed that because prism
adaptation responders fared better after ET surgery than did control subjects, prism
adaptation had a positive therapeutic effect, one could use the same logic to state that because
prism adaptation nonresponders fared worse after ET surgery than did control subjects, prism
adaptation was harmful. In reply to this comment, Repka and co-authors [28] stated that the
group of nonresponders probably did more poorly because they have poor sensory fusion, not
because of an adverse effect of prism adaptation. Furthermore, the non-prism-adapted group
did more poorly than the prism responders because patients who are nonresponders were
included.
In conclusion, the value of preoperative prism adaptation in the management of acquired
ET was crowned by many authors [13,17-26,28,32,33,37]. Nevertheless, the PAT was
condemned by several other authors [34-36].
Mechanisms of Prism Adaptation
Various authors proposed different explanations for the mechanism of prism adaptation:
how it affects the angle of ET and the binocular status.
Bagolini [38] stated that the binocular adaptational phenomena occurring in strabismus
are purely sensorial, such as suppression and anomalous retinal correspondence (ARC), and
sensorimotorial, such as anomalous movements.
Sensorial Adaptational Phenomena

Suppression prevails in large-angle deviation, while anomalous correspondence, with
achievement of an anomalous binocular vision, prevails in relatively small-angle deviation
(usually between 15 and 30 Δ) (38,39).
Sensorimotorial Adaptational Phenomena

Various authors have investigated the behaviour of the angle of ET, which has been
corrected by base-out prisms (16,38,40,41). In patients with ARC, an increase in the
esodeviation during prism adaptation (―eating up prisms‖) has frequently been described
[42]. The angle sometimes increases, compensating the full correction of the esotropic angle,
and in many cases strong prismatic overcorrection could also be compensated for with a
remarkable increase of the angle of deviation (41). This phenomenon is considered a
sensorimotorial sequel of strabismus (40}. It is also considered to be an expression of

anomalous fusional movements in the sense that the retinal elements of the deviated eye tend
to change not only their directional localisation in binocular vision (ARC) but also their
motorial value (41).
The aim of these ocular movements appears to be that of bringing the two retinal images,
of the object the patient is looking at, over retinal areas in the two eyes which have become
anomalously correspondent (having therefore acquired the same directional localisation).
Directional localisation and motorial value in patients with strabismus, however, do not
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necessarily change at the same time, as can be demonstrated in some cases that show ARC
but do not compensate for prisms or more rarely vice versa. The fact that base-out prisms
tend to determine convergent movements while base-in prisms elicit divergent movements
and vertical prisms vertical movements seems to prove the fusional nature of these anomalous
movements (AM) (38).
Anomalous movements are slow in comparison with normal fusional movements, and
they cannot be seen with the naked eye. They can be detected only after base-out prisms have
been worn for minutes or hours. Their detection becomes possible only by the cover test,
when prismatic correction or overcorrection has been partially or totally compensated for by
an increase in the strabismic deviation (38).
Two tests have been described to study AM; namely: the ―Prism Adaptation Test‖ (PAT)
[16] and the ―Progressive Prism Compensation Test‖ (PPT) [38].
Prism Adaptation Test

The PAT has been devised as an objective method for determining whether the sensorial
status is anomalous. In this test, the angle of ET is corrected with prisms and is observed
from several minutes to one or two hours. When there is compensation of the prism
correction, this is proof of an anomalous sensory status (16).
Progressive Prism Compensation Test

When the PAT is positive, the strength of the AM is studied by the PPT. This is done by
progressively overcorrecting with base-out prisms the angle of ET up to an amount where the
patient is no longer capable of compensating by overconverging. If the patient compensates
for large prismatic overcorrection of their original ET, AM are strongly developed (38).
Jampolsky [16] and Bagolini [38] have concluded that the presence of ARC in certain
patients with ET is the cause of increase of the angle of deviation with prism adaptation. This
was due to the fact that these patients try to regain the retinal image location to which they
were accustomed. It is, therefore, expected that in esotropes with normal retinal
correspondence, prism correction should lead to binocular single vision and an increase in the
angle of squint is unlikely (43). The authors [43], however, reported an increase in the angle
of ET during prism adaptation in patients with normal retinal correspondence (i.e. patients
with normosensoric ET). Similarly, Herzau and Schoser [33] reported an increase in the
initial deviation under prisms most often when normal retinal correspondence was present.
Langenthal and co-workers [43] investigated patients with normosensoric ET for an
increase in the angle of ET with prism adaptation. All patients had their ―basic deviation‖
first determined by the ACPT, then they underwent prism adaptation followed by occlusion
of the deviating eye for approximately 6 hours. The authors compared the squint angles
measured with the ACPT with those after prolonged prismatic correction of the squint angle
and with those after prolonged occlusion of one eye. All patients showed an increase of the
squint angle after prism adaptation. The angle was generally smaller after diagnostic
occlusion of one eye than after prism adaptation.
Langenthal and co-workers [43] concluded that the increase in the angle after PA could
not be interpreted as a latent esodeviation because the angle was much smaller under
diagnostic occlusion. Instead, they interpreted the increase of the angle under prisms in these
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normosensoric esotropes as an anomalous fusional movement, in analogy to the phenomenon

in patients with anomalous retinal correspondence. This anomalous sensorial relationship
between the two eyes, however, was not detected with the usual psychophysical tests
performed before prism adaptation.
Langenthal and co-workers [43] suggested the following hypothesis to explain why the
anomalous connections, present in these patients with apparently normosensoric ET, were not
detected with the synoptophore. A suppression of the central retina of the squinting eye may
have been present under casual seeing conditions, while ARC may have been present in the
peripheral retina. When tested with the synoptophore, only the central 6-8 degrees of the
retina were stimulated. Under these conditions, the suppression of the central retina may have
resolved so that normal retinal correspondence with fusion was demonstrated. During the
PAT, the entire retina was stimulated, suppression of the central retina occurred and fusional
movements, based on anomalous correspondence of the peripheral retina, were elicited.
As an alternative interpretation of the increase in the angle under prisms, Langenthal and
co-workers [43] suggested that a convergence spasm might have been triggered by the
prisms. Pseudomyopia and miosis, however, were not observed in these patients.
Effect of Anomalous Movements on Surgery
Bagolini [38] stated that AM are elicited in ET by base-out prisms. The adequate
stimulation is a displacement of the retinal images. The same retinal displacement occurs
after corrective surgery. An increase in medial rectus tonus is, therefore, expected either by
using base-out prisms or by carrying out corrective surgery. The author added that if the AM
are very powerful and thus make the patient compensate for very strong prisms, less surgical
corrective effect is expected. The author also suggested that when AM are present and
powerful, they are an expression of an irreversible anomalous sensorial and sensorimotorial
situation. Similarly, Bagolini and co-workers [40] concluded that normal binocular vision
could never be attained when ―anomalous movements‖ (during PA) are strongly developed.
Langenthal and co-workers [43], on the other hand, argued against the conclusion of
Bagolini [38] and Bagolini et al. [40]. They suggested that the preoperative PAT simulates
the postoperative change of the location of the retinal image. Using the PAT, postoperative
sensomotoric reactions could be predicted. Unlike Bagolini [38], Langenthal et al. [43] based
the amount of surgery performed for most cases on the angle found after prism adaptation.
They, therefore, demonstrated that orthotropia with normal binocular single vision and good
stereoacuity can be achieved despite a marked increase in that angle of squint during prism
adaptation.
Delisle and co-authors [25] proposed the following explanations for a positive response
to the prism adaptation test:
Active Adaptation
Using fusional convergence, a patient with normal orthophoric eyes will adapt to base-
out prisms to avoid crossed diplopia. On the cover test, however, an exophoric movement
will rapidly be evident behind the prisms. Similarly, in patients with esotropia who have
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abnormal retinal correspondence, the PAT causes fusional convergence efforts to find and
maintain the initial basic deviation in order to eliminate the sensorial exodeviation and
crossed diplopia [25].
Passive Adaptation
In patients who still have normal binocular vision despite large esophoria, intermittent
esotropia or recent-onset esotropia, the real angle is masked by divergence efforts. The
amplitude of the divergence becomes abnormally large because of sustained effort [25].
These patients, therefore, have strong tonic fusional divergence, and they may have a latent
deviation much larger than is determined by alternate cover testing [44]. The PAT allows the
patient to relax the divergence and the eyes then deviate passively towards the true angle
[25]. Preoperative prism adaptation, thus, can be helpful in disclosing the full latent deviation
[44,45].
If this was the only explanation, a simple occlusion test would show the same angle as
that found with the PAT; however, this is not the case. A third explanation is, therefore,
needed [25,43].
Changes in the Afferent Impulses to the Oculogyric Centres

Measuring the deviation with base-out prisms allows binocular vision, as opposed to the
cover test, which measures the deviation under monocular conditions. Looking with both
eyes at the same time over a period of time may change the pattern of the afferent impulses
reaching the oculogyric centres, which alters efferent impulses, causing the increase of the
angle [25]. Several authors suggested that one of these centers lies in the cerebellum [46-48].
Milder and Reinecke [46] stated that the phoria adaptation response is based on the
integrity of the cerebellum, visual cortex and brainstem. The visual cortex is the first site at
which inputs from each eye are compared [46]. The cerebral cortical visual areas project to
the dorsolateral region of the pontine nuclei [49]. Ultimately, the alignment of the visual axes
is determined by the activity of ―oculomotor‖ nuclei within the midbrain and pons. The
cerebellum may be able to sample brainstem efferent outflow indirectly via mossy-fibre input
[46], as cells in the dorsolateral pontine nuclei project heavily to the caudal posterior lobe of
the cerebellum [50]. In a study performed by Baizer and co-workers [49] on rhesus monkeys,
an induced cerebellar lesion involving the dorsal paraflocculus and uvula abolished
completely the normal prism adaptation for the arm ipsilateral to the lesion. It was, therefore,
concluded that the region of the cerebellar cortex that receives mossy-fibre visual information
relayed via the pontine nuclei from the cerebral cortex is a critical site for prism adaptation.
Zhang and Gamlin [48] reported that studies performed on rhesus monkeys have revealed
the presence of neurones related to vergence and ocular accommodation within a

circumscribed region of the posterior interposed nucleus (IP) and the posterior fastigial
nucleus of the cerebellum. Neurones related to vergence and accommodation are also present
in the midbrain in the vicinity of the oculomotor nucleus [51-54]. The neurones of the
posterior interposed nucleus of the cerebellum are related to divergence and accommodation
for far (the far-response); these are, therefore, termed far-response neurons [48]. On the other
hand, the posterior fastigial nucleus of the cerebellum contains near-response neurons [55].
The midbrain region contains mostly near-response neurones, with only 25% being far-
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response neurons [48]. Neurones within the fastigial and IP nuclei of the cerebellum have
reciprocal connections with the midbrain near-response region [56]. It is, therefore, possible
that the fastigial near-response region and the interposed far-response region represent a
push-pull system for modulating vergence and accommodation. Increases in the tonic activity
of the vergence-related cells of the cerebellar near-response region combined with decreases
in the tonic activity of the vergence-related cells of the cerebellar far-response region would
result in an overall increase in the convergence signal, but not the accommodation signal,
impinging on the midbrain near-response region. A signal of this nature could underlie the
ability of individuals to show phoria adaptation to prisms and would be consistent with the
report of Milder and Reinecke [46] that indicated that cerebellar damage compromises this
ability [48].
Kono and co-authors [57] in 2002 reported an impairment of phoria adaptation to vertical
prism disparity in patients with cerebellar dysfunction. They concluded that their results
bolstered the hypothesis that phoria adaptation is a cerebellar-determined response.
Combination of Mechanisms
A combination of the first and third or the second and third mechanisms has been
suggested [25].
Use of Prisms in Horizontal Incomitant

Ocular Deviations
Patients with acquired nerve palsies develop an incomitant strabismus and encounter the
annoying symptom of binocular diplopia. In case of acquired sixth nerve palsy, the patient
develops an incomitant esotropia that is maximal in gaze towards the paralyzed lateral rectus
muscle and minimal, reaching to zero degrees, in case of gaze away from the paralyzed
lateral rectus muscle (Figure 18). In addition, a patient with sixth nerve palsy develops
diplopia that is binocular, horizontal and homonymous. In case of acquired third nerve palsy,
the patient develops an incomitant exotropia, but diplopia is absent if the patient has
ipsilateral complete ptosis. If the lid, however, does not occlude the pupil (Figure 19), the
patient will complain of binocular diplopia. As an adaptive mechanism, patients with
acquired nerve palsy either close one eye or acquire a compensatory head posture to abolish
this diplopia (Figure 20). The patients turn their heads in the direction of action of the
paralyzed muscle. That is, in right sixth nerve palsy, with a paralyzed right lateral rectus
muscle, the patient turns his/her head to the right (Figure 18a). In case of right third nerve
palsy, the patient turns his/her head to the left.
Spontaneous cure of acquired nerve palsy may occur in many cases. Accordingly,
strabismus surgery has to be postponed for at least six months to wait for spontaneous cure.
Meanwhile, to relieve binocular diplopia, a neutralizing prism may be prescribed. This
abolishes the need to close one eye or acquire a compensatory head posture.
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Figure 18. Right traumatic sixth nerve palsy with right incomitant esotropia. a. Face turn to the right. b. Right
esotropia on straightening the head. c. Gaze to the right: limited abduction of right eye. d. Gaze to the left:
normal motility.
Figure 19. Right third nerve palsy (due to diabetes mellitus) with incomitant exotropia and partial ptosis.
Figure 20. Patients abolishing binocular diplopia by: a. Closing one eye, b. Acquiring a compensatory head
posture.
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Figure 21. Neutralizing Fresnel press-on prism used to abolish diplopia resulting from acquired right sixth
nerve palsy. a. Face turn to the right to abolish diplopia. b. Neutralizing Fresnel press-on prism over right eye
to abolish diplopia. c. Neutralizing press-on prism resulted in head straightening.
Neutralizing prisms prescribed in case of incomitant horizontal deviations may be of

Fresnel press-on type (Figure 21) or regular ground-in prisms in the patient‘s spectacles
(Figure 22). In case of deviations that are 10 Δ or less, ground-in prisms are more practical as
they provide less compromise to the visual performance than press-on prisms. Moreover, the
problems of press-on prism soilage, discoloration or loss are abolished. In case of larger
deviations, press-on prisms are preferred to avoid unacceptably thick spectacle lenses with
ground-in prisms. As a general rule, the neutralizing prism is placed over the paretic eye,
base-out to correct an incomitant ET (sixth nerve palsy) and base-in to correct an incomitant
XT (third nerve palsy).
The prism power to be prescribed is determined in two ways. In case of presence of a
compensatory head posture, the prism bar is placed over the eye with paralyzed muscle, base
away from the deviation. As an example, the prism bar is placed base-out over the right eye
in case of a right sixth nerve palsy. The prism power is gradually increased till the least
power that straightens the head is reached. Another method uses the Maddox rod. The
Maddox rod is placed horizontally (to produce a vertical red line) over the normal eye. The
prism bar is placed over the eye with the paretic muscle to neutralize the deviation present. A
penlight is viewed by the patient. As an example, the prism bar is placed base-in over the
right eye and the Maddox rod is placed horizontally over the left eye in a patient with right
third nerve palsy. The prism power is gradually increased till the patient sees vertical red line
overlapping the light spot. The prism power obtained by these two methods is tried in the trial
frame for 10 minutes, then these procedures are repeated. If an increase of prism power
needed to abolish the diplopia present is encountered, the higher prism power is again tried.
Eventually, the least stable prism power that abolishes diplopia and straightens the head is
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prescribed. The patient is followed at 1 to 2 weeks intervals, and prism power is decreased
accordingly in case of gradual cure.
Figure 22. Neutralizing ground-in prism used to abolish diplopia resulting from acquired right sixth nerve
palsy. a. Face turn to the right to abolish diplopia. b. Neutralizing ground-in prism over right eye abolished
diplopia, resulting in head straightening.
Figure 23. Determining the prism power to be prescribed by measuring the degree of compensatory head
posture. a. Face turn to the right due to right sixth nerve palsy. b. Measuring the degree of right face turn
using base-out prisms. The prism power at which the head is straightened is prescribed.
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Figure 24. Determining the prism power to be prescribed using the Maddox rod. Patient with acquired right
third nerve palsy wearing a neutralizing base-in trial Fresnel prism over the affected right eye, and a Maddox
rod over the left eye. The patient views a penlight. The prism power that results in overlap of the light spot
and the red line, seen by the patient, is prescribed.
Neutralizing prisms prescription to relieve diplopia in acquired nerve palsies pose the
risk of preventing spontaneous cure and developing ipsilateral antagonist muscle contracture.
As a precaution against this, the least prism power, that relieves diplopia and straightens the
head, is prescribed. Larger powers should be avoided to maintain vergence efforts of the
paralyzed muscle to straighten the eye. The author conducted a previous study, where
neutralizing prisms were prescribed to seven patients presenting within 6 months from the
onset of acquired sixth nerve palsy. Spontaneous recovery of the palsy ensued in all 7
patients [58].
In conclusion, prisms are indispensable tools in the diagnosis and treatment of horizontal
comitant and incomitant ocular deviations.
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Chapter III
MRSA (Methicillin-resistant
Staphylococcus Aureus)
and Eye Infection
Theodora Tsirouki, Sofia Androudi and Evangelia Tsironi

University Eye Clinic, Larissa, Greece
Abstract
Staphylococcus Aureus (SA) suggests one of the major human pathogens. It is a

common cause of disease, both in healthcare facilities, as well as in the community. SA
constitutes such a problem to physicians, throughout the world, due to its great virulence
[1]. It causes an extensive variety of human diseases, many of which are life threatening
[2]. Actually, SA is estimated to be responsible for 20-40% of mortality in bacteremias
[3]. It spreads from human to human, and it is easily adapted in a variety of
environmental conditions [4]. However, the most important problem in its management is
its capability to develop resistance to almost every antibiotic.
Staphylococcus is a genus of gram positive bacteria. There have been described 32
species and 8 sub-species of genus Staphylococcus. SA is the most virulent species, and
has been thoroughly investigated over the years [4, 5]. To date, the SA genome databases
have been completed for 7 strains; 8325, COL, MRSA, MSSA, N315, Mu50, and MW2
[4].
SA normally colonizes human nose and skin, a fact that influences the pathogenesis
of staphylococcal infection. Less often, SA is found on the respiratory or urinary tract [6,
7]. Healthy individuals carry the bacteria from weeks to years, without manifesting a
disease. It is estimated that 20% of individuals are persistent carriers, who carry almost
always one type of SA. Another 20% almost never carry it, whilst the rest of the human
population carries SA intermittently [8].
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Copyright © 2012. Nova Science Publishers, Inc. All rights reserved. May not be reproduced in any form without permission from the publisher, except fair uses permitted under 70 Theodora Tsirouki, Sofia Androudi and Evangelia Tsironi
SA Virulence
SA causes disease by toxin production, or through cell penetration. It is responsible for a

series of infections in humans that vary in severity, from minor to life threatening. It can
cause skin infections, pneumonia, endocarditis, meningitis, osteomyelitis, bacteramia and
sepsis [2]. Actually, in a European study by the HARMONY collection, it was estimated that
SA was responsible for 20-40% of mortality in bacteremias between 1981 and 1998 [3].
Similar results are evaluated by another study that was realized by the SENTRY program,
and involved the United States, Canada, and Latin America. The latter estimate that SA is
responsible for 22,6% of bloodstream infections during 1997-1999 [8].
The ability of SA to lead to infections to numerous body sites depends on synthesis of
large number of virulence factors. These include cell wall components (peptidoglycan,
lipoproteins, lipoteichoic acid), and proteins (such as coagulase, SpA, alpha-, beta-, gamma-,
and delta-toxin, and leukocidin) [9]. The importance of gram-positive cell wall components
in inflammation is highlighted. Metabolically inactive organisms, cell walls, and individual
envelope components (peptidoglycan, lipoteichoic acid, and capsular polysaccharide)
stimulate inflammatory reactions [10, 11]. SA causes severe infections in the eye that may
result to even loss of vision. This devastating result is partly due to the production of toxins
that cause direct damage at the tissues, or induce inflammatory response. SA is estimated to
produce 35 exotoxins, many of which are considered to participate significantly at the
virulence of SA [11].
MRSA Resistance
SA is difficult to manage, because of the resistance that it has created against various
antimicrobial factors. Since the emergence of the first MRSA strains, in the early 60s, the
spread of MRSA has been reported worldwide.
Many decades before, during the early 40s, physicians noticed that penicillin, the
antibiotic used until then, was no longer effective on some strains of Staphylococcus [12, 13].
Specifically, it was noticed, in 1944, that certain strains of Staphylococcus were able to
produce a protease, penicillinase (a β-lactamase) that inactivated penicillin [12, 14, 15]. That
was the beginning of the observation that Penicillinase producing Staphylococcus alleviates
effectiveness of all β-lactame antibiotics.
Methicillin is a semi-synthetic penicillin, that was firstly presented in 1961 [16].
Resistant strains of Staphylococcus Aureus to methicillin (Methicillin-Resistant

Staphylococcus Aureus, MRSA) arose very quickly [16]. Actually, only one year after the
introduction of methicillin, Jevons and Knox presented the MRSA. Until the 70s,
Staphylococcus had developed resistance to erythromycin, streptomycin, tetracyclines,
neomycin, chloramphenicol, sulphonamides. The overwhelming majority of these strains was
resistant to many of the antimicrobial agents mentioned above at the same time [17]. By
1998, 50% of MRSA isolates identified at National Nosocomial Infection Surveillance
(NNIS) system in hospitals in USA were susceptible only to vancomycin [18, 19]. Currently,
the term MRSA is used to describe Staphylococcus resistant to all β-lactame antibiotics.
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Copyright © 2012. Nova Science Publishers, Inc. All rights reserved. May not be reproduced in any form without permission from the publisher, except fair uses permitted under MRSA (Methicillin-resistant Staphylococcus Aureus) and Eye Infection 71
Antibiotic resistance occurs when a microbe acquires a plasmid, which allows the
microbe to inactivate the antimicrobial activity of the antibiotic [20]. MRSA owes its
capability to resist action of Methicillin to a large genetic element, known as staphylococcal
cassette chromosome mec (SCCmec) [20, 21]. This fragment of DNA is mobile, and carries a
gene, Mec A, responsible for the encoding of an altered protein, PBP2a (penicillin binding
protein).This results in decreased affinity for most β-lactam antibiotics, including penicillins,
cephalosporins, β-lactam/β-lactamase inhibitor combinations, monobactams, and
carbapenems [22, 23]. Thus, the term MRSA is used to describe SA resistant to all β-lactame
antibiotics.
Resistance to antibiotics increases every year. According to a study concerning hospital
infection in the UK, Methicillin resistance in SA increased from 1.7% to 3.8% from 1990 to
1993, to 32% in 1997 and to 34% in 1998 [24]. In the US hospitals, 30%–40% of SA strains
were MRSA in 1997, and its occurrence was growing higher every year (45% after 2 years).
In Europe, methicillin resistance was evaluated from 12.8% in the early 1990s to 26.3%
during 1998 [8].
However, differences are evaluated in MRSA prevalence among different geographic
areas. This fact could be attributed to differences in antimicrobial usage and in infection
control practice in every country. The SENTRY surveillance program observed the
occurrence and antimicrobial susceptibility of SA strains collected in the United States,
Canada, Latin America, Europe, and the Western Pacific countries. For example, methicillin
resistance rates are high in the nations of southern Europe (e.g., Italy, Greece, Portugal, and
Turkey), and they are even higher in Asian countries (Taiwan, Singapore, Japan, and Hong
Kong) [8].
Hospital Acquired Methicillin-Resistant Staphylococcus Aureus (HA MRSA)
MRSA is responsible for a large proportion of nosocomial infections, and it was

observed only in hospitalized patients until before 2 decades. Its incidence has been reported
greater in long term care facilities [25, 5]. Colonization of MRSA has been found in a
minimal proportion of the general population, compared to patients exposed to a healthcare
facility [26].
The survival of SA is dependent on the cells ability to adapt to environmental changes. It
has evolved many mechanisms to overcome such changes, particularly in an infection [4]. SA
can survive for hours to days, weeks, or even months on dry environmental surfaces. There
have been conducted various studies that prove the ability of MRSA to survive on various
surfaces, such as glass, aluminum foil, polyvinyl chloride, bed rails and stethoscopes, and
generally fabrics and plastic [27].This explains why infections can be spread through contact
with objects such as towels, sheets, clothing, or athletic equipment used by an infected
person, or by skin-to-skin contact with an infected person. Other studies indicate that
colonized patients are the major reservoir for MRSA and that most transmissions occur via
the hands of hospital personnel [28, 29].
As MRSA hospital infections continuingly increase in interest, the need to control these
infections has been expressed. According to the SENTRY Antimicrobial Surveillance
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Program, a survey conducted in the United States, Canada, Latin America, Europe, and the
Western Pacific countries, between 1997- 1999, there has been reported a steady increase in
methicillin resistance among nosocomial SA isolates in every country [8]. All around the
world it is clear that measures must be recommended, so as to suppress the spread of
multidrug-resistant staphylococci. It has been demonstrated that it is urgent to control overuse
and misuse of antibiotics. There is also need to establish certain precautions in hospitals
worldwide [1, 8].
These measures include information and training of personnel; most cases of
transmission of HA-MRSA are proven to be attributed to contaminated healthcare workers
[30]. Hand hygiene may be the single most important way for controlling transmission of
MRSA [12]. Other measures are isolation of MRSA patient in separate room; use of masks,
gloves, caps; disinfection and cleaning measures of medical instruments; thorough cleaning
of the room and bed of the patient [20]. Unfortunately these measures have a high cost, are
time-consuming, and need careful organization and control [31].
Another measure that has been suggested for the limitation of MRSA nosocomial
infections, is screening for MRSA, and giving MRSA prophylaxis to all patients having
history of previous hospital admission, if they are planned for any surgical procedure [26].
Community Associated Methicillin-Resistant Staphylococcus

Aureus (CA MRSA)
Until the 90s, resistant strains of Staphylococcus to multiple antibiotics were reported,
only within hospital care. Patients infected by MRSA in the community, had previously been
hospitalized, or had a contact with a hospitalized patient [32, 33]. During the last two
decades, it has been confirmed that MRSA causes severe infections to persons with no
association to hospitalization [25, 32]. MRSA strains that interfere with community (CA
MRSA), have considerably different genoma than hospital acquired MRSAs, a fact that was
confirmed by Molecular analysis (pulse-field gel electrophoresis analysis) [32 , 35]. NIH
(National institute of Health of USA) published in 2008 the opinion that the vast majority of
CA MRSA infections worldwide, are caused by strains belonging to only 5 clonal lineages
[32]. In the United States there are two MRSA clones commonly found at community
infections, pulsed field types USA300 and USA400 [12]. The USA300 clone carries genes
for Panton–Valentine leukocidin (PVL), a virulent toxin, and is also commonly found in
community associated infections globally [36]. PVL-positive MRSA strains most commonly
cause skin and soft tissue infections. Rarely, they have been reported to be responsible for
necrotizing pneumonia, necrotizing fasciitis, and fatal sepsis [37].

Since 1982, when CA MRSA was first reported among intravenous drug users in Detroit
[34], the incidence of the infection is increasing worldwide [35]. CA MRSA was described in
Polynesian populations in Western Australia, and in pediatric populations in the Southern and
Midwestern United States [38]. Between 1997 and 1999, four Pediatric deaths from CA
MRSA were described in Minnesota and North Dakota. The MRSA strains isolated from the
last patients appear to be different from typical nosocomial MRSA strains in antimicrobial
susceptibility [19].
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CA MRSA infection needs to be differentiated from hospital acquired infection.

Definition of CA MRSA is established by certain facts. Firstly, diagnosis of MRSA has to be
made in the outpatient setting or, if it concerns a hospitalized patient, by a culture positive for
MRSA within 48 hours of admission to the hospital. The patient has to be of clear past
medical history of MRSA infection or colonization. The patient has to not have medical
history in the past year of any type of hospitalization (including admission to a nursing home
or nursing facility, dialysis, surgery). Finally, the patient has to have no permanent indwelling
catheters or percutaneous medical devices [39].
CA MRSA predominantly causes skin and soft-tissue infections [40]. Some population
groups are observed to have greater incidence in manifesting CA MRSA. Patients with CA
MRSA are often younger than patients with nosocomial infection. They are usually children
(particularly those in day care centers), soldiers, prisoners, homeless persons, intravenous
drug users, people of low socioeconomic status, or people who live in crowded quarters. This
could be attributed to lack of personal hygiene, and lack of basic infection-control principles.
Other risk factors for the development of CA MRSA infection are considered the recent
exposure to antibiotics, recent hospitalization, contact with others who have skin and soft
tissue infections [12, 19], previous MRSA infection, diabetes, chronic hepatitis C, cancer,
and HIV infection [12]. Finally, certain ethnic groups have been associated with CA MRSA
infection (Pacific Islanders, Native Americans, Alaska Natives, and Pacific and Canadian
aboriginals). In one study of necrotizing fasciitis caused by CA MRSA, four of fourteen
patients had no identifiable risk factors [12].
SA and the Eye
A fact that is described by many articles, during the past 20 years, is the increase of
MRSA prevalence, which concerns hospital strains, as well as community strains. Inevitably,
international interest has been directed towards MRSA infections, and consecutively towards
MRSA ocular infections. Ophthalmologists are obligated to keep in mind that MRSA is a
potential virulent in many ophthalmological infections: Conjunctivitis, Blefaroconjunctivitis,
Cellulitis, Keratitis, Endophthalmitis, Dacryocystitis, Blebitis. Certainly some of these
infections are extremely rare, but MRSA infection can have devastating results to the eye and
even be life-threatening.
Many hospitals have adopted measures to control the spread of MRSA. MRSA is a
common cause of nosocomial infections, especially in hospital wards where it is endemic. A
problem that often arises is MRSA postoperative morbidity. Postoperative endophthalmitis is
a potentially devastating complication of cataract surgery. Cultures of conjunctival scrabs of

clinically healthy conjunctivas preoperatively, results to high rates of Gram-positive cocci,
and in many cases MRSA [41]. This is why the Department of Health in the UK has
established screening, and decolonization of patients undergoing cataract surgery,
specifically to those patients who have risk factors to manifest an MRSA infection.
MRSA infections attract interest of ophthalmologists not only due to the increasing
prevalence, and the gradually increasing appearance of community strains, but also due to the
continuous development of resistance of MRSA to various antibiotics. Numerous studies
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explore resistance of MRSA to specific antibiotics. For example, lately, there has been
extensive interest to the increase of resistant MRSA strains to fourth generation
Fluoroquinolones [42, 43, 44].
Freidlin et al (1998-2006) examines the spectrum of eye disease caused by MRSA. They
support the general tendency that the prevalence of SA infections, resistant to methicillin, is
increasing (the proportion of MRSA increased from 4.1% in 1998 to 1999 to 16.7% in 2005
to 2006).The isolates of MRSA were also sensitive to the oxacillin, bacitracin, vancomycin,
ciprofloxacin, erythromycin, sulfisoxazole, and tetracycline, highlighting the difficulties for
the treatment of MRSA infections. A total of 78.0% of patients with MRSA had
blepharoconjunctivitis, 2.4% had cellulitis, 2.4% had dacryocystitis, 14.6% had keratitis, and
2.4% had endophthalmitis [45].
VA Shanmuganathan et al (1997-2001) calculates as MRSA 3% of the isolates, among
548 external ocular infections caused by SA, and also supports the tendency of MRSA
proportion to constantly increase. The MRSA infected patients manifested conjunctivitis (six
patients), keratitis (four patients), dacrocystitis (three patients), conjunctival socket infection
(three patients), and one patient had a plomb abscess following retinal detachment surgery
[25].
1. Conjunctivitis
SA is one of the most common isolates that cause acute bacterial conjunctivitis. It is the
most common bacterial isolate obtained from infected conjunctivas, and also commonly
found to asymptomatic conjunctivas and eyelids [46]. It is responsible for infections in all age
groups. Nevertheless, the spectrum of conjunctival bacteria varies among age groups. It is
higher in the oldest (>70) and the youngest patients (<1) [47]. MRSA conjunctivitis has been
associated with long-term care units [25, 48], especially in patients with neurologic
impairment [49], nurseries [49], neonatal ICUs [50], and healthcare workers [51, 48, 12].
Its action to conjunctiva and cornea is caused by the production of exotoxins that lead to
varying symptomatology from the eyes, depending on the severity and the chronicity of the
infection.
MRSA conjunctivitis manifests the same symptoms and signs as all the common acute
bacterial conjunctivitis. Tearing, grittiness, foreign body sensation, photophobia, diffuse
conjunctival hyperemia and chemosis are observed. Specifically, abundant, thick, fibrinous
discharge is produced, and is characterized purulent and mucopurulent. This causes stuck
eyelids, and the patients describe it difficult to open their eyes at waking. Bacterial infection
exudates consist mostly of polymorphonuclear leukocytes. In more severe inflammations,

there are formed phlyctenules, a characteristic sign of staphyloccocal infection. These are
small, whitish collections of polymorphonuclear leukocytes in the stroma of conjunctiva,
surrounded by small visible vessels. Finally, inflammatory membranes may be present. They
are formed by combination of the fibrous exudates, mucous secretions and inflammatory cells
[52, 53].
SA is strongly contagious via the infected secretions, usually by eye-hand contact. The
infection commonly affects one eye and can be transmitted to the other after 1-2 days [43].
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Acute bacterial conjunctivitis caused by bacteria is usually self-limiting within 5-8 days.
Nevertheless, treatment is recommended. Therapy has been shown to reduce the symptomatic
period, to alleviate the possibility of recurrence, and to limit rare but potentially serious
complications. Treatment also prevents the spread of the infection, protecting from a possible
epidemic. The choice of the management of conjunctivitis is based on the clinical
examination, the age, and the environment of the patient, without performing lab
investigation. The suggested treatment is a broad-spectrum antibiotic topically (ointment or
eye drops), that is effective not only against MRSA, but also against other possible gram
negative and positive bacteria that are usually responsible. Moreover, the patient needs to
follow hygienic instructions, so as to avoid the transmission of the infection; frequent hand
washing, avoiding direct contact with the eyes, not sharing of pillows and towels [43].
Chronic infection is considered conjunctivitis that lasts more than 15 days, and it can
continue for months. Commonly, chronic SA conjunctivitis is concurrent with an infection of
the margins of the eyelids, which produces a specific clinical image, the angular
blepharoconjunctivitis. This condition often presents an epithelial punctuate keratitis at the
lower half of cornea, caused by an intense allergic reaction to the powerful SA exotoxins.
More severe chronic conditions are associated with marginal corneal infiltrates, ulcerations,
peripheral corneal infusions and the formation of pannus [52].
2. Ophthalmia Neonatorum
Ophthalmia Neonatorum is the term used for conjunctivitis of a neonate that arises
during the first 15 days of life. It is the most common infection at this age. Many pathogens
are responsible for this infection, including SA. During the last decade, a great increase of
MRSA infections in neonates has been noticed. SA neonatal conjunctivitis is a concerning
situation, because of systemic complications that may emerge, including sinusitis,
pneumonia, osteomyelitis. Lack of immunity and anatomical reasons (absence of lymphatic
tissue of conjunctiva) predispose to the latter situations. For this reason, prophylactic
treatment is used one hour after their birth. Various treatment modalities are used globally for
prophylactic purpose; silver nitrate, erythromycin, tetracycline, gentamicin, povidone iodine
solution [55, 52].
The infection can be of early-onset, transmitted vertically from the mother to the infant
during birth. SA can also be responsible for late-onset infections. Those occur after 3 days of
life, and are horizontally acquired from parents, or other contacts, such as healthcare
personnel [50]. However, mother is the usual source, since the bacterial strains are often the
same between mothers and infants (in an occurrence of 68% according to a prospective study
of 2003) [56].There is a decline in carriage rate of neonatal conjunctivitis after the 8th week
of life in infants, which reflects the development of an immune response, and subsequent
eradication of carriage [56].
Clinical signs include erythema and edema of the eyelids and the conjunctiva, and
purulent discharge. SA usually produces uncomplicated infections that do not proceed to the
cornea when treated appropriately.
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Outbreaks of MRSA in Neonatal Intensive Care Units have been continuingly and
substantially increasing during the last two decades, worldwide [50, 57] . It is surprising, that
some of these MRSA infections have been attributed to MRSA strains responsible for
infections in the community. These strains were introduced only two decades before, and are
genetically and phenotypically distinct from MRSA strains that cause hospital infections.
These are usually MRSA types USA300 and USA400 [50]. The dangers of CA MRSA
infection in neonatal nurseries should be highlighted; it is easily transmitted and it colonizes
in the nasopharynx for several months. After that space, it relapses, or causes secondary
infections among families [58].
Infants that are mostly reported to be infected by MRSA, gather one or more
characteristics. Often, they are of low birth weight (under 1000 g). This can be correlated to
the fact that these neonates have immature immune system, prolonged hospital stays, and
exposure to invasive devices and procedures 50. Additional attention should be given to the
use of steroids, as it can suppress the immune system, and can increase the chance of
infection [26].
3. Keratitis
Bacterial keratitis is an ophthalmologic emergency. It is the most common reason leading

to corneal blindness in developing countries, as it can have devastating consequences.
Common signs of staphylococcal keratitis are initially corneal epithelial defects. Focal
infiltrates are surrounded by stromal edema and conjunctival injection. Corneal ulcer is
central, white, circular or oval. A small abscess may develop in the frontal stroma. In more
severe cases, hypopyon is evident. Finally, the progression of the corneal ulcer may lead to
perforation of cornea and endophthalmitis [52, 53].
Virulence of SA in SA keratitis is associated to the production of exotoxins, particularly
a-toxin [11], that cause direct damage at the tissues, or induce inflammatory response.
Cell wall components and toxins, produced by SA, stimulate inflammatory reactions to
the cornea. This condition is combined with blepharoconjunctivitis caused by SA. It is
characterized by marginal infiltrates. These are the result of the formation of complexes of
antigens that derive from the tear film, and the antibodies that reach the cornea through the
blood circulation. Secondarily, lymphocytes are present to the inflammation. Cultures from
the infiltrates can be negative, but the clinical presentation, combined with the presence of
SA to the lid margins, poses the diagnosis. Hypersensitivity reaction to the cell wall of SA
may lead to the formation of phlyctenules at the limbus, which may progress into the cornea.
This type of SA keratitis is also associated with conjunctivitis and blepharitis. It can be
associated with systemic conditions as well, such as tuberculosis, or rosacea [52, 53].
Staphylococcus Aureus is one of the most common pathogens that lead to keratitis.
Prevalence of keratitis caused by SA varies in different geographical areas [59, 60, 61].It is
the leading pathogen that leads to bacterial keratitis in the Northern and Northeastern United
States, California, Canada, and in London. In Texas, South Florida, Germany, Netherlands and
Switzerland, it is the second most common cause of bacterial corneal ulcers [46, 62].
Similarly, resistance of various strains of SA to antibiotics differs around the world.
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Methicillin-sensitive isolates are responsible for the majority of SA corneal isolates [46].
However, MRSA prevalence is continuingly increasing, as well as the resistance for SA
strains to various antibiotics (quinolone, aminoglycosides, ciprofloxacin, levofloxacin, and
tobramycin) [63, 64, 44.] Corneal MRSA induces an alerting medical situation. Corneal
perforation caused by MRSA corneal ulcer can develop in less than one day [62]. MRSA is a
usual pathogen for keratitis in children as well [65].
Treatment of MRSA keratitis presupposes receiving of samples from the cornea,
conduction of microbiological cultures, and investigation of sensitivity of the microbe. After
the diagnosis is posed, MRSA treatment is with vancomycin, since there is an increasing
resistance to fluoroquinolones, in certain reports even to the fourth-generation
fluoroquinolones. Topical antibiotics are used, except in certain advanced situations; when
keratitis has proceeded to corneal perforation, or to complication of sclera. Possible
concurrent infection may require treatment with oral tetracycline.
3a. Risk Factors for Keratitis

Risk factors that predispose to microbial keratitis vary among different age groups.
Contact lens wear is the most common risk factor in young patients. Contact lens–related
keratitis is usually associated with soft lenses, and is related to poor hygiene [61, 64, 26].
Traumatic keratitis is also more common in younger patients. These cases usually include
men in a substantial percentage, and are caused by organic or metallic material. It is
noteworthy that keratitis related to corneal trauma in developing countries is mostly due to
agricultural accidents [61].
Keratitis associated with previous ocular surgery, or ocular surface disease, is more
common in older patients. Bacterial keratitis after a recent surgery is usually associated to a
stitch abscess from cataract extraction, or penetrating keratoplasty. Keratitis related to an old
surgery is typically observed after penetrating keratoplasty [61]. More rare types of surgery
that may lead to keratitis are trabeculectomy, retinal detachment repair, photorefractive
keratectomy (PRK), lid procedures, conjunctival flap, strabismus surgery, and vitreous biopsy
[66]. Appropriate follow up for postoperative surgical patients is necessary, and so is removal
of all corneal sutures. Particular attention should be given to identifying loose or broken
sutures [66].
Ocular surface disease that leads to MRSA keratitis, includes a variety of ocular
conditions; the most usual is Herpes simplex keratitis. Other cases are keratoconus, eyelid
abnormalities, history of penetrating keratoplasty or other intraocular surgery, corneal
thinning or pannus, neurotrophic keratitis, neovascular glaucoma, bullous keratopathy, atopic
keratoconjunctivitis, blepharitis, ocular rosacea, corneal dystrophies, corneal ulcer, band
keratopathy, orbital cellulitis, and orbital fractures [46, 64]. Disorder of the lacrimal system
limits the function of defence of the ocular surface, and may result to corneal infection. Dry
eye, dacryocystitis, deficiency in tears, obstruction of the nasolacrimal duct and generally
local corneal trauma may lead to bacterial keratitis [67]. Topical steroid use is an important
risk factor for the emergence of bacterial keratitis. It is associated with larger corneal lesions
and poorer vision [66].
Systemic diseases and conditions can constitute risk factors for bacterial keratitis. These
include diabetes, autoimmune diseases, hepatitis C, HIV, cocaine use [64]. Stevens-Johnson
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syndrome is related to microbial keratitis. It leads to the expression of corneal intraepithelial

infiltrations [25].
Finally, MRSA keratitis has been described in healthcare units, at patients that had not
been subjected to surgery [25, 12].
In ages below twelve, the commonest risk factor for development of MRSA keratitis is
trauma. Moreover, systemic disease, ocular disease and previous ocular surgery predispose to
children‘s keratitis. Systemic diseases include hypoxic encephalopathy, cerebral palsy,
Marshall-Smith syndrome, atrial septum defect and pulmonary stenosis. Ocular diseases
include epidemic keratoconjunctivitis, trichiasis, blepharitis, phthisis bulbi. Finally, ocular
surgeries predisposing to keratitis are surgery for ptosis, corneal laceration, and strabismus
[65]. Infants more easily develop bacterial keratitis when they have epidemic
keratoconjunctivitis, compared to adults. This probably is explained due to immaturity of
neonatal immune system [26, 56, 68, 59].
3b. MRSA Postopperative Keratitis

Suture-related keratitis caused by MRSA, following uncomplicated cataract surgery, is a
condition of great concern. It can be associated with a loose suture from cataract surgery
conducted even years ago [69]. Risk factors for the development of infection include loose or
broken sutures, corticosteroid use, persistent epithelial defect, contact lens use,
keratoconjunctivitis sicca, and a history of herpes simplex virus keratitis.
Severe MRSA keratitis, which may result to endophthalmitis, can emerge after multilayer
amniotic membrane transplantation (AMT) [70]. MRSA sclerokeratitis has also been
observed after pterygium excision [71]. Frequently, infectious scleritis after pterygium
excision is associated with adjunctive b-irradiation, mitomycin C application, and excessive
cauterization, but pterygium excision alone can also induce this condition [71].
Post operative MRSA concern has emerged since photorefractive surgery has been
introduced. Refractive surgery is considered one of the safest ophthalmic surgeries. However,
one of the most serious complications is the potentially sight threatening development of
infectious keratitis. The incidence of corneal infection has been calculated to be 0.01% to
0.8% after PRK, and 0.01% to 0.31% after LASIK 72. Gram-positive bacteria, in particular
Staphylococcus species, are the most common organisms causing infectious keratitis
following PRK, while MRSA keratitis is a serious and increasing complication following
refractive surgery [73], that may even end up to corneal transplantation [74]. Patients exposed
to a hospital environment, and subjected to ocular surgery, are recommended to receive
prophylaxis against MRSA [72, 73, 74].
3c. Contact Lenses and Keratitis

Contact lens using is the most severe predisposing factor for the development of MRSA
infection of cornea, specifically in young patients, in the Western world [7]5. Staphylococcus
Aureus is the second commonest pathogen causing keratitis related to contact lens use,
following Pseudomonas aeruginosa. Symptomatology and severity vary, and may lead to
potentially sight threatening infections [76], if not treated properly. Keratitis of CLs can be
considered a public health problem, because CL use is continuingly increasing [77].
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CL use causes hypoxia and mechanical stress on the cornea [78], which lead to epithelial
edema, and superficial punctate keratitis, which may predispose to corneal infection [67].
High-oxygen-permeable lenses permit less bacterial adhesion to corneal cells than less
permeable lenses [77]. Microtrauma of the cornea may occur with the use of contact lenses,
allowing bacteria to adhere to the surface of the cornea. Contact lens use also causes reduced
tear exchange and dryness, depriving the corneal epithelium of normal tear flushing, and
consequently from the non-specific humoral immune mechanisms [67].
Pathogenic organisms are transmitted to the contact lens usually after colonization of the
contact lens storage case, which is commonly the most contaminated item [79, 80]. Lens
cases are contaminated in a high percentage of asymptomatic CL wearers, despite good
hygienic compliance. Contact lenses are also found to harbor microorganisms in
asymptomatic wearers [80, 81].
Extended research has led to the information that offers details concerning the types of
CLs more often related to keratitis. Daily-wear soft lenses are usually responsible for
infections of cornea. Silicone hydrogel lenses are reported to decrease possibility of
contamination compared to lenses made of traditional hydrogel materials. Hydrophilic and
roughest CLs reduce the possibility of bacterial retention and transmission [77].
Noncompliance of CL users to the hygienic instructions is the most common cause of
CL–related microbial keratitis [82]. Appropriate and effective contact lens disinfection and
storage is fundamental to their safe use. Therefore, numerous studies compare contact lens
disinfection methods and efficacy of contact lens solutions [79, 81, 83], storage cases, or
generally lens care accessories [80].
Extended wear is also associated with higher risk of microbial keratitis [76, 67]. The
incidence of microbial keratitis is greater in patients who wear their lenses continuously [78],
since extended use of CLs is more likely to lead to hypoxia and microtrauma of the cornea.
Contact lenses used by occasional wearers were reported to present a higher contamination
rate. CLs that are left in the case, without being used for a period of time, provide a more
appropriate environment for the attachment of microorganisms [80]. New contact lens users
seem more compliant with the recommended hygiene guidelines, compared to long-term
users, as they feel less experienced and follow the instructions for disinfection. Thus, they
present limited risk of developing infection [83]. On the other hand they are less skilful in
wearing CLs. Hands and lower eyelids are sources of microbes [84], therefore contaminants
from these sites may easily be transferred onto contact lenses and lens cases ]80, 85].
High rates of CL keratitis emphasize the need for the development of new methods to
protect contact lenses and lens care accessories from contaminants. These could include
modified lens care solutions with more effective antimicrobial agents, and new contact lens
or lens case materials that are resistant to the attachment of microorganisms [80]. Patients
who wish to wear lenses continuously should be educated about the potential risks of
bacterial corneal infection [78].
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4. Cellulitis
Preseptal cellulitis is an infection of subcutaneous tissues. It is located anterior to the

orbital septum, thus it is not considered an orbital infection. However, it can rapidly lead to
orbital infection, behind the orbital septum, orbital cellulitis. Orbital cellulitis is a much rarer
condition, but it is a life-threatening situation, and needs immediate treatment. Preceptal
cellulitis is a clinical entity of great concern to ophthalmologists, since if it is not correctly
treated, the infection may proceed towards the orbit, the brain and the blood circulation and
cause devastating visual, intracranial, and systemic infections. SA is one of the most usual
pathogens causing this infection [86]. Nosocomial or community acquired MRSA occurrence
is constantly rising, representing a great percentage of SA isolates [87, 88, 39].
Preseptal cellulitis is usually associated with skin topical trauma, periocular infections or
after haematogenous transmission from an upper respiratory infection, or an otitis. Periocular
conditions include ethmoiditis, hordeolum, impetigo, dacryocystitis, and tooth abscess [86].
Preseptal cellulitis typically has a benign clinical course, when appropriate antibiotic
treatment is given. However, in some cases, it may lead to complications, such as lid abscess
and orbital cellulitis [86]. The majority of orbital cellulitis occurs as secondary spread from
paranasal sinusitis. The anatomical proximity of the sinuses and the orbit, predispose to
infections of the paranasal sinuses that spread to the orbit [89]. Other local infections may
also lead to orbital cellulitis, such as dacryocystitis, and odontogenic MRSA sinusitis, via
maxillary sinusitis [89]. Other causes are penetrating trauma through the orbital septum, and
periocular surgery (orbital, lacrimal or retinal surgery) [39, 90]. There has also been
described development of orbital cellulitis after deep posterior subtenon triamcinolone
injection [91]. Finally, SA infection to the orbit can manifest due to haematogenous spread.
The infection is unilateral. Preseptal cellulitis is an infection anterior to the orbital
septum, characterised by acute onset of lid edema, tenderness, erythema, warmth and
chemosis [86]. Severe orbital inflammation can progress to an orbital compartment
syndrome, resulting in compression of orbital soft tissues [39], and leading to additional signs
that assist the differential diagnosis of the two conditions (preseptal and orbital infection).
Compression of the optic nerve and extraocular muscles compose the clinical image. The
additional signs observed are potentially severe visual loss pain, limited bulbar motility,
afferent pupillary defect, proptosis. Increase of intraocular pressure and exposure keratopathy
are possible complications. Occlusion of central retinal artery and vein, endophthalmitis and
orbital abscess are more serious but rarer manifestations of orbital cellulitis. Often, the
infection produces general features, malaise, fever, risen white blood cells, and erythrocyte
sedimentation rate (ESR) [87, 92].
Within the orbit, this infection is extremely virulent, and resistant to most antibiotics,
causing severe damage [93]. It may lead to complications such as cavernous sinus
thrombosis, meningitis, and multiple brain abscesses [88]. Subperiosteal abscess is also a
serious infection that may proceed into the cranial cavity.
Orbital infections with MRSA usually occur in individuals with well established risk
factors, such as a recent hospital admission, multiple antibiotic treatment, or chronic illness.
Other predisposing factors are diabetes, cancer, liver disease (usually hepatitis C),
intravenous drug abuse, or surgical procedures [12], alcoholism, insect bites, thorn injury,
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and impetigo [86]. Known risk factors for transmission of CA MRSA include end stage renal
disease, recent hospital admission, an outpatient visit, nursing home admission, antibiotic
exposure, chronic illness, and close contact with a person with risk factors, including health
care contacts [ 92].
Community-acquired MRSA (CA MRSA) causing orbital cellulitis is rare, but there has
been an increase in incidence [93]. These CA MRSA strains have different genetic
background and antibiotic susceptibility profiles than hospital strains. They affect patients
that do not belong to the common risk groups. They affect prison inmates, athletic teams,
military recruits, and children attending day care [93]. Nevertheless, community-acquired
MRSA has been noticed to infect children with no risk factors for MRSA soft-tissue infection
[94, 95].
Treatment of preseptal cellulitis is with oral antibiotics, rarely intramuscular. Orbital
cellulitis requires an immediate and organized treatment modality. Admission to the hospital
is necessary for the investigation of optic nerve function, blood tests, CT of the orbit, brain
and sinuses. Therapy is with intramuscular or intravenous antibiotics. In addition, the orbit
usually is drained. In case there is no response to treatment, and the condition is progressing,
or if there has been formed an abscess, it is necessary to proceed to surgical treatment [53].
5. Endophthalmitis
Endophthalmitis is the term used to define any intraocular infection. It is classified as

either endogenous or exogenous, depending on the route of acquisition of the infection.
Exogenous endophthalmitis occurs when certain situations allow the pathogen to insert into
the intraocular spaces. Such situations are intraocular surgery, penetrating injury, or a corneal
ulcer. It most commonly occurs as a postoperative complication of cataract surgery.
Endogenous (metastatic) endophthalmitis, occurs when organisms reach the eye via the
bloodstream. Pathogens enter the internal ocular spaces by crossing the blood–ocular barrier,
resist host defenses, and multiply within the eye. Endogenous bacterial endophthalmitis
(EBE) is less common than exogenous bacterial endophthalmitis and accounts for only 2–6%
of all cases of endophthalmitis [96, 97, 42].
Endophthalmitis is a therapeutically challenging and often sight-threatening infection.
The poor prognosis is related in part to the delay in diagnosis, inadequate treatment, and the
virulence of the organism [97, 98].
Staphylococcus aureus is a frequent cause of endophthalmitis, most commonly associated
with cataract surgery, which is the most reported cause of endophthalmitis in general [42].
SA is a highly virulent pathogen leading to loss of vision when implicated with

endophthalmitis [97]. The production of numerous toxins by SA in situ in endophthalmitis,
influences the course of the infection [10, 11]. Moreover, intraocular infections from SA have
devastating results due to an anatomical reason; intraocular space is characterized by lack of
arteries and veins, which leads to no significant immunological reaction at the early stages of
bacterial infection, and the expression of rapid and severe infection [11]. MRSA prevalence
leading to endophthalmitis is constantly rising, a fact that expresses the global increase of the
entity of MRSA infections [99].
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A significant proportion of SA cases not only were resistant to methicillin, but those
resistant isolates also were resistant to the commonly used fourth-generation
fluoroquinolones [42]. MRSA endophthalmitis has been observed to patients that have
received topical antibiotic prophylaxis with fluoroquinolones prior to cataract surgery and in
the postoperative period [100]. The recent emergence of increased antibiotic resistance
among staphylococcal strains, threatens to further increase the rate of treatment failure for
this organism [11]. MRSA endophthalmitis is manifested with a more severe clinical
presentation, related to MSSA. A tendency for worse visual acuity in the MRSA cases is
observed, showing limited to no improvement.
Symptoms of endophthalmitis include ocular pain, blurred vision, floaters, photophobia,
and headache. Clinical presentation of endophthalmitis is expressed with swollen eyelids,
injected and chemosed conjunctiva. In more severe cases, anterior chamber inflammation, iris
nodules or plaques, elevated IOP with associated corneal edema. MRSA infections are
presented with a significant hypopyon and visible fibrinous exudates in the anterior chamber
[42, 53]. The posterior segment presents a reduced or absent red reflex. There may be
observed retinal infiltrates, vitreous haze, retinal necrosis or poor fundal view secondary to
intraocular inflammation [96, 53]. The patient usually presents systemic signs with fever.
SA endophthalmitis is associated with poor visual outcome (defined here as best-
corrected visual acuity of 20/400 or worse) in more than 52% of reported cases [11].
Bacterial endophthalmitis is a concerning postoperative complication, due to its devastating
results. Various measures have been suggested globally for prophylaxis against infection.
Pre-operative conjunctival irrigation with 5% povidone iodine is indicated. Topical
antibiotics (fluoroquinolone) are instilled up to 3 days before surgery. Injections of
intracameral cefuroxime and subconjunctival antibiotics are proposed after surgery.
However, cefuroxime is not effective against MRSA. Additionally, resistance of MRSA to
fluoroquinolone is also proven to be emerging [101,102]. Any type of ocular infection has to
be treated before surgery (conjunctivitis, blepharitis, dacryocystitis).
Risk factors for MRSA endophthalmitis include advanced age, recent hospitalization,
antibiotic use, immunosuppression, treatment with an immunosuppressive agent. Other
factors that are associated to this severe ocular infection are intravenous drug use, HIV
infection, autoimmune disease, blood malignancy, alcohol abuse, asplenia 96,42,12. Invasive
MRSA, however, may still occur in patients with no established health care risk factors, and
may be associated with both community and health care origin.
Bacterial endophthalmitis is associated with underlying medical conditions such as
diabetes, cardiac disease, and malignancy. It also occurs as a result to medical interventions,
such as intravenous access, surgery, invasive therapeutic or diagnostic procedures, and
hemodialysis. Patients may present with systemic infections, by hematogenous spread, from
an infection from liver, lungs, central nervous system, endocardium, renal and urinary tracts.
However, it can also manifest in isolation [96].
Factors that induce bad prognosis are delay in diagnosis, use of inappropriate antibiotics,
diffuse infection of the vitreous and retina, or panophthalmitis, low visual acuity at
presentation, infection with virulent organisms [96, 97, 98].
Prognosis of endophthalmitis is poor. Patients may result with count fingers vision or
better, they may end up blind, or require evisceration or enucleation. There is also a mortality
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rate with 5% of patients, dying as a result of associated extraocular infection [96]. Other
complications concern post-operative endophthalmitis, and lead to loss of vision, such as
secondary glaucoma, retinal detachment, chronic uveitis, macular edema, phthisis,
panophthalmitis.
Aqueous and vitreous samples must be received and undergo microbiological
examination. Blood culture is the most reliable way of establishing the diagnosis of
endogenous endophthalmitis [96]. Finally, it is fundamental to investigate for other potential
inflammations, meningitis, endocarditis, arthritis or other locations such as catheter tips, skin
wounds, and abscesses [53].
Treatment of post operative endophthalmitis is with intravitreal antibiotics. Usually,
second generation cephalosporins are combined with vancomycin. Oral antibiotics are added,
as well as steroids, for the limitation of the inflammatory process. Vitrectomy is performed
when visual acuity is light perception at presentation [53].
Endogenous endophthalmitis necessitates the treatment of the systemic infection, based
on the findings from the cultures and sensitivity tests. Chosen antibiotics are usually
ceftazidime and vancomycin intravenously [53].
6. Dacrocystitis
Dacryocystitis is a common infection of the lacrimal sac. Among the bacterial isolates
found during dacryocystorhinostomy, SA is one of the most common, if not the commonest
[103,10]4. Increased frequency of MRSA infections is observed at this type of ocular
infection as well [105]. MRSA dacryocystitis usually develops in cases with a history of
nasolacrimal duct obstruction [106]. In a small proportion, dacryocystitis is associated with
previous maxillofacial trauma or maxillofacial surgery, with chronic use of antibiotics, and
with immunosuppression [105,40]. CA MRSA has also been reported as the responsible
pathogen in many cases during the last decade [12,107,40].
MRSA dacryocystitis may be chronic, acute or subacute and can be associated with
concurrent conjunctivitis [108]. MRSA is suggested to cause acute infections more
frequently, when compared with chronic. MRSA infections are more aggressive and less
possible to respond to initial antibiotic treatment [105].
Dacrocystitis may be painful, but rarely result to limitation of vision, or to life-
threatening situations [108]. A child colonized with CA MRSA chronic dacyrocystitis is at
high risk for developing an MRSA wound infection, either postoperatively or secondary to
trauma, since CA MRSA is related to soft tissue and skin infections [40]. Rare but
devastating outcomes are at risk to manifest after progress of dacryocystitis to orbital

cellulitis or orbital abscess, meningitis, or cavernous sinus thrombosis [105]. Accepted
therapies for dacryocystitis include antibiotics, lacrimal incision and drainage, and lacrimal
surgery (dacryocystorhinostomy).
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Conclusion
SA is one of the most common causes of ocular infection worldwide, responsible for
infections in the hospital environment, and during the last two decades, in the community as
well. Constantly emerging SA strains, resistant to multiple antibiotics, alarm
ophthalmologists globally, and this highlights the need of introduction of national and
international guidelines for strict infection control. These guidelines should target to
limitation of transmission of the pathogens, and to controlled use of the antibiotics.
Suppression of transmission of MRSA infection should involve specific instructions to
patients and healthcare workers. These instructions could include frequent hand washing and
antisepsis between contacts with different patients; cleaning of ophthalmological instruments
and surfaces that patients have been in contact with; isolation measures for patients known to
be infected by MRSA or other resistant strains; use of gloves and masks by healthcare
workers when they are in contact with infected patients.
Early and accurate identification of presence of resistant strains of SA is essential, to
prevent their spread. This is achieved with rapid and safe laboratory results. New diagnostic
methods are constantly proposed. However, empirical therapy is often imposed, and it should
include antibiotics effective on resistant strains of SA. In addition, CA MRSA infection
should be suspected when there is contact with facilities where it is usually spread (prisoners,
soldiers, team athletes), and where there are manifested predisposing factors for CA MRSA
infection.
Specific recommendations to ophthalmologists, worldwide, need to be introduced, and
aim to the control of misuse of antibiotics. Limited or excessive dose and duration of therapy
leads to development of resistance more easily, consecutively, treatment modalities should be
strictly appropriate in dose and duration. An example of debate between ophthalmologists is
the routine prophylactic use of vancomycin before intraocular surgery, for the prevention of
bacterial endophthalmitis. Many consider it overuse and are not convinced that routine
vancomycin prophylaxis is necessary. They propose alternative measures, like preoperative
povidone iodine antisepsis of the ocular surface [109].
Ocular MRSA prevalence is continuingly increasing [46] , as sensitivity of SA has
declined over the past decade, globally [43, 54]. The need for the investigation of new agents
targeted for ocular use, leads to novel broad-spectrum antibiotics, which are favourable for
the treatment of MRSA infections [43].
Increasing resistance of SA to ciprofloxacin and levofloxacin is reported, which leads to
the conclusion that newer fluoroquinolones will also end up ineffective for the treatment of
such infections. Fourth-generation quinolones, such as moxifloxacin and gatifloxacin are
recently introduced against MRSA, but already resistant strains of MRSA from systemic
infections have arisen. Other antibiotic drugs recently introduced and effective against SA are
oxazolidinones such as linezolid. However, resistance to linezolid has also already been
reported. In addition, its use must be controlled, due to severe adverse effects, such as
myelosuppression. Chloramphenicol use is also restricted, due to the possibility of
development of aplastic anemia. Teicoplanin, another glycopeptide antibiotic, is less effective
against SA than vancomycin, especially in cases of reduced vancomycin susceptibility. Both
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Account: ns004409
vancomycin and gentamicin remain at least 95% effective, against corneal and conjunctival
SA isolates [1, 12, 8].
In countries where strict isolation measures and infection control are followed, there has
been successfully controlled dissemination of MRSA. Global strategies for the prevention of
the spread of new resistant strains of SA need to be suggested and imposed.
Ophthalmologists need to be capable of treating resistant strains on time, but also to
participate to the global effort to suppress the emergence and spread of resistant strains.
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Chapter IV
Proteomics and Metabonomics of the

Vitreous Fluid as a New Approach to
Identify New Candidates in the
Pathogenesis of Diabetic Retinopathy
Rafael Simó* and Cristina Hernández

CIBERDEM (CIBER de Diabetes y Enfermedades Metabólicas Asociadas) and
Diabetes and Metabolism Research Unit. Vall Hebron Institut de Recerca (VHIR),
Universitat Autónoma de Barcelona, Barcelona, Spain
Abstract
Diabetic retinopathy remains a leading cause of blindness and visual impairment

among adults aged <40 years in the developed world. Investigation into the processes
involved in DR and the testing of new therapies are limited because the unavailability of
human retina samples and the lack of diabetic animal models that faithfully replicates the
features of human DR. Vitreous fluid obtained from diabetic patients undergoing
vitreoretinal surgery is currently used as a surrogate for the retina in clinical research.
However, the volume of vitreous fluid obtained after vitrectomy is approximately 1 ml
and, therefore, only a few peptides or metabolites can be analyzed simultaneously. The
recent development of high-throughput techniques such as proteomics and metabonomics
has made it feasible to analyze both the protein and metabolite profiles in a massive
manner. In recent years we have been able to demonstrate by proteomic analysis that
several proteins such as complement factors (C3, C4b, C9 and factor B), Apo A1 and
Apo-H were higher in the vitreous fluid from patients with proliferative DR (PDR) than
non-diabetic patients, whereas the interstitial retinol-binding protein (IRBP) was lower.
In addition, mRNA levels in the retina run in parallel with proteins, thus suggesting that
* Correspondence: Dr. Rafael Simó, Diabetes Research Unit. Institut de Recerca Hospital Universitari
Vall d‘Hebron, Pg. Vall d‘Hebron 119-129. 08035 Barcelona. Spain, Telephone: 34934894172. FAX:
34934894032, e-mail: rsimo@ir.vhebron.net
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Copyright © 2012. Nova Science Publishers, Inc. All rights reserved. May not be reproduced in any form without permission from the publisher, except fair uses permitted under 94 Rafael Simó and Cristina Hernández
the intraocular production of these proteins participates in the physiopathology of DR.

Furthermore we have recently identified by proteomic analysis genuine proteins (ie.
hemopexin and clusterin) differently expressed in the vitreous fluid from patients with
diabetic macular edema. Finally, by using metabonomics we have been able to shown
that, apart from a higher abundance of both lactate and glucose, significant deficits of
galactitiol and ascorbic acid also exist in the vitreous fluid from PDR patients. The
potential role of all these candidates in the pathogenesis of DR will be discussed in this
chapter. In summary, proteomics and metabonomics of the vitreous fluid are useful not
only for identifying potential candidates in the development of DR, but also for
designing new approaches leading to a more efficient pharmacologic treatment of this
devastating complication of diabetes.
Introduction
Diabetic retinopathy (DR) remains the leading cause of blindness and vision loss among
adults aged under 40 years in the developed world. Whereas proliferative diabetic retinopathy
(PDR) is the commonest sight-threatening lesion in type 1 diabetes, diabetic macular edema
(DME) is the primary cause of poor visual acuity in type 2 diabetes. Because of the high
prevalence of type 2 diabetes, DME is the main cause of visual impairment in diabetic
patients [1, 2]. Although tight control of both blood glucose levels [3, 4] and hypertension [5]
are essential to prevent or arrest progression of the disease, the recommended goals are
difficult to achieve in many patients and, consequently, DR develops during the evolution of
the disease. When DR appears laser photocoagulation remains the main tool in the
therapeutic armamentarium [6]. The objective of laser photocoagulation is not to improve
visual acuity but to stabilize DR, thus preventing severe visual loss. When laser
photocoagulation is indicated in time, the risk of blindness during the following 5 years is
reduced by 90%, and the loss of visual acuity is reduced in 50% in those patients with
macular edema. However, timely indication is often passed and, therefore, the effectiveness
of laser photocoagulation in current clinical practice is significantly lower. In addition, laser
photocoagulation destroys a part of the healthy retina and, in consequence, side effects such
as loss in visual acuity, impairment of either dark adaptation and colour vision, and visual
field loss may appear. Vitreo-retinal surgery could be indicated in advanced stages of DR (ie.
hemovitreous, retinal detachment) [7]. However, this therapeutic option requires a skilful
team of ophthalmologists, is expensive and fails in more than 30% of cases. With this
scenario it seems clear that new treatments based on the understanding of the
pathophysiological mechanisms of DR are needed.
Investigation into the processes involved in DR and the testing of new therapies are
limited because of the unavalaibility of human retina samples and the lack of diabetic animal
models that faithfully replicate the features of human DR.
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Copyright © 2012. Nova Science Publishers, Inc. All rights reserved. May not be reproduced in any form without permission from the publisher, except fair uses permitted under Proteomics and Metabonomics of the Vitreous Fluid … 95
Analysis of Vitreous Fluid in the Study

of Diabetic Retinopathy
The posterior chamber of the eye has an approximately spherical shape, and is filled with
vitreous humor, a transparent material with viscoelastic properties [8]. Besides providing an
unhindered path for light to reach the retina, the vitreous has the important mechanical roles
of supporting the eye shape, promoting the adherence of the retina to the choroid (the
vascular layer between the retina and the sclera), and also acting as a diffusion barrier
between the anterior and posterior segments of the eye [9].
Vitreous fluid obtained from patients undergoing vitreoretinal surgery is currently used
as a mirror of the in vivo metabolic events that are taking place in the retina. However, there
are three main confounding factors that could lead to misinterpretation of the results [10].
First, vitreous hemorrhage, which often occurs in PDR can produce a massive influx of
plasma metabolites into the vitreous fluid, thus precluding the usefulness of vitreous fluid
when studying intraocular metabolite production. In order to minimize this problem blood
contamination should be ruled out by selecting vitreous samples with detectable hemoglobin.
Secondly, the disruption of the blood-retinal barrier that occurs in diabetic retinopathy
produces an increase of proteins in the vitreous body of diabetic patients. Therefore, the
elevated intravitreal level of any particular protein does not necessarily mean its intraocular
production, and might only reflect the non-specific increase of proteins due to serum
diffusion. Finally, laser photocoagulation can modify transcriptional activity in the retina and,
for this reason, samples from patients in whom laser photocoagulation had been performed in
the preceding 6 months should be excluded. However, the volume of vitreous fluid obtained
after vitrectomy is 1 ml and, therefore, only a few peptides can be analyzed simultaneously.
The recent development of proteomics and metabonomics have made it feasible to
analyze protein profiles in various cells, tissues and body fluids using only a small sample
[11]. In fact, both proteomics and metabonomics, as well as genomics/transcriptomics, are
high-throughput enabling technologies, giving us information quickly about different
biological levels by measuring transcripts, proteins and metabolites [12-14]. While
genomics/transcriptomics involves the study of gene expression and proteomics the
expression of proteins, metabolomics investigates the consequences of the activities of these
genes and proteins. The progress of this systems based approach is in a large part dependent
on developments in biostatistics and bioinformatics to integrate high-dimensional data.
This review examines the application of proteomic and metabonomic techniques for the
analysis of vitreous fluid which have permitted us to identify new candidates in the
pathognesis of DR and, therefore, potential targets for DR treatment.
Vitreous Proteomics
Proteomics is the research area revealing the temporal dynamics of proteins expressed in
a given biological compartment at a given time. One widespread proteomic strategy involves
the use of 2D gel electrophoresis followed by enzymatic degradation of isolated protein spots
and identification using mass spectrometry (MS). There are a number of different types of
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MS systems current in use [15]. Two of the most widely used systems are matrix assisted
laser desorption/ionization-time of flight (MALDI-TOF) and LC-tandem MS (MS/MS).
Altough the development of proteomic analysis has made it feasible to analyse protein
profiles in body fluids using only a small sample [11], the information drawn from proteomic
analyses using human vitreous fluid in the setting of DR is limited [16-25]. Characterization
of the vitreous proteome in patients with diabetic retinopathy is likely to provide new insights
into the factors and mechanisms responsible for its development.
Proteomic Analysis in Diabetic Retinopathy
Proliferative Diabetic Retinopathy

There is little information about the intravitreal proteins differently expressed in PDR
patients in comparison with non-diabetic subjects using proteomic analysis [16-19, 21-24].
We have previously reported the differentially produced proteins in PDR patients in
comparison with non-diabetic patients by using fluorescence-based difference gel
electrophoresis (DIGE) [23]. This technique provides accurate quantitative comparisons and
we have further optimized it in order to avoid potential confounding factors. Indeed, we
facilitated the identification of potential candidates by depleting, by affinity chromatography,
the two most abundant proteins (albumin and IgG) prior to electrophoresis. This is important
because, albumin and IgG account for more than 80% of whole-vitreous protein, and the
large spots of these proteins overlap other small spots, corresponding to less abundant
proteins, so precluding their identification. Furthermore, all the candidates identified by
proteomic analysis were independently confirmed by ELISA or western blot analysis.
However, a limitation of this technique is that proteins of very high or low molecular mass
are frequently precluded from analysis by the electrophoresis procedure itself.
By means of proteomic analysis we and others have identified several proteins differently
expressed in the vitreous fluid of patients with PDR in comparison to non-diabetic subjects
(Table 1 and Figure 1). Among these proteins, the most directly involved in the pathogenesis
of DR are the following:
Table 1. Proteins differently expressed in vitreous fluid from patients with PDR
Increased Decreased
Actin cytoplasmic 1 [24] Amyloid beta A4 protein [24]
Alpha-1-acid glycoprotein [24] Amyloid-like protein 2 [24]
Alpha-2-HS-glycoprotein [24] Calsyntenin 1 [24]
Alpha-1-antitrypsine [24] Collagen alpha-3(V) chain [24]

Angiotensinogen [24] Complement C1q subcomponent subunit C [24]
Antithrombin III [24] Ectonucleotide pyrophosphatase/phosphodiesterase 2 [24]
Apolipoprotein A1 [23, 24] Extracellular SOD [24]
Apolipoprotein A-II [24] Hemoglobin subunit alpha [24]
Apolipoprotein H [23] Hemoglobin subunit delta [24]
Apolipoprotein C-III [24] Interphotoreceptor matrix proteoglycan 2 [24]
Apolipoprotein A-IV [24] Interstitial retinol-binding protein [23, 24]
Beta-2-microglobulin [24] ITIH2 [23]
Carbonic anhidrase 1 [24] Neuroserpin [24]
C1 [24] PEDF [23]
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C3 [23, 24] Protease serine 3 [24]

C4b [23] Spondin 1 [24]
C9 [23] Steerin3 protein [24]
Catalase [18] Vacuolar ATP synthase subunit S1 [24]
Factor B [23, 24]
Factor XII [24]
Fibrinogen A [23]
Histidine-rich glycoprotein [24]
IGHG1 protein [24]
Leucine-rich alpha-2 glycoprotein [24]
Neuron-especific enolase [18]
Peroxiredoxin [24]
Prothrombin [24]
Vit. D-binding protein [24]
Zinc-2 glycoprotein [23]
Figure 1. Upper panel: superimposed images in pseudocolor from Cy3 (green, PDR vitreous proteins) and
Cy5 (red, macular hole [MH] vitreous proteins) labeled samples run on a 2D-DIGE gel. The range of the
horizontal dimension is pI=3 (left) to pI=10 (right). The vertical dimension ranges from ~ 15 kDa (bottom) to
~ 200 kDa (top). The position of the spots corresponding to two proteins increased and decreased in
abundance are marked. Flanking the 2-D gel image are the three-dimensional images of these spots,
corresponding to the Cy5 image (control MH, left panel for each protein), and the PDR Cy3 image (PDR,
right panel for each protein). Lower panel: Standardized abundance plots for each of the protein spots
marked above. Each graph displays the abundance observed for the spot in each of the four gel images
corresponding to control MH samples (C) (blue circles) and PDR samples (red circles), after standardizing
the values using the internal standard pool images (Cy2) of each of the four gels. The line links the average
abundance values for each group of samples (crosses). Calculation of a t-test for the difference in abundance
between each two groups results in p values < 0.05 in all cases shown.
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PEDF (Pigment Epithelium Derived Factor)

PEDF is mainly secreted by retinal pigment epithelial (RPE) cells in the eye [26]. Apart
from its neuroprotective properties, there is growing evidence to suggest that PEDF is one of
the most important natural inhibitors of angiogenesis and that it is the main factor accounting
for the antiangiogenic activity of vitreous fluid where it is found in abundant quantities [26,
27]. Two of the most importnant components of the diabetic milieu, high glucose levels and
hypoxia downregulate PDF expression in RPE cells [10].
The relevance of PEDF to PDR development is supported by studies showing that
decreased levels of PEDF in the vitreous are associated with PDR [28, 29]. PEDF has been
also reported as a novel endogenous anti-inflammatory factor in the eye, and its decreased
ocular levels may contribute to inflammation and vascular leakage in diabetic retinopathy
[30]. In addition, it should be noted that the upregulation of PEDF in proliferating retinal
pigment epithelial cells may, in part, account for the success of panretinal photocoagulation
in reducing neovascularization [31].
In 2006, the human Transport Secretion Protein-2.2 (TTS-2.2)/independent phos-
pholipase A2 (PLA2) , a novel lipase critical for triglyceride metabolism (also known in
mice as adipose triglyceride lipase -ATGL, desnutrin, and patatin-like phospholipase domain
containing protein -PNPLP2), was identified as a specific receptor for PEDF (PEDF-R) in the
retina [32]. In addition, it has been suggested that antiangiogenic and neurotrophic activities
reside in separate regions of the molecule, thus suggesting that more than one receptor exists
[33].
PEDF can successfully be delivered to the eye by viral vectors. As an alternative to viral-
mediated gene transfer, transplantation of autologous cells transfected with plasmids
encoding for PEDF delivers therapeutic doses of PEDF to the eye. Another method of
delivering PEDF to the eye is to exploit its endogenous availability or production. It seems
likely that much of the endogenous PEDF in the eye is bound to extracellular matrix
molecules and thus may not be active. Drugs that release PEDF from these matrix molecules
could increase free PEDF to therapeutic levels. In addition, levels of PEDF mRNA and
secreted protein could be increased by either dexamethasone or retinoic acid [34].
Therefore, there are good reasons for proposing PEDF as a serious candidate for DR
treatment and new strategies for diabetic retinopathy treatment based on PEDF activation are
warranted.
IRBP (Interstitial Retinol-Binding Protein)

By means of proteomic analysis we have been able to demonstrate for the first time that
lower intravitreous IRBP levels exist in patients with non-proliferative DR in comparison
with non-diabetic patients [23]. Furthermore, we have found lower expression and content of
IRBP in the retinas from diabetic donors without fundoscopic abnormalities in comparison
with non-diabetic donors [35]. Therefore, a low production of IRBP does exist in the diabetic
eye. IRBP is a large glycoprotein (Mr ~140 kDa) that constitutes approximately 70% of the
protein component of the interphotoreceptor matrix, in which it has a highly restricted tissue-
specific expression. IRBP mRNA is present in the photoreceptor cells of the retina, primarily
in rod cells [36].This localization, and the observation that the isomeric form of retinoid
bound to IRBP varies with light and dark adaptation, led to the suggestion that IRBP plays a
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major role in the visual cycle [37, 38]. Apart from participating in the visual cycle, IRBP is
important in fatty acid transport and is essential to the maintenance of the photoreceptors [39,
40]. In this regard, a reduction of IRBP may precede the loss of photoreceptors seen in some
animal models of hereditary retinal degeneration [41, 42]. In addition, ―knockout‖ (IRBP-/-)
mice revealed a loss of photoreceptors and profound changes in the structural integrity of the
receptor outer segments [40, 43]. Furthermore, a homozygous missense mutation in the IRBP
gene (RBP3) has been recently associated with autosomal recessive retinitis pigmentosa in
children [44]. It is well-known that visual deficits occur in diabetic patients before changes
could be detected in the fundoscopic examination, and IRBP deficit might participate in these
events. The IRBP content in the retina is regulated by vitamin A [45]. Granado et al [46]
reported that type 1 diabetic patients showed lower serum retinol status, and this deficit
persisted after intensive insulin therapy [47]. Although there are no reports on the retinal
content of retinol in either diabetic or non-diabetic patients, it could be postulated that the
systemic deficit of vitamin A observed in diabetic patients contributes to the lower
intravitreous IRBP levels observed in PDR patients. Therefore, the need for retinol
supplementation should be evaluated in diabetic patients in order to prevent IRBP deficit. In
addition, a defect in retinol could also contribute to down-regulated PEDF levels [48].
Apoa1 (Apolipoprotein A1)

Apo A1 has been proposed as a key factor for intraretinal reverse transport of lipids, thus
preventing lipid accumulation in the retina [49]. In a proteomic analysis of human vitreous
fluid we found that apoA1 concentration was higher in patients with proliferative DR in
comparison with non-diabetic subjects [23]. Furthermore, we confirmed that apoA1
overexpression does indeed exist in the human diabetic retina (cadaveric eyes) not only in
terms of mRNA levels but also in protein content [50, 51]. Apo A1 was expressed in all the
layers of the retina and it was more abundant in RPE than in the neuroretina in both diabetic
and nondiabetic donors [51].
The reason why apoA1 is overexpressed in the diabetic retina needs to be elucidated but it is
possible that the diabetic milieu could stimulate apoA1 production by the retina. In this regard,
Kawai et al. [52] observed an increased secretion of apoA1 from the main lacrimal gland in
patients with DR, but it was not detected in healthy subjects. In recent years new insights have
been gained into the transport of lipids within the retina [53], thus allowing us to hypothesize
that the mechanisms regulating intraretinal lipid transport rather than serum levels are more
important in the pathogenesis of DR [49, 54]. In this regard, ABCA (ATP binding cassette
transporter A1) and apoA1 have been found in several layers of monkey retina, thus
suggesting the existence of an intraretinal mechanism to export HDL-like particles [49].
Ishida et al. have demonstrated that HDL stimulates efflux of radiolabelled lipids, of
photoreceptor outer segements origin, from the basal surface of RPE cells in culture [55]. The
role of this HDL-based intraretinal lipid transport could be important in preventing
lipotoxicity. The fact that the retina is the only neural tissue that has a direct and frequent
exposure to light presents a significant problem. This is because many lipids, especially
polyunsaturated fatty acids (which are mainly located in the photoreceptor outer segments)
and cholesterol esters are highly susceptible to photo-oxidation and these oxidized lipids
become extremely toxic to retinal cells [56]. In DR, this problem could be aggravated by the
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increase of oxidative stress and lipid peroxidation associated with diabetes. Apart from
preventing or arresting lipotoxicity, apoA1 is a potent scavenger of reactive oxygen species
[57, 58] and, therefore, it could play an important role in protecting the retina from the
overall oxidative stress due to diabetes. In this regard, it is should be noted that retinal
damage has been associated with apoA1 deficiency of genetic origin [59, 60].
Taken together, it seems reasonable to hypothesize that apo A1 is increased in diabetic
patients as a compensatory mechanism to prevent the development of DR. That is, those
patients with less capacity for apo A1 production by the retina are more prone to develop
lipid deposition (hard exudates) in the retina and, in consequence, to initiate DR. These
findings open up new strategies addressed to promoting the overexpression of apo A1 in
order to prevent or arrest the development of DR.
ZAG (Zinc--2 Glycoprotein)

ZAG is a 41-kDa soluble protein identified by Sanchez et al [61] as a fat-depleting factor
and it is related to class I histocompatibility complex molecules (MHC). Although its
biological functions have not been completely understood it seems a novel adipokine, which
may be involved in the local regulation of adipose tissue function [62]. Thus, ZAG is a lipid
mobilizing factor expressed in mouse adipose tissue and it is markedly upregulated in mice
with cancer cachexia [63]. In addition, ZAG induces loss of body fat in vivo [64], and it has
been proposed as a candidate for regulation of body weight [65]. In fact, we provide evidence
that a down-regulation of ZAG in adipose tissue and liver exists in obese patients [66]. ZAG
has been also identified in the vitreous fluid from PDR patients by proteomic analysis [17,
18]. The reason why ZAG is increased in the vitreous of diabetic patients with PDR remains
to be elucidated, but it should be noted that hyperglucemia has been involved in ZAG
expression [65], and gene expression of ZAG has been found up-regulated in mouse with
diabetic nephropathy [67].
The role of ZAG in the pathogenesis of DR is far from being understood but it has been
reported that ZAG hinders cell proliferation and reduces cdc2 expression (a rate-limiting step
in the cell cycle) [68] and, therefore, it might be a reactive limiting factor for PDR
progression. Obviously, the mechanisms involved in the intraocular production of ZAG and
its role in the pathogenesis of DR require further investigation.
Complement Factors
In recent years there has been cumulating evidence which indicates that inflammation is
an important event in the pathogenesis of diabetic retinopathy [69-71]. In our study, several
components of the complement system (C4b, Factor B, C3, and C9) have been found for the
first time simultaneously increased in the vitreous fluid from PDR patients in comparison
with control subjects [23]. Although complement activation could be secondary to the
inflammatory process, Zhang et al [72] have recently demonstrated in human and
experimental non-proliferative diabetic retinopathy an early complement activation
associated with a prominent and selective decrease in the levels of CD55 and CD59, two
glycosylphosphatidylinositol anchored complement inhibitors. In addition, these authors have
shown that C1q and C4, the complement components unique to the classical pathway, were
not detected in diabetic retinas, thus suggesting that the alternative pathway is the main
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mechanism of complement activation in the early stages of diabetic retinopathy. Apart from
Factor B, which is an specific component of the alternative activation pathway, we have
detected higher intravitreal levels of C4 in PDR subjects. Therefore, classical pathway
activation also seems involved in the advanced stages of diabetic retinopathy. In fact,
intravitreal C4 has been found previously increased in patients with non-diabetic proliferative
vitreoretinopathy (PVR) [73]. Activation of the complement cascade can both compound and
initiate thrombosis, leukostasis, and apoptosis, all processes involved in vascular lesions of
diabetic retinopathy. Interestingly, it has been reported that the inhibition of aldose reductase
prevents the activation of complement in the wall of retinal vessels and decreased levels of
complement inhibitors in diabetic rats [74]. Therefore, since several ways of specifically
manipulating the complement system already exist, they could represent a possible
therapeutic approach in DR.
Carbonic Anhydrase
Using mass spectroscopy–based proteomics, Gao et al. [24] found elevated levels of
extracellular carbonic anhydrase-I (CA-I) in the vitreous from individuals with diabetic
retinopathy, and suggest that retinal hemorrhage and erythrocyte lysis are main contributers
to the diabetic vitreous proteome. Regarding the clinical relevance of CA-I they show that the
presence of extracellular CA-I inside either the blood-retinal or blood-brain barrier can
induce vasogenic edema. Furthermore, the release of erythrocyte CA-I may account for
increased vascular permeability and edema in DR. In this regard, it has been reported that the
magnitude of CA-I‘s effect on retinal vascular permeability is equivalent to that of VEGF,
and the kallikrein-kinin pathway mediates these effects [75].
Diabetic Macular Edema

To the best of our knowledge only two proteomic studies using the vitreous fluid from
patients with non-proliferative DR or DME have been reported [20, 25].
Ouchi et al [20] analyzed the vitreous fluid of diabetic patients with non-proliferative
DR, and reported that six proteins (PEDF, Apo A4, Apo A1, Trip-11, plasma retinol-binding
protein, and vitamin D binding protein) were more abundants in vitreous fluid of diabetic
patients with DME than in patients without it, and Apo H was not expressed in patients with
DME (Table 2).
Table 2. Proteins differently expressed in vitreous fluid from patients with DME
Increased Decreased
Apolipoprotein A1 [20] Apolipoprotein H [20]

Apolipoprotein A4 [20] Beta crystalline S [25]
Hemopexin [25] Clusterin [25]
PEDF [20] Transthyretin [25]
Plasma retinol binding protein [20]
Trip-11 [20]
Vit. D-binding protein [20]
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We have found four proteins specifically associated with DME (hemopexin, beta
crystalline S, clusterin and transthyretin) (Table 2). Hemopexin abundance was significantly
increased in the vitreous fluid of patients with DME in comparison with PDR and control
subjects. By contrast, beta crystalline S, clusterin and transthyretin were significantly lower
in DME patients than in PDR patients and control subjects. In view of the current
information, hemopexin and clusterin seems to be more directly related to the development of
DME.
Hemopexin is the best-characterized permeability factor in steroid-sensitive nephrotic
syndrome (SSNS) [76]. T-cell–associated cytokines like tumor necrosis factor-alpha (TNF-
alpha) are able to enhance hemopexin production in mesangial cells in vitro and this effect is
prevented by corticosteroids [77]. It should be noted that proinflammatory cytokines have
been involved in the development of DME and, therefore, hemopexin might be a mediator of
the disruption of the blood-retinal barrier.
Clusterin is associated with protection from apoptotic retinal cell death [78]. Recently,
Kim et al. [79] demonstrated that clusterin effectively inhibited vascular endothelial growth
factor-induced hyperpermeability in human retinal microvascular endothelial cells
(HRMECs) and in retinal vessels from streptozotocin-induced diabetic mice. Since clusterin
plays an essential role in restoring tight junctions and limiting the inflammatory response
after injury (two capital features in the pathogenesis of DME), it seems reasonable to propose
clusterin deficit as a contributor to DME development.
Vitreous Metabonomics
The general aim of metabolomics is to identify, measure and interpret the complex time-
related concentration, activity and flux of endogenous metabolites in cells, tissues and
biofluids. It is based on the concept that metabolic properties of tissues predispose to or are
altered by disease processes, and these changes can be reflected in characteristic patterns
(metabolomic profile) in blood, urine, or other body fluids. Metabolites include not only
small molecules that are the products and intermediates of metabolism but also
carbohydrates, peptides and lipids. Because the metabolome is downstream from the
proteome and transcriptome it represents a more sensitive level of organization than the
proteome or transcriptome do for understanding a complex biological system [80-82].
While genomic and proteomic techniques have been useful in generating useful data and
novel hypotheses, they are ultimately dependent on a candidate gene/protein approach, which
is costly and time consuming for the study of significant numbers of patients. In contrast,
metabolomics is low cost, reproducible and, with bioinformatic analysis of the data, easily
translated into a clinical test that could inform future therapy. Furthermore, because a
metabolic profile is summative of all the biochemical processes occurring in the body at a
given time, it makes no presumption about the relative importance of these processes and so
is more likely than a candidate approach to be able to highlight differences between disease
groups and to identify changes occurring during therapy.
The importance of the complex array of metabolites in human physiology has long been
recognised, and as early as 1971 Linus Pauling developed methods based on gas–liquid
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chromatography to separate 280 metabolites in human urine [83]. Recent technical advances
in nuclear magnetic resonance (NMR) and mass spectrometry have allowed extremely high-
density data sets to be constructed from individuals by examining the changes in hundreds or
thousands of low-molecular-weight metabolites in intact tissues or biofluids.
Metabolomics most often combines either high-field NMR or mass spectrometry of
biofluids with pattern recognition (principal components analysis) within the resulting
dataset. NMR-based metabolomics offers several distinct advantages in a clinical setting
since it is relatively quick and can be carried out on standard preparations of blood cells,
serum, or other body fluids. NMR spectroscopy is less sensitive than mass spectroscopy, but
it has the advantage of being applicable in vivo in the eye [84], thus allowing a new strategy
for monitoring DR.
Proteins in serum can interfere with the quality of the spectrum derived from low-
molecular-weight metabolites, and so removal of these by filtration can significantly enhance
the quality of the NMR spectrum derived [85]. Separation of samples into hydrophilic, water-
soluble metabolites and those which are hydrophobic or bound to proteins [85] also allows a
greater depth of information to be derived from a particular sample. NMR spectra of biofluids
are highly complex, containing signals from hundreds of metabolites that represent many key
biochemical pathways. To make the spectra tractable for analysis it is usual to segment the
spectra into small regions [86] to allow processing using a number of statistical approaches.
Pattern recognition methods (principal component and partial least-squares analysis, see
below) allow the complex biofluid/tissue NMR data to be reduced and analysed
quantitatively to provide pattern recognition maps that can assist in disease classification.
Metabolomic diagnostics can be extremely sensitive for the detection of low-level damage in
a variety of organ systems and is potentially a powerful new adjunct to conventional
pathological procedures and to assist in functional genomics problems [87].
Principal components analysis (PCA) is a particularly useful statistical technique for the
analysis of complex datasets such as metabolite or transcript profiles. Partial least-squares
analysis discriminant analysis (PLA-DA) is a method of partial least-squares regression (PLS
regression), which bears some relation to PCA; however, instead of finding the hyperplanes
of maximum variance, it finds a linear model describing some predicted variables in terms of
other observable variables.
Metabonomic Analysis in Diabetic Retinopathy
Recently, we have analyzed for the first time metabolite fingerprints in vitreous fluid
from patients with PDR [88], using high-resolution 1H-nuclear magnetic resonance
spectroscopy. This study revealed that the concentrations of several retinal metabolites
involved in different pathways varied between PDR patients and non-diabetic controls. Apart
from glucose levels, these biochemical processes included glucolytic products (lactate), -
oxidation (ketone body-derived metabolite: acetate), aldose reductase or polyol pathway
(galactitiol), and ascorbic acid (AA) defense against oxidative stress. (Figura 2).
We have found that lactate was the most abundant metabolite and it was higher in the
vitreous fluid of PDR patients. Interestingly, a previous analysis using in vivo NMR
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spectroscopy identified lactate as a dominant metabolite in human vitreous [84]. There are
several reasons accounting for this finding. Lactate has been found to be a major product of
glucose metabolism [89], and elevated lactate levels reflect increased tissue acidosis and
anaerobic glycolysis. In addition, the retina is the part of the body that proportionally
consumes more oxygen and, therefore, altered microcirculation in conjunction with hypoxia
(hallmarks of PDR) can trigger lactate production by anaerobic glycolisis in an attempt to
compensate for reduced oxidative ATP production. Furthermore, inflammation, which is also
involved in the pathogenesis of DR [90] can also stimulate lactate production.
After removing the lactate peak at 1.35 ppm, we were able to obtain a model that
differentiates control and PDR samples with 86% sensitivity and 81% specificity (Figura
metab). The main metabolites involved in this specific pattern recognition of NMR spectra
were galactitiol (also named dulcitol) and AA. The lower levels of galactitiol detected in the
vitreous fluid from PDR patients can be attributed to the activation of the polyol pathway, a
metabolic pathway involved in the pathogenesis of DR [91]. Aldose reductase (AR) is the
first and rate-limiting enzyme in this pathway and both glucose and galactose are substrates
that compete for this enzyme while being reduced to sorbitol and galactitiol respectively.
Because AR has a higher affinity for galactose than for glucose, under physiological
conditions (normoglycemia) glucose is poorly reduced by AR to sorbitol and, therefore,
galactitiol production is favored. By contrast, when intracellular glucose levels are elevated
the polyol pathway of glucose metabolism becomes active and sorbitol rather than galactitiol
is produced (Figure 3) [91, 92]. The reason why we did not find higher levels of sorbitol in
the vitreous of PDR patients could be because it is metabolized to fructose, fructose-3-
phosphate and 3-deoxyglucosone [91]. By contrast galactitol is not further metabolized or
transported and thus accumulates inside AR expressing cells [93].
AA, which is an essential substance in humans, acts as an antioxidant and/or free radical
scavenger [94-96]. Although most animals can synthesize vitamin C from glucose, humans
can only acquire the vitamin from dietary sources because they lack gluconolactone oxidase,
the enzyme required for AA biosynthesis. Vitamin C exists in two major forms. The charged
form, ascorbic acid (AA), is taken into cells via sodium-dependent facilitated transport. The
uncharged form, dehydroascorbate (DHA), enters cells via glucose transporters (GLUT) and
is then converted back to AA within these cells. Retinal cells appear to be dependent on
GLUT-1 transport of DHA rather than sodium-dependent AA uptake [97]. DHA uptake
through facilitative glucose transport is competitively inhibited by D-glucose. In fact, the
molecule of AA is very similar to D-glucose. Therefore, chronic hyperglycemia of long-
standing diabetes reduces DHA transport via GLUT1 at the blood retinal barrier (BRB).
Exclusion of DHA from cells by hyperglycemia deprives the cells of the central antioxidant
action of AA, thus favoring accumulation of reactive oxygen species [98]. It should be noted
that the retina is the only neural tissue that has a direct and frequent exposure to light, thus
leading to free radical production due to photo-oxidation which becomes extremely toxic to
retinal cells [99]. AA is present in the retina at a high concentration compared with its
presence in other organs in humans, and it is able to protect the retina against oxidative
damage [95, 99, 100].
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Figure 2. Typical spectra obtained from control (top) and PDR (bottom) vitreous humour showing
differences in lactate, acetate and galactitiol. Only the aliphatic part of the spectra is shown, inserts focusing
in the 3 to 4 ppm region where most of the sugar resonances appear. Spectra have been scaled to show equal
glucose peaks; assignations are as follows: 4 Lactate, 6 Acetate, 8 Succinate, 10 Glucose, 15 Galactitiol.
Each peak corresponds to a proton nucleus. Thus, a single molecule may appear as a group of peaks.
In fact, we have found 20 fold higher levels of AA in the vitreous fluid than in serum. Given
that oxidative stress is a key factor in the pathogenesis of DR, the significant lower levels of
AA detected in the vitreous fluid of PDR patients, point to this deficit as a crucial factor in
determining DR development. AA is a required cofactor for several intracellular
hydroxylases, including proline hydroxylase and dopamine hydroxilase [95]. Therefore, AA
deficit could also participate in the impairment of neuropetide production and, therefore, in
neurodegeneration, a key element in the pathogenesis of DR [101, 102]. In addition, it has
been demonstrated that AA has antiangiogenic properties [103, 104] and, consequently, the
lower levels that exist in diabetic patients can contribute to neovascularization, the hallmark
of PDR. There are several reasons for explaining the lower levels of AA detected in the
vitreous fluid of PDR patients. First, the competitive inhibition mediated by hyperglycema in
AA transport from systemic circulation to the retina. Second, the AA consumption that exists
in the diabetic retina to compensate for elevated degree of oxidative stress. Third, the lower
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serum levels of AA that we have detected in PDR patients in comparison with control group
might also contribute to the lower levels reaching the retina. Our results agree with a previous
report showing that diabetic patients with microangiopathy had lower AA serum
concentrations than those without microangiopathy and control subjects [105]. This is
probably due to a high metabolic turnover reflecting increased consumption of AA by
oxidation to DHA. Taken together, AA can be contemplated as a new therapeutic target.
Prospective trials using diet supplements of vitamin C for preventing or arresting DR, and
experimental studies addressed to increasing AA transport across BRB in the presence of
hyperglycemia are needed.
Figure 3. Mechanism by which there are lower concentrations of galactitiol in the vitreous fluid of diabetic
patients. Aldose reductase (AR) is the rate-limiting enzyme in the polyol pathway and both glucose and
galactose are substrates that compete for this enzyme while being reduced to sorbitol and galactitiol
respectively. Because AR has a higher affinity for galactose than for glucose, under physiological conditions
(normoglycemia) glucose is poorly reduced by AR to sorbitol and, therefore, galactitiol production is
favored. By contrast, when intracellular glucose levels are elevated the polyol pathway of glucose
metabolism becomes active and sorbitol rather than galactitiol is produced.
Conclusion
Given the layered, evolutionary complexity of biological systems, it will not be possible
to understand them comprehensively on the basis of hypothesis-driven research alone.

Likewise, it will not be possible to do so solely through free-hypothesis through the ‗omic‘
studies of genes, proteins and other molecules in aggregate. Rather, the two modes of
research are complementary. In the setting of DR, proteomic and metabonomic analysis
facilitate the identification of new potential candidates involved in the development of both
PDR and DME. Further studies addressed to the evaluation of the precise role of these
candidates in the pathogenesis of DR and their potential as therapeutic targets are needed.
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Account: ns004409
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Chapter V
Measuring Cornea
Cristina M. Oliveira and S. Franco

Centre of Physics, University of Minho, Braga, Portugal
Abstract
By virtue of its remarkable mechanical strength and optical transparency, the cornea
serves as the major refractive element of the eye while protecting its contents. These
properties are directly attributable to the cornea‘s structural architecture of collagen
fibrils and surrounding hydrated matrix containing proteoglycans, glycoproteins, other
soluble proteins, inorganic salts and keratocytes.[1,2]
The maintenance of corneal shape and transparency is crucial for its optical function.
The optical properties of the cornea are mainly governed by its collagen fibril
organization, which ensures that the cornea maintains correct surface shape under the
action of intraocular pressure on its internal surface.[3]
1. Cornea’s Shape and Optical Properties
The cornea has a fundamental role in retinal image quality, as it is responsible for two-
thirds of the eye‘s focusing power. The optical properties of this tissue are determined by
three aspects: a) its transparency, b) its refractive index and c) its surfaces' shapes,
predominantly that of the anterior surface. The expressiveness of the optical action of the
cornea's anterior surface is justified by its highly curved surface and the considerable
difference in refractive index at the interface between the air and itself.
Accordingly, changes in the corneal shape caused by pathological conditions or
refractive surgery/therapy may impact its optical quality and refractive power of the eye.
Subtle imperfections in cornea‘s shape cause light rays to be improperly focused in the retina,
resulting in focusing errors known as optical aberrations. [4]
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Copyright © 2012. Nova Science Publishers, Inc. All rights reserved. May not be reproduced in any form without permission from the publisher, except fair uses permitted under 116 Cristina M. Oliveira and S. Franco
The shape of the cornea can be assessed by topography. Keratometry was the first
technique in widespread clinical use for measuring corneal topography. Although still
commonly used, this technique only measures the curvature of the central cornea (within
3mm in diameter). [5]
Modern corneal topography techniques are able to provide shape information of the
entire cornea, instead of simply measuring its center. Furthermore, techniques like slit-
scanning and rotating Scheimpflug imaging enabled the assessment of the posterior corneal
surface. Such complete information can be leveraged to compute a wavefront aberration
function that describes the optical properties of the cornea. [6-10]
This section reviews the technologies commonly employed to evaluate corneal shape and
optics and concepts useful for understanding the outputs of these instruments.
1.1. Current Topographic Technologies
1.1.1. Placido-Disk Based Imaging

Placido-disk imaging is based on the projection of an illuminated pattern of rings onto
the cornea. Systems employing this technology directly measure the slope of the anterior
corneal surface through the analysis of the specularly reflected image of the rigs on the tear
film. From these data, corneal radius of curvature and refractive power can be derived, as
well as elevation. The latter analysis, however, requires geometrical assumptions about the
cornea shape as the slope data is fitted to a predefined (spherical, aspherical or conical)
mathematical model. Therefore, the true corneal shape cannot be provided by these
reflection-based topographers.13
Besides not directly computing corneal elevation, Placido disk-based systems only
measure the AC shape using a measurement axis that is not the line of sight.
1.1.2. Slit-Based Imaging

Topographic systems based on slit imaging overcome some of the drawbacks of the
Placido-disc systems, as they provide direct measurements of both anterior and posterior
corneal surfaces elevation with full coverage.
These systems acquire multiple optical sections of the cornea through the projection of
the light from a slit onto the cornea. The data points acquired from the analysis of anterior
and posterior edges of each section are used to reconstruct a 3-D model of the cornea,
providing the true topography of both surfaces. Analyses of corneal pachymetry, topography
and thickness of crystalline lens and anterior chamber are also available with these systems.
Slit-Scanning Imaging
The Orbscan II is the only commercially available device employing slit-scanning
technology. In actually, the Orbscan systems combine both Placido disk and slit scanning
technologies to provide a noninvasive analysis of the anterior segment of the eye. The
purpose of the Placido disk system was already discussed. The slit-scanning technology is
based on two vertical scans performed by projecting 40 optical slits, 20 from the right and 20
from the left, on the cornea at a fixed angle (45 degrees) to the instrument axis. [14,15 ]
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Copyright © 2012. Nova Science Publishers, Inc. All rights reserved. May not be reproduced in any form without permission from the publisher, except fair uses permitted under Measuring Cornea 117
Scheimpflug Imaging
Tomography systems employing Scheimpflug imaging accomplish wide depth of focus,
providing sharp images from the anterior corneal surface to the posterior crystalline capsule.
Table 1 is a comparison of instruments employing Slit-scanning and Scheimpflug
imaging. Some technical specifications and features of these devices are presented.
Table 1. Technical specifications of the tomography systems (information provided

by the manufacture)
ORBSCAN II/IIZ PENTACAM/HR GALILEI SIRIUS

SYSTEM CHARACTERISTICS
Ziemer CSO
Manufacturer Bausch & Lomb Oculus
Ophthalmology Co.
Rotational scan of
Parallel scanned Rotational scan of
Rotational scan of Scheimpflug slit
Measuring slit images and Dual-Scheimpflug
Scheimpflug slit images and
Principle Placido disk slit images and
images Placido disk
images Placido disk images
images
Top view 1024 x 786 pixels 640 x 480 pixel
_ _
camera CCS
20 monochrome 22 monochrome
40 monochrome
Placido disk _ rings, 200mm rings, 0.4 to 12
rings
diameter mm diameter
Slit Blue LED (475nm, Blue LED (470 nm, Blue LED (475
White flash light
Illumination UV free) UV-free) nm, UV-free)
Contact/Non- Non-contact
Non- contact Non-contact Non-contact
contact
ACQUISITION CHARACTERISTICS
Photographic
Parallel Scan 0º to 180º 0º to 180º 0º to 180º
Range
40 images (20 slit N/A
25 to 50 images/up
No. of images from the right and 15 to 60 images
to 100
per scan 20 slit from the (settable by user)
(settable by user)
left)
No. of Placido N/A
3 images _ N/A
images
No. measure ≈ 100.000 points
data points per ≈ 9.000 points >25.000 points >122.000 points
scan
Time of a full 2 sec
1.5 sec ≈ 2 sec 1-2 sec
scan
Total Area Limbo to limbo
N/A Limbo to limbo 14 mm diameter
covered
N/A – Inormation not available
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1.2. Topographic Display
1.2.1. Color-Coded Maps

Topographic systems enable the assessment of the corneal characteristics on each point
along its surfaces. A wide range of color-coded maps are drawn from the captured data
points, in order to represent specific features of the cornea. Color-coded maps enable a
qualitative display of the topographic data, allowing easy pattern recognition. Moreover, this
concept markedly facilitates the clinician‘s interpretation through the assignment of colors to
the values of certain corneal characteristic like axial and instantaneous curvature, elevation
and pachymetry.
Curvature/Power Maps
Two sets of curvature readings are provided by the current topography systems: a) the
radius of curvature measured in millimeters, b) the same, but in diopters.
The measure of axial radius of curvature simplifies the optical properties of the cornea by
assuming that all rays reaching the cornea are paraxial. The axial map is provided by
measuring the distance from each surface point to the optical axis along the normal. 16
Although appropriately providing central corneal curvature, the axial approach produces
inaccurate curvature values for peripheral portions of the cornea. This approach does not
encompass the effects of spherical aberration and is, thus, less accurate in the corneal
periphery and for irregular corneas. [17]
The instantaneous radius of curvature differs from the axial radius of curvature in that it
calculates a true radius of curvature independent of the optical axis. Instantaneous curvature
maps display the local curvature of the cornea at each point, considering different references
axes for each point. These maps typically show more marked changes in corneal curvature
over smaller regions, while providing more accurate measurements of corneal curvature and
better representation of local irregularity.
Each type of radius of curvature measurement can be converted in power measurement,
axial power or instantaneous power, through the application of the formula:
P= (n – 1)/r
where n is 1.3375 (standardized value for keratometric index of refraction). The Standard
keratometric index (SKI) is not the true refractive index of the cornea. It is an approximation
index to yield the total corneal power as a single refracting surface compensating for the
negative power of the posterior surface.18,19 This index assumes the curvature of the posterior
surface to be normal, a constant relationship between anterior and posterior corneal

curvatures and a normal corneal thickness, which can lead to inaccuracies when describing
cornea. [20]
Table 2 describes the advantages and disadvantages of axial and instantaneous curvature
measurements.
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Elevation Maps
Typically, corneal elevation maps are depicted in relation to a reference shape. These
maps display how the actual corneal raw elevation deviates from a known shape, such as a
best-fit-sphere (BFS), best-fit ellipse and a best-bit-toric ellipsoid. [21-23] For clinical
applications, the reference shape is often a sphere, selected such that it provides the best
mathematical approximation of the actual cornea (BFS). Points on the cornea above the
reference sphere are shown with warmer colors and those below are represented by cooler
colors. The color green is usually used specifies points on the cornea coincident with the
sphere.
Elevation maps are extremely important in refractive surgery because they provide a
direct analysis of the tissue removed and help calculate the ablation depth and optical zones.
This approach is also more accurate than curvature map in detecting corneal irregularities,
such as cone location in ectatic diseases and decentered ablation, which allows better
diagnosis. [24]
Table 2. Comparison of axial and instantaneous measurements of corneal shape
Advantages Disadvantages
Axial Curvature Provides a good description of the Dependent on the choice of the
overall corneal shape reference axis
Value are directly comparable to Inaccurate representation of the
keratometry peripheral topography
Underestimates abrupt changes in local
curvature
Instantaneous Measures the local curvature More affected by noise
Curvature
Sensitive to marked changes in local Less repeatable
curvature
More accurate representation of Does not provide a simple interpretation
peripheral curvature of the cornea topography
Make local irregularities (such as
ectasias) more obvious
Pachymetry Maps
Slit-scanning and Scheimpflug imaging technologies yield pachymetry data, that it, the
distance between anterior and posterior surfaces. Thickness information is represented by
color coded maps. Other topographic systems are used for this purpose will be presented in
the next section, which discusses the measurement of corneal thickness.
1.2.2. Topographic Indexes

Although the color-coded map provides a useful representation of the quantitative
measurements obtained from corneal topography, such maps do not provide numerical values
that can be used in clinical practice.
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Several topographic indexes have been developed to mathematically describe the

cornea‘s features qualitatively. Many indexes are now available in commercial. Table 3
presents some examples of these indexes.
Table 3. Commonly used topography indexes
Shape and power indexes

Simulated keratometry (SimK) [25,26] Simulates the curvature measures given by
keratometry, including power and axes of the steepest
(SimK1) and flattest (SimK2) meridians.
Corneal eccentricity index (CEI) [27] Measures the eccentricity of the cornea.
Surface asymmetry index (SAI) [25,26] Measures the difference in corneal powers at every
ring.
Average corneal power (ACP) [28] Area-corrected average corneal power ahead of the
entrance pupil.
Standard deviation power (SDP) [29] Calculated from the distribution of all corneal powers.
Optical quality indexes
Surface regularity index (SRI) [25,26] Measures the impact of corneal irregularities on
optical quality
Fourier series transformation [30,31] Provide ACP, amount and axis of astigmatism and
terms to estimated irregular corneal astigmatism
Ray Tracing Approach [32,33[ Calculate images resulting from individual corneal
with irregular astigmatism
Keratoconus detection indexes
Inferior-superior (I-S) values [34] Measure power differences between the superior and
inferior paracentral corneal regions
Keratoconus predictability index (KPI)Derived from several topographic indexes to represent
[]35 specific features of the keratoconus
1.3. Corneal Wavefront Aberration
1.3.1. Principles of Wavefront Analysis

The elevation data obtained with corneal topography is used to compute corneal
wavefront aberration. [6-10] Videokeratoscopy only provides measurement of anterior
surface aberrations, while elevation-based topography allows anterior, posterior and total
corneal aberrations measurement.
Corneal wavefront analysis can be based on the interpretation of optical aberrations as
errors of optical path length (OPL). This approach expresses the behavior of light rays in
terms of OPL relying on Fermat‘s principle of least time, which states that the light from an
object point will reach an image point taking the shortest optical path.
A wavefront is a conceptual surface that connects all the light rays emerging from a
source with the same OPL, thus, in phase with each other. Considering the eye a single
surface system composed by a perfect cornea, all light rays travelling from an object point
will reach a unique image point at the same phase. Their optical path difference (OPD) will
be null, so a spherical wavefront centered on the image point is originated. In reality
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however, the presence of corneal aberrations implies that the marginal light rays arrive at the
image point in different phases relative to the axial ray. These rays originate an aberrated
wavefront that deviates from the ideal shape.
Accordingly, the determination of the corneal wavefront function can be based on the
OPD calculated as the difference between the axial and marginal rays path lengths.
Another simple method to compute corneal aberrations is to determine the difference
between its shape, assuming it to be the actual wavefront, and an aberration free wavefront.
This ideal wavefront is generated by fitting the corneal elevation data to a Cartesian oval
surface aligned with the sighting center. The OPD is thus calculated as the residual surface
profile multiplied by the refractive index difference between air and cornea. [36]
1.3.2. Zernike Polynomial Decomposition

The wavefront aberrations are usually described mathematically by the infinite Zernike
polynomial series. These series comprise orthogonal terms defined in the unit circle, that is,
they are mathematically independent of each other.
The orthonormal nature of the Zernike series is essential for modeling the wavefront
shape, because it allows each polynomial function to represent a particular component of the
wavefront‘s aberration. [37] These components are grouped into lower (defocus and
astigmatism) and higher-order aberrations that are represented by a Zernike term.[38]
1.3.3. Metrics of Optical And Visual Quality

Objective descriptors of the optical quality can be derived from the wavefront aberration
data. Optical quality metrics, such as the wavefront variance or root mean squared (RMS)
error and the peak-to-valley (PTV) difference, quantify the overall optical quality of the
cornea. The commonly used RMS is the standard deviation of the wavefront error at various
positions in the surface, while PTV difference represents the difference between the
maximum and minimum height of the wavefront. [39-41]
Image quality metrics like the point-spread function (PSF) and the optical transfer
function (OTF) describe the effect of the optical aberrations on the image optical quality in
the fovea. The PSF analyzes image quality for point objects in the special domain, whereas
the OTF analysis the effect of aberration for granting objects in the frequency domain.
[39,40]
2. Corneal Thickness
Pachymetry is the measurement of the corneal thickness (CT). It is one of the most direct
measures of corneal health and has a fundamental role in several fields of ophthalmology.
Accurate corneal pachymetry is required for preoperative judgment and planning of the
most types of refractive surgery, particularly keratorefractive procedures. This parameter
allows the identification of infeasible candidates due to reduced preoperative corneal
thickness or the presence of early ectatic disorders. It is thus a useful tool to avert serious
postoperative complications such as corneal ectasia or corneal perforation.
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In the contactology field, corneal thickness is important for contact lens fitting and
monitoring corneal changes after extended wear. Additionally, it is useful for studying new
types of contact lenses, by analyzing the swelling of the cornea due to hypoxia. Finally,
corneal pachymetry is also a useful tool in the diagnosis and follow up of several ocular and
corneal diseases such as Keratoconus and glaucoma.
Currently, a wide range of techniques are available to assess corneal thickness. These
techniques can be grouped according to the methodology employed, namely techniques based
on optical principles, such as standard optical pachymetry, optical coherence tomography
(OCT), optical low-coherence reflectometry, confocal microscopy through-focusing and
specular microscopy, and on ultrasonic principles.
2.1. Measurement of the Corneal Thickness
2.1.1. Ultrasound Technology

Ultrasound (US) imaging relies on the propagation of sound waves through tissues and
their reflections produced at the boundaries between tissues with different acoustic
impedance. The ophthalmic ultrasound systems consist of a piezoelectric transducer located
at the face of the probe that, when stimulated by an electromagnetic pulse, generates an
ultrasonic wave. The ultrasonic wave propagates through the eye and is differentially
reflected at ocular tissues‘ interfaces. These reflections, referred to as echoes, return to the
transducer and its mechanical vibrations induce the production of a detectable electric signal.
The frequency of the transducer determines the resolution and depth of penetration of the
ultrasonic waves into the tissue. Transducers with higher frequencies have an increased
spatial resolution with a reduction in tissue penetration.
Ultrasound Pachymetry
In conventional US pachymetry, the probe containing a static transducer is applied
perpendicularly to the cornea along a specific line, after topical anesthesia. The transducers
used have frequencies between 10 MHz to 20 MHz, which means that a frequency pulse of
15 MHz to 20 MHz is produced by the piezoelectric element of the transducer.
The ultrasonic pulse generated by the stimulation of the piezoelectric element in the
transducer propagates through the cornea and is reflected by the Descemet‘s membrane back
to the transducer, producing an A-scan. The A-scan provides a one-dimensional, time-
amplitude representation of the ultrasonic echoes data. The distance between echo vertical
spikes represents the time required for the ultrasonic wave to reach the posterior corneal
surface and for its corresponding echo to return to the probe. The time between the two echo
spikes can be converted into the cornea‘s thickness by using the formula:
Corneal thickness = (total travel time x speed of sound in cornea)/2
Current ultrasound pachymetry systems are handheld, which offer portability, relative
ease use and low cost. However, it is an invasive technique that requires corneal contact and,
thus, the use of anesthesia. The edema resulting from the topical anesthesia, the compression
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of the cornea by the probe and the displacement of the tear film potentially limited the
accuracy of the measurements, leading to erroneous readings of the cornea thickness. [42-
45] One final factor that may influence the accuracy of pachymeter readings is the speed of
sound in corneal tissue. The current standard value for the speed of sound in the cornea is of
1640 m/s. [44,46,47] However, the assumption that all corneal tissue conducts sound at the
same speed can also add a degree of uncertainty to ultrasound pachymetry, as in reality there
are slight variations of the speed of sound in healthy and diseased tissue. [48-50]
Moreover, it is a spot measurement technology and the manual placement of the probe
increases the potential variability of the measurements. Since the precision relies on precise
probe corneal center, a variability in CCT measurements has been observed when more than
one person takes the measurements and when the same person takes the CCT measurements.
[44,46,49,51-54]
This technique has also been used to measure the thickness at the periphery of the cornea
[55-60]. In some works the placement of the probe was performed subjectively, taking the
pupil center as a reference. Others developed methods to ensure the exact positioning of the
probe improving the precision of the measurement. Owens and Watters [55] integrated a
corneal topographer cone with the microscope of a biomicroscope and posted light-emitting
diodes over the cone for the patient fixation. Parafita and colleagues [60,61] constructed a
fixations system composed by a series of concentric circles. However, there is a reduction of
the precision at the peripheral region related with the subjective probe's placement on the
cornea. [62]
Despite these drawbacks, the US pachymetry remains the reference standard for the
measurement of CCT, as this system proved to have better reproducibility and lower inter-
observer variability than the optical pachymetry. [49,51]
Ultrasound Biomicroscopy
Ultrasound biomicroscopy (UBM), also referred as high- or very high-frequency
ultrasound biomicroscopy, uses high frequency transducers (50-100 MHz) to provide high-
resolution cross-sectional images of the anterior segment of the eye. Depending on the choice
of the frequency, UBM grants images with a minimum of lateral and axial resolution of 50
μm and 25 μm, respectively, with a tissue penetration of approximately 4-5 μm. As its
superficial location requires lesser tissue penetration, higher frequency ranges can be used for
corneal imaging, allowing higher resolution. UBS is able to measure regional differences in
corneal thickness, including the corneal epithelium, the stromal component of the flap, and
the stromal surface of the posterior flap.
These systems produce A-scan signals that are converted and displayed as B-scan data,
which provides bi-dimensional imaging of the cornea. [63] A variant of the instrumentation
employed is the arc scanner that moves the transducer the transducer in an arc following the
cornea‘s curvature. [64]
In UBM, the piezoelectric transducer requires the eye to be immersed in a saline or
methylcellulose solution usually housed in an eye cup that is directly placed on the ocular
globe. This requires the use of topical anesthesia and the patient to be lying down fixing a
point placed vertically on the contralateral eye. More recent applications of the USB
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incorporate self-contained water-bath probes which, applying gel between the globe and the
probe, can be used with the patient in the sitting position.
UBS enables precise pachymetric evaluation and values in agreement with those of
conventional US pachymetry. [54,65,66 ]
2.1.2. Standard Optical Pachymetry

Although it has been losing competitive ground to newer techniques, optical pachymetry
is still the most common technique for determining corneal thickness.
Optical pachymetry is usually performed with a Haag-Streit slit lamp device. The
pachymeter contains a biprism responsible for breaking the image of the corneal optical
section provided by the slit-lamp into two parts. Two parallel glass plates are placed one
upon the other in front of the lens of the microscope and the upper one swivel. The upper
glass plate is used to move the upper half of the corneal image so that the endothelium can be
aligned with the epithelium in the lower half. When this is done, the apparent thickness of the
cornea is found. The true thickness is calculated from the upper plate‘s rotation angle, the
refractive index and thickness of the plate and refractive index and curvature of the anterior
corneal surface.
The two surfaces can be aligned either by overlap or juxtaposition. A comparison of the
two methods concluded that the values of corneal thickness determined through juxtaposition
were statistically higher than those found when surfaces were aligned through overlapping
[67].
2.1.3. Specular Microscopy

Specular microscopy leverages the specular reflection to examine the corneal endothelial
mosaic, qualitatively and quantitatively, at high magnification. In particular, it can also
determine the corneal thickness considering the reflections of both corneal surfaces. For this
application, the specular reflections from the front and back surfaces are simultaneously
focused, and the distance between two sets of points is calculated.
Two kinds of specular microscopes are available: contact or non-contact microscopes.
Their difference resides in the requirement of anesthesia, inexistent in the most recent latter
variety. Contact microscopes present a dipping cone that is pushed against the cornea and
adjusted (by rotation) until it‘s possible to focus on the corneal endothelium. The amount of
displacement effected (x) is determined through direct reading on a micrometer scale and is
converted to a measure of the corneal thickness (CT) using the equation: [68]
CT (mm) = [100 - (x + 30)] / 100

Compared with the optical pachymeter, with or without modifications, this type of
specular microscope showed significantly lower intra-session variability.68 Besides that, it
also has the advantage of providing readings on the density and morphology of endothelial
cells. However, this technique has a few weaknesses. The cornea has to be anesthetized and
the technique should be performed by a experienced observer to avoid epithelial damage.
Recent specular microscopes do not need corneal contact and allow the determination
corneal thickness in peripheral points. A comparison of the two specular microscopy
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techniques found significantly higher values of central corneal thickness (CCT) with the
contact microscope either in normal corneas (difference of about 100 μm) or in post-
keratoplasty (difference of approximately 90 μm). [69,70] In the latter situation, the
difference in values could be explained by the presence of distortions and changes in optical
density of the cornea. [71] The difference in the normal corneas could be due to the fact that
the images obtained with non-contact specular microscopy had been affected by anterior
corneal refractive power.
Studies comparing non-contact microscopes‘ CCT measurements with those provided by
US techniques found differences between their results (the non-contact microscopes produce
lower values). [71-73] On the other hand, this technique showed small inter-observer
variation, meaning that can be operated by different technicians without compromising the
results. [71, 74] However, contradicting this result, a study of several eyes concluded that
non-contact specular microscopy showed higher variability than either ultrasonic pachymetry
or ultrasonic biomicroscopy. [54]
2.1.4. Confocal Microscopy

In confocal microscopy, the illumination and observation systems are focused at the same
focal point. [75] This gives the ability to eliminate information from out-of-focus planes
above or below the focal plane, producing a marked increase in both lateral and axial
resolution.
In confocal microscopy light goes through diaphragm D1 and is focused on the cornea‘s
focal plane by lens L1. Reflected and scattered light from the cornea is focused again by lens
L1 towards diaphragm D2. The two diaphragms share the focal point that coincides with the
point being both illuminated and observed. The scattered light on planes other than the focal
one (dashed lines) is limited by diaphragm D2, preventing the formation of its image in the
observation system (camera or observer).
The principle of confocal imaging makes confocal microscopy a powerful tool for
microscopic evaluation of the cornea structure and measurement of its layers thickness. [76]
Tandem Scanning Confocal Microscope (TSCM)

TSCM uses a modified Nipkow disk containing several sets of conjugate
(source/detector) pinholes arranged in Archimedean spirals. [77] A bright illuminating light
source, such as a Xenon or Mercury arc lamp, is required to compensate for the poor light
transmission of the confocal microscope. [78]
The light from a broadband source passing through the pinholes on a portion of the
Nipkow disk is focused into the cornea, providing multiple spot illuminations. Then, the light
reflected back from the cornea passes through the optically conjugate pinholes on the
opposite side of the disk, which prevents light originated from points not illuminated by the
first set of pinholes to reach the observation system. The rotation of the disk allows scanning
of the whole cornea.
Combined with the technique of confocal microscopy through focusing (CMTF), TSCM
allows the measurement of epithelial and corneal thickness, by continuously moving the focal
plane of the objective lens through the entire cornea. [79] CMTF, also known as Z-scan
mode, results on equally spaced two-dimensional images from which depth intensity profiles
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can be generated by averaging pixel intensity in each image. Sublayer corneal pachymetry is
estimated as the difference between peaks corresponding to the epithelial and endothelial
layers.
McLaren and colleagues [80] found that confocal microscopy underestimated the CCT
values. Contrarily, Patel et al. [81] obtained similar values between these technique and US
pachymetry for both CCT and peripheral thickness.
Slit Scanning Confocal Microscope (SSCM)

SSCM uses vertical slits for illumination and in-vivo observation of the cornea, instead
of point scanning. [82] The slits‘ heights control the depth of the optical section acquired.
This system allows many points along the slit axes to be scanned simultaneously, leading to
significantly lower scanning times compared to TSCM. Another advantage of SSCM is that
the slits ensure greater illumination of the cornea. Therefore, a lower intensity source can be
used, potentially allowing longer examination times. Also, contrast is greater with SSCM in
comparison to TSCM.
The main drawbacks of SSCM are that the confocal principle is only conserved in the
axis perpendicular to the slit height, and that it provides lower transverse and axial resolution
than TSCM.
The Confoscan series, namely Confoscan 2, Confoscan 3 and Confoscan 4 (Nidek,
Gamogori, Japan), is the most commonly used SSCM in the published literature.
Laser Scanning Confocal Microscope (LSCM)

The Heidelberg Retina Tomograph (HRT) is a laser LSCM that integrates the laser
scanning for retinal imaging with the Rostock Cornea Module (RCM) microscope. The RCM
performs confocal microscopy of the cornea and measures its thickness by changing the focal
plane within the cornea. High-contrast and high quality images of the cornea are produced by
using laser light at a wavelength of 670 nm.
Corneal thickness measurements using LSMC remain to be validated.
2.1.5. Low Coherence Interferometry-Based Techniques

Low-coherence interferometry is based on the principle that light split from one beam
into two different paths will produce interference patterns dependent on the phase difference
introduced by discrepancies in the path length when recombined. Optical coherence
tomography (OCT) emerged from low-coherence interferometry. [83] These systems
typically employ a Michelson interferometer to measure the echo time delay and intensity of
reflected and backscattered light using a reference beam of known path length and time
delay.
Two main approaches are used in OCT instrumentation. Conventional Time-domain
OCT (TD-OCT) provides depth scans (A-scan) obtained by changing the reference arm. [84]
In Frequency-domain OCT (FD-OCT), a spectral fringe pattern is recorded either by a
spectrometer or by wavelength tunable lasers.
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Optical Low-Coherence Reflectometry (OLCR)

OLCR uses of a low-coherence light source and an optical interferometer to match the
optical path length of the light reflected from the cornea with a reference. The coherence
properties of light reflected provides information on the time-of-flight delay from the
reflective surfaces in the cornea. Delay information is then used to determine its thickness.
This technique is limited to point measurements, but was later extended into optical
coherence tomography (OCT) scanner that provides area measurements.
TD-OCT
TD-OCT provides non-invasive, in-vivo high-resolution cross-sectional images of the
anterior segment structures.
The optical path length difference between the reference mirror and the sample in the
Michelson interferometer is mechanically modulated in time. The A-scans are produced by
the movement of the mirror in the reference arm during the acquisition. Thus, the imaging
speed is mechanically limited by the time required to acquire each B-scan (two-dimensional
scan constructed from the A-scan series at several transverse positions). [85,86] The image‘s
axial resolution is limited only by the coherence length of the light source.
There are currently two commercially available TD-OCT devices, namely the standalone
Visante OCT (Carl Zeiss Meditec Inc., Dublin, California, USA) and the slit-lamp adapted
OCT (SL-OCT; Heidelberg Engineering GmbH, Heidleberg, Germany), an AS-OCT system
attached to a Haag-Streit slit lamp. Both devices provide automatic and manual measurement
of CCT, through non-contact imaging of the anterior segment of patients sitting in the upright
position. These systems use light from 1310 nm, which provides relatively good penetration
through the sclera allowing anterior segment imaging.
These systems enable pachymetry evaluation with good repeatability and reproducibility,
[87] and are consistent with US pachymetry. [87,88]
FD-OCT
The recent introduction of FD-OCT brought significant improvements in both resolution
and imaging speed. Although similar in principle to TD-OCT, the mirror in FD-OCT is
stationary and the A-scan can be performed in two ways: SD-OCT and SS-OCT. In SD-OCT,
the interference pattern generated by light reflected from the stationary mirror and the cornea
is acquired using a spectrometer equipped with an array of photodetectors (charge-coupled
device and CMOS). [89,90] A-scans are obtained by applying the Fourier transform to the
spectrum of the reflected/backscattered light. Several scans are acquired along a transverse
plane through the eye and assembled into B-scans, similarly to TD-OCT. RTVue employs
SD-OCT technology and is 65 times more fast than the current TD-OCT (Table 4).
SS-OCT imaging is based on the analysis of a spectral fringe pattern recorded by a
detector with a high speed wavelength tunable laser source. Tomey Corp., launched the first
SS-OCT system, the Casia SS-1000, with a 30 kHz acquisition speed. [91] Recently, Gora
and colleagues presented a potential next generation of SS-OCT systems, working at a
repetition rate of 200 kHz. [92]
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Table 4. Comparison between TD-OCT and FD-OCT features
TD-OCT FD-OCT
(SL-OCT™) (RTVUE)
Scan Range
7 mm 2mm to 2.3mm
Depth
Transverse 15 mm 2mm to 12mm
No. of images per scan ≈2000 A scans/second 26,000 A-scan/second
Frame rate 512 to 256 A-scan/Frame 256 to 4096 A-scan/Frame
Optical Resolution
<25 μm (max) 5.0µm
Axial
Transverse <100 μm (max) 15µm
2.1.6. Slit-Based Imaging

As discussed above, slit-scanning and Scheimpflug technologies can also be used to
obtain the pachymetry. The Orbscan II system has shown to underestimate the corneal
thickness values, whereas Scheimpflug systems, such as Pentacam and Galilei, are usually
more accurate given the proximity of their results to the US pachymetry‘s. [15,93,94]
3. Biomechanical Properties of the Cornea
The field of corneal biomechanics concerns with the study of its function and structure
through the application of mechanical principles.
The cornea‘s internal structure and thickness determine its intrinsic biomechanical
properties, granting it a viscoelastic behavior. These properties are the basis of the cornea‘s
topography and optical quality. The stability of the shape of the cornea (and consequent
optical aberrations) results from a dynamic steady-state balance between its intrinsic
characteristics and pressures exerted on its surfaces. The intraocular pressure is the most
influent extra-corneal factor and it acts on the posterior corneal surface. Other factors include
the atmospheric pressure, extra-ocular muscles, ciliary muscle during accommodation and the
eyelids tension. [95]
Corneal procedures/diseases induce structural changes that weaken or strengthen the
cornea at a biomechanical level, which consequently disturbs its homeostasis. New
equilibriums in the cornea/environment system result in changes to the corneal
geometry/optics and the eye‘s optical quality.
Interest in corneal biomechanics is increasing due to the importance of these properties

on the measurement of IOP, diagnostic of diseases and outcomes of keratorefractive
procedures.
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3.1. Biomechanical Features of the Cornea
3.1.1. Corneal Biomechanical Metrics

The cornea has a viscoelastic behavior, since it exhibits both elastic and viscous
characteristic when undergoing deformation due to stress. [95]
Elastic materials are characterized by a linear relationship between stress and strain
(relative deformation), with no time dependence. As an elastic material, cornea returns to its
original shape after the exerted stress responsible for its deformation is removed. The elastic
modulus measures the material stiffness and can be quantified using Young‘s modulus.
The elastic modulus of the cornea can be assessed with ex-vivo extensiometry techniques
that measure force required during steady axial elongation of the cornea. A cornea with low
modulus of elasticity will exhibit a greater deformation for a given stress than a material with
high modulus.
Viscoelastic behavior, on the other hand, has a time-dependent component, which means
that in a viscoelastic material the magnitude and rate of an applied force affects the
biomechanical response. The cornea quickly assumes a new shape under stress but does not
return immediately to its original shape when the stress is removed. Viscoelastic properties
are usually described by metrics such as hysteresis, stress relaxation and creep.
3.1.2. In-Vivo Measurement of Corneal Biomechanical Properties

A new era is drawing in biomechanical research with the development of techniques
capable of measuring biomechanical properties of the cornea in vivo. Interferometric
techniques and dynamic corneal imaging had been used to characterize corneal elastic
properties in vivo by analyzing the electronic speckle pattern interferometry or the change in
pattern of the reflected Placido topographic‘ mires during central indentation of the cornea,
respectively. However, these system remained in a prototype stage at the present due to the
necessity of improve the data analysis.
The only commercially available system for assessing corneal biomechanical properties
is the Ocular Response Analyzer (ORA; Reichert Ophthalmic Instruments, Buffalo, NY). It‘s
a non-contact tonometer developed to simultaneously assess and compensate for corneal
biomechanical properties on IOP measurement using a dynamic bidirectional applanation
process. This process consists of a collimated air pulse applying pressure on the cornea and
causing it to move inward, past applanation, and into slight concavity. After milliseconds, the
air pumps shut off, the pressure decreases, and the cornea begins to return to its original
convex shape. An electro-optical infrared system monitors the inward and outward corneal
bending responses by detecting the corneal applanation as a peak in the reflected signal
intensity. This approach provides two applanation pressure values determined from the
inward and outward events.
The measurement result consists of a graph with a green curve, which corresponds to the
air pulse pressure, and a red curve, which corresponds to the corneal movement. The last one
has two principal peaks, the inward and outward applanation, which correspond to points P1
and P2 in the green curve. The first peak on the red curve coincides with the pressure
required to applanate the cornea inward (P1). The second peak on the red curve coincides
with the pressure exerted by the cornea as it moves out, under the decreasing pressure of the
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air pulse (P2). As a result of the viscoelastic nature of the cornea, the air pressure (green
curve) at the initial applanation (P1) is different from the pressure at recovery applanation
(P2).
In addition to the IOP measurement, the ORA provides measures of the biomechanical
properties of the cornea, characterized by the corneal hysteresis (CH) and the corneal
resistance factor (CRF). CH is the property related to the viscoelasticity and is calculated
from the ORA waveform as the difference the two air pressures at the two applanation events
(P1 and P2). The CRF indicates the overall resistance of the cornea and is calculated as a
linear function of P1 and P2. Two measures of the IOP are generated, the Goldmann-
correlated IOP (IOPg) and the corneal-compensated IOP (IOPcc). The IOPg is the average
between the P1 and P2 and is a Goldmann-correlated IOP measurement. On the other hand,
the IOPcc is design to provide more accurate measurement of the IOP, through the linear
combination of P1 and P2. This measure is less affected by the biomechanical properties of
the cornea, such as CCT and corneal hysteresis.
Conclusion
Corneal topography has become essential for assessing the features of the cornea. The
clinical understanding and interpretation of the several topographic data is indispensable for
diagnosis and management of clinical cases.
As our understanding of the optical and mechanical properties of the cornea improves, so
will our ability to effectively plan refractive surgeries, to define strategies for further
improving the predictability of keratorefractive surgery and to manage postoperative
complications and ocular diseases.
Acknowledgment
This work was supported by the Fundação para Ciência e a Tecnologia (grant
PTDC/SAU-BEB/72220/2006). Cristina Oliveira was supported by grant Uminho/BI/141
/2008 from Fundação para a Ciência.
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Chapter VI
Retinal Control of the Refractive

State of the Eye
Francisco J Carrillo-Salinas1 and Jaime Tejedor*1,2

1
Faculty of Medicine, Universidad Autónoma de Madrid, Spain
2
Department of Ophthalmology, Hospital Ramón y Cajal, Madrid, Spain
Abstract
In this chapter, we summarize advances in the understanding of factors that control

refractive development and refractive state of the eye, particularly by retinal mechanisms.
When the normal refractive development process towards emmetropia (emmetropization)
fails, it leads to refractive errors, such as myopia or hyperopia, which are highly
prevalent in the general population. Different studies have reported a regulatory effect of
the retina in the refractive state of the eye, through a cascade of neurotransmitters or
growth factors signals, in which the involvement of different systems, including
muscarinic, glucagonergic and dopaminergic, has been demonstrated. The main findings
described in the literature will be presented. Transcription factors of neuromodulators,
whose expression is known to change with variations in light conditions, in particular
Egr-1, could be of vital importance in regulating refractive error. Different genetically
modified mouse strains showing the potential role of neurotransmitters and transcription
factors, and the experimental studies showing this effect, will be described. Other studies
stress the importance of the ionic and water balance and content of the outer retina and
choroid as controlled by the retinal pigment epithelium, and its role in the mechanisms of
myopic and hyperopic changes. The influence of peripheral versus central retina in the
development of myopia has been a matter of debate, with contradictory conclusions
regarding the importance of retinal periphery and peripheral vision. We will finally deal
with this controversy, as well as introduce potential therapeutic implications of some
involved described regulatory elements.
* Email: jtejedor.hrc@salud.madrid.org
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Copyright © 2012. Nova Science Publishers, Inc. All rights reserved. May not be reproduced in any form without permission from the publisher, except fair uses permitted under 138 Francisco J Carrillo-Salinas and Jaime Tejedor
1. Introduction
One of the most important challenges for researchers is to understand how neural circuits
process information, and one of the most studied is the retina.
The retina has a functional architecture described widely. It is designed to transmit all the
potential visual information of the environment, using as little space as possible and the
minimum energy (Balasubramanian & Sterling, 2009).
The normal refractive state of the eye is called emmetropia, in which the parallel rays
from the infinity are focused exactly on the retina. This process is called emmetropization.
When it fails, a refractive error (or ametropia) appears. The most common refractive errors
are myopia, hyperopia and astigmatism. Infants are born with hyperopia, and require a period
of time so that the eyes tend to emmetropization.
Idiopathic myopia affects 25% of the western population and more than 30% of the
population in eastern countries. In Hong Kong, the prevalence has increased from 30% to
70% in a single generation, which is probably the result of an environmental causal factor
(Goh & Lam, 1994). In the United States of America, approximately 25% of children become
myopic (Sperduto et al., 1983), although at present the prevalence of myopia in this country
has increased (Vitale et al., 2009). European countries may have a similar prevalence. A
much smaller percentage suffers a significant hyperopia, and most people develop little or no
refractive error and therefore they are emmetropic. The prevalence of myopia is increasing
worldwide. A curious fact is that damage to the photoreceptors or RPE produces axial growth
inhibition and hyperopia (Westbrook et al., 1995).
Until the last decade, it was considered that myopia was primarily genetic in origin due
to the increased incidence of myopia among children of myopic parents and the enormous
difference in the prevalence of myopia in ethnic groups (Mutti et al., 2002). This view is
weakened by the discovery of the homeostatic control of refractive errors in animals,
including primates. This gave credence to the epidemiologic evidence accumulated over
decades of environmental factors may contribute to myopia in humans.
Currently, it is unclear whether the myopic refractive errors are developed as defective
physiological processes in the regulation of ocular growth or as an adaptive process due to
the huge visual demands of modern societies (Stone & Khurana, 2010).
For the study of myopia, there are different ways to induce experimentally. The form-
deprivation myopia (FDM) is the elongation of the eyeball due to the use of diffusers (allow
in light but not the perception of shapes projected on the retina) or to the completion of an
eyelid suture. It is also possible for a less invasive way to induce myopia or hyperopia, with
the imposition of positive [lens-induced hyperopia (LIH)] or negative lenses [lens-induced
myopia (LIM)].
2. Emmetropization
Crewther (2000) suggested that emmetropization is an event that can be divided into
three general processes. The first process describes the mechanisms responsible for
perceiving the blurred image. The second process is based on the transmission of the signal
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Copyright © 2012. Nova Science Publishers, Inc. All rights reserved. May not be reproduced in any form without permission from the publisher, except fair uses permitted under Retinal Control of the Refractive State of the Eye 139
of defocus through the retina, retinal pigment epithelium (RPE) and choroid, to control the
growth of the sclera. The third process is the change induced in axial length and scleral
growth by effectors.
2.1. Detection of Blurred Image
The blur detection must be located in the retina, and it can respond to extremely low
contrast (Schaeffel & Diether, 1999). The neurons responsible for detecting the blurred image
(amacrine and bipolar cells) are contiguous to the photoreceptors, with the assembly of neural
circuits with inhibitory and excitatory input signals. These connections establish a center-
periphery antagonism in receptor fields (Lawrence & Azar, 2002), that through complex
mechanisms (and currently unclear), it is possible to detect the blurred image.
2.2. Transmission of the Defocus Signal
To transmit the blur stimulus, the photoreceptor sends a signal to adjacent cells (e.g.,
bipolar and amacrine cells). The signaling molecule must diffuse through the retina, RPE,
choroid and sclera to increase the growth rate when the image is blurred or to decrease this
rate in the opposite case. Dopamine is considered one of the major signaling molecules
involved in the defocus signaling. Another candidate is the vasoactive intestinal peptide
(VIP), or the relationship between glucagon and insulin, among others. This issue is treated in
more detail later.
2.3. Effectors
Whatever the signaling molecule released by the retina, the process ends with the change
in elongation of the eye. In most animals, the elongation involves a proteoglycan and/or
collagen synthesis in the sclera in chicks (Christensen & Wallman, 1991; Rada et al., 1991,
2006), probably with an intermediate regulation. But several studies indicate that in primates
and humans there is a negative correlation between the rate of proteoglycan synthesis and the
rate of elongation of the vitreous chamber in experiments with marmosets (Rada et al., 2000,
2006). This contradiction suggests the existence of other local processes of regulation of
emmetropization.
3. Molecules That Could Participate

in the Control of Refractive Errors
Visual input signal could lead to a response in the retina, displaying a cascade of signals
to regulate axial elongation, which could be related other layers of the eye such as choroid or
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RPE (Rymer & Wildsoet, 2005). There are many studies showing that the sclera is a dynamic
area, and it can induce changes in extracellular matrix composition due to different visual
stimulus to regulate ocular size (Rada et al., 2006). The retina regulates this process by
signaling factors (dopamine, retinoic acid, nitric oxide, among others), as we described
below.
3.1. Dopamine
Dopamine belongs to the family of catecholamines, is one of the main neurotransmitters

in the retina and is involved in signal transmission in the visual system. In the inner nuclear
layer (INL) of the retina, in most species there is a network of dopaminergic neurons, the
amacrine cells (Djamgoz & Wagner, 1992; Klitten et al., 2008). These cells are responsible
for the release of dopamine. It has been shown that retinal dopamine is involved in the
regulation of electrical coupling between horizontal cells (Weiler et al., 2000) and the
retinomotor movement of the photoreceptors (Djamgoz & Wagner, 1992).
FDM leads to ocular elongation, and the retinal levels of dopamine and its metabolites
are decreased in FDM (Stone et al., 1989; Iuvone et al., 1991; Mao et al., 2010). It has also
been seen that the concentration of dopamine is a controlled light-dependent process, i.e. high
levels during the day and low at night, in contrast there is a decrease in dopamine retinal
levels during the day with FDM [Bloomfield & Völgyi, 2009, both in chicken (Megaw et al.,
2006) and rabbits (Ribelayga & Mangel, 2010)]. This process is affected by flickering, and
retinal dopamine depletion could be related to the loss of contrast sensitivity and high spatial
frequency in deprived chick eyes (Feldkaemper et al., 1999). Repeated intravitreal injections
of dopamine completely prevented the myopic shift in rabbits (vitreous chamber elongation
and axial elongation due to eyelid suture), which may be associated with increased retinal
dopamine due to intravitreal injection (Gao et al., 2006). Intraperitoneal injection of L-DOPA
could cause an increase in retinal dopamine content (Mao et al., 2010), and has a retarding
effect of myopia. Apomorphine (dopaminergic agonist) also blocks the axial elongation in
chickens and monkeys (Iuvone et al., 1991; Drumheller et al., 1995) and other more recent
studies have shown that dopamine levels are related to the axial growth by the use of agonists
and antagonists of dopamine (McCarthy et al., 2007).
Exposure to light of the ON-pathway stimulates dopamine release (Voigt & Wässle,
1987). Dopamine and DOPA synthesis increases to compensate the massive release of the
neurotransmitters by exposure to light, so that, the baseline concentration of dopamine does
not change (Nir et al., 2000, Pardue et al., 2008; Pozdeyev et al., 2008).
3.2. Insulin
Insulin and insulin receptor plays an important role in the development of cognitive and
memory processes in the CNS (Zhao et al., 2004). Insulin concentration in ocular tissues is
relatively low. Specifically, the retina has a low expression of insulin-degrading enzymes
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(Varandani et al., 1982). Insulin acts on the anterior eye segment, and has a myopic effect
(Feldkaemper et al., 2009).
After studies in different animal models, insulin may appear to have a neuromodulatory
role in the retina, rather than in regulating glucose metabolism (Reiter et al., 2003; Reiter &
Gardner, 2003), but it is unclear their role in the retina. There are conjectures (Cordain et al.,
2003) that overexpressing insulin metabolic diseases (e.g. hyperinsulinemia) can cause
myopia, but there is insufficient data to support this assumption. Jacobsen et al. (2008)
suggested glucose as one of the first mediators in the myopic shift. We need to investigate
further the role of insulin and glucose in the regulation of ocular growth.
3.3. Glucagon
Glucagon is a potent stimulator of cAMP production in the retina in different animal

models (Schorderet et al., 1981; Longshore & Makman, 1981), and activates protein kinase A
(PKA). Moreover, glucagon shows a bidirectional modulation in eye growth, possibly as a
sign of stop-and-go (Buck et al., 2004), and affects the RPE cells (Koh & Chader, 1984) [like
VIP, another retinal neurotransmitter involved in myopia (Seltner & Stell, 1995)]. In the
retina were detected higher levels of glucagon and glucagon receptor than in the choroid
(Buck et al., 2004). Glucagon may exert its action on receptors linked to the retinal side of
RPE. Other studies show that insulin may interact with glucagon mRNA when the retinal
image is blurred (Feldkaemper et al., 2009).
Glucagon significantly inhibits the development of FDM in chicks, and there is a
choroidal thickening (Vessey et al., 2005), so that the retina moves forward, where the
focused image is projected. It has also been observed that the glucagon receptor antagonist
Phe6-antagonist inhibits the development of hyperopia in experimental eyes treated with plus
lenses (Vessey et al., 2005).
The role of glucagon in the visual control of eye growth in mammals is uncertain,
because it is not detectable or is there a small amount in the mammalian retina (Tornqvist &
Ehinger, 1983). The glucagon precursor is proglucagon, and many proteins can be originated
from proglucagon. Perhaps other peptides of the glucagon superfamily also have done their
job (Brand et al., 2005; Brand et al., 2007). It is possible that changes in the expression of
ZENK (Egr-1, shown below) lead to the synthesis and/or release of glucagon, because ZENK
is expressed in glucagonergic amacrine cells. Further studies are needed to unravel the true
role of glucagon in the mammal retina, perhaps as part of an insulin-glucagon regulation of
axial growth.
3.4. VIP
There are several studies showing a higher level of retinal VIP when myopia is induced
(Koh & Chader, 1984; Seltner & Stell, 1995), both in chicken and primate. But later studies
make uncertain the relationship between retinal VIP and eye growth regulation, because VIP
levels remain high after the end of the FDM (Stone et al., 1988).
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It seems that VIP and glucagon may be related to the EPR, as an intermediate layer
between the retina and choroid/sclera, which regulate ocular growth in part by controlling the
ion flow of the RPE and the exchange of fluids between the subretinal space and choroid. In
recent years, was given importance, but seems not very important in the regulation of ocular
growth. More research is needed to clarify these hypotheses.
3.5. Growth Factors
There are specific growth factors that control the extracellular matrix remodeling in the
sclera. There are some more effective than others to stimulate proteoglycan synthesis in the
sclera, particularly in experiments in porcine sclera in organ culture, in which Luebke &
Rada (2003) saw that insulin-like growth factor I (IGF-I) was effective at upregulating the
synthesis of proteoglycan compared with other growth factors such as basic fibroblast growth
factor (b-FGF, Rohrer & Stell, 1994), transforming growth factor-beta (TGF-, Rohrer &
Stell, 1994), or retinoic acid, which had no significant effects.
TGF- is distributed in the outer segment of photoreceptors (Lutty et al., 1991) and RPE
(Tahinara et al., 1993). Honda et al. (1996) suggest that a decrease in the TGF- levels is
followed by an axial elongation in chicks. TGF- induces the differentiation of
myofibroblasts in the sclera, to have an increase in the cell-mediated contraction of the scleral
matrix (Jobling et al., 2009).
It is unlikely that TGF- isoforms have an important role in signal transmission from the
retina to the sclera, and the signs must be based probably in different ways when it is
necessary to control the eye growth due to refractive errors.
3.6. Retinoic Acid
Retinoic acid is related to the cascade of signaling between the retina and the sclera,
which controls the process of ocular growth (Mertz & Wallman, 2000). Retinoic acid also
regulates changes in the extracellular matrix in different connective tissues (Maden, 1985;
Vincenti et al., 1996). Retinoic acid receptor was located in amacrine cells, choroid, and
chondrocytes and fibroblasts in the sclera of the chick eye (Fischer et al., 1999a)
Retinoic acid levels with or without myopia varies according to which animal models
were used. For example, in chicks with induced myopia the retinal retinoic acid level was
increased, while the choroidal retinoic acid levels decreased. In models of eutherians
mammals [group of mammals that do not include monotremes (platypus) or marsupials], e.g.
guinea pig and marmoset, both retinal and choroidal retinoic acid levels increased with
myopia (Rada et al., 2006). These differences may be due to the enormous structural
differences in the eyes of eutherians and chicks (Mertz & Wallman, 2000; McFadden et al.,
2004). In contrast, a recent study denies the involvement of retinoic acid receptor alpha in the
development of refractive errors (Veerappan et al., 2009). More research is needed to clarify
the ambiguity of the role of retinoic acid.
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3.7. Muscarinic System, Atropine and Pirenzipine
Until now, there are only five muscarinic acetylcholine receptors (mAChR) described
(M1 to M5): M1, M3 and M5 mAChR are coupled to heterotrimeric G-proteins (are
associated with the Gq-alpha subunit), and its activation usually produce an excitatory result.
However, M2 and M4 mAChRs are associated with the Gi-alpha subunit, and tend to have
inhibitory actions (Strang et al., 2010).
Many muscarinic receptor antagonists have been studied, as atropine (a nonselective
mAChR antagonist; McBrien et al., 1993b), pirenzipine (an M1-selective antagonist;
Cottriall & McBrien, 1996), himbacine (a M2 and M4-selective antagonist; Cottriall et al.,
2001; Luft et al., 2003), oxyphenonium (a nonselective mAChR antagonist; Luft et al.,
2003). These molecules inhibit the synthesis of glycosaminoglycans in the sclera and slow
the progression of myopia, based on the results obtained in different studies (Stone et al.,
1991; McBrien et al., 1993ab; Schwahn et al., 2000; Luft et al., 2003; Duncan & Collison,
2003; Strang et al., 2010). After studying the mAChR antagonists, only pirenzepine and
oxyphenonium were effective to inhibit ocular growth in chickens due to FDM (Luft et al.,
2003), but not scopolamine (a competitive antagonist at muscarinic acetylcholine receptors,
specifically M1 receptors).
Atropine may cause an indirect action in the retina, causing the release of dopamine or
other neurotransmitters (Schwahn et al., 2000). In RPE cells, atropine also blocks the
muscarinic acetylcholine receptors, thereby inhibiting the expression of TGF- (Tan et al.,
2010).
In view of the results obtained by Liu et al. (2007) in guinea pig, the fact that FDM
causes an increase in mRNA expression of M1 and M4 receptors in the sclera seems to be
definitive. These data contradict the results of McBrien et al. (2009), although they suggest
that it may be due to the methodology used, and it is possible that the changes observed by
Liu et al. (2007) do not reflect a causal relationship related to eye growth, but probably
subsequent changes to this growth (McBrien et al., 2009).
If the muscarinic system acts in the choroid and induce changes in ocular growth rate,
muscarinic agonists should have the effect of inducing axial eye growth and thinning of the
choroid (Nickla & Wallman, 2010). Although the five subtypes of muscarinic acetylcholine
receptors are expressed in the retina (human: Collison et al., 2000), choroid and sclera, there
is no significant change in the expression of these receptors during eye growth in guinea pig
(McBrien et al., 2009). It possibly indicates an indirect action of the acetylcholinergic system
in myopic development.
3.8. Nitric Oxide
Nitric oxide is a gaseous short-lived neurotransmitter, usually degraded or reacted within

a few seconds, and a potent relaxant of peripheral vascular smooth muscle. It has numerous
locations in the eye, as intrinsic choroidal neurons (Flugel et al., 1994), RPE (Goldstein et
al., 1996) and retinal neurons (Fischer & Stell, 1999), among others. NO travels from
postsynaptic neuron back to presynaptic neuron which activates guanylyl cyclase, the enzyme
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that catalyzes cGMP production. If nitric oxide synthase (NOS) is inhibited with L-NAME
(nonselective inhibitor of nitric oxide synthase, which is used to induce hypertension; Nickla
& Wildsoet, 2004) or L-NMMA (nonselective inhibitor of all NOS isoforms, analogue of L-
arginine; Nickla et al., 2009), and thus NO formation, prevents the thickening of the choroid
due to a myopic blur
Nickla and Wallman (2010) research suggests that NO is part of the cascade of signals
related to inhibition of ocular growth in response to a myopic blur. NO from a neural source
can also lead to a thickening of the choroid, promoted by contractions and relaxations of non-
vascular smooth muscle.
3.9. Serotonin
This neurotransmitter is found in different subpopulations of amacrine cells (Vaney,

1986), and is the precursor of melatonin. Several studies have demonstrated the role of
melatonin in the eye, mainly related to the regulation of circadian rhythms (activation of rod
shedding, Besharse & Dunis, 1983; regulation of dopamine release, Dubocovich, 1983;
among others).
It is possible that the modification of the dopaminergic system by serotonin inhibits a
mild myopia (serotonin has a similar effect to apomorphine, a dopamine agonist), although it
is unclear (George et al., 2005). Everything seems to indicate that serotonin/melatonin is not
directly involved in the regulation of ocular growth due to refractive errors (Hoffmann &
Schaeffel, 1996; George et al., 2005; Rada & Wiechmann, 2006).
3.10. Egr-1
Egr-1 (Early Growth Response Protein 1) is a zinc-finger protein and is localized in the
nucleus (Cao et al., 1990). It is also called Krox-24 and NGFI-A, but probably its orthologue
ZENK is best known because of his numerous studies in chicks. In this chapter we use ZENK
when the experiments have been done in the chick model, and Egr-1 when mammals were
used.
Following the completion of a microarray study (Fu et al., 2003), some target genes of
this transcription factor were confirmed, such as TGF-1 (Khachigian et al., 1996; Liu et al.,
1996), PDGF-A (Wang & Deuel, 1992), PDGF-B (Khachigian et al., 1996), bFGF (Hu &
Levin, 1994), adhesion proteins (collagen, Chen et al., 2006) among others.
ZENK-immunoreactive cells are located in INL, so it is likely that ZENK expression is

induced in Müller cells, bipolar cells, or both. But ZENK has a very clear expression pattern
in glucagon expressing amacrine cells, with a positive correlation with the defocus sign
induced by the projection of an image on the retina (Fischer et al., 1999b; Bitzer & Schaeffel,
2002).
The use of Egr-1 knockout mice has helped to unravel the role of this protein, although it
is not clear yet. The comparison of results from wild-type and heterozygous mice suggests
that Egr-1 is probably involved in the regulation of ocular growth. An increase in the levels
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of ZENK causes enhanced release of neurotransmitters in glucagon containing amacrine cells

in chicks (Vessey et al., 2005) and the lack of Egr-1 induces a decrease in the levels of
several transcription factors (e.g., bFGF and TGF-) and levels of scleral collagen, causing
thinning of the sclera and giving it more flexibility, and thereby causing ocular growth
(Schippert et al., 2007). In recent studies, Ashby et al. (2010) compared the ZENK and
preproglucagon (PPG) RNA transcript levels in retina during periods of induced axial growth
in chicken FDM and LIM, obtaining results that show a possible link between the ZENK
expression and synthesis and release of glucagon.
The Figure 1 suggests the action of these molecules in different compartments of the eye,
and their role after an induction/inhibition of axial growth.
Figure 1. A) Axial growth inhibition; B) Axial growth induction. Egr-1: Early Growth Response Protein 1;
IGF-I: Insulin-like Growth Factor 1; bFGF: basic Fibroblast Growth Factor; NO: Nitric Oxide; VIP:
Vasoactive Intestinal Peptide.
4. Gene Expression in Myopia
There have been many studies looking for candidate genes that may favor the
development of refractive errors, especially related to myopia (particularly high myopia, less
than -6D), rather than genes associated with hyperopia (Baird et al., 2010).
Numerous studies have provided a long list of candidate genes involved in the
development of refractive errors, among which are TGF-1 (Lin et al., 2006), TGF-2 (Lin et
al., 2009a), collagen 1A1 (COL1A1; Inamori et al., 2007; Mutti et al., 2007), hepatocyte
growth factor (HGF; Han et al., 2006; Veerappan et al., 2010), lumican (LUM; Chen et al.,
2009), transforming growth b-induced factor (TGIF; Lam et al., 2003), bone morphogenic
protein 2 (BMP2; Liu et al., 2009), muscarinic acetylcholine receptor 1 gene polymorphisms
(CHRM1; Lin et al., 2009b), matrix metalloproteinases (MMP-1, -3 and -9; Hall et al., 2009),
among many others.
Some studies in primates with FDM showed that many different genes expression varied
significantly, but almost 70% of these genes were related to cell proliferation. Later it was
found that there is a population of neuroprogenitor cells in the peripheral retina, that increase
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in number when there is an elongation of the vitreous chamber due to eyelid suture
(Tkatchenko et al., 2006). Among the differentially expressed genes with induction of
refractive errors, those with more interest now are prepro-urotensin II-related peptide (prepro-
URP, McGlinn et al., 2007), glucagon and Egr-1 (Fischer et al., 1999b; Bitzer & Schaeffel,
2002; Simon et al., 2004).
Other studies associate the HGF gene with the hyperopia development (Veerappan et al.,
2010), and their participation in the control of ocular growth, although more research is
needed to clarify.
It is necessary to replicate the experiments in this research, because false positives can
mask the data, or true positives can not be verified and thus give a specific and truthful result.
Although it is only a conjecture, possibly genetic contribution to the development of
refractive errors is the sum of many small factors, little changes in the expression of many
genes that in turn alter the regulation of other genes, rather than very obvious changes in the
expression of few genes or a gene family in particular.
5. Central vs. Peripheral Retina
Nearwork-induced transient myopia (NITM) is one of the main factors that cause
myopia. It produces a small transient pseudomyopic change in the eye after a sustained
period of nearwork, in which the lens is unable to reduce its power quickly, thus
demonstrating an accommodative effect induced by the sympathetic and parasympathetic
systems (Ciuffreda & Vasudevan, 2008; Vasudevan et al., 2009; Ciuffreda & Vasudevan,
2010) (Figure 2). It seems that progressive myopic eyes are more susceptible to NITM than
stable myopic eyes, although no conclusive results have been reported (Vasudevan &
Ciuffreda, 2008).
Smith III studies show that peripheral vision can have a regulation on the central
refractive development. Specifically in monkeys, the results have shown that can be induced
central axial growth (myopic) due to relative peripheral hyperopia, although projecting sharp
images focused on the central retina. It seems that with discordant visual signals between the
central and peripheral retina, the ultimate direction of axial growth is controlled by the
peripheral retina (Smith III et al., 2009b). Instead, it appears that in chicks the peripheral
retina not necessarily induces central refractive development (Schippert & Schaeffel, 2006).
Although, this contradiction may be due to methodological differences. All studies to date
suggest a dominance of the peripheral retina on the central retina when it comes to
controlling the refractive development. As Smith III suggested, this regulation is a much
conserved process in evolution, and very effective in species without foveae.

Recently, retinal blur and perception blur on the peripheral retina to the central retina has
been considered as a myopigenic factor (Smith III et al., 2005; Ciuffreda et al., 2007).
It was observed that myopic eyes have a relative hyperopia in the peripheral retina (Singh
et al., 2006) when it is compared with the refractive state of the central retina, due to a
difference in the refractive plane of the peripheral retina and the back of the eye, i.e.,
peripheral vision may influence the shape of the eye by local mechanisms, in particular axial
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length, in an independent way of central retina visual signals (Ciuffreda & Vasudevan, 2010;
Smith III et al., 2010).
Figure 2. Nearwork-induced transient myopia (NITM). A) Example of visual perception when the lens is
accommodated for the approach of a nearby object; B) The nearby object is focused in the central retina, but
is blurred in the peripheral retina (peripheral hyperopia). The retina tends to focus before peripheral blur, so
there is an axial growth and the eye gets more elongated form, thus inducing myopia.
Figure 3. The traditional use of negative-powered spectacle lenses results in an increase in relative peripheral
hyperopia, thus enhancing the myopic effect on the peripheral retina, increasing the axial growth.
It would be important to know how the optical defocus affects ocular shape. In monkeys
(Huang et al., 2009) and humans (Atchison et al., 2005), spherical-equivalent refractive
errors can be altered with eccentricity (flattening in the curve of the eye, Smith III et al.,
2010). Indeed, human with myopia and monkeys with experimentally induced myopia have
relatively elongate eyes, with peripheral hyperopia (Millodot, 1981; Mutti et al., 2000;
Atchison et al., 2004). East Asian moderate myopes have more relative peripheral hyperopia
than Caucasians whites, and therefore a more elongated eye, with a similar refractive error of
the central retina (Kang et al., 2010). It seems that the traditional negative-powered spectacle
lenses increase the amount of relative peripheral hyperopia in myopic eyes, also causing axial
elongation (Tabernero et al., 2009, Lin et al., 2010) (Figure 3).
Currently, there are fewer supporters of the fact that a global mechanism acts when it
comes to regulating refractive development, and the idea that emmetropization is given by
local processes (Diether & Schaeffel, 1997; Smith III et al., 2009a) is more correct than the
other.
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Conclusion
In the future we must try to improve existing techniques to reduce measurement error. It
would also be good for the future to meet a daunting task: to determine the genes involved in
the regulation of axial growth in response to refractive errors, and clarify the relationship
between central and peripheral retina. The movement of molecules from a location to another
one in the eye attaches great importance to the RPE today.
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Chapter VII
Proliferative Vitreoretinopathy:
Signaling Mechanism Involved
and Available Treatments
Bose Karthikeyan, Venkataraman Deepak

and Sangiliyandi Gurunathan*
Division of Molecular and Cellular Biology, Department of Biotechnology,
Kalasalingam University, Virudhunagar District, India
1. Abstract
Proliferative vitreoretinopathy (PVR) is the most common cause of visual loss after
retinal detachment. It is clinicaly characterized by uncontrolled proliferation of Retinal
pigment epithelial (RPE) cells in the vitreous and the concomitant effect of membrane
formation on bothside of the retina. In normal adult eye, blood retinal barrier (BRB)
comprises of non-proliferating RPE cells which are also essential for the survival of
photoreceptors. Continuous damage to the BRB causes numerous growthfactor release
into the vitreous and often precedes clinically recognisable PVR. Both retinal injury and
BRB damage enhances RPE cell migration into vitreous and transforms fibroblast like
cells. The early cellular cascade includes various growth factors which involve in
autocrine or paracrine loop is a key factor in PVR. Although several growthfactors are
involved in PVR condition, vascular endothelial growth factor (VEGF) and interleukin1-
β are the major key factors which play important role in cellular proliferation. Significant
evidence suggests that multiple intracellular pathways regulate inflammatory phase of
PVR. Better understanding of pharmacological agents and elucidation of the exact
signaling mechanism involved in the PVR associated metabolic alterations may reveal
novel therapeutic approaches. Several inhibitors were tested for efficient treatment of
* Corresponding Author: Dr.G.Sangiliyandi, Professor and Head, Department of Biotechnology, Kalasalingam

Univeristy, Krishnankoil-626190, Virudhunagar District., E.mail: lvsangs@yahoo.com ,Tel.: +91 4563
289042, Fax: +91 4563 289322.
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Copyright © 2012. Nova Science Publishers, Inc. All rights reserved. May not be reproduced in any form without permission from the publisher, except fair uses permitted under 158 Bose Karthikeyan, Venkataraman Deepak and Sangiliyandi Gurunathan
PVR but many of them were unsuccessful. Although some had satisfied a few of criteria,
the place of a promising agent for the potential treatment of PVR still remains void.
Keywords: RPE,VEGF, IL-1β, BRB, proliferative vitreo retinopathy
Proliferative vitreoretinopathy (PVR) is specified as uncontrollable growth and

contraction of cellular membranes on both sides of the retinal surfaces and within the vitreous
cavity. Proliferative vitreoretinopathy can also be termed as, an abnormal wound healing and
scarring process (Miller et al., 1986; Wiedemann et al., 1988; Scott et al., 1989) that appears
with some retinal detachments, the most common cause of failed repair of a primary
rhegmatogenous retinal detachment (RRD) (Ryan et al., 1993). Initially, PVR is characterized
by the formation of epiretinal membrane formation by various kinds of cells which includes
retinal pigment epithelial cells (RPE), glial cells, fibroblast like cells, macrophage resembling
cells and also some other inflammatory cells. Retinal detachment and the associated vitreal
alterations are considered as some important factors in the development of PVR. (Raymond
et al., 1982; Martini 1992). The sequelae of proliferative diabetic retinopathy and break down
of blood retinal barrier leads to non-neoplastic intraocular proliferation, represented some of
the most important causes of blindness in developed countries (Ryan et al., 1985). Based on
the analysis of the data reported previously in the literature, PVR cannot be separated as a
specific clinical entity; rather, it can be considered as the end stage of a number of intraocular
diseases with various stimuli. Normally, ocular wound healing involves a tightly coordinated
series of events including recruitment and activation of inflammatory cells, release of
cytokines, activation and proliferation of ocular cells, secretion of extracellular matrix, tissue
remodeling and repair (Cordeiro et al., 1999; Campochiaro 2001). The mechanisms of
protracted wound healing process, as is found in PVR, are unknown but are presumed to
include either sustained or dysregulated growth factor responses.
2. Clinical Notes
Generally all the age groups are affected with PVR, but the incidence of PVR is more in
patients with age groups more than 50. PVR has an incidence rate of 5-10% of all retinal
detachments. Studies show that approximately 1600 new cases of PVR are being reported in
USA every year. Development in science and sophistication in treatment over the past
decades resulted in the evolution of several surgical techniques aimed at preventing and
bringing down the incidence rate of PVR. However, statistical analysis from 1990‘s to till
date suggests that the frequency of incidence of PVR remain almost the same (Gartry et al.,
1993; Girard et al.,1994; Duquesne et al., 1996; Heimann et al., 1996). Reports says that
though anatomical success rates of PVR is about 75-90%, only 40-50% of the eyes resulted
in better visual activity. When retinal detachment remains untreated there is 100% possibility
for the occurrence of PVR (Ivanisevic., 1997). In penetrating trauma PVR is estimated to
occur in between 10 and 45% of eyes with a mean incidence of approximately 25% (Morton
et al., 1978; Goffstein et al., 1982; Cardillo et al., 1997; Spiegel et al., 1997; Mittra et al
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Copyright © 2012. Nova Science Publishers, Inc. All rights reserved. May not be reproduced in any form without permission from the publisher, except fair uses permitted under Proliferative Vitreoretinopathy 159
1999; Wong et al., 1999; Colby 1999; Sobaci et al., 2000). PVR has become an important
complication in retinal translocation surgery. Incidences of 18–23% have been reported
where a 360 degree retinotomy has been undertaken to allow maximum foveal translocation
(Claes C, Bartz-Schmidt K, Eckardt C. Advanced Vitreoretinal Course, Antwerp, Belgium
16–18 March 2001). The frequency of PVR appears to be lower where an extensive
retinotomy is not performed (Pieramici et al., 2000). Treatment of PVR still remains a
challenge despite of the recent technical advances in medical field.
Because of the often disappointing results of PVR surgery its justification has been
questioned. Patients who had undergone the surgery, however, often report that they
considered the surgery to be worthwhile (Andenmatten et al., 1993; McCormack et al., 1994.
Schwartz et al., 1998) documented that over 50% of these eyes had vision threatening
pathology and approximately 75% of these had had rhegmatogenous events (retinal tears or
detachment). Forty-seven per cent of eyes with vision threatening pathology had a final
visual acuity of 20/50 or worse and over half of these had a final visual acuity of 20/250 or
less. Five per cent of the entire group ended with no light perception vision in the fellow eye.
Given the uncertain visual prognosis in the fellow‘s eye and patients reporting satisfaction
with surgery, it appears that at present PVR surgery remains justified.
3. Mysteries to be Revealed in PVR
Understanding and identification of high risk factors would result in the primary
prevention while strategies to control the biological process involved in proliferation and
retinal wound healing would help not only in controlling the pathogenesis of the disease but
also helps in managing other forms of posterior segment diseases such as ocular trauma and
age-related macular degeneration. The poor visual outcome of many PVR cases requires
further investigation to define both its causes and potential therapeutic measures.
4. Retinal Pigment Epithelium (RPE)
Blood retinal barrier is composed of various kinds of cells. Among them one is a kind of
pigmented cells called as retinal pigment epithelium (RPE) which is present as a
monolayer(Steinberg 1985;Bok 1993; Rizzolo 1997; Marmorstein 2001).To be precise RPE
cells are localized between the neural retina and the choriod of eye (Fig.1). The side which
faces the photoreceptor outer segments is the apical segment where long apical microvilli
forming a complex of close structural interaction with the surrounding light-sensitive outer
segments. The other side of the monolayer which faces the Bruch‘s membrane is the
basolateral membrane, which separates the RPE from fenestrated choriocapillary endothelial
layer. RPE cells play an pivotal role in the survival and normal functioning of photoreceptors
which includes synthesis and transportation of many substances, such as vitamin A
metabolites and phagocytosis of shed outer segments of rods and cones. In order to improve
the sharpness of the image and to reduce the scattering of light during vision, RPE cells and
differentiated melanocytes, produces a light absorbing pigment called melanin (Strauss
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2005). Normally in adult there will be no growth in RPE cells, but during some pathological
conditions, like proliferative vitreoretinopathy (PVR), RPE cells migrate into the vitreous due
to the blood-barrier breakdown and start to proliferate on the retinal layer and also within the
vitreous.
5. Factors Influencing PVR
Vitreous Modulating RPE Function
During the conditions of PVR, RPE cells are found to proliferate and present in the
vitreous of eye. Here they loose their typical columnar epithelial morphology and become
more fibroblast like structures. Similar conditions have been reported in in vitro conditions
too when the RPE cells are treated with vitreous humor. Therefore the addition of vitreous
and studying the expression of genes could give an idea of the types of proteins expressed
during the proliferation in eye.
Retinal detachment one of the late events in PVR, can be induced due to the contraction
of epiretinal membranes (ERMs). ERM cellular components include retinal pigment
epithelial (RPE) cells, glial cells, fibroblasts, and inflammatory cells (Charteris 1995). Since
during PVR the BRB breaks and RPE cells get into contact with vitreous, the investigation of
the effect of vitreous when treated with RPE cells becomes important (Fig. 2). Many of the
RPE cells in ERMs have have been found to undergo a morphologic transformation and show
a fibroblastic phenotype (Charteris 1995; Hiscott et al., 1999) . RPE cells are reported to play
a pivotal role in the development and contraction of ERMs during PVR (Hiscott et
al.,1999),and a better understanding of the changes in the phenotype of RPE cells in this
disease would be of great help in the design of therapeutic approaches for PVR. Vitreous is
made up of number of components and it was hard to determine the factors that are
responsible in the changes in the morphology. In a study, when RPE cells are exposed to
vitreous a sustained increase in the membrane-associated prostaglandin E synthase (mPGES)
expression and transient increase in cyclooxygenase (COX)-2. Both of these enzyme are
involed in conversion of arachidonic acid to prostaglandins and are induced by inflammatory
mediators. Although the experiments suggested that besides collagen, some other components
of vitreous are needed to induce mPGES and COX-2, but the experiment did not conclude
that collagen has no role to play. Therefore, there is every possibility of involvement of other
factors along with collagen or proteolysis of collagen by proteases induced by vitreous for the
release of components that is actively reponsible for the development and progression. The
increased production of components of the prostaglandin pathway in RPE cells exposed to

vitreous suggests a role for prostaglandins in the development of PVR (Parapuram et al.,
2003). Therefore, prostaglandins are also involved in PVR. Besides normal vitreous there are
other growth factors that are involved in the development of PVR. Many of them are detected
in the vitreous of the PVR patients.
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Figure 1. Normal eye anatomy and the location of RPE cells. ( Figure adopted and modified from strauss
2005).
Figure 2. Confluent primary RPE cells (A,B). Primary RPE cells treated with vitreous, for 24 hours (C) and
48 hours (D). (Karthikeyan and Gurunathan, Unpublished data).
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Serum Factors in PVR
Besides vitreous blood also act as an potential inflammatory factor and also a major
source of growth serum factors. Since during vitreous hemorrhage which is cosidered as an
important risk factor in the pathogenesis of PVR, there is every chance of growth factor
present in blood/serum reach the RPE cells (Scott 1989; Weller et al., 1990; Wiedemann
1992). Since patients with vitrous hemorrhages have high incidence of PVR, it can be very
well established that serum factors also influence the pathogenesis of PVR. During some
other complications like disbetic retinopathy (DR), vitreous hemorrhages are common and all
such conditions are not found to induce PVR. In DR too similar growth factors that are
implicated in PVR are also involved. (Schultz et al., 1991), but the development of PVR in
all such conditions is unlikely as retinal break should occur for the development of PVR,
which is missing in most cases of DR. Since serum contains all the growth factors the
exposure of RPE to serum is a potential risk factor for PVR.
6. Role of Cytokines and Growth Factors

Involved in PVR
It was speculated that the effect of vitrous in PVR through the prostaglandin pathway in
RPE cells might be because of the growth factors present in it. During PVR some of the
growth factors are found to be at increased levels. They include Transforming growth factor –
β (TGF- β), connective tissue growth factor (CTGF), Platelet derived growth factor (PDGF),
Hepatocyte growth factor (HGF), Vascular endothelial growth factor (VEGF), Interleukin – 1
β (IL-1β) etc., Along with various growth factors, epithelial mesenchymal transition and
retinal detachment leads progression of PVR (Fig 3) .
TGF- β is considered to be one of the important growth factors that is found at the
elevated conditions during PVR. TGF- β is a prime contributor of tissue fibrosis and the
growth factor is usually in inactivated state in vitreous at normal conditions.(Connor et al.,
1989; Pfeffer et al., 1994) Thrombospondin-1 is also likely to be an activator of TGF- β
within tissue (Bornstein et al., 2001; Koli et al., 2001) .
CTGF is one of the downstream mediator of TGF – β in fibroblasts, where it was found
to induce the proliferation and cell matrix deposition(Frazier et al., 1996). Therefore CTGF
may be the major mediator of retinal fibrosis. CTGF belongs to CCN (ctgf/cyr61/nov) family
of genes which participiate in the basic biological processes like wound healing and fibrosis.
Although previously CTGF has not been implicated in PVR, recent reports suggests a strong
role of CTGF in PVR (Suzuma et al., 2000; Hinton et al., 2002).
HGF exerts pleiotropic biological functions as mitogenic, motogenic, morphogenic, and
angiogenic factors in the target cells (Rosen et al., 1990; Uhargava et al., 1992). It primarily
exhibits its actions by the activation of the HGF receptor (HGFR), which is a high-affinity
tyrosine kinase receptor (Park et al., 1987). The HGFR is reported to be expressed in many of
the cell types, especially the cells of epithelial origin (Lashkari et al., 1999). HGFR is also
found to be strongly expressed in ERMs during PVR. Therefore the expression of HGFR
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abundantly in RPE cells suggest that HGF may play a major role in normal RPE development
in humans and during the pathogenesis of diseases like PVR.
Figure 3. Events in PVR. Inset figure, Retinal detachment, epithelial mesenchymal transition and fibrosis
formation ( Figure adopted and modified from Saika et al., 2009).
Interleukin-1-beta (IL-1β) is shown to be involved in the acute-phase of PVR. Many cell

types chemotactically respond to IL-1β like RPE cells monocytes, and neutrophils in vitro,
and proliferative responses are stimulated by it in many kinds of tissues (Dinarello et al.,
1986; Kirchhof et al., 1989). Many reports previously has shown that IL-1β is produced by
cells in eye including corneal cells and RPE cells (Shams et al., 1989; Alexander et al.,1990)
and had been detected at hihgher levels in ocular fluids during PVR, trauma and
inflammation (Grabner et al., 1983; Davis et al., 1988). Moreover, when an in vitro model
was tested for retinal membrane contraction as found in PVR, the process was modulated by
IL-1 β (Hunt et al., 1993).
In addition, many reserchers have studied the secretion of VEGF and RPE cells have
been found to express VEGF receptor too. VEGF is primarily identified as Vascular
permeability factor and considered as a cell specific mitogen to endothelial cells. But later it
was also found that VEGF also induces proliferation in RPE cells also (Guerrin et al., 1995;
Chen et al., 1997; Schneeberger et al., 1997). Previously during many ocular complications,
VEGF is found to be upregulated and involved in the breakdown of blood-retinal-barrier.
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Since BRB breakdown is the major factor for the development of PVR, VEGF also has some
role to play in development of PVR.
PDGF is considered as one of the important growth factor involved in the pathogenesis
of PVR. PDGF is a dimer and three different isoforms are available for it viz. PDGF-AA,
PDGF-AB and PDGF-BB. All the three isoforms have different functions but are mainly
involved in the pathogenesis of PVR (Andrews et al., 1999). PDGF induces proliferation in
RPE cells and also permeability. PDGF is considered to be the main factor for the
pathogenesis of PVR as PDGF is a potent migration inducing factor for RPEs (Campochiaro
et al., 1985).
7. Molecular Mechanism Involved in the

Development of PVR
Any potential treatment to any kind of disease requires a thorough knowledge of the
signaling molecules involved inside the cells besides the regulators outside the cell.
Therefore, studying the signaling mechanism involved in the progression of PVR becomes
very much crucial. There are significant evidences that several pathways are upregulated in
the inflammatory phase of PVR. For. E.g. Rho-kinase pathway was found to be involved in
Bovine RPE cells when the cells were induced by TGF-β in collagen gel contraction model
(Miura et al., 2006). Whereas, IL-1β and TGF- β induced collagen gel contraction by acting
through protein kinase and expression of Integrin-α2 in human RPE cells(Hunt et al., 1993,
1994; Kupper and Ferguson, 1993; Raymond and Thompson, 1990). When BRPEs were
treated with tranilast, TGF-β secretion was blocked and collagen gel contraction was very
much reduced (Yasukawa et al., 2002). In another model, when ARPE cell lines were used
and stimulated with PDGF, MAP kinase and PI3K pathways were found to be activated
(Bando et al., 2006). This is due to the enhanced expression of integrin α1 and α2, that is
mediated by MAP kinase. Moreover, a seperate mechanism was involved by PI3K for the
regulation of contraction of gel (Bando et al., 2006; Yamakawa et al., 1989). The
fundamental to the traction phase is the contration and the interaction between the RPE cells
and collagen matric plays a major role. There are many varities of integrin isoforms are
available which play a pivotal role in the interaction resulting in the integrin engagement,
activation and activation of various signaling molecules (Xia et al., 2004; Moulin and
Plamondon, 2002; Langholz et al., 1995). Integrin α1β1 and α2β1, the collagen binding
isoforms have been shown to be involved in the binding of RPE cells with the collagen
matrix and its contraction (Bando et al., 2006; Moulin and Plamondon, 2002; Langholz et al.,
1995; Carver et al., 1995; Cooke et al., 2000; Kieffer et al., 1995; Robbins et al.,1994; Zhang
et al., 2006).
When the interaction with collagen through integrin was inhibited the collagen gel
contraction was decreased. During treating the collagen gels by crovidisin (a collagen
binding protein derived from snake) resulted in the reduced BRPEs mediated collagen matrix
contraction where crovidisin is an anti-adhesion compound (Yang et al., 1997). Alternatively,
the induction of contraction vary with the type of cells used and also the factors used for the
treatment.
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One of the most important pathways that have been reported is the combination of PI3K,
PLCγ and Src kinase which are all important for PDGF induced chemotaxis (Rosenkranz ET
AL., 1999). The Src family kinases are one among the cytosolic tyrosine kinases and PLC γ
is responsible for the generation of diacylglycerol and inositol triphosphate from
phosphatidylinositol-4,5-bis phosphate (Heldin et al., 1998). Both the generated molecules
are potent second messengers (Rhee et al., 1989). PI3K enzyme family have the potential to
phosphorylate phospholipids which further activates several signaling cascades (Rameh et
al.,1999).
There are several signaling molecules which have been reported to be activated by
several grwoth factors in PVR. Instead of blocking the effector molecules if the numero uno
of the signaling cascades is blocked then it can bring an full stop for developing PVR. PI3K
pathway is considered to be the most important pathway and as in every disorder cross talk
may also be involved. Src kinase is also a molecule that involves in the activation of PI3K
and blocking Src kinase is also a viable strategy for the treatment of PVR.
8. Therapeutic Agents and

Antiproliferative Molecules
Based on the above said mechanisms some anti-proliferative and anti-inflammatory

agents are tried for the treatment that block the activation of the various intracellular
signaling molecules. Many kinds of drugs that were used are aclacinomycin A, daunomycin,
fluorouracil, retinoic acid, paclitaxel, camptothecin, corticosteroid, genistein and
daunorubicin. These drugs were tested for their potency to hinder the progression of traction
retinal detachment in various models of PVR. But most of the drugs were found to have very
limited appications for the patients as they are highly toxic, lack of efficiency when used for
the treatment which required multiple injections and vitreous infusion.
Recent development in vitrectomy along with the tamponade like Silicone Oil (SiO) is
used for the treatment of most severe and end stage PVR cases has enhanced the success rates
of surgery. But, no evidence has been further obtained that SiO blocks PVR (Fastenberg et
al., 1983). Despite meticulous removal of membrane by surgical means and usage of SiO as
tamponade, failure occured in larger propotion of cases due to incomplete removal of
membranes formed and continous regrowth of membrane. Therefore, in order to address this
fearsome vision affecting problem various experiments have been conducted with various
anti-proliferative separately or with combination of vitreo-retinal surgery (Tano et al., 1981;
Stern et al.,1983; Blumenkranz et al., 1984; Daniels et al., 1990; Wiedemann et al., 1991;
Kirmani et al., 1993). But since these agents were toxic at slightly higher concentrations and
safety margins being very narrow it was very hard for the use of treatment. Moreover, these
agents used were water soluable and difficulty arised when used with SiO. Here Retinoic acid
(RA) was the choice since the agent was soluable in SiO and it had anti-proliferative effect in
various cell types especially epithelial cells (Lotan 1980). Previous studies have shown that
RA has a potential anti-proliferative effect on RPE cells (Doyle et al., 1992). When RA was
used along with SiO it was shown to have dose-dependent effect on the prevention of retinal
detachment (Manzanas et al.,1990).
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Usually, the surgery for PVR succeeds when the break closes and releiving the the
traction of the retina by any means (including the removal if epiretinal membrane formation).
This is followed in order to minimize the recurrence of traction and to prevent the recurrence
of PVR. Although several agents have been suggested for the treatment, none of them have
been allowed to use for day-to-day treatment as they are toxic to retina. Some of the agents
that are suggested for the treatments are 5-fluorouracil (5FU), 2‘-Benzoyl-
oxycinnamaldehyde, Minoxidil etc., a mixture of 5-FU and low molecular weight heparin
(LMWH) is one of the few agents used for humans (Asaria et al., 2001; Scheer et al., 2005;
Garcia et al., 2007) . Addition of 5-FU and LMWH to the infusion fluid may result in a
favorable outcome in the patients with severe PVR (Gurunadh et al., 2010). 2‘-Benzoyl-
oxycinnamaldehyde (BCA) is a derivative of 2‘-hydroxycinnamaldehyde which was
originally isolated from the stem bark of Cinnamomum cassia Blume, and inhibits farnesyl-
protein transferase (Kwon et al., 1996; Lee et al.,1999). BCA has been shown to inhibit
growth of various tumor cells in vitro and in vivo. In the present study, we investigated
whether BCA can induce apoptosis in human RPE (hRPE) cells and effectively inhibit
proliferation and migration of hRPE cells in vitro.
Minoxidil, a piperidinopyrimicline derivative, is a potent anti-hypertensive drug, that has
been proven to reverse male pattern baldness. Previously, minoxidil was found to inhibit the
proliferation of human retinal pigment epithelial (RPE) cells in culture (Handa et al., 1993).
Minoxidil also suppresses the activity of lysyl hydroxylase, an enzyme essential for the
formation of stable interrnolecular collagen cross-links.1 Since RPE cells are involved in the
pathophysiology of proliferative vitreoretinopathy (PVR) which is characterized by unwanted
cell proliferation and fibrous tissue formation (Glaser et al., 1989), treating PVR with
Minoxidil will prove to be fruitful. Amidst all these pros, the cytotoxicity of Minoxidil is a
potential impedance in this thereapy. Interestingly, structural analogues of Minoxidil such as
its hydroxy derivatives (formed by the action of lysyl hydroxylase) have been reported to
posses more potent anti-angiogenic effect than Minoxidil itself and also was found to posses
less cytotoxicity than Minoxidil. The structural modification arising due to hydroxylation is
found to increase the efficacy and efficiency of the Minoxidil (Handa et al., 1994).
Although these drugs glimmer a positive ray of hope in PVR treatment, various side
effects associated with their application proves to be less encouraging. PVR, which is
characterized by the release of numerous growth factors, still remains elusive when these
drugs are applied since they do not eliminate the root cause of PVR. This is less encouraging
than surgical methods, which by itself is unreliable. This forces us to throw the limelight in
various other alternative therepeutic strategies, which could prove to be a panacea for PVR
and its complications. One such promising spectrum of therepeutics is the employment of
biologically synthesized Nanoparticles in the treatment of PVR.
Gold Nanoparticles – Novel Therapeutic Molecule for the Treatment of PVR
Nano refers to anything at the size 10-9 of SI units. Recently, the technology at this scale
has gained significance due to the potential applications in several fields. Nanoparticles
(NPs) can be used to detect and enhance various signals in sensors, added in the ink to
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improve the quality, in the imaging to get the precise pictures, in the couture for new colours
to the cloths etc. Medically NPs have great number of applications. They are shown to posses
potential antimicrobial activity, anticancerous activity (silver nanoparticles), for drug delivery
(gold nanoparticles) etc., NPs are highly advantageous since they can travel to any part of the
body without any problem because the capillaries are in the range of microns. Therefore NPs
can easily reach any part of the human body without any occlusion. If larger particles are
used there is every chance of occlusion. Although there are several NPs, many are not
suitable for applications as they are highly toxic to the cells. So, for treatment researchers are
trying to use non-toxic NPs (Pan et al., 2007). Gold nanoparticles (AuNPs) are one such non-
toxic nanoparticles which are suggested for treatment and delivering the drug in various
diseases (Mukherjee et al., 2005). But here also a problem arises, eventhough AuNPs are
suggested to be safe, they are also found to be toxic at smaller sizes that is in the range of 1-
20nm. Therefore, great care has to be taken while preparation of NPs for treatment.
There are several methods that are used for the preparation of NPs. But they can be
broadly classified into physical, chemical and Biological methods. But the latter two methods
are often followed as they produce nearly uniform size particles at ease. In both the chemical
and biological method, Gold ion (Au+) is converted to (Au0) state by an reducing agent and
leads to the formation of crystals. But chemical methods witnesses the use of chemicals
which are toxic both to the humans and to the environment as they have to be disposed to the
environment with great care. Moreover, a stabilizer has to be added in oder to prevent the
NPs from agglomeration. But all these steps can be prevented in biological methods and it is
relatively easy for the synthesis of NPs at desired sizes (Kalishwarala et al., 2010).
Figure 4. Gold nanoparticles (Au-NPs) inhibits VEGF and Il-1β induced BRPE cell spreading (Karthikeyan
et al., 2010).
Recently a report has shown that AuNPs effectively inhibited the proliferation of BRPE
cells induced by VEGF and IL-1β in vitro at a maximum safe dose of 300nM. AuNPs also
prevented the migration and cell spreading which are the two important events that are
involved in the proliferation and epiretinal membrane formation. Therefore AuNPs can be
effectively used for the treatment of PVR (Karthikeyan et al., 2010). As mentioned above,
study of the signaling mechanism by which AuNPs inhibit the proliferation would give a
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clear idea of the potential of the NPs. Here AuNPs are shown to inhibit the proliferation,
migration and cell spreading (Fig. 4) by blocking the Src pathway.
Owing to all these potential anti-proliferative effects associated with the Gold
nanoparticles, it is wise to say that Nanoparticle treatment for PVR certainly holds an
attractive edge over other approches like surgery and therepeutical application of drugs.
Since Gold nanoparticles were found capable of inhibiting the proliferative effects of
molecules like VEGF and IL-1β, it would be interesting to analyse their effects over
molecules like PDGF, TGFβ in which researches are still underway. If the outcome such
studies are motivating, it would certainly pave a competent remedy in the treatment of PVR.
Future Insights
Considering all the available modes of treatment, the current scenario requires even more
limelight and focus for effective treatment of PVR. Since the complications associated with
PVR vary from person to person, it is difficult to zero one mode of approach as the prime
solution to PVR. Strategies should be framed in such a way that the cons associated with one
mode of approach is counter-balnced by the pros of the other. Having said this, researches are
still on the verge of designing a combination therapy, that contains the essence of all
advantages associated with each treatments. This will eventually lead us to a world where
treatment for vision impairment becomes a threatfree remedy.When such researches are given
an impetus, it will certainly foster advancement in the treatment of PVR taking a big leap
from conventional methods of treatments which involves much complications. On a
concluding note, it is worthy enough to say that the cure for PVR is not very far.
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Chapter VIII
Neuropathology of Cultured Retinas:

Degenerative Events
and Rescue Paradigms
Camilla Mohlin1, Ingela Liljekvist-Soltic1, Jenny Olofsson1

and Kjell Johansson*1,2
1
School Natural Sciences, Linnéus University, Kalmar, Sweden
2
Department of Clinical Medicine, School of Health and Medical Sciences,
Örebro University, Sweden
Abstract
This review examines the potential of retinal explant cultures as a model system for
neuroprotective strategies aiming at the treatment of retinal diseases. Early studies on
explanted retinas were initially concentrated on developmental and differentiation events
of different cell subtypes. A growing number of studies have examined different
neuroprotective strategies including growth factor supplementation as well as coculture
with progenitor and stem cells. Most studies have focused on neuroprotection of
photoreceptors, but protection of axotomized ganglion cells have also been explored .
Both cell types are injured during the culture procedure, and show an injury-sequence
that is morphologically comparable to the ones described in other experimental models
and human retinal dystrophies. Injury-related responses include photoreceptor sprouting
as well as neuronal remodeling, glial cell activation, and neuroinflammatory responses.

In general, the explant models offer several advantages: 1) degenerative pathways and
remodeling events are rapid, 2) different neuroprotective strategies are possible and can
be, compared to in vivo experiments, rapidly evaluated, 3) a controlled in vitro
environment, and 4) an easy experimental procedure that is cost-effective.
* Corresponding author: Kjell Johansson, PhD , Department of Clinical Medicine, Örebro University, SE-701 82
Örebro, Sweden. , E-mail:kjell.johansson@oru.se
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Copyright © 2012. Nova Science Publishers, Inc. All rights reserved. May not be reproduced in any form without permission from the publisher, except fair uses permitted under 178 Camilla Mohlin, Ingela Liljekvist-Soltic, Jenny Olofsson et al.
Introduction
The mammalian retina is a complex, layered structure and an appealing model in

morphological, developmental, and physiological research on the central nervous system. As
the retina is the most accessible part of the CNS, cell and tissue transplantation as well as
neuroprotective issues have been addressed in numerous studies. Most of the retinal cell
types are known, including their development, morphology, and in some cases, also their
physiology. The flow of information in the form of electrical impulses comes from the
sensory photoreceptor cells, interneuron processing and moves towards the retinal ganglion
cells, which are the projection neurons of the retina. Information about the visual world is
conveyed in electrical impulses in the optic nerve to the brain for perception. In retinal
disorders like retinitis pigmentosa, rod photoreceptors are the first to degenerate followed by
the death of cones, which eventually results in severe visual impairment.
The retina belongs to the nervous system and is afflicted by several degenerative
diseases. As mammalian retinal cells have no capacity to regenerate, the loss of cells results
in visual impairment and/or blindness. Subsets of retinal cells (e.g. photoreceptors and
ganglion cells) degenerate, and the loss of photoreceptors in particular results in neuronal and
glial remodeling events that eventually destroy the retinal organization (Jones and Marc,
2005; Fariss et al., 2000). Increased glial remodeling in injured retina not only threatens
retinal layering, but may also impair the outcome of cell replacement strategies.
Organotypic culturing of mammalian retina have become an experimental approach that
allows manipulations under controlled conditions. In contrast to single cell cultures of retinal
cells, most cell-cell contacts and interactions among different cell types are retained in
explants. Recent studies show considerable injury-related responses in sensory, neuronal, and
glial cell populations of the explanted retina. For instance, injuries are induced during
explantation as the ganglion cells are axotomized and the photoreceptors are separated from
the pigment epithelium. However, these features and the photoreceptor cells lose their
function and morphological integrity in primary cell cultures, indicating a clear advantage of
using intact retina for experimental purposes.
Initial studies on cultured retinas were focused on developmental issues and techniques
that improved tissue handling in vitro. Later explant studies evaluated potential retinal
protection strategies using growth factor supplementation to cultured retinas with mutated
photoreceptors (Caffé et al., 2001; Ogilvie et al., 1999). More recent studies have used
explants as a transplantation model system to study interactions between retinal tissue and
progenitor/stem cells (Englund-Johansson et al., 2010; Johnson et al., 2009; Johnson and
Martin, 2008; Liljekvist-Larsson and Johansson, 2005; 2007). As in vivo stem cell
transplantations are evaluated after 1-6 months and require a substantial amount of animals,
similar interactions between retinal tissue and progenitor/stem cells can rapidly be done
under controlled forms of in vitro. Interactions between explants and grafted cells can usually
be evaluated within a week, suggesting that various efficacy improvements of progenitor
/stem cell grafting are less time consuming. A variety of retinal disease and injury models are
known and host-graft interactions in pre-clinical studies of retinal diseases like age-related
macular degeneration, glaucoma, retinitis pigmentosa, and retinal detachment can be
conducted.
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Copyright © 2012. Nova Science Publishers, Inc. All rights reserved. May not be reproduced in any form without permission from the publisher, except fair uses permitted under Neuropathology of Cultured Retinas: Degenerative Events and Rescue Paradigms 179
The present study examines progenitor/stem cell approaches in retinal explant models,
particularly in the context of neuroprotection. Both ganglion cells and photoreceptor cells are
considered in this review since they show neurodegenerative conditions that have a poor
diagnosis and pose a major threat to vision. Secondary injuries following photoreceptor
degeneration will also be reviewed.
Degenerative Events and

Neuroprotective Strategies
The neuropathology of explanted retinas starts shortly after explantation and proceeds at
least during 5 days of culture. Depending on culture medium composition (Johnson and
Martin, 2008) and/or culture set-up (Kaempf et al., 2008; Kobuch et al., 2008), longer culture
periods are possible. Juvenile animals are used in most studies for screening of
neuroprotective substances and drugs. Retinas from older animals may be used, but they have
rather low viability under normal culture conditions. Although photoreceptor and ganglion
cells degenerate in the explants, electrophysiological data suggests the possibility of short-
term functional activity in various cell types (Koizumi et al., 2007).
Neuroprotective strategies of photoreceptors based on various growth factors have been
evaluated in several animal models in vivo and are beyond the scope of this review. Although
photoreceptor degeneration still remains unclear, induced or inherited death pathways in vitro
parallel those described in vivo. In vitro studies may be concurrent with data that reveals
death pathways, both in photoreceptors and ganglion cells, and demonstrates how cell death
can be limited.
Death of Photoreceptor Cells In Vitro
Rat and mouse retinas are rod dominated with a low percentage of cone photoreceptors.
Both photoreceptor types develop in explants (Arango-Gonzalez et al., 2010; Liljekvist-
Larsson et al., 2003; Mack et al., 2003; Rohrer and Ogilvie, 2003), although the experimental
focus is on rods and their survival. Shortly after explantation, rod photoreceptor outer
segments start to degenerate. Despite this, photoreceptors are viable and shuttle transduction
proteins in a light-dependent manner (Reidel et al., 2006). As the outer segments degenerate,
rhodopsin accumulates in the membrane throughout the rod photoreceptor (Liljekvist-Larsson
et al., 2003). However, photoreceptors in explants have rudimentary outer segments, but
retain some physiological properites and respond to illumination (Bandyopadhyay and
Rorher, 2010). Judging from immunohistochemical (Johansson and Ehinger, 2005) and
transmission electron microscopic data (Pinzón-Duarte et al., 2000), rod and cone photo-
receptors establish synaptic contacts in the outer plexiform.
Shortly after explantation, caspase-3 and -12 immunolabeled photoreceptors appear in
the outer nuclear layer (ONL). The density of caspase immunoreactive photoreceptors peaks
between 48-72 h in vitro, which results in a considerable decreases of ONL width. The
caspase-12-dependent degeneration shows a correlation to aberrant rhodopsin accumulation
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and ER-stress (Mohlin and Johansson, 2011), while the upstream trigger for caspase-3-
dependent degeneration is undetermined. In vitro, caspase-3 activation may occur through
hypoxia and/or serum-starvation. The latter stresses both cone and rod photoreceptors in
explanted retinas and the photoreceptors respond by producing reactive oxygen species
(Bhatt et al., 2010). Oxidative stress contributes to photoreceptor death in vitro and in vivo
(see Sanz et al., 2007; Carmody and Cotter, 2000), but if reactive oxygen species also affects
proteins and elicits a stress response has yet to be determined.
Endoplasmic reticulum stress (er-stress) has recently been associated with photoreceptor
apoptosis in animal models with different death mechanisms. Accumulation of mutated (Lin
et al., 2007) and normal (Mohlin and Johansson, 2011) rhodopsin as well as abnormal Ca2+
influx (Yang et a., 2007) triggers er-stress. ER-stress in photoreceptors is an unfolded protein
response that involves signaling via the chaperone Grp78/BiP and different downstream
pathways. Virus-based chaperone therapy successfully promotes the survival of
photoreceptors with mutated rhodopsin (Gorbatyuk et al., 2010), and retinal explants can be a
good model for evaluation of chaperone-therapies in vitro.
Table 1. Neuroprotection by growth factors in vitro. Quantitative assessment

of neuroprotective effects are either by measurements of the outer nuclear
layer width or by cell counts of cells labeled by a ¨death marker¨
Animal Factors Cell type Marker Reference

Normal rat BDNF/NT4/citicoline RGC TUNEL/caspase-3/-9 Oshitari et al., 2010
Normal rat VEGF RGC TUNEL Nishijima et al., 2007
rd1 mouse BDNF+CNTF photoreceptors ONL width Ogilvie et al., 1999
BDNF+CNTF photoreceptors ONL width Caffé et al., 2001
BDNF+CNTF photoreceptors ONL width/TUNEL Azadi et al., 2007
LEDGF photoreceptors ONL width Ahuja et al., 2001
Normal rat BDNF photoreceptors TUNEL Pinzón-Duarte et al., 2004
Normal rat VEGF+TIMP photoreceptors cc-3/TUNEL Liljekvist-Soltic et al., 2008
Normal rat Several photoreceptors calpain/cc-3/TUNEL Englund-Johansson et al., 2010
Abbreviations: BDNF brain-derived neurotrophic factor, cc-3 cleaved caspase-3, CNTF ciliary
neurotrophic factor, LEDGF lens epithelium-derived growth factor, NT-4 neurotrophin-4, RGC
retinal ganglion cell, TIMP tissue inhibitor of matrix metalloproteinase, VEGF vascular endothelial
growth factor.
Neuroprotection of photoreceptors in different in vitro model systems have been focused

on growth factor supplementation, a strategy most effective when a combination of growth
factors are used (see table 1 for details). For instance, BDNF and CNTF together effectively
limit photoreceptor degeneration in the rd1 mouse (Ogilvie et al., 1999). In normal rat retina,
a combination of VEGF and TIMP limits caspase-3-dependent photoreceptor degeneration
(Liljekvist-Soltic et al., 2008). Even though substantial protection occurs, other death
markers indicate the presence of subpopulations of unprotected photoreceptors (Liljekvist-
Soltic et al., 2008). Reduction of photoreceptors in the ONL may also lead to hyperoxia and
an oxidative damaging environment (Komeima et al., 2006; Yu et al., 2004). This suggests
that other mechanisms, not only apoptotic, leading to photoreceptor degeneration are yet to
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be revealed. Successive treatment strategies may thus be multifactorial, for example

consisting of a combination of growth factors and antioxidants.
As photoreceptor degeneration is rapid in vitro, interactions between an employed rescue
paradigm and its effect on death pathways can be studied in detail. Neuroprotective effects
have mostly been given by quantification of limited photoreceptor death; counts of caspase-3,
caspase-12, calpain, or TUNEL labeled cells. Irrespective of death executioner,
neuroprotective effects are most likely through the activation of specific receptors. The
receptors may be localized to separate cell types and may show a temporal expression
pattern, and once activated elicit complex tissue responses. CNTF and BDNF increased not
only intracellular pathways for survival but also FGF2 mRNA expression in explanted mouse
retinas (Azadi et al., 2007). This suggests that neuroprotecitve factors may activate an
endogenous repair mechanism and prolong photoreceptor survival. Application of GDNF to
rd1 retinal explants is neuroprotective, perhaps via a glutamate transporter (GLAST) in
Müller cells (Delyfer et al., 2005). Thus, neuroprotection via Müller cells may be pivotal in
injured in vitro retinas as well (see below).
Figure 1. Schematic drawing illustrating the explant culture procedure, retinal tissue and retinal cell
morphology pre- and post-culturing. A) The optic nerve is cut, followed by removal of the sclera and
vitreous body and dissection of the retina. The retinal tissue is explanted without the retinal pigment
epithelium onto a culture plate insert and cultured with the photoreceptors facing down on the insert
membrane. In coculture experiments, the retina is cultured with or without human neuronal progenitor cells
that are seeded at the bottom of the culture dish. B) Normal retinal tissue (control) is usually thicker
compared to cultured retina, but the latter display normal layering. C-E) Schematic comparisons between
ganglion cell, Müller cell and rod photoreceptor, respectively, in normal and cultured retina. C) Ganglion
cells degenerate rapidly via apoptosis after optic nerve transaction. D) Compared to normal Müller, the
activated Müller cell are hypertrophic with outgrowth of cell processes. E) A rod photoreceptor before and
after culturing. Most obvious is the degeneration of the outer segment.
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Death of Ganglion Cells In Vitro
Rescue of injured retinal ganglion cells (RGC) and the optic nerve is desirable as a
treatment of sight-impairing conditions like glaucoma and diabetic retinopathy. Apoptotic
death mechanisms have been suggested as a cause of RGC death, and are most likely
triggered by elevated intraocular pressure, oxidative stress, ischemic injuries, and high
glucose. Protection of RGC in vitro is possible, and is focused not only on increased survival
but also on their capability to regenerate axons. Similar to photoreceptor protection,
limitations of RGC death in vitro have mostly been based on growth factor/drug
supplementation. A generality is that explantation results in a rapid and massive apoptotic
death of RGC.
During eunucleation of the eye, the ganglion cells are axotomized and deprived of their
post-synaptic targets in the lateral geniculate nucleus. Additional experimental approaches
include the addition of glutamate to the explant and study excitotoxicity and its
pharmacological treatment (Ruscher et al., 2007; Xin et al., 2007). Immunohistochemical
analysis shows that RGC death following explantation is an apoptotic process, executed by
caspase-3 (Engelsberg et al., 2004). Caspase-12 and other markers for er-stress are expressed
in RGC in response to experimental glaucoma and diabetic retinopathy (Ha et al., 2011;
Biermann et al., 2010), and may also contribute to cell death in vitro.
Neuroprotective strategies to limit caspase-dependent death pathways in RGC in vitro are
possible and have been addressed in some studies. The role of VEGF as a neuroprotectant for
photoreceptor cells is known and this growth factor inhibits RGC apoptosis in postnatal
explants (Nishijima et al., 2007). This effect appears to be temporally restricted, as VEGF has
no effect on RGC degeneration in adult explants exposed to high glucose (Oshitari et al.,
2010). Both BDNF and NT-4 had significant effects on apoptotic pathways in RGC exposed
to high glucose. A recently suggested RGC neuroprotective factor is interleukin-2, which is
receptor-mediated and features active intracellular survival pathways (Marra et al., 2011).
Remodeling Events
Neural remodeling in vitro can be detected shortly after explantation and are most
obvious in the plexiform layers where synaptic contacts are established. Rod photoreceptors
react to explantation and enter a degenerative process, which also includes sprouting and/or
retraction of axonal terminals. The outcome of this is that synaptic contacts between
photoreceptors and interneurons are lost and functions are also lost. Formation of ectopic
neuropil areas also occurs. Similar phenomena are known from human retinitis pigmentosa
retinas and the end result is a morphologically and functionally corrupt retina (Jones and
Marc, 2005).
In vitro, the remodeling process starts within 24 hours and thereafter the secondary
interneurons start to display signs of plastic changes. The explanted retina is layered, but
horizonal cells as well as rod and cone bipolar cells are affected and there is a loss of these
cells (Johansson and Ehinger, 2005; Mohlin and Johansson, unpublished observarions).
Ectopic neuropil areas are formed and are invaded by glial and neural processes (Johansson
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and Ehinger, 2005). Rosette formation increases with time in vitro, suggesting that tractional
forces are induced. Thus, remodeling events in vitro show on a morphological basis several
similarites to injuries in retinitis pigmentosa and detached retinas. Neural remodeling results
in a functionally corrupt retina that appears to be initiated by the loss of photoreceptors.
Further studies have to show that increased photoreceptor survival limits neural remodeling.
Glial Cell Responses
Müller cells are the radial glia of the retina, extending between the inner and outer
limiting membrane and hence present in all retinal layers. Müller cells not only maintain
ionic and osmotic homeostasis in normal retina, but also protect retinal neurons after injury.
Neuroprotection by Müller cells is most likely accomplished by the secretion of growth
factors in an endogenous repair mechanism (Harada et al., 2002; Wahlin et al., 2000).
Receptors for different neuroprotective agents are indeed expressed on the Müller cells.
During longer injuries, activated Müller cells may alter their protective function and support
tissue degeneration as gliotic tissue develops. Early signs of an activated state are the
upregulation of glial fibrillary acidc protein (GFAP), hypertrophy, and altered expression of
inwardly rectifying potassium channels (Kir). Processes from activated Müller cells
eventually form epi- and subretinal membranes, surrounding the injured retina.
Explantation is accompanied with gliotic events including an activation of Müller cells
and microglia. Similar to injured in vivo retina, there is an increased accumulation of GFAP
and growth of cell processes into the plexiform layers. GFAP expression is usually
substantial after 5 days of culture and subretinal protrusion of cell processes are observed at
the same time (Johansson and Ehinger, 2005; Winkler et al., 2002). Alterations of Kir-
channels have also been detected, suggesting that explants can be used to study gliotic events
(Kuhrt el al., 2008). For instance, Müller cell activation in vitro increases in the presence of a
blood-derived mononuclear fraction (Fernandez-Bueno et al., 2008).
Microglia are resident cells in the retina and are activated in response to various injuries.
Activated microglia cleans the tissue from cell debris derived from degenerated cells, and the
activated microglia can be detected with antibodies against the lysomal enzyme (ED-1)
and/or by lectin staining. Microglia are rapidly activated in vitro and are, after a few days,
found in all retinal layers (Engelsberg et al., 2004, Mertsch et al., 2001).
The initial protective role of Müller cells and/or microglia in vitro has not yet been
thoroughly evaluated, although these cells may play a pivotal role in degeneration. In a
recent study, Delyfer and colleagues (2005) evaluated neuroprotection by glial cell line-
derived neurotrophic factors (GDNF) on explanted rd1 retinas, and observed effects on
photoreceptors via the Müller cells. Other data shows that various cytokines are produced in
the explants, including TNF-, IL-6, and MCP-1 (Mertsch et al., 2001). The former two may
in turn induce suppressors of cytokine-signaling proteins (SOCS), a group of proteins that
can be induced in explants by IFN- and/or insulin (Liu et al., 2008). Interestingly, SOCS
proteins initially reduce neuroinflammation and promote neuroprotection, but later inhibit the
action of neurotrophic factors. Other immune molecules with neuroprotective capabilities are
IL-4, which protects rod photoreceptors directly via IL-4 receptors (Adáo-Novaes et al.,
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2009). In addition, Müller cell activation and interleukin-8 production increases in retinas co-
cultured with choroidal melanoma cells (Enzmann et al., 2000). This interaction may be
responsible for retinal detachment that is associated with choroidal tumors. As glial cell
activation in vitro is very rapid as compared to in vivo, the shift from a neuroprotective to
degenerative function in Müller cells and microglia may be identified on a molecular basis.
Progenitor Cell-Based Neuroprotection
From previous studies on animal models with photoreceptor degeneration, it became

evident that transplanted progenitor and stem cells provided support to photoreceptors and
limited degeneration. The neuroprotective effect is pronounced in the vicinity of the
transplanted cells, suggesting that protection was accomplished by diffusible factors. A later
study confirmed that growth factors are derived from the progenitor cells and provided
neuroprotection to photoreceptor cells (Gamm et al., 2007).
Progenitor and stem cell-derived neuroprotections have been addressed and analyzed
using retinal explant cultures. The set-up is based on retinal tissue and various progenitor or
stem cells that are cocultured under normal in vitro conditions. Two slightly different
approaches have so far been used; progenitor/stem cells are either used as a feeder layer or
directly grafted onto the explant. Irrespective of approach, explantation is rather simple and
rapid and rescue effects can be evaluated after 5-7 days of culture. As the feeder layer is
separated from the tissue, diffusible factors in the medium can be analyzed.
In a series of experiments, progenitor/stem cells have been shown to significantly limit
photoreceptor degeneration cocultured mouse and rat retinas (Englund-Johansson et al.,
2010; Liljekvist-Soltic et al., 2008). Both human and rat progenitor cells of neuronal origin
showed a neuroprotective capacity, and biochemical screening suggested that a multitude of
growth factors were involved. An even more interesting finding was that the progenitor/stem
cells reduced calpain-, caspase-3-, and caspase-12-dependent photoreceptor degeneration.
Further studies have to evaluate the most potent combination of beneficial factors as well as
their interactions with target receptors and cells in the injured retina.
Other Approaches
Cocultures consisting of progenitor/stem cells and retinal explants have been used as
models for cell transplantation studies, evaluating the migration/integration of transplanted
cells into a tissue. This approach is used to study and improve techniques that enhance
functional integration of transplanted cells. Progenitor/stem cells with different origins and
with various pretreatments to enhance the functional integration and/or differentiation into
retinal cell fates have been tested. Irrespective of origin, the grafted cells migrated into
several layers of the explanted retina. In general, successful integration increases when
dissociated cells are used and migration of retinal progenitor cells clustered in neurospheres
is very limited (Liljekvist-Larsson and Johansson, 2007; 2005). Integration of Müller cell
progenitors and mesenchymal stem cells have also been studied (Johnson and Martin, 2008).
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Most encouraging for further studies is that the grafted cells behaved similarly in vivo and in
vitro.
Hippocampus-derived stem cells pretreated with retinoic acid for differentiation into
neurons integrated well, but only a few adopted a retinal cell fate (Akita et al., 2002). Grafted
retinal progenitors treated with growth factors showed some differentiation after migration
into the retina (Tomita et al., 2006). The latter study also showed that retinal progenitor cells
are to be preferred compared to bone marrow-derived cells. Other strategies to accomplish
functional integration include gene transfer of transcription factors and homeobox genes that
are related to photoreceptors and/or retinal cell fates. Mouse embryonic stem cells carrying
the homeobox gene Rx/rax not only migrated into the explants, but also extended processes
that were immunopositive for some neural markers (Tabata et al., 2004). By gene transfer of
various transcription factors related to photoreceptors, Akagi and colleagues (2005) showed
that adult iris-derived cells could adopt photoreceptor-like characteristics. The cells migrated
and survived in explants at appropriate locations, but in low numbers.
Apart from growth factor-related experiments, drug screenings and/or toxicity tests based
on retinal explant techniques are so far very rare. One possible explantation for this may be
that many drugs are diluted in dimetylsulphoxid and/or alcohol, and longer culture periods
may lead to cell injuries (Johansson unpublished). For limitations of ganglion cell death,
valproic acid shows neuroprotection and regeneration in vitro and in vivo (Biermann et al.,
2010). Limited RGC degeneration in vitro has also been attributed to caspase-3 inhibitors
and inhibitors of translation and transciption (Manabe et al., 2002). Usage of retinal explants
in toxicity tests of the VEGF inhibitor Bevacizumab has recently been published, and initial
drug screening was accomplished within 3 days of culture (Kaempf et al., 2008). Substantial
drug screening for RGC neuroprotection was recently published, and further validates that the
explant model can be used for efficient and reliable drug screening of retinal neuroprotective
therapies (Bull et al., 2011).
Conclusion
Photoreceptors and perhaps also ganglion cell death in explanted retinas mimic
corresponding degenerative events described in other experimental models and human retinal
disorders, and the explant models are suitable for the evaluation of treatment strategies.
Different neuroprotective interventions, ranging from growth factor supplementation to
progenitor/stem cell coculture, limit photoreceptor degeneration in vitro irrespective of the
death pathway. As neurotrophic factors may have undesirable side effects, experiments
aiming at an activation of endogenous repair systems may be approached in explants.
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Chapter IX
Non-Invasive Imaging of Retinal

Gliosis for Screening Neurotoxicity
and Neuroprotection in Mouse Models
of Human Diseases
Lang Zhuo* and Saravana Kumar

Institute of Bioengineering and Nanotechnology, Biopolis, Singapore
Abstract
Reactive gliosis, a hallmark of neurotoxicity, is triggered by injury or insult to the

central nervous system (CNS) due to various factors such as drugs, chemicals, trauma
and diseases [1]. Reactive gliosis involves the transformation of glial cells, such as
astrocytes in the brain and Müller cells in the retina, from a quiescent to an activated
state. Glial fibrillary acidic protein (GFAP), expressed in astrocytes, is widely regarded
as the ideal biomarker for astrogliosis due to its sensitivity and specificity [1]. As such, a
non-invasive detection and quantification of GFAP transcriptional activity would not
only facilitate the objective assessment of neurotoxicity from compounds but also enable
the timely detection of CNS related diseases [Alzheimer‘s disease (AD), Parkinson‘s
disease (PD), multiple sclerosis (MS), diabetic retinopathy (DR), etc.] and development
of effective treatment strategies which intervene at an earlier stage.
This is now possible with the development of a transgenic GFAP-GFP mouse model [2]
where green fluorescent protein (GFP-S65T) was expressed under the control of human
GFAP promoter as shown in Fig. 1. Confocal imaging of ex vivo tissue samples confirmed
* Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, The Nanos, #04-01, Biopolis, Singapore
138669, E-Mail: lzhuo@ibn.a-star.edu.sg
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Copyright © 2012. Nova Science Publishers, Inc. All rights reserved. May not be reproduced in any form without permission from the publisher, except fair uses permitted under 192 Lang Zhuo and Saravana Kumar
the presence of the GFAP-GFP transgene and its co-localization with endogenous GFAP in
the brain, optic nerve and retina [2].
Figure 1. Transgenic GFAP-GFP construct.
The GFAP-GFP transgenic mouse model enables us to study the in vivo dynamic
changes of glial cells during development, and in response to physiological and pathological
conditions. Fig. 2 shows the non-invasive measurement of GFAP-GFP transgene from the
saline and 2′-CH3-MPTP treated neonatal mouse brain at postnatal day 4 (PD4) using the
IVIS Imaging System 100 Series (Xenogen Corp, Alameda, CA, USA) [3-5]. As early as 4
hours post treatment, the 2′-CH3-MPTP treated transgenic (Tg) neonatal mouse has
significantly higher GFP fluorescence emitted from the brain over the saline treated
transgenic mouse. However, direct imaging of GFAP-GFP transgene from the brain is limited
to the study of developmental neurotoxicity in neonatal mice and this is attributed in part to
the presence of fur and thicker skin as well as skull in adult mice which impedes the
excitation of and GFP signal emission from the site of interest.
Alternatively, the retina would be an ideal in vivo channel for visualizing glial cells real-
time in adult transgenic GFAP-GFP mice. The retina is, after all, an extension of the CNS
with its own population of neurons and GFAP positive glial cells i.e. astrocytes and Müller
cells. It is also visually accessible given the relatively clear optical medium of the eye.
Retinal imaging of the adult transgenic GFAP-GFP mice with a confocal scanning laser
ophthalmoscope (cSLO): Heidelberg Retina Angiograph 2, HRA 2 (Heidelberg Engineering,
Dossenheim, Germany) confirmed the in vivo transgenic expression in the astrocytes [6]. In
order to allow more light to access the retina, the small mouse pupils were dilated with 0.5%
Cyclogyl® (cyclopentolate hydrochloride, Alcon, Puurs, Belgium) and the 30o focal lens was
replaced with a 55o wide angle objective which reduces the laser beam diameter of the cSLO
to 1.7mm. Topical eye drops (Tears Naturale II, Alcon, Puurs, Belgium) and custom-made
polymethylmethacrylate (PMMA) hard contact lenses (Cantor & Nissel, Northamptonshire,
UK) were also applied on the mouse eye to minimize dehydration of the cornea during
imaging and correct for spherical optical aberration. But despite these modifications, the in
vivo retinal images have a high level of thermal and shot noise (as observed in the optic disc
region of the retina in Fig. 3A) which cannot be removed via direct frame averaging without
compromising the inherent cellular resolution of the GFP+ cells (see Fig. 3B). This is due to
the continuous and sometimes rapid motion of breathing and heartbeat of the anesthetized
animal. We addressed this problem by developing a non-linear frame averaging algorithm
called ―averaging via rank matching‖ (ARM) [6, 7] which significantly reduces the noise
level while retaining inherent cellular resolution such that individual GFP+ glial cell bodies
are visible as shown in Fig. 3C.
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Copyright © 2012. Nova Science Publishers, Inc. All rights reserved. May not be reproduced in any form without permission from the publisher, except fair uses permitted under Non-Invasive Imaging of Retinal Gliosis for Screening Neurotoxicity … 193
CONTROL (saline) 2′-CH3-MPTP (12 mg/kg)
0 hr
nTg Tg nTg Tg
2 hr
4 hr
6 hr
8 hr
x 106 photons/sec/cm2/sr X 106 photons/sec/cm2/sr
Figure 2. In2.vivo
Figure brainbrain
In vivo gliosis in PD4
in PD4
gliosis neonatal
neonatal treated
micemice with with
treated neurotoxicant 2′-CH2′-CH
neurotoxicant 3-MPTP. Mice were
3-MPTP.
treated with saline (control) or 12 mg/kg of 2′-CH3-MPTP and imaged from 0 to 8 h. For imaging each pair
of un-anesthetized mice, the transgenic (Tg) mouse was positioned on the right and the non-transgenic (nTg)
mouse on the left using tape. Transgenic mouse treated with the neurotoxicant displays enhanced GFP signal
from 4 to 8 h, when compared to transgenic mouse treated with saline. Units are in photos/s/cm2/sr.
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A B C
50um
Figure
Figure3.3.Comparison
Comparison of ARM against
of ARM direct direct
against frame averaging method in method
frame averaging noise suppression
in noise and preservation
suppression and
of GFP+ cellular resolution. (A) Noisy in vivo GFP fluorescence image from the transgenic GFAP-GFP
preservation
mouse of GFP+
retina centered cellular
around resolution.
the optic disc (B)(A) Noisy in image
Composite vivo GFP
fromfluorescence
conventionalimage fromofthe
averaging 55 transgenic
noisy in
vivo GFP fluorescence
GFAP-GFP mouse retinaimages (C)around
centered Composite image
the optic from
disc (B) ARM of 55image
Composite noisy from
in vivo GFP fluorescence
conventional averaging
images.
of 55 noisy in vivo GFP fluorescence images (C) Composite image from ARM of 55 noisy in vivo GFP
fluorescence images alignment process is based on a pixel rank matching criterion where the
The non-linear
rank of a pixel denotes the order of that pixel in an image neighborhood assuming all pixels
in that neighborhood have been sorted in ascending order based on their intensity values. If
image A is to be aligned to image B, the criterion requires the pixel values in image A to be
updated such that the rank of the center pixel in every local neighborhood of image A is
identical to that of the center pixel from a corresponding neighborhood of image B. This is a
reasonable requirement since the rank of both pixels should not differ despite there being
variations in the average intensity level in their neighborhood. However, this assumption is
only valid in the absence of noise which could significantly alter the pixel ranks. As such,
ARM comprises two sequential stages where the first stage ensures noise robustness by
deconvolving each frame, Ii, and using the deconvolved set to guide the alignment of Ii
whereas the second stage describes the non-linear alignment.
1) Noise robustness: In order to ensure the noise robustness of our rank matching
criterion, we utilize the rank information obtained from the a-priori estimates Ji, i =
1, 2 …, 55 of the underlying fluorescence signal corresponding to each retinal frame
Ii, i = 1, 2, …, 55 from the original sequence. The a-priori estimates are obtained by
deconvolving each image Ii (Huygens Essential Software, Scientific Volume
Imaging BV) [8] in the sequence. The deconvolution software computes a theoretical
point spread function (PSF), resembling a 2–D Gaussian profile in the x – y plane,
based on the ophthalmoscope device settings. Also, it automatically estimates the
image SNR and uses this as a regularization parameter to control the sharpness of the
deconvolved image. In doing so, photon noise is effectively removed.
2) Non-linear alignment: The non-linear alignment is implemented sequentially as

shown in Eq. 1.
I ir x, y   I i xo , yo , i  2 (1)
where the pixel (xo, yo) is located within a local (2Wr + 1)×( 2Wr + 1) neighborhood centered
r r
at (x, y) and Wr is the width and the superscript r in I i denotes that I i has been
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ref
non-linearly aligned such that the ranks of its pixels match those of J i 1 as defined in Eq. 2.
Rank I i xo , yo   Rank J iref

1 x, y  (2)
J iref is defined as
J ref

i  1J iref
1  J ia ,r
(3)
i
i
where J1ref  J1 and J i is computed as follows:

r
J ir x, y   J i xo , yo  (4)
subject to
Rank J i xo , yo   Rank J iref

1 x, y  (5)
Finally, the result of averaging i number of aligned images in the sequence is given by
I iref 
i  1I iref1  I ia ,r
(6)
i
where I1ref  I1 . Eqs. 1–6 are implemented in a cyclic fashion where for every cycle, i is
ref
incremented before proceeding to the next cycle where Eqs. 1–6 are recomputed and I N
denotes the final averaged composite image. Fig. 1 compares the performance of our ARM
method against Heidelberg‘s deconvolution algorithm and the HRA 2‘s averaging module.
Although the Huygen‘s deconvolution result in Fig. 1(b) appears to have significantly
enhanced both SNR and cellular resolution, it is processed from a single frame in the
sequence and, as a result, the fluorescence expression, especially of the cells peripheral to the
optic disc, is not as complete as the ARM processed result in Fig. 1(c) which combines both
deconvolution and non-linear alignment from a sequence of time-lapse images. It is clear here
that both components play complementary roles in ARM. In contrast, HRA 2‘s averaging
module shows significantly poorer cellular resolution compared to the other two approaches.
We have also successfully applied ARM to quantify the upregulation in retinal GFAP-
GFP transgene, in vivo, due to the systemic administration of the kainic acid (KA)
neurotoxicant [9]. Fig. 4 shows the representative non-invasive longitudinal retinal images,
centered around the optic disc, of transgenic GFAP-GFP mice (saline vs. kainic acid, KA).
As observed, the fluorescence intensity (FI) from the saline mouse remained fairly stable
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from day 0 through day 14 (Figs. 4A–D) but the FI from the KA-treated mouse showed a
gradual increase at day 3 which peaked at day 7 (Fig. 4G) due to KA-induced neurotoxicity.
Figure 4. In vivo transient gliosis in KA treated mice. Representative non-invasive longitudinal retinal
imaging of transgenic GFAP-GFP mice (saline vs. KA, 25mg/kg intraperitoneal administration). Eight-week-
old male transgenic mice injected with saline or KA were subject to retinal imaging on 0, 3, 7, and 14 days
after injection. Each composite image was assembled using our algorithm from a total of 45 raw images. As
illustrated in (A), most of the FI was from the astrocytes within the optic disc (circle). Retinal blood vessels
(arrowheads), associated retinal glial cells in the optic disc (long arrow), and surrounding region (short
arrows) were all visible at the NFL. This pattern stayed fairly constant from day 0 to day 14 in the saline
group (A–D). However, in the KA group (E–H), an increase in FI was observed in the retinal glial cells (long
arrows) within the optic disc on day 7 (G). In addition, FI and the number for glial cells (short arrows)
labeled by GFP in the surrounding retina were also significantly elevated on day 7 (G). The increase in FI on
day 7 seemed to subside by day 14 (H). Scale bars, 100 µm.
Retinal gliosis is quantified based on the fluorescence intensity (FI) from the astrocytes
around the optic disc of the mouse retina. Each astrocyte has a ―spot-like‖ profile with its
respective peak fluorescence intensity whereas FI is the fluorescence intensity value averaged
over all astrocytes in the image. The accurate detection of the astrocytes is therefore an
important precursor to the FI quantification. Given the ―spot-like‖ profile of the astrocytes in
the ARM processed images, we employ a gray-scale morphological operation known as
―extended maxima‖ transform [10] to locate the intensity maximas (peak intensities) which
correspond to the centroids of the astrocytes in these images. The extended maxima transform
denotes the regional maxima of the Hmaxima transform where H = 2 and the regional maxima
are 8-neighborhood connected components of pixels with a constant intensity value, and
whose external boundary pixels all have a lower value. We demonstrated that the KA-
induced gliosis (an elevation in GFAP–GFP fluorescence intensity, FI) can be non-invasively
detected starting on day 3, and peaked at day 7 ( p  0.05 ) as quantified from the optic disc
astrocytes. The quantified values were expressed as mean FIs ± SEM, and statistically tested
using Friedman‘s non parametric comparison on repeated FI measures as shown in Fig. 5.
Our non-invasive observation on retinal gliosis was consistent with previously published
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studies, which documented an increase in the GFAP in both Müller cells and astrocytes after
administration of KA [11].
Figure 5. Quantification of fluorescent signal. FI for the optic disc astrocytes was measured from processed
images of the saline and the KA group (n = 5) respectively. The measurements for day 0 were normalized to
1, as a reference for comparing the later longitudinal time points. A significant increase in FI for the retinas
of KA-treated mice was noticed on days 3 and 7 (*p < 0.05). The quantified values were expressed as mean
FIs ± SEM, and statistically tested using Friedman‘s non parametric comparison on repeated FI measures.
Figure 6. High magnification view of retinal whole-mounts, from transgenic GFAP-GFP mice, stained for
GFAP. A and B are the merged images of GFP (green) and GFAP (red) staining for saline and KA-treated
retinas on day 7 respectively. The retinal vasculature (arrow heads) were seen ensheathed by astrocytes (long
arrows). The punctated spots (short arrows) of retinal glia are also visible in the retina surrounding the optic
disc. It was obvious that both the GFP and GFAP staining for the KA-treated retina on day 7 were more
intense, indicative of KA neurotoxicity. Scale bar = 50 µm.
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In order to verify that the noninvasively collected fluorescence signal was from the GFP
in the retinal glia, we performed immunohistochemistry (IHC), for the GFAP protein, on
retinal whole-mounts isolated from mice treated with KA or saline at day 7. From Fig. 6, both
hypertrophy and hyperplasia of retinal glia can be observed in the KA-treated retina (6B)
compared to the control (6A) where the unstained GFAP-GFP transgene shows green
fluorescence whereas the endogenous GFAP is stained here in red.
Our non-invasive observation on retinal gliosis was consistent with previously published
studies, which documented an increase in the GFAP in retinal glial cells after administration
of KA [11, 12]. And critically our data showed that the KA-induced gliosis in the
hippocampal areas (CA1, CA3 and dentate gyrus) was tightly coupled to the retinal gliosis.
Hence in the current study, we have established a proof-of-concept for using molecular
retinal imaging not only to study retinopathies, but also to study neurodegeneration in the
brain, since it is well established that many neurodegenerative disorders, including
Parkinson‘s disease modeled after MPTP [13], are underlined by neurotoxicity and inevitably
triggering gliosis in the CNS.
Perhaps another equally important application of the current method is to screen for
compounds with neuroprotective properties in the retina which correlate with pathologies
deep within the diseased brain. Parkinson‘s disease (PD) is the second most frequent
degenerative disorder after Alzheimer‘s disease. It is characterized by the depletion of
mesenchephalic dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc)
and striatal dopamine. Oxidative stress plays a major role in the pathogenesis of Parkinson‘s
disease where DA neurons are especially vulnerable to excessive production of reactive
oxygen species. We have quantitatively evaluated, via this imaging platform, the efficacy of a
synthetic antioxidant, 1,3-bisbenzylimidazolium bromide (DBZIM) [14, 15] as shown in Fig.
7, in attenuating retinal gliosis [16, 17].
Figure 7. Chemical structure of 1,3-bisbenzylimidazolium bromide (DBZIM).
As observed in Fig. 8, saline alone had no influence on gliosis over the course of 4 days
(Figs. 8A and D). However, a systemic (i.p.) administration of 2′-CH3-MPTP caused a drastic
increase in GFP fluorescence on the mouse optic disc at 4 days post-dosing (comparing Figs.
8B and E). But interestingly, mice receiving two doses of DBZIM (i.p., 20 mg/kg) 24 h
before and after the 2′-CH3-MPTP injection showed no apparent gliosis (comparing Figs. 8C
and F), suggesting a neurotoxicity-suppressing and neuro-protective effect by DBZIM.
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Figure 8. Neurotoxicity suppressing effect of DBZIM in live mice. Representative retinal images acquired
from the same eye of a saline-treated mouse at 0 h (A) and 96 h (D). Retinal images from the same eye of a
2′-CH3-MPTP-treated mouse at 0 h (B) and 96 h (E). Retinal images from the same eye of a mouse treated
with DBZIM and 2′-CH3-MPTP, respectively at 0 h (C) and 96 h (F). The retinal vein (arrow head) and
blood vessels (empty arrow heads) are clearly recognizable for the same eye acquired at different times (0 vs.
96 h in B and E). It is noted that the maximal GFP fluorescence is seen in astrocytes (arrows) around the
fringe of the optic disc (A). Treatment with 2′-CH3-MPTP triggered a significant increase in the GFP signal
(E) at 96 h (or 48 h after the neurotoxicant dosing), when compared to the basal level (B). Co-treatment of
the mouse with DBZIM abolished the 2′-CH3-MPTP-induced toxicity (F). Scale bar? = 100 μm.
Figure 9. Retinal whole-mount staining (merged green and red) from mice with different treatments. (A) In
mouse treated with saline, retinal astrocytes and Müller cells expressed basal level of GFP (green) and GFAP
(red). (B) In the 2′-CH3-MPTP-treated mouse, the retinal glia expressed significantly higher level of GFP
and GFAP, when compared the saline control. (C) Treatment with DBZIM abolished the 2′-CH3-MPTP-
induced signal increase. Scale bar = 200 µm.
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We performed IHC on the retinal tissues ex vivo and merged confocal images of retinal
whole-mount stained for GFAP (red) and GFP (green) showed a basal signal intensity in the
saline group (Fig. 9A), whereas the whole-mount from the 2′-CH3-MPTP group had a much
higher staining intensity (Fig. 9B). In line with the in vivo observation, the gliosis
(neurotoxicity)-suppressing effect of the DBZIM was also observed in the retinal whole-
mount (Fig. 9C), as judged by the dimming staining intensity comparable to that in the saline
control (Fig. 9A).
We also observed the protection of DA neurons (stained for TH) by DBZIM in the
substantia nigra pars compacta (SNpc) of Fig. 10B, placing the number of DA neurons
between the saline group (Fig. 10A) and the 2′-CH3-MPTP group (Fig. 10C).
Figure 10. IHC staining for tyrosine hydroxylase (TH) in DA neurons of mice with different treatments. IHC
using TH antibody clearly identified many DA neurons in the SNpc. On contrast, the number of DA neurons
was greatly decreased in the 2′-CH3-MPTP-treated SNpc, whereas the number of DA neurons in the
DBZIM/2′-CH3-MPTP group was between the saline and the 2′-CH3-MPTP group.
Currently several drugs are available for PD management. However all these drugs are
symptomatic by nature and associated with significant side effects. This situation underlines
the fact that an unmet medical need persists for this major neurological disorder. With recent
technological advancement, a number of non-invasive methodologies have been developed to
aid the study and therapeutic development of PD. Notably, the advanced 3-dimensional
imaging tools, such as MRI and PET, have proved to be rather effective for studying
neurotoxicity and developing therapeutics in models of PD [18]. However the requirement
for using a contrast agent or a labeled ligand and the cost associated with these intricate
instruments limit their wide-spread and routine use in an independent laboratory setting.
In search for an alternative non-invasive imaging method to investigate the ocular

consequence in Parkinsonism or other neurological disorders, we developed a molecular
retinal imaging system, comprised of a GFAP-GFP mouse [2], a confocal scanning laser
ophthalmoscope, and an image processing algorithm [6, 9, 17]. Results from the current study
showed that this imaging system can be applied to study retinopathy in a neurotoxicant (2′-
CH3-MPTP)-induced mouse PD mode. In particular, we are able to longitudinally image and
quantify gliosis in the retinal glia in response to a known Parkinsonian toxin 2′-CH3-MPTP.
This technical achievement proves the concept for using this method to primarily screen for
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compounds with neuroprotective property by simply measuring the transgenic GFP signal,
which is an established surrogate for the endogenous pathological marker GFAP. The
theoretical basis for this approach is based on an early concept by O‘Callaghan [1] and a
large body of literatures by others in subsequent years that GFAP can be used as an early and
sensitive biomarker for neurotoxicity.
With the establishment of molecular retinal imaging method, several aspects of the drug
development could be improved. Firstly, the success of the 2′-CH3-MPTP-induced toxicity
and DA neuronal loss in the brain could be indirectly estimated from the real-time live retinal
imaging, since the retinal neurotoxicity (or gliosis) was tightly coupled to neurotoxicity in the
striatum and DA neuronal loss in the SNpc. In other words, the parkinsonian neurotoxicant
2′-CH3-MPTP not only caused damages in the brain, but also imprinted a pathological
signature on the retina. Only mice that have been successfully induced as a PD model with a
correct dosing scheme in an appropriate genetic background among other factors (sex and
age, etc.) were further used to test anti-PD candidate compounds. In this way, false positive
results and data variation were minimized. Secondly, the potential neurotoxicity of a
candidate compound could also be independently assessed. Drug compounds (intended for
nervous or non-nervous system) ought to be systematically tested for potential neurotoxicity
in vivo. Under the dosing scheme used in this study, DBZIM itself showed no neurotoxicity
in the retina and the brain. Thirdly, the efficacy of the candidate compounds could be
concurrently evaluated using retinal imaging as well.
In contrast to 2′-CH3-MPTP and KA treatment, a more gradual and chronic increase in
GFAP is observed in diabetic mice over a 5 week study [19]. Fig. 11 shows representative
retinal images at week 0 (11A and 11C) and week 5 (11B and 11D) for both the control (11A
and 11B) and STZ-treated diabetic mice (11C and 11D). Interestingly, new astrocytic cell
bodies are observed in Fig. 11D (white arrow-heads) of the STZ-treated mouse at the same
locations (dashed white circle) in the base-line image of Fig. 11C. In contrast, the number of
astrocytic cell bodies, at both time points, is comparable in the control mouse. Our in vivo
data correlates with clinical reports with regards to retinal gliosis-related inflammatory
response during early diabetic retinopathy [20]. This opens up the possibility of using in vivo
molecular imaging of retinal glial cells as a platform for monitoring the efficacy of anti-DR
drug candidates which intervene at an early stage.
The Müller cell end feets in the diabetic mice, though activated due to STZ treatment, are
below the 10 mm resolution limit of the cSLO. In fact, the upregulation of the GFAP-GFP
transgene in the diabetic mice, as observed in the in vivo retinal images of Figs. 11 is clearly
due to retinal astrocytes. Although the current cSLO setup has a limited resolution of 10μm,
recent developments in the use of customized [21] or adaptive optics [22] for retinal imaging
could lead to significant improvements in resolution such that fine glial processes can be
visualized.
Although preclinical in its scope, the retinal glia imaging method is an effective platform
for non-invasive screening of neurotoxicity and therapeutic efficacy of compounds against a
wide range of CNS related diseases. Future development of non-toxic small molecular probes
for a specific cellular target should open up the possibility of conducting molecular retinal
imaging on humans for the early diagnosis of and early therapeutic intervention against these
diseases in a clinical setting.
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Figure 11. Qualitative evidence of increase in the cell number of retinal astrocytes and increase in the GFAP-
GFP transgene expression levels from both astrocytic cell bodies and processes in STZ-treated F1 hybrid
mice at week 5 relative to the base-line. Magnified in vivo GFP images of the control (A and B) and STZ-
treated (C and D) F1 hybrid (FVB/N × C57BL/6J) mouse retina centered around the optic disc at week 0 (A
and C) and week 5 (B and D). The white arrow-heads in D indicate the presence of new astrocytic cell bodies
in the STZ-treated mouse retina at the same locations indicated by dashed white circles in the base-line
image (C). The number of astrocytic cell bodies, at weeks 0 and 5, is comparable in the control mouse as
shown in A and B. The long arrows in D indicate the astrocytic cell processes and again these are more
distinct here at wek 5 compared to the base-line in C.
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Chapter X
Proteomic Approach to Chronic

Eye Diseases
Takashi Kanamoto*
Department of Ophthalmology and Visual Sciences,
Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan
Introduction
Proteomic analyses have been used to study different aspects of various systemic and
ophthalmic diseases, and the number of proteomics publications on eye diseases has been
rapidly increasing. Proteomic has been used to investigate chronic eye diseases, such as dry
eye, cataracts, glaucoma, diabetic retinopathy, and aged-related macular degeneration. It is
expected that the results of this proteomic approach can be used to investigate the mechanism
and diagnosis of diseases, and that information will help in developing new treatments.
We shall present the summary of proteomic research for several chronic eye diseases and
show the up-dating of them.
1. Tears
1.1. Tear Film

The tear film has several functions, e.g., lubrication, protection of the eye, and
nourishment of the cornea [1]. Tears contain lipids, proteins, mucins, and other chemicals.
The tear film over the cornea consists of three layers; an outer layer containing lipids, a
* Department of Ophthalmology and Visual Sciences, Graduate School of Biomedical Sciences, Hiroshima
University, 1-2-3, Kasumi, Minami-ku, Hiroshima, Japan. Postal-code: 734-8551. Tel: +81-82-257-5247, Fax:
+81-82-257-5249, E-mail: tkana@hiroshima-u.ac.jp
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Copyright © 2012. Nova Science Publishers, Inc. All rights reserved. May not be reproduced in any form without permission from the publisher, except fair uses permitted under 206 Takashi Kanamoto
middle layer that is an aqueous layer containing proteins, and an inner layer covering the
surface of the corneal epithelial cell layer and is a mucin layer [2].
The major tear proteins are lysosomes, lactoferrin, secreted immunoglobulin, serum
albumin, transferrin, lipocalin, and lipophilin [3-8]. Immunoassay-based methods have
identified phospholipid transfer protein, growth factors, matrix metalloproinases, bradykinin,
phospholipase-A2, and lactate dehydrogenase in the tears in the tears [9-13]. The recent use
of mass spectrometry has markedly increased the number of proteins in the tear film, and one
mass spectrometry study reported that approximately five hundreds proteins were present in
the tears [14]. In addition, 97 proteins including zinc-alpha2-glycoprotein, proline rich
protein, cystatin S, lacritin, and haptoglobin, have been identified in normal human tear. The
most categorized proteins were the immune-responsive proteins [15]. The difference between
reflex tear and normal eye tear has also been pointed out [16].
These suggest that the protein profile of tear by proteomics method showed various kinds
of proteins present in the tear film. Furthermore, tear collection is easier than blood
collection. Interestingly, it has been proposed that the tear proteomic pattern might be useful
for the diagnosis of breast cancer [17].
1.2. Dry Eyes
Recent studies have demonstrated that the composition of the tear film is different in eyes
with ocular surface diseases such as conjunctivitis [18], keratoconus [19], and dry eye
syndrome. The dry eye syndrome is an ocular surface disease characterized by tear secretory
deficiency or excessive tear evaporation [20]. A dry eye rabbit model has been used to
examine the proteins in the tears [21]. Tears from patients with the dry eye syndrome have
also been investigated by proteomic analyses, and the results showed that α-enolase,
calgranulins, and cagizzarin were up-regulated, while prolactin-inducible protein, lipocalin-1,
lactoferrin, and lipophilins were down-regulated [22-23].
Reduced levels of lactoferrin and lipocalin were also found in the profile of tears of
patients with Sjögren syndrome [24-25]. Sjögren syndrome is an autoimmune-mediated
inflammatory disease characterized by progressive lymphocytic infiltration into the lacrimal,
parotid, and salivary glands. The infiltration leads to a destruction of these glands thus
reducing or eliminating their secretory proteins [26-27]. The diagnostic criteria for primary
Sjögren syndrome include dry eye, dry mouth , lymphocytic infiltration into exocrine glands
as shown by pathological investigations, and the presence of autoantibodies against nuclear
factors SS-A and SS-B [28-29]. It was recently suggested that a profile of the proteins
associated with Sjögren syndrome would be helpful in designing diagnostic tests to replace
tissue biopsies or blood tests. Besides lactoferrin and lipocalin, the down-regulated proteins
also included globet cell-specific mucin [30], prealbumin, lysozyme [31], peroxidase,
amylase, α1-antitrypsin [32], and clusterin [33]. Defensins and aquaporins were included in
the profile of abnormal proteins in the tears of eyes with dry eye syndrome [34].
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Copyright © 2012. Nova Science Publishers, Inc. All rights reserved. May not be reproduced in any form without permission from the publisher, except fair uses permitted under Proteomic Approach to Chronic Eye Diseases 207
2. Cataracts
2.1. Crystallins
The lens of the eye is made up of two cell types; the fiber cells in the lens nucleus and
the epithelial cells in the lens membrane. Mature fiber cells contain crystallins which can be
disrupted resulting in unfolding and aggregation, and ultimately to lens opacity or a cataract
[35-36]. Proteomic analysis of the crystallins in animal lenses showed that with aging there is
a degradation of α-crystallins and β-crystallins leading to insoluble lens fibers [37]. Also, the
β-crystallins in rats were modified post-translationally during cataract formation [38]. Post-
translational modification of proteins was also confirmed in human moderate cataracts by
mass-spectrometry which detected 155 post-translational modification sites in the crystallins
[39]. These findings confirmed that cataractogenesis is accompanied by alteration of the lens
crystallins.
2.2. Lens Fiber Cells
More than two hundred proteins were identified by membrane proteomic analyses of
mouse fiber cells including Mip1, Lim2, Gja3, Gja8, and Gje1 [40]. Gja3 is a lens-specific
connexin protein which is associated with the development of cataracts [41]. Comparative
proteomic of the lenses of Gja3-deficient mice showed changes in the expression of 63
proteins including chaperonin subunit 6A, mortalin, Erp29, and syntaxin-binding protein 6,
and it was suggested that heat shock protein 27/25 and /or Erp29 were the major modifying
factors for cataractogenesis [42] However, a proteomic profile of human lens fiber cells has
not been reported.
3. Glaucoma
3.1. Aqueous Humor
Glaucoma is an ocular disease with characteristic damages of the optic nerve fibers and
defects in the corresponding visual fields. The damage to the nerve fibers results from an
abnormal elevation of the intraocular pressure (IOP), and the elevation is caused by excessive
synthesis of aqueous humor or a decrease in the aqueous outflow from the eye [43].
However, how the aqueous humor proteins and trabecular meshwork are related to the
pathogenesis of glaucoma has not been definitively determined. It has been found by
proteomics that human aqueous humor obtained during cataract surgery is abundant in
antioxidant, immune-regulatory, and anti-angiogenic proteins [44]. However, the protein
content of the aqueous humor has not been analyzed thoroughly. The trabecular meshwork
has not been also completely analyzed by proteomics, however 255 protein spots in two
dimensional gels derived from human eyes were identified, and they included proteins
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involved in metabolic processes, cell adhesions, anti-apoptosis, cell mobility, and signal
transduction [45].
It is well-known that the healing of a filtration bleb after trabeculectomy is mediated by
TGF-β signaling. Eight proteins were identified by functional proteomics in failed filtration
bleb, and the expression of ribosomal S6 kinase 2 is probably associated with wound healing
in filtration blebs [46].
3.2. Glaucoma Induced Retinal Degeneration
Proteomic analysis for glaucomatous retinal degeneration has been performed only in a
mouse model of glaucoma. DBA2J mice, a model for hereditary pigment glaucoma, have
been analyzed by functional proteomics. Eighteen proteins were identified, and among of
them, Integrin β7 was found to be associated with retinal ganglion cell death in vitro [47].
Functional proteomic analyses of excitatory amino acid carrier 1-deficient mice, which have
glaucomatous retinal degeneration, identified 13 proteins [48] including the GABA-A
receptor β1 subunit. This subunit is also associated with retinal ganglion cell death [49].
Quantitative proteomic analysis of primary culture of glaucomatous rat retinal ganglion cells
showed an 8% up-regulation and 13% down–regulation of the 268 proteins in the retinal
ganglion cells [50].
Table 1. Profiles of alternative expression-changed proteins in glaucoma animal models.
Okumichi, et al. 48) Mouse retina Up-regulated proteins Gamma-aminobutyric acid (GABA-A) receptor subunit beta1, p300 transcriptional cofactor JMY,
LEK1, activator of Rho-dependent signaling, platelet-derived growth factor receptor α
Down-regulated proteins Galnt7, microfibrillar associated protein 3
46)
Kanamoto, et al. Mouse retina Up-regulated proteins T cell receptor, pottasium channel, glautamine transaminase K, glautamine synthetase,
creatinine kinase
Down-regulated proteins Mitofusin, sepiapterin reductase, tRNA synthetase, integrin β7, tyrosine phosphatse, aldolase,
recoverin,
Crabb, et al. 50) Rat retinal ganglion cells Up-regulated proteins Aconitate hydratase, endoplasmic reticulum protein, poly(U)-binding-splicing factor,
aldose reductase, Ubiquitin
Down-regulated proteins Purine-rich, element-binding, protein gamma, scaffold attachment, factor B1, prothymosin alpha,
guanine nucleotide-binding protein (Transducin beta), tRNA synthetase, pyridoxal kinase
4. Diabetic Retinopathy
Diabetic retinopathy is the most common microvascular complication caused by diabetes

mellitus [51]. At the early stage, microvascular occlusions in retina progresses to retinal
neovascularization. With time, the diabetic retinopathy reaches the proliferative stage.
Several proteomic analyses on the retina obtained from animal models of diabetic retinopathy
have been published, and there have also been several reports on human samples obtained
from eyes with diabetic retinopathy.
The retinas of streptozotocin-induced diabetic rats with hyperglycemia have been
frequently used for proteome analyses. While 24 proteins, including heat shock protein
70.1A, HSP 8, and platelet activating factor, were uniquely expressed in diabetic retinopathy,
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37 proteins were found to have disappeared [52]. A time course study during the diabetic
process showed a differential up-regulation of the protein isoforms of retinal crystallin 53).
Retinal proteomic analyses of diabetic mice treated with the angiotensin 1 antagonist,
candesartan, demonstrated that 24 proteins related to metabolism were up- or down-
regulated, and the expressions of 5 proteins associated with apoptosis were also changed [54-
55].
Table 2. Profiles of alternative expression-changed proteins in diabetic animals.

Gao, et al. 54) Mouse retina Up-regulated proteins Prefoldin, coatomer, eukaryotic translation factor, sarcolemmal membrane protein
tRNA synthetase, heat shock protein, AP2-, AP3--associated protein, dynamin-like protein,
protein phosphatase 3, ribosomal protein,, fatty-acid-CoA ligase, estradiol dehydrogenase,
retinol dehydrogenase 12, cytochrome reductase, apoptosis inhibitor, ubiquinone oxidoreductase,
Down-regulated proteins ubiquitin enzyme, protein phosphatase, FK506-binding protein, astrocytic phosphoprotein
Wang, et al. 55)

Rat retina Down-regulated proteins Gln synthetase, stress-induced-phosphoptotein, Leucine aminopeptidase 3, α-enolase
arginine dimethylaminohydrolase, glyceraldehyde-3-phosphate dehydrogenase, lipocortin-1
pyridoxal phosphate phosphatase, serine proteinase inhibitor, retinal dehydrogenase
Quin, et al. 52) Rat retina Up-regulated proteins Heat shock protein 70 1A, aldehyde reductase, platelet activating factor, phosducin, 14-3-3 protein
glyceraldehyde-3-phosphate, β-catenin, glucose regulated protein, ATP synthase, Calreticulin
Down-regulated proteins Peroxiredoxin 2, nucleoside diphosphate kinase, calreticulin, reg 1 binding protein, aldolase A
chaperonin Subunit 2, tyrosine oxygenase, phoshoducin, guanine nucleotide binding protein,
phosphatidylethanolamine, ndufa 1, heterogenous nucleoriboprotein, heat shock protein 84
pyruvate dehydrogenase, glutamate ammonia ligase, fructose bisphosphate aldolase, ldh3a ,
malate dehydrogenase, glucose regulated protein, GTP-ase binding protein, proteasome alpha
phosphoglycerate mutase,iIsovaleryl coenzyme A dehydrogenase, succinyl CoA Ligase, Tpi1,
Sumo 1, calbindin2, DRP2, Apo-cellular retinoic acid binding protein, retinoic acid binding protein,
arginine dimethylaminohydrolase, aminoacyclase 1, profilin , guanine nucleotide binding protein,
Fort, et al. 53) Rat retina Up-regulated proteins αB-, βB1-crystallin, βB3-crystallins, β-crystallin A3, β-crystallin 5, γ-crystallin D, γB-crystallin
Samples of human vitreous from patients with proliferative diabetic retinopathy were
analyzed by comprehensive proteomic to determine the diabetes-induced alterations in the
vitreous protein. Angiotensinogen, complement C3, complement factor 1, pro-thrombin, α1-
antitrypsin, anti-thrombin 3, factor XII, and peroxiredoxin 1, were up-regulated in the
vitreous of proliferative diabetic retinopathy, whereas calsyntensin 1, inter-photoreceptor
retinoid binding protein, neuroserpin, and extracellular superoxide dismutase, were down-
regulated [56].
The proteomic profile of retinal pigment epithelium, dissected from diabetic human
donor eyes, was also determined. A majority of the up-regulated proteins belonged to a
functional group of chaperones, including mitochondrial translation factor Tu, GRP75, heat
shock protein 71, and protein disulphide isomerase A3, aldehyde dehydrogenase, cathepsin
D, β-actin, retinaldehyde binding protein, selenium binding protein 1, and sterol carrier
protein. The down-regulated proteins were in a group involved in energy metabolism, e.g., γ-
enolase, phosphoglycerate mutase 1, and succinyl CoA [57].

Alterations in the proteins in the vitreous, retina, or pigment epithelium, would be
expected to be associated with the diabetic retinopathy, but a specific effect of the identified
proteins on retinal neovascularization and neuronal degeneration has not been determined.
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5. Age-Related Macular Degeneration
In the early stage of age-related macular degeneration (AMD), lipoproteinaceous

deposits are formed between the retinal pigment epithelium and choroid, and the eye can
progress to either the dry type or wet type of AMD. The dry type is associated with
degeneration of the photoreceptors and retinal pigment epithelium cells resulting in central
geographic atrophy. The wet type develops abnormal blood vessels originate from the
choriocapillaris and pass through Bruch‘s membrane into the subretinal space. These
abnormal blood vessels form the choroidal neovascularization which characterize the wet
type AMD [58].
AMD has been extensively investigated pathologically, and classified depending on the
location of the lesion. Based on the pathological features, the proteomic profile for AMD has
been classified as retinal endothelial cells, retinal pigment epithelium cells, or choroidal
endothelial cells.
Proteomic analyses of normal human eyes without AMD or any ocular disease have
shown that several proteins including inorganic pyrophophatase, protein disulfide isomerases,
calretuculin , peroxideredoxin 4, serpin B9, F-actin capping protein subunit β, coactosin-like
protein, vimentin, cathepsin B, and high molecular weight form of annexin A3, were
expressed more abundantly in the retinal than choroidal endothelial cells. On the other hand,
gluthatione peroxidease 1, ubiquitin calboxyl-terminal hydrolase isozyme L1, heat-shock
protein β1, superoxide dismutase, nucleotide diphosphate kinase A, and low molecular
weight form of annexin 3, were expressed more abundantly in the choroidal than retinal
endothelial cells [59]. Proteomics of mitochondria of the human retinal pigment epithelium
from eyes with AMD identified expression changes of some proteins including α-, β-, and δ-
ATP synthases, cytochrome C oxidase, mitofilin, mitochondrial heat shock protein 70, and
mitochondrial translation factor Tu. These changes suggested that AMD is associated with
mitochondrial dysfunction [60].
Overall, the results of tissue-specific proteome of the retina and choroid suggested a
differential expression of proteins associated with retinal and choroidal angiogenesis,
although there were a few proteomic analyses of human AMD samples.
Conclusion
At present, proteomics studies on the eye have been performed mainly on ocular surface
diseases because diseased tissue samples are more easily collected than tissues from
intraocular diseases. Proteomics for intraocular diseases have been carried out using samples
derived from animal models, but it is essential that similar proteomic analyses be performed
on human tissues. Proteomic studies to investigate the role of proteins associated with eye
diseases are necessary, and those data should help in determining the pathophysiological
mechanisms involved in the disease processes. Based on these molecular findings, new
targets for clinical therapy by molecular agents should develop.
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heterogeneity related to tissue of origin. Zamora DO; Riviere M; Choi D; Pan Y;
Planck SR; Rosenbaum JT; David LL; Smith JR. Mol Vis. 2007 Oct;13:2058-65.
[61] Mitochondrial proteomics of the retinal pigment epithelium at progressive stages of
age-related macular degeneration. Nordgaard CL; Karunadharma PP; Feng X; Olsen

TW; Ferrington DA. Invest Ophthalmol Vis Sci. 2008 Jul;49(7):2848-55.
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Chapter XI
Lens Plasticity and Intralenticular

Translocation of Crystallins
Jeffrey O. Henderson and Larry J. Takemoto*

Division of Biology, Kansas State University, Ackert Hall, Manhattan, KS, US
Abstract
Very high concentrations of ―crystallin‖ proteins in the human lens are necessary for
refractile properties of this tissue, and abrupt changes in refractive indices within the lens
due to aggregation of these proteins result in scattering of light and subsequent loss in the
ability of the lens to focus light upon the retina. Such a condition called cataractogenesis
is prevalent in patients greater than approximately 60-65 years of age, and is currently
one of the most common causes of blindness in the world. We have previously
demonstrated weak, heterologous interactions of alpha and gamma crystallins. We now
hypothesize that such weak interactions contribute towards the plasticity of heterologous
interactions, facilitating a uniform refractive environment that will minimize light
scattering in a tissue of very high protein concentration. Loss of this lens plasticity, due
to covalent interaction of lens crystallins and formation of strong heterologous
interactions, would result in changes in refractive indices and subsequent lens
opacification.
Keywords: lens plasticity, cataract
The vertebrate lens is a highly specialized organ whose only known function is to focus
incident light onto the retina. The lens is composed of a very high cytosolic concentration of
crystallin proteins which make up approximately 90% of total water-soluble protein.[1] This
very high concentration of crystallin molecules produces non-covalent, short-range, protein-
* Corresponding Author. Tel.: +1 785 532 6636; fax:+1 785 532 6799, E-mail address: takemlj@ksu.edu (L.J.
Takemoto)
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Copyright © 2012. Nova Science Publishers, Inc. All rights reserved. May not be reproduced in any form without permission from the publisher, except fair uses permitted under 216 Jeffrey O. Henderson and Larry J. Takemoto
protein interactions that are proposed to reduce the scattering of incident light, resulting in a
consistent refractive index and concomitant visual acuity.[2-5] Globally, cataract is the
leading cause of blindness.[6] As such, understanding the mechanism(s) by which lens
transparency is lost due to ageing, genetic predisposition, co-morbidity factors (e.g. diabetes
and hypertension) or a combination of the above-mentioned factors, is a vigorous area of eye
research.[7]
Significant advances have been made in our understanding of the biochemical and
molecular genetic events that accompany cataractogenesis.[8,9] Fundamentally, cataract
appears to be a disease of protein aggregation, disrupting short-range interactions of
crystallins, leading to a loss of transparency through fluctuations in the refractive index of the
lens.[4, 7,10,11] However, the exact mechanism of how aggregation occurs with attendant
light scattering is unclear.
Protein aggregation, as a causative agent of cataract, is thought to occur over time due in
part to environmental insults such as ultraviolet radiation and oxidative stress.[7,12] Because
one of the unique properties of the lens is the virtual lack of protein turnover in differentiated
fiber cells, these proteins have been present for the lifetime of the individual.[1,13] These
long-lived proteins undergo several post-translational modifications that fall into at least two
categories; those that occur during lens development including C-terminal degradation[14,15]
and phosphorylation of alpha-crystallin[16], and those that are observed on crystallin
molecules primarily in the aged (>40 years) lens including acetylation, oxidation and
deamidation.[5,12] What role these modifications play in cataractogenesis is not entirely
clear, though undoubtedly some of these modifications lead to crystallin aggregation.
Takemoto and Sorenson [5] have put forth a model where large-scale distribution of alpha-
and gamma- crystallin aggregates would alter light transmission through increased light
scatter leading to opacification. It should be noted that if the aggregates formed do not result
in changes in protein density, light will not be scattered and no loss of transparency will
result.
Considering the longevity of, and many age-related changes to, crystallin proteins,
combined with the environmental insults to the lens that occur from birth, it might be
expected that the incidence of cataract would be higher. Is there a physiological mechanism
to correct for inappropriate aggregation of crystallin molecules due to insult to the lens, other
than the well documented role of alpha crystallins as a molecular chaperone?[17] Previously,
it was thought that communication between fiber cells of the lens only occurred through gap
junctions. Gap junctions exclude the movement of molecules with a molecular weight greater
than approximately 1.5 kDa, abrogating their role in the movement of large macromolecules
such as crystallins. However, several lines of evidence point to an as yet unknown
mechanism(s) by which large molecular weight molecules, including green fluorescent

protein (GFP), albumin and alpha-crystallin can move intralenticularly.
Shestopalov and Bassnett [18] utilized large fluorescent proteins to demonstrate the
presence of a functional syncytium in the developing chick lens nucleus. Autofluorescent
proteins initially expressed within a single fiber cell in the lens nucleus traveled to adjacent
fiber cells during embryonic development creating a diffuse pattern of fluorescence in the
lens nucleus. These data, obtained by two-photon microscopy, suggest a previously
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Copyright © 2012. Nova Science Publishers, Inc. All rights reserved. May not be reproduced in any form without permission from the publisher, except fair uses permitted under Lens Plasticity and Intralenticular Translocation of Crystallins 217
undescribed cell-cell membrane fusion pathway for the movement of macromolecules from
one fiber cell to another.
Bassnett and colleagues extended their findings in the chick to mammals using two
different transgenic mouse models that express GFP, either constitutively[19], or upon
induction with tamoxifen.[20,21] Using the TgN(GFPU)5Nagy mouse strain, constitutively
expressing GFP, Shestopalov and Bassnett [19] demonstrated the establishment of a
macromolecular permeable pathway in the lens nucleus during development, confirming the
presence of a functional syncytium in the mammalian lens similar to that observed in the
avian lens. This pathway was termed the large molecule diffusion pathway (LMDP) and
appears to operate in parallel with gap-junction-mediated cell-to-cell communication.
Utilizing a tamoxifen inducible transgenic mouse strain (Cre-ERTM;Z/EG), Bassnett‘s group
[21] was able to analyze the LMDP in a temporally regulated manner in adult animals. Here
they were able to confirm the intralenticular movement of the heterologous protein, GFP,
among mature and differentiating fiber cells of the lens cortex, but not between the cortex
and the lens nucleus. This is consistent with radiocarbon dating demonstrating that the
proteins in the nucleus of the human lens are as old as the individual [13] indicating the
absence of movement of lens proteins between the cortex and the nucleus. Additionally, they
identified the claudin-like membrane protein, Lim2, as being necessary for LMDP
operation.[20,21] By utilizing these genetic models the authors were able to demonstrate the
intralenticular movement of a macromolecule, GFP, without perturbing the lens by injection
of a fluorophore or other tracking molecule.
Ex vivo experiments, utilizing whole rat lens in culture, demonstrated that alpha-
crystallin, a large macromolecular complex (600-900 kDa), after uptake by lens epithelial
cells, could pass into superficial cortical lens fiber cells and subsequently into deeper layers
of the lens cortex.[22] Movement of a molecule this size could not be mediated by gap
junctions but instead might be facilitated by regions of fiber cell/fiber cell membrane fusions
similar to those observed by Bassnett and colleagues.[18,20] The observation that large
molecular weight proteins could pass intralenticularly in culture was extended by Sabah and
colleagues [23] who showed, in vivo, that alexa-conjugated albumin could pass between fiber
cells of the intact rat lens. These data, taken together, suggest that lens homeostasis is
maintained by a robust system that allows for the intralenticular passage of preexisting and/or
newly synthesized lens proteins including alpha-crystallin or other repair enzymes.
We propose a model in which the protein constitution of differentiated lens fiber cells is
not static, but is instead present in a dynamic environment where newly synthesized and/or
preexisting crystallin molecules can move intralenticularly to areas of protein damage. This
would complement and extend the hypothesis put forth by Horwitz [17] that the chaperone
activity of alpha-crystallin could resolve inappropriate condensation events. Upon insult to

the young lens (< 40 years), local damage to crystallin proteins in the lens cortex or nucleus
would be resolved by translocation of crystallins from adjacent regions of the lens to sites of
damage (Figure 1, left column); concomitantly, damaged proteins would be moving to other
regions of the lens diluting out the changes in protein density of the affected area and the rest
of the lens. Therefore, changes in refractive index would be transient and not result in
cataract. Note that in this scenario lens proteins are able to move within the nucleus as not all
crystallins have been consumed.[24] This proposed mechanism of dilution of damaged
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proteins is not mutually exclusive from the proposed in vivo chaperone activity of alpha-
crystallin, but instead could work in tandem. Translocated alpha-crystallin to the insult site
could interact with damaged proteins to prevent condensation, conversely, damaged proteins
that have moved out into unaffected areas of the lens could interact with alpha-crystallins at
the arrival point.
By contrast, in the aged lens (Figure 1, right column), upon insult, protein redistribution
would not occur to an extent capable of diluting out damaged protein. Coupled with the fact
that by age 40-50 years alpha-crystallin is unavailable for binding to newly damaged
crystallin molecules, inappropriate condensation of crystallin protein leading to cataract
formation would occur. It is worth noting that in our model of the aged lens not only is the
movement of protein within the nucleus inhibited leading to nuclear cataract, but we
hypothesize that selected regions of the lens cortex are also refractory to the translocation of
crystallin protein possibly producing cortical punctate opacifications.
Though we limited our discussion to crystallin proteins, the model outlined above would
also accommodate other proteins such as repair enzymes (e.g. superoxide dismutase,
glutathione peroxidase, glutathione reductase). Nevertheless, we envision several lines of
experimental evidence that would support our hypothesis. First, a systematic microscopic
analysis of lens fiber cell membrane structure should be done to demonstrate the presence of
membrane structures able to facilitate passage of large molecular weight complexes. Our
model is partially supported by the work of Shestopalov and Bassnett [18]; these workers,
using confocal microscopy, created an image stack demonstrating a 30 μm septum between
adjacent lens fiber cells of the chick lens nucleus. Due to technical limitations these authors
were unable to observe cell fusion of less than 5 μm. Nevertheless, an opening of at least 0.5
μm in diameter would be sufficient to accommodate passage of any known crystallin or
crystallin complex.[25] However, a caveat with using microscopy is the difficulty in
observing ephemeral structures such as those that might be induced by the temporal insult we
have proposed. Nonetheless, we would predict that structures large enough to accommodate
translocation of crystallin molecules between lens fiber cells would be either unique, or
present in greater numbers in young lenses versus aged lenses.
As briefly described above, movement of protein from fiber cell to fiber cell has been
demonstrated ex vivo in a whole rat lens cell culture model and in vivo in the developing
chick and mouse lens and adult rat and mouse lens.[18-20,22,23] Each of the above
mentioned studies has provided important proof-of-principle evidence that proteins can
translocate intralenticularly in the vertebrate lens. We propose that these studies be extended
to other vertebrate species that more closely approximate the human lens. Protein
translocation in young lenses, but not aged lenses, would provide further evidence that this
process could be of physiologic importance in the human lens. Identification of membrane

structures large enough to accommodate crystallin molecules, coupled with data from other
lens systems demonstrating intralenticular passage of high molecular weight proteins in the
young lens, but not the aged lens, would provide support for our model of lens plasticity.
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Figure 1. Role of intralenticular movement of crystallin on resolving cataract. Left column is a young lens (<
40 years) with insult represented by a lightning bolt (top panel). Middle panel, potential cataract is
represented by filled dark red circles, with movement of crystallin proteins indicated by solid arrows. Solid
circle shows that nucleus of lens is closed to movement of proteins. Bottom panel demonstrates the
resolution of inappropriate condensation events in the young lens. Right column depicts an aged lens
undergoing insult (top panel) with subsequent cataract formation. Movement of crystallin molecules into and
out of cataract is impeded (middle panel, solid arrows with X). Bottom panel demonstrates the eventual
formation of lens opacities (filled dark red circles) in lenticular regions lacking plasticity, including the
nucleus (medium red).
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Acknowledgment
This work was supported by a grant from the NIH to L.J.T.
References
[1] Bloemendal H. The vertebrate eye lens. Science 1977;197:127-138.

[2] Bettelheim FA, Siew EL. Effect of change in concentration upon lens turbidity as
predicted by the random fluctuation theory. Biophys J. 1983;41:29-33.
[3] Delaye M, Tardieu A. Short-range order of crystallin proteins account for eye lens
transparency. Nature 1983;302:415-417.
[4] Ponce A, Sorensen C, Takemoto L. Role of short-range protein interactions in lens
opacifications. Mol Vis. 2006;12:879-884.
[5] Takemoto L, Sorensen CM. Protein-protein interactions and lens transparency. Exp
Eye Res. 2008;87:496-501.
[6] World Health Organization. Visual impairment and blindness. Fact Sheet No282. 2009
[7] Wang S-S, Wu JW, Yamamoto S, Liu H-S. Diseases of protein aggregation and the
hunt for potential pharmacological agents. Biotechnol. J. 2008;3:165-192.
[8] Andley UP. Crystallins and hereditary cataracts: molecular mechanisms and potential
for therapy. Expert Rev Mol Med. 2006;8:1-19.
[9] Horwitz J. Alpha-crystallin. Exp Eye Res. 2003;76:145-153.
[10] Benedek GB. Theory of transparency of the eye. Appl Opt. 1971;10:459-473.
[11] Benedek GB. Cataract as a protein condensation disease. Invest Ophthalmol Vis Sci.
1997;38:1911-1921.
[12] Horwitz J. The function of alpha-crystallin. Invest Ophthalmol Vis Sci. 1993;34:10-22.
[13] Lynnerup N, Kjeldsen H, Heegaard S, Jacobsen C, Heinemeier J. Radiocarbon dating
of the human eye lens crystallines reveal proteins without carbon turnover throughout
life. PLoS ONE 2008;1:e1529.
[14] Takemoto L. Quantitation of C-terminal modification of alpha-A crystallin during
aging of the human lens. Exp Eye Res. 1995;60:721-724.
[15] Takemoto L, Gopalakrishnan S. Alpha-A crystallin: Quantitation of C-terminal
modification during lens aging. Curr Eye Res. 1994;13:879-883.
[16] Takemoto L. Differential phosphorylation of alpha-A crystallin in human lens of
different age. Exp Eye Res. 1996;62:499-504.
Horwitz J. α-Crystallin can function as a molecular chaperone. Proc Natl Acad Sci
[17]
USA 1992;89:10449-10453.
[18] Shestopalov VI, Bassnett S. Expression of autofluorescent proteins reveals a novel
protein permeable pathway between cells in the lens core. J Cell Sci. 2000;113:1913-
1921.
[19] Shestopalov VI, Bassnett S. Development of a macromolecular diffusion pathway in
the lens. J Cell Sci. 2003;116:4191-4199.
[20] Shi Y, Barton K, De Maria A, Petrash JM, Shiels A, Bassnett S. The stratified
syncytium of the vertebrate lens. J Cell Sci. 2009;122:1607-1615.
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[21] Shi Y, Bassnett S. Inducible gene expression in the lens using tamoxifen and a GFP
reporter. Exp Eye Res. 2007;85:732-737.
[22] Boyle DL, Carman P, Takemoto L. Translocation of macromolecules into whole rat
lenses in culture. Mol Vis. 2002;8:226-234.
[23] Sabah JR, Davidson H, McConkey EN, Takemoto L. In vivo passage of albumin from
the aqueous humor into the lens. Mol Vis. 2004;10:254-259.
[24] Friedrich MG, Truscott RJW. Membrane association of proteins in the aging human
lens: Profound changes take place in the fifth decade of life. Invest Ophthalmol Vis Sci.
2009;50:4786-4793.
[25] Harding JJ, Crabbe MJC. The lens: Development, proteins, metabolism and cataract.
In: Davson H, editor. The Eye. Vol IB. Orlando: Academic Press; 1984:201-492.
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Index
Alaska Natives, 73
A
albumin, 96, 211, 216, 217, 221
alcohol abuse, 82
Abnormal fibroblasts, vii, 2
alcoholism, 80
access, 82, 192
algorithm, 192, 195, 196, 200
accommodation, 60, 68, 128, 149, 155
allele, 7
accounting, 98, 104
allergic reaction, 75
acetylation, 216
alpha-tocopherol, 110
acetylcholine, 143, 145, 151, 154
alters, 60, 154
acid, 13, 23, 70, 96, 98, 99, 109, 113, 140, 142, 152,
Amazon Rain Forest, 31
155, 160, 165, 185, 186, 195, 203
amblyopia, 55, 67
acidic, xi, 191, 202
ametropia, 138, 152
acidosis, 104
amine, 155
acute infection, 83
amino, 203, 208, 213
adaptation, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,
amino acid, 203, 208, 213
66, 67, 68, 94, 98
aminoglycosides, 77
adenine, 25
amplitude, 60, 122
adhesion, vii, 1, 2, 18, 19, 79, 85, 144, 164, 174, 175
amylase, 206
adhesions, 208
anatomy, 161
adhesives, viii, 2, 3
angiogenesis, 3, 7, 9, 18, 98, 108, 114, 210
adipose, 98, 100, 111
anhydrase, 101, 112
adipose tissue, 100, 111
antagonism, 139, 214
adjunctive therapy, viii, 2, 3
antibiotic, viii, 69, 70, 71, 75, 80, 81, 82, 83, 84, 86,
adjustment, 52, 91
87, 88, 89
adults, viii, 48, 66, 78, 91, 93, 94, 211, 214
antibiotic resistance, 82, 86
advancement, 168
antibody, 8, 200
adverse effects, 84
antigen, 12
aetiology, 148
anti-inflammatory agents, 165
age, xi, 4, 6, 8, 13, 24, 25, 55, 57, 74, 75, 77, 82, 87,
anti-inflammatory medications, 13
148, 158, 159, 173, 178, 201, 210, 213, 214, 215,
antioxidant, 104, 198, 207
216, 218, 220
anti-VEGF treatment, viii, 2, 8
aggregation, xi, 207, 215, 216, 220
apex, 37, 38, 39, 41, 42, 50
agonist, 140, 150
aplastic anemia, 84
agriculture, 5
apoptosis, vii, 2, 9, 14, 21, 22, 101, 166, 180, 181,
alanine, 20
182, 186, 208, 209
Alaska, 73
apoptotic pathways, 182
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Copyright © 2012. Nova Science Publishers, Inc. All rights reserved. May not be reproduced in any form without permission from the publisher, except fair uses permitted under 224 Index
aqueous humor, 207, 213, 221 biomarkers, 202, 212

ARC, 57, 58, 59 biomaterials, 85
arrest, 94, 100 biomechanics, 128
arteries, 81 biopsy, 77
artery, 80 biosynthesis, 104
arthritis, 83 birth weight, 76
ascorbic acid, ix, 94, 103, 104, 113, 114 blepharitis, 76, 77, 78, 82
Asian countries, 71 blindness, viii, xi, 76, 93, 94, 158, 178, 215, 216,
assessment, xi, 3, 5, 12, 116, 118, 174, 180, 191 220
astigmatism, vii, 1, 5, 48, 120, 121, 132, 138 blood, x, 6, 14, 18, 76, 80, 81, 82, 94, 95, 101, 102,
astrocytes, x, 191, 192, 196, 197, 199, 201, 202 103, 104, 107, 112, 113, 157, 158, 160, 162, 163,
astrogliosis, xi, 191 183, 196, 199, 206, 210
asymmetry, 120 blood circulation, 76, 80
asymptomatic, 74, 79 blood flow, 14
athletes, 84 blood pressure, 107
atmospheric pressure, 128 blood retinal barrier (BRB), x, 104, 157
ATP, 24, 96, 99, 104, 210 blood vessels, 6, 196, 199, 210
atrophy, 210 blood-brain barrier, 101
attachment, 79 bloodstream, 70, 81
autoantibodies, 206 body fat, 100, 111
autoimmune diseases, 77 body fluid, 95, 96, 102, 103
autosomal dominant, 5 body weight, 100
autosomal recessive, 99, 110 bone, 20, 145, 185, 189
avian, 217 bone marrow, 185, 189
axons, 182 bradykinin, 206
brain, x, 80, 81, 113, 178, 180, 189, 191, 192, 193,
198, 201, 203
B
brain abscess, 80
brainstem, 60
backscattering, 135
breakdown, 112, 160, 163
bacteria, viii, 69, 74, 75, 78, 79, 87, 89, 90
breast cancer, 206, 212
bacterial conjunctivitis, 74, 75, 87
breathing, 192
bacterial infection, 81
bacterial strains, 75
Barbados, 4, 5, 30 C
barriers, 113
basal cell carcinoma, 14 cachexia, 100, 111
basal lamina, 16 Cairo, 37, 67
basal layer, 3 calcium, viii, 2, 9, 22
base, 37, 38, 39, 41, 42, 46, 47, 48, 53, 57, 58, 59, cancer, 73, 80, 100, 111, 172
60, 63, 64, 65, 201, 202 cancerous cells, 24
Beijing, vii, 1 candidates, ix, 56, 94, 95, 96, 106, 108, 121, 201
Belgium, 159, 192 capsule, 117

bending, 129 carbohydrates, 102
beneficial effect, 53, 55 carbon, 220
benefits, 55 carcinoma, 19
benign, 80 cartilage, 153
biochemical processes, 102, 103 casein, 10
bioinformatics, 95 cataract, 13, 73, 77, 78, 81, 82, 89, 90, 91, 132, 135,
biological processes, 162 207, 213, 215, 216, 217, 218, 219, 221
biological systems, 106, 107 cataract extraction, 77
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Copyright © 2012. Nova Science Publishers, Inc. All rights reserved. May not be reproduced in any form without permission from the publisher, except fair uses permitted under Index 225
catecholamines, 140 clinical application, 119

catheter, 83 clinical examination, 75
caucasians, 19, 147 clinical presentation, 76, 82, 90
causal relationship, 143 clinical trials, 25
cauterization, 78 clone, 20, 72, 87, 172
cavernous sinus thrombosis, 80, 83 clothing, 71
cDNA, 20 cocaine, 77
cell adhesion, vii, 1, 18, 208 coding, 20
cell culture, 12, 21, 178, 218 codon, 151
cell cycle, vii, 2, 21, 100 coenzyme, 3, 24, 25
cell death, 9, 21, 22, 102, 113, 179, 182, 185, 186, coherence, 122, 126, 127, 133, 135, 136
187, 188, 208, 213, 214 collagen, ix, 4, 12, 18, 20, 22, 115, 130, 139, 144,
cell division, 21 145, 148, 160, 164, 166, 168, 169, 170, 171, 172,
cell fate, 184, 185, 188 173, 175
cell fusion, 218 colon, 19
cell line, 22, 164, 170, 183 colonization, 73, 79, 88
cell migration, vii, x, 1, 20, 22, 157, 171 color, 118, 119
cell signaling, 18 combination therapy, 168
cell surface, 22 commercial, 120
cellulitis, 74, 77, 80, 81, 83, 90 communication, 216, 217
central nervous system (CNS), x, 9, 35, 82, 140, 178, community, viii, 69, 72, 73, 76, 80, 81, 82, 84, 86,
191, 192, 198, 201, 203 87, 88, 90, 91
cerebellum, 60, 68 comparative analysis, 213
cerebral cortex, 60 compartment syndrome, 80
cerebral palsy, 78 compensation, 58, 66, 68, 132, 135
challenges, 138 complement, ix, 93, 100, 111, 209, 217
chaperones, 209 complexity, 106
chemicals, x, 167, 191, 205 compliance, 79
chemokines, 14 complications, viii, 2, 3, 75, 78, 80, 83, 107, 113,
chemosis, 74, 80 121, 130, 162, 163, 166, 168
chemotaxis, 22, 165, 173 composition, 4, 140, 179, 206
chicken, 140, 141, 145, 148, 152 compounds, xi, 6, 191, 198, 201, 203
childhood, 66, 90 compression, 80, 122
children, 48, 66, 67, 73, 77, 78, 81, 87, 89, 99, 138, computer, 132
151, 152 computing, 116
China, 4, 5, 30, 34, 88 condensation, 217, 218, 219, 220
cholesterol, 24, 99, 110 conduction, 77
choroid, x, 95, 137, 139, 141, 142, 143, 144, 149, conjunctiva, vii, 1, 2, 4, 7, 12, 13, 18, 20, 21, 22, 23,
152, 173, 210 24, 74, 75, 82
chromatographic technique, 211 conjunctivitis, 74, 75, 76, 82, 83, 86, 87, 88, 206
chromatography, 96, 112 connective tissue, 142, 162, 174
chromosome, 5, 22, 71 consumption, 105

chronic illness, 80 contamination, 79, 89, 95
cigarette smoke, 6, 24 contracture, 65
circadian rhythm, 144, 153 contradiction, 139, 146
circulation, 105 contrast sensitivity, 140
civilization, 149 control group, 13, 106
classification, 103, 174 controlled studies, 8
cleaning, 48, 72, 84 controversial, 11
cleavage, 9 convergence, 59, 61, 68
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cornea, vii, ix, 1, 2, 3, 4, 5, 6, 9, 12, 13, 14, 74, 75, dehydration, 192
76, 77, 78, 79, 115, 116, 118, 119, 120, 121, 122, dendritic cell, 3
123, 124, 125, 126, 127, 128, 129, 130, 132, 134, deposition, 15, 100, 162
135, 136, 192, 205 deposits, 210
corneal laceration, 78 deprivation, 138, 149, 150, 151, 152, 153, 154, 155
corneal transplant, 24, 78 depth, 103, 117, 119, 122, 125, 126
corneal ulcer, 76, 77, 81, 88 derivatives, 166
correlation, 139, 179, 202 dermatology, 173
cortex, 60, 217, 218 desorption, 96
cortical neurons, 189 destruction, 4, 9, 16, 18, 206
corticosteroids, 102 detachment, 20, 158, 159, 160, 163, 172
cost, x, 55, 56, 72, 102, 122, 177, 200 detectable, 22, 95, 122, 141
covering, 49, 206 detection, xi, 7, 14, 16, 58, 103, 120, 139, 191, 196
creep, 129 developed countries, 76, 77, 158
crown, 46 deviation, viii, 37, 39, 42, 43, 44, 45, 48, 49, 50, 51,
crystalline, 101, 102, 116, 117 52, 53, 54, 55, 56, 57, 58, 60, 63, 120
crystals, 167 diabetes, ix, 62, 73, 77, 80, 82, 94, 100, 104, 107,
culture, x, 11, 73, 83, 99, 110, 142, 166, 177, 179, 108, 110, 111, 113, 208, 209, 214, 216
181, 183, 184, 185, 187, 188, 189, 208, 217, 221 diabetic nephropathy, 100
culture conditions, 179 diabetic patients, ix, 93, 94, 95, 96, 98, 100, 101,
culture medium, 179 105, 106, 110, 114, 150
cure, 61, 64, 65, 168 diabetic retinopathy, vii, xi, 93, 94, 95, 96, 98, 100,
cycling, 2, 20 101, 107, 108, 109, 110, 111, 112, 113, 153, 158,
cyclooxygenase, 160 174, 182, 191, 201, 204, 205, 208, 209, 214
cysteine, 9 diabetic retinopathy (DR), xi, 191
cytochrome, 6, 210 diacylglycerol, 165
cytokines, 2, 14, 18, 19, 102, 158, 171, 183 diagnostic criteria, 206
cytology, 3 dialysis, 73
cytometry, 21 diaphragm, 125
cytoplasm, 23 diet, 106
cytotoxicity, 166, 172 differential diagnosis, 80
diffraction, 48
diffusion, 95, 217, 220
D
diplopia, 53, 55, 59, 61, 62, 63, 64, 65
direct measure, 116, 121
dacryocystitis, 74, 77, 80, 82, 83, 87, 91
discomfort, 13
damages, 201, 207
discriminant analysis, 103
data analysis, 129
disease model, 198, 204
data set, 103
diseases, viii, x, xi, 7, 69, 77, 78, 88, 119, 122, 128,
deaths, 72, 86
141, 149, 158, 159, 163, 167, 171, 173, 178, 191,
decay, 155
201, 205, 206, 210
decolonization, 73
disinfection, 72, 79
decomposition, 131
disorder, vii, 2, 16, 17, 165, 198, 200
deconvolution, 194, 195
dispersion, 48, 135
defects, vii, 1, 2, 5, 17, 20, 76, 207
displacement, 38, 59, 123, 124
defence, 77
dissociation, 51
deficiency, 6, 77, 100, 110, 111, 206
distortions, 47, 48, 125
deficit, 99, 102, 105
distribution, 68, 120, 216
deformation, 129
divergence, 60, 68
degenerate, 178, 179, 181
DNA, vii, 2, 5, 6, 19, 21, 31, 33, 71, 148, 173
degradation, 4, 12, 14, 95, 207, 216
PHILIPPINES
Account: ns004409
DNA damage, 21 endothelium, 124, 173

DNA repair, 5, 6 energy, 25, 138, 209
dominance, 146 England, 86
donors, 98, 99 enlargement, 149, 152
dopamine, 105, 140, 143, 144, 149, 150, 151, 152, environment, xi, 6, 75, 78, 79, 84, 128, 138, 167,
153, 154, 155, 198 180, 215, 217
dopamine agonist, 144 environmental change, 71
dopaminergic, ix, 137, 140, 144, 198, 202 environmental conditions, viii, 69
dosage, 56, 91 environmental factors, 19, 138
dosing, 198, 199, 201 enzyme, 10, 11, 12, 14, 24, 25, 104, 106, 143, 155,
down-regulation, 21, 100 160, 165, 166, 183, 211
drainage, 83 enzyme-linked immunosorbent assay, 10
drawing, 129, 181 enzymes, 9, 12, 13, 22, 23, 24, 110, 140, 173, 217,
drug abuse, 80 218
drug delivery, 167 epidemic, 75, 78, 85, 86
drug screenings, 185 epidemiology, 2, 4, 19, 21, 85, 87, 88
drugs, viii, x, 2, 84, 165, 166, 168, 171, 179, 185, epigenetics, 12, 19
191, 200 epiretinal membrane, 158, 160, 166, 167, 169, 174
epithelia, 16
epithelial cells, viii, 2, 3, 4, 7, 11, 14, 15, 16, 18, 19,
E
21, 22, 23, 85, 98, 110, 154, 158, 165, 169, 170,
171, 172, 175, 207, 217
East Asia, 147
epithelial mesenchymal transition, vii, 2, 6, 15, 162,
E-cadherin, vii, 1, 18, 19
163
ECM, 9, 18
epithelium, vii, x, 2, 6, 10, 14, 16, 18, 21, 22, 79,
ECM degradation, 18
108, 109, 110, 123, 124, 134, 137, 139, 149, 151,
edema, ix, 13, 75, 76, 79, 80, 82, 83, 94, 101, 108,
153, 159, 168, 169, 170, 172, 173, 174, 178, 180,
122
181, 186, 187, 188, 209, 210, 213, 214
editors, 88
EPR, 142
education, 24
equipment, 71
Egypt, 37, 68
erythrocyte sedimentation rate, 80
elastin, 18
ESI, 22
electromagnetic, 122
esotropia, 39, 41, 42, 46, 49, 52, 53, 55, 56, 59, 60,
electron, 179
61, 62, 66, 67, 68
electrophoresis, 72, 95, 96, 107, 108, 211, 213, 214
ESR, 80
ELISA, 10, 14, 96
ester, 24, 152
elongation, 129, 138, 139, 140, 142, 146, 147, 150,
ethmoiditis, 80
152
ethnic groups, 73, 138
elucidation, x, 157
Europe, 71, 72, 85
e-mail, 93
evaporation, 206
embryonic stem cells, 185, 189
evidence, viii, x, 2, 8, 9, 13, 18, 23, 24, 98, 100, 138,
emergency, 76
150, 154, 157, 165, 202, 216, 218

emission, 192
evolution, 86, 87, 94, 146, 158
employees, 88
examinations, 8
employment, 166
excision, 2, 5, 8, 22, 25, 78, 89
encephalopathy, 78
excitation, 192
encoding, 71, 98
excitotoxicity, 182
endocarditis, 70, 83
exercise, 12
endocardium, 82
exotoxins, 70, 74, 75, 76
endothelial cells, 7, 14, 102, 109, 124, 150, 163, 210,
exotropia, 39, 41, 46, 49, 61, 62
214
PHILIPPINES
Account: ns004409
exposure, 4, 5, 14, 19, 21, 73, 76, 80, 81, 99, 104, fungi, 89
134, 140, 162 fusion, 49, 51, 52, 53, 54, 55, 57, 59, 154, 217
expressiveness, 115
extracellular matrix, 12, 20, 98, 140, 142, 158, 175
G
extraction, 112
extraocular muscles, 80
GABA, 208, 214
eye movement, 68
ganglion, x, 154, 177, 178, 179, 180, 181, 182, 185,
186, 188, 208, 213, 214
F gel, 72, 95, 96, 97, 107, 124, 164, 168, 169, 172,
173, 175, 211
factories, 5 gelatin zymography, 11, 12, 14
false positive, 146, 201 gene expression, vii, 2, 15, 20, 95, 100, 109, 111,
families, 76 148, 151, 152, 171, 213, 221
fat, 100 gene promoter, 23
fatty acids, 4, 99 gene transfer, 98, 185
fever, 80, 82 genes, vii, 1, 5, 6, 19, 20, 23, 24, 72, 95, 106, 144,
fiber, 18, 207, 213, 216, 217, 218 145, 146, 148, 150, 160, 162, 173, 185
fibers, 4, 18, 207 genetic background, 19, 81, 201
fibrin, viii, 2, 3, 22 genetic predisposition, vii, 1, 151, 216
fibroblast growth factor, 18, 142, 150, 153, 174 genetics, 2
fibroblast proliferation, 21 genome, viii, 69, 112
fibroblasts, vii, 2, 10, 11, 12, 15, 18, 19, 20, 21, 23, genomics, 95, 103, 112
24, 142, 160, 162, 169, 172, 175 genotype, 7
fibrosarcoma, 151 genus, viii, 69
fibrosis, 162, 163, 169 geometry, 128
fibrous tissue, 8, 22, 166 Germany, 76, 87, 127, 192
fibrovascular proliferation, vii, 1 gland, 99, 211
filtration, 103, 208 glasses, 46
fingerprints, 103, 112 glaucoma, xi, 77, 83, 122, 178, 182, 205, 207, 208,
fixation, 42, 48, 49, 51, 54, 55, 123 213, 214
flexibility, 145 glia, 183, 187, 189, 197, 198, 199, 200, 201
flight, 96, 127 glial cells, x, 158, 160, 191, 192, 196, 198, 201
floaters, 82 Glial fibrillary acidic protein (GFAP), xi, 191
flora, 91 glucagon, 139, 141, 142, 144, 145, 146, 148, 151,
fluctuations, 216 153
fluid, viii, ix, 2, 93, 95, 96, 98, 99, 100, 101, 102, glucose, ix, 3, 25, 94, 98, 103, 104, 105, 106, 107,
103, 104, 105, 106, 110, 111, 166, 169, 211, 212 113, 141, 182, 188
fluorescence, 96, 192, 194, 195, 196, 198, 199, 216 GLUT, 104
fluoroquinolones, 77, 82, 84, 88 glutamate, 181, 182, 186, 189
force, 129 glutathione, 5, 25, 154, 218
formation, vii, x, xi, 1, 2, 3, 6, 7, 12, 13, 15, 16, 17, glycolysis, 104
18, 19, 21, 22, 23, 24, 75, 76, 125, 144, 153, 157, glycoproteins, ix, 115
158, 163, 166, 167, 183, 207, 215, 218, 219 glycosaminoglycans, 143
formula, 118, 122 goblet cells, 11
Fourier analysis, 132 gold nanoparticles, 167, 171, 172
fovea, 39, 40, 41, 42, 121 grants, 123
fractures, 77 graph, 97, 129
free radicals, 25 Greece, 69, 71
fructose, 104 growth, vii, ix, x, 1, 3, 7, 11, 14, 21, 102, 110, 137,
functional architecture, 138, 148 138, 139, 140, 141, 142, 143, 144, 145, 146, 147,
PHILIPPINES
Account: ns004409
148, 149, 150, 151, 152, 153, 154, 155, 157, 158, hybrid, 202
160, 162, 164, 166, 169, 170, 171, 172, 173, 174, hybridization, 10
177, 178, 179, 180, 182, 183, 184, 185, 186, 188, hydroxyl, 3, 24
206, 211 hygiene, 72, 77, 79
growth factor, vii, ix, x, 1, 3, 7, 14, 21, 102, 110, hyperemia, 8, 74
137, 142, 145, 150, 155, 157, 158, 160, 162, 164, hyperglycemia, 104, 106, 208
166, 169, 170, 171, 172, 173, 174, 177, 178, 179, hyperinsulinemia, 141
180, 182, 183, 184, 185, 186, 188, 206, 211 hypermethylation, 19
growth rate, 139, 143 hyperopia, ix, 137, 138, 141, 145, 146, 147
guidance, 9 hyperplasia, 198
guidelines, 79, 84 hypertension, 94, 144, 216
hypertrophy, 183, 198
hypothesis, 6, 14, 18, 54, 59, 61, 106, 217, 218
H
hypoxia, 79, 98, 104, 122, 180
hysteresis, 129, 130
haptoglobin, 206
haze, 82
headache, 82 I
healing, 158, 172, 208
health, 81, 82, 121 ICAM, 26
health care, 81, 82 ideal, xi, 4, 121, 191, 192
heat shock protein, 207, 208, 209, 210 identification, 84, 95, 96, 106, 121, 154, 159, 173,
height, 121, 126, 131 213
hemodialysis, 82 IFN, 183
hemoglobin, 95 illumination, 125, 126, 179
hemorrhage, 101 image, 38, 39, 41, 42, 52, 58, 59, 66, 75, 80, 97, 115,
hepatic stellate cells, 203 116, 120, 121, 124, 125, 126, 127, 131, 138, 139,
hepatitis, 73, 77, 80 141, 144, 148, 159, 194, 195, 196, 200, 201, 202,
herpes, 78 203, 218
herpes simplex, 78 images, 39, 42, 57, 59, 97, 117, 120, 123, 125, 126,
heterogeneity, 214 127, 128, 146, 153, 192, 194, 195, 196, 197, 199,
high density lipoprotein, 110 200, 201, 202, 203
hippocampus, 186 immune response, 12, 75
history, 72, 73, 77, 78, 83, 171, 174 immune system, 76, 78
HIV, 73, 77, 82 immunity, 75
HLA, 12, 26, 29, 33 immunofluorescence, 16
homeostasis, 24, 128, 183, 217 immunoglobulin, 206
Hong Kong, 71, 89, 138, 150 immunohistochemistry, 7, 10, 11, 13, 21, 198
hordeolum, 80 immunoreactivity, 11
hospitalization, 72, 73, 82 immunosuppression, 82, 83
host, 81, 178, 188 immunosuppressive agent, 82
human, vii, viii, ix, x, xi, 1, 2, 4, 5, 10, 12, 14, 24, impetigo, 80, 81
69, 85, 93, 94, 96, 98, 99, 100, 102, 104, 107, impregnation, 90
108, 111, 112, 133, 135, 143, 147, 149, 150, 151, improvements, 127, 178, 201
153, 154, 164, 166, 167, 168, 169, 170, 171, 172, impulses, 60, 178
174, 177, 181, 182, 184, 185, 187, 191, 204, 206, in situ hybridization, 10
207, 208, 209, 210, 211, 213, 214, 215, 217, 218, in vitro, x, 4, 11, 102, 111, 153, 154, 160, 163, 166,
220, 221 167, 169, 170, 171, 172, 177, 178, 179, 180, 181,
human body, 167 182, 183, 184, 185, 186, 187, 188, 208, 213
human ocular surface disease, vii, 1 in vitro environment, x, 177
Hunter, 111
PHILIPPINES
Account: ns004409
in vivo, x, 3, 4, 95, 100, 103, 111, 112, 129, 135, intraocular, ix, 77, 80, 81, 82, 84, 94, 95, 100, 115,
153, 166, 172, 177, 178, 179, 180, 183, 184, 185, 128, 135, 158, 174, 182, 187, 207, 210
187, 192, 194, 195, 200, 201, 202, 203, 204, 217, intraocular pressure, ix, 80, 115, 128, 182, 207
218 intravenous antibiotics, 81
incidence, 71, 72, 73, 78, 79, 81, 88, 107, 138, 158, intravenously, 83
162, 174, 216 in-vitro evidence, viii, 2
indentation, 129, 133 iodine, 75, 82, 84
India, 88, 157 ionization, 96, 108, 211
individuals, viii, 19, 61, 69, 80, 101, 103 ipsilateral, 60, 61, 65
Indonesia, 4, 5, 28 IRC, 170
inducible protein, 155, 206 iris, 82, 185
induction, 14, 23, 145, 146, 151, 164, 217 irradiation, 14, 78
infancy, 88 irrigation, 82
infants, 48, 75, 86, 88 ischemia, 187
infection, viii, 69, 71, 72, 73, 74, 75, 76, 77, 78, 79, Islam, 29, 155
80, 81, 82, 83, 84, 85, 87, 88, 89, 91 isolation, 72, 82, 84, 85, 86
inflammation, 2, 9, 11, 12, 13, 14, 18, 21, 70, 76, 80, isozyme, 210
82, 98, 100, 104, 111, 113, 163 issues, 18, 23, 173, 178
inflammatory cells, 3, 11, 74, 158, 160 Italy, 71
inflammatory disease, 206
inflammatory mediators, 160
J
inflammatory responses, vii, 2
inheritance, 5
Japan, 56, 71, 126, 205
inhibition, 9, 11, 19, 101, 105, 138, 144, 145, 155,
justification, 159
174
inhibitor, 9, 14, 22, 23, 71, 108, 144, 152, 168, 180,
185 K
injections, 8, 68, 140, 150, 165
injuries, 169, 172, 174, 178, 179, 182, 183, 185 keratinocytes, 175
injury, x, 80, 81, 102, 151, 157, 174, 177, 178, 183, keratoconjunctivitis, 77, 78, 86
188, 191 keratoplasty, 77, 125
inmates, 81 kidneys, 111
inositol, 165, 173
insulin, 3, 21, 99, 107, 110, 111, 139, 140, 141, 142, L
153, 154, 155, 183, 188, 214
insulin resistance, 111, 188 lack of control, 8
insulin signaling, 153 lactate dehydrogenase, 206
integration, 184, 185 lactate level, 104
integrin, 19, 164, 169, 171, 172, 175 lactoferrin, 206
integrins, 169, 171, 173 L-arginine, 144, 152
integrity, 60, 99, 178 lasers, 126
intensity values, 194 Latin America, 70, 71, 72, 85

intensive care unit, 88 lattices, 171
interface, 115, 172 layering, 178, 181
interference, 126, 127 LDL, 3, 24
interleukin-8, 184 lead, 13, 14, 18, 21, 56, 58, 70, 74, 76, 77, 78, 79,
interneuron, 178 80, 83, 95, 118, 139, 141, 144, 168, 180, 185,
interneurons, 182 201, 216
interstitial retinol-binding protein (IRBP), ix, 93 leakage, 98
intervention, 2, 201 learning, 155
PHILIPPINES
Account: ns004409
LED, 117 mammals, 141, 142, 144, 217

lens, vii, xi, 45, 46, 47, 77, 78, 79, 89, 90, 116, 122, management, vii, viii, 8, 56, 57, 66, 67, 69, 75, 86,
124, 125, 132, 134, 138, 146, 147, 148, 149, 180, 107, 130, 169, 172, 200
192, 207, 213, 215, 216, 217, 218, 219, 220, 221 mapping, 107, 131, 134
lesions, 4, 5, 12, 13, 68, 77, 101, 113 mass, 95, 101, 103, 107, 108, 206, 207, 211, 212
leucocyte, 12 mass spectrometry, 95, 103, 107, 108, 206, 211, 212
lifetime, 5, 216 mast cells, 4
ligand, 200 materials, 79, 129
light, vii, viii, ix, xi, 1, 5, 16, 18, 25, 37, 38, 39, 45, matrix, vii, ix, 1, 2, 3, 5, 6, 9, 14, 15, 19, 22, 96, 98,
48, 63, 65, 83, 95, 98, 99, 104, 113, 115, 116, 110, 115, 142, 145, 150, 155, 162, 164, 168, 175,
117, 120, 123, 125, 126, 127, 135, 137, 138, 140, 180, 206, 211
148, 149, 159, 179, 187, 189, 192, 215, 216 matrix metalloproteinase (MMPs), vii, 1, 2, 3, 9, 11,
light conditions, ix, 137 12, 13, 14, 16, 18, 110, 145, 150, 155, 168, 180,
light scattering, xi, 215, 216 211
light transmission, 125, 216 matrix reorganization, vii, 1
light-emitting diodes, 123 matter, x, 137
limbal basal epithelial stem cell disorder, vii, 2 maxillary sinus, 80
linear function, 130 MCP-1, 183
linear model, 103 measurement, 44, 45, 51, 66, 116, 118, 119, 120,
lipid peroxidation, 100, 110 121, 123, 125, 127, 128, 129, 130, 133, 135, 136,
lipids, 11, 99, 102, 205 148, 192
lipolysis, 111 measurements, 51, 56, 113, 118, 119, 123, 125, 126,
lipoproteins, 70, 110 127, 133, 135, 180, 197
liquid chromatography, 103, 211 mechanical properties, 130
liver, 24, 80, 82, 100, 111 mechanical stress, 79
liver disease, 80 media, 12
localization, 16, 17, 23, 98, 108, 151, 154, 192 medical, 72, 73, 77, 82, 89, 159, 200
locus, 152 medical history, 73
longevity, 216 medication, 13
low-density lipoprotein, 24, 110 medicine, 107
lying, 45, 123 melanin, 159
lymphocytes, 4, 13, 76 melanoma, 184, 186
lysis, 101 melatonin, 144, 153
lysozyme, 206, 212 mellitus, 110, 208
membranes, 7, 46, 74, 158, 165, 173, 183
memory, 140, 155
M
memory processes, 140
meningitis, 70, 80, 83
machinery, 107
Mercury, 125
macromolecules, 216, 217, 221
mesangial cells, 102, 112
macular degeneration, xi, 8, 159, 173, 178, 205, 210,
mesenchymal stem cells, 184
214
messengers, 165
magnetic resonance, 112, 113, 153
meta-analysis, 3
magnetic resonance imaging (MRI), 153, 200
metabolic, 112
magnetic resonance spectroscopy, 112
metabolic pathways, 24
magnitude, 101, 129
metabolism, 23, 24, 25, 98, 102, 104, 106, 141, 148,
major histocompatibility complex, 111
149, 152, 209, 221
majority, 70, 72, 77, 80, 209
metabolites, ix, 6, 93, 95, 102, 103, 104, 140, 159
malaise, 80
metabolome, 102, 112
malignancy, 82, 172
metalloproteinase, 5
mammal, 141, 152
PHILIPPINES
Account: ns004409
meter, 38 mortality, viii, 69, 70, 82

methodology, 122, 143 mortality rate, 83
methylation, 19 mosaic, 124
methylcellulose, 123 mRNA, viii, ix, 2, 11, 18, 24, 93, 98, 99, 141, 143,
MHC, 100 148, 155, 181, 188
mice, 4, 98, 99, 100, 102, 109, 111, 144, 148, 153, mucin, 15, 21, 206, 212
192, 193, 195, 196, 197, 198, 199, 200, 201, 202, mucus, 11
203, 207, 208, 209, 213 multidimensional, 213
microcirculation, 104 multiple sclerosis (MS), xi, 191
microenvironments, 4 muscarinic receptor, 143, 149, 151, 152
micrometer, 124 muscles, 128
microorganisms, 79 mutation, 99, 109
microscope, 123, 124, 125, 126 mutations, 112
microscopy, 3, 122, 124, 125, 126, 135, 216, 218 myofibroblasts, 142
midbrain, 60, 68 myopia, ix, 132, 137, 138, 140, 141, 142, 143, 144,
migration, vii, x, 1, 11, 18, 20, 22, 23, 157, 164, 166, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154,
167, 171, 173, 184, 185 155
military, 81
miosis, 59
N
misuse, 72, 84
mitochondria, 210
nanoparticles, 167, 168, 171
mitogen, 163
nasopharynx, 76
mitomycin C (MMC), viii, 2, 3
Native Americans, 73
MMP, 5, 9, 10, 11, 12, 14, 16, 23, 27, 28, 30, 32, 33,
necrosis, 82
35, 145, 211
necrotizing fasciitis, 72, 73
MMP-2, 9, 10, 12, 14, 16, 35
neonates, 75, 76, 86, 88
MMP-3, 9, 10, 11, 12, 14
neovascularization, 98, 105, 173, 208, 209, 210
MMP-9, 10, 11, 12, 14, 16, 28, 30, 35
nephroblastoma, 173
model system, x, 177, 178, 180
nephrotic syndrome, 102
models, ix, x, 11, 15, 93, 94, 99, 141, 142, 151, 154,
nerve, 3, 61, 62, 63, 64, 65, 181, 207
155, 165, 172, 177, 178, 179, 180, 184, 185, 200,
nerve fibers, 207
208, 210, 217
nervous system, 178, 201
modifications, viii, 2, 124, 192, 213, 216
Netherlands, 76, 90
modulus, 129
neurodegeneration, 105, 113, 198, 202
molecular biology, 2
neurodegenerative disorders, 198
molecular mass, 96
neurogenesis, 154
molecular medicine, 107
neuroinflammation, 183
molecular weight, 12, 22, 166, 210, 216, 217, 218
neuronal apoptosis, 188
molecules, vii, 2, 3, 9, 18, 19, 22, 23, 25, 98, 100,
neurons, 60, 68, 139, 140, 143, 150, 154, 178, 183,
102, 106, 139, 143, 145, 148, 164, 165, 168, 183,
185, 187, 188, 192, 198, 200
215, 216, 217, 218, 219
neuropathy, 111
MOM, 113, 189

neuropeptides, 150
Mongolia, 4
neuroprotection, x, 177, 179, 181, 183, 184, 185, 186
monocyte chemoattractant protein (MCP), 3, 14, 23,
neuroprotective agents, 183
111, 183
neurotoxicity, x, 191, 192, 196, 197, 198, 200, 201,
monolayer, 159
202, 203, 204
Moon, 172
neurotransmitter, 141, 143, 144
morbidity, 73, 90, 216
neurotransmitters, ix, 137, 140, 143, 145, 211
morphogenesis, 16
neurotrophic factors, 183, 185, 188
morphology, 5, 124, 160, 178, 181
neutral, 24
PHILIPPINES
Account: ns004409
neutrophils, 10, 11, 163

P
New Zealand, 89
next generation, 127
Pacific, 71, 72, 73, 85
nicotinamide, 25
Pacific Islanders, 73
Nigeria, 88
paclitaxel, 165
nitric oxide, 140, 144, 152
pain, 13, 80, 82
nitric oxide synthase, 144, 152
Panama, 68
nodules, 82
parallel, ix, 11, 43, 50, 93, 124, 138, 179, 217
non-diabetic patients, ix, 93, 96, 98
parents, 75, 138
nuclear magnetic resonance (NMR), 103, 104, 112
parotid, 206
nuclei, 23, 60, 61, 68
partial least-squares, 103
nucleus, 23, 60, 68, 105, 144, 182, 207, 216, 217,
participants, 5, 7, 19
218, 219
partition, 112
null, 120
pathogenesis, vii, viii, ix, 1, 2, 10, 11, 24, 25, 69, 87,
nursing, 73, 81
94, 96, 99, 100, 102, 104, 105, 106, 111, 113,
nursing home, 73, 81
154, 159, 162, 163, 164, 170, 198, 207, 214
pathogens, viii, 69, 75, 76, 80, 84, 86, 89
O pathology, vii, 1, 2, 7, 12, 13, 15, 18, 22, 25, 159
pathophysiological, 94, 210
obesity, 111 pathophysiology, 166, 173, 174
obstruction, 77, 83, 87, 91 pathways, x, 22, 23, 24, 103, 157, 164, 165, 168,
occlusion, 58, 60, 167 174, 177, 179, 180, 181, 182, 186
occupational groups, 4 pattern recognition, 103, 104, 118
ocular diseases, 130 PCA, 103
ocular movements, 57 PCR, 10, 11, 14, 188
oculomotor, 60, 68 penicillin, 70, 71, 86
oedema, 13 peptide, 23, 146
oil, 169, 172, 174 peptides, ix, 22, 93, 95, 102, 141
opacification, xi, 215, 216 perforation, 76, 77, 121
opacity, 207 perfusion, 187
operations, 5 permeability, 101, 102, 112, 163, 164
optic nerve, 80, 81, 178, 181, 182, 192, 207 permit, 79
optical density, 125 personal hygiene, 73
optical properties, ix, 66, 115, 116, 118 PET, 200
oral antibiotic, 81 phagocytosis, 159
orbit, 80, 81, 86 pharmacological treatment, 182
organ, 12, 103, 142, 187, 188, 189, 215 phenotype, 15, 112, 160
organism, 81, 82 phenotypes, 18, 112, 186
organs, 104 Philadelphia, 29, 66, 131
osteomyelitis, 70, 75 phosphate, 3, 25, 104, 165
overlap, 65, 96, 124 phosphatidylcholine, 23
oxidation, 99, 103, 104, 106, 216 phospholipids, 165

oxidative damage, vii, 2, 25, 104 phosphorylation, 23, 24, 153, 216, 220
oxidative stress, 13, 100, 103, 105, 110, 182, 214, photodetectors, 127
216 photophobia, 13, 74, 82
oxygen, 79, 104, 180, 189 photorefractive keratectomy, 77, 89, 211
physicians, viii, 69, 70
physiological, 34
physiology, 102, 107, 178
physiopathology, ix, 94
PHILIPPINES
Account: ns004409
pigmentation, 5 proliferative vitreoretinopathy (PVR), x, 157, 158

pigs, 151 proline, 105, 206
pioglitazone, 24 promoter, 5, 19, 155, 191
plasma proteins, 14 propagation, 122
plasmid, 71 prophylactic, 75, 84
plasticity, vii, xi, 215, 218, 219 prophylaxis, 72, 78, 82, 84, 88, 91
platelet activating factor, 208 proptosis, 80
platform, 45, 198, 201, 203 prostaglandins, 13, 160
PLS, 103 protection, x, 22, 102, 177, 178, 180, 182, 184, 188,
PMMA, 192 200, 205
pneumonia, 70, 72, 75 protective factors, 5
point spread function, 194 protective role, 183
polarity, 172 protein family, 14, 22
polarized light microscopy, 18 protein structure, 12
polyacrylamide, 107, 211 protein-protein interactions, 216
polyamines, 20 proteins, vii, ix, xi, 1, 2, 11, 12, 15, 22, 70, 93, 95,
polymethylmethacrylate, 192 96, 97, 101, 102, 103, 106, 107, 108, 115, 141,
polymorphisms, vii, 1, 5, 6, 145, 150, 151 143, 144, 149, 160, 171, 175, 179, 180, 183, 188,
polypeptide, 154 202, 205, 206, 207, 208, 209, 210, 211, 212, 213,
polysaccharide, 70 215, 216, 217, 218, 219, 220, 221
polyunsaturated fat, 99 proteinuria, 112
polyunsaturated fatty acids, 99 proteoglycans, ix, 115
polyvinyl chloride, 46, 71 proteolysis, 160, 213
pons, 60 proteolytic enzyme, 11, 14
population, vii, viii, ix, 1, 4, 24, 69, 71, 73, 85, 107, proteome, 96, 101, 102, 108, 208, 210, 211, 212,
137, 138, 145, 174, 192 213, 214
population group, 73 proteomics, ix, xi, 93, 95, 101, 107, 205, 206, 207,
portability, 122 208, 210, 212, 213, 214
Portugal, 71, 115 prototype, 129
positive correlation, 144 pseudomonas aeruginosa, 15, 78, 90
postoperative outcome, 55 pterygium, vii, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
potassium, 183 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,
predictability, 56, 120, 130 27, 28, 29, 30, 31, 32, 33, 34, 35, 78, 89
preparation, 167 ptosis, 61, 62, 78
preservation, 194 public health, 78
prevention, 25, 84, 85, 87, 159, 165, 172 pulmonary stenosis, 78
primate, 141, 150, 153, 154 pumps, 129
principles, 65, 73, 122, 128 PVC, 46, 47, 48
prisoners, 73, 84
PRK, 77, 78
Q
probability, 20
probe, 122, 123, 124

quantification, xi, 3, 181, 191, 196
progenitor cells, 181, 184, 185, 186, 189
quartile, 24
prognosis, 54, 67, 81, 82, 159
pro-inflammatory, 13, 23
project, 60 R
prolactin, 206
proliferation, vii, x, 1, 2, 7, 9, 18, 21, 22, 23, 24, Rab, 34
100, 111, 145, 157, 158, 159, 160, 162, 163, 164, race, 55
166, 167, 169, 171, 174, 175 radiation, 2, 13, 30, 33, 216
PHILIPPINES
Account: ns004409
radicals, vii, 2 retinal disease, x, 170, 174, 177, 178, 187

radius, 116, 118 retinal pigment epithelial (RPE), x, 157
raman spectra, 4 retinitis, 99, 110, 113, 178, 182, 183, 187
raman spectroscopy, 3, 4 retinitis pigmentosa, 99, 110, 113, 178, 182, 183,
RANTES, 23 187
reactions, 6, 59, 70, 76 retinol, ix, 93, 96, 99, 101, 109, 110
reactive oxygen, 13, 100, 104, 180, 198 retinopathy, viii, 93, 94, 100, 108, 111, 113, 158,
reading, 124 162, 170, 172, 174, 200, 208, 209, 214
real time, 10 rhodopsin, 109, 179, 180, 187, 188
reality, 120, 123 rings, 116, 117
receptors, 9, 24, 110, 141, 143, 149, 153, 169, 170, risk, 4, 5, 7, 13, 21, 54, 65, 73, 77, 78, 79, 80, 81, 82,
173, 179, 181, 183, 184, 214 83, 85, 87, 94, 107, 150, 159, 162, 169, 170
recession, 52, 55 risk factors, 4, 73, 77, 80, 81, 82, 159, 169, 170
recognition, 103 RNA, 145, 148
recommendations, 84 rodents, 186
recovery, 65, 130 rods, 159, 179
recurrence, 5, 7, 8, 75, 166 root, 121, 166
redistribution, 218 rosacea, 76, 77, 211
refractive index, 46, 115, 118, 121, 124, 215, 216,
217
S
regenerate, 178, 182
regeneration, 185, 186, 188
safety, viii, 2, 88, 165
regression, 8, 24, 103
salivary gland, 206
regression model, 24
salts, ix, 115, 203
regrowth, 165
Saudi Arabia, 5
relapses, 76
scatter, xi, 159, 215, 216
relatives, 110
school, 152
relaxation, 129
school achievement, 152
relevance, 25, 98, 101
science, 158
repair, 77, 158, 181, 183, 185, 217, 218
sclera, 7, 8, 77, 95, 127, 139, 140, 142, 143, 145,
researchers, viii, 2, 8, 25, 138, 167
149, 151, 153, 181
resection, viii, 2, 3
scope, 179, 201
resistance, viii, 14, 69, 70, 71, 72, 73, 76, 77, 82, 84,
second generation, 83
85, 86, 87, 88, 91, 130
secrete, 170
resolution, 103, 122, 123, 125, 126, 127, 135, 192,
secretion, 99, 154, 158, 163, 164, 175, 183, 211
194, 195, 201, 203, 219
selenium, 209
response, 22, 53, 54, 55, 59, 60, 61, 67, 68, 70, 76,
sensation, 13, 74
81, 85, 102, 129, 139, 144, 148, 152, 154, 175,
sensitivity, xi, 77, 83, 84, 91, 104, 148, 191
180, 182, 183, 186, 188, 192, 200, 201
sensors, 166
retardation, viii, 2
sepsis, 70, 72
reticulum, 180, 188, 189
septum, 78, 80, 218
retina, ix, x, xi, 52, 59, 82, 93, 94, 95, 98, 99, 100,
sequencing, 20
104, 105, 109, 110, 113, 115, 137, 138, 139, 140,
serine, 24, 96
141, 142, 143, 144, 145, 146, 147, 148, 149, 150,
serotonin, 144, 150, 154
151, 152, 153, 154, 155, 157, 159, 166, 173, 174,
serum, 16, 24, 95, 99, 103, 105, 110, 112, 162, 180,
178, 180, 181, 182, 183, 184, 185, 186, 187, 188,
206
189, 191, 192, 194, 196, 197, 198, 201, 202, 203,
serum albumin, 206
204, 208, 209, 210, 213, 214, 215
sex, 24, 55, 201
retinal detachment, x, 74, 77, 83, 94, 108, 157, 158,
shape, ix, 46, 47, 95, 115, 116, 119, 121, 128, 129,
162, 165, 169, 170, 171, 173, 174, 178, 184
132, 146, 147, 148, 150, 152
PHILIPPINES
Account: ns004409
shock, 210 stress, vii, ix, 2, 13, 22, 24, 100, 105, 129, 137, 150,
showing, ix, 82, 98, 105, 106, 137, 140, 141 180, 182, 186, 187, 188, 189, 198
side effects, 94, 166, 185, 200 striatum, 201
signal transduction, 208 stroma, viii, 2, 3, 7, 15, 18, 74, 76, 134
signaling pathway, 12, 13, 23, 175, 189 structural changes, 128
signalling, 9 structure, 109, 125, 128, 130, 178, 186, 198, 218
signals, ix, 23, 103, 123, 137, 139, 144, 146, 147, style, 132
148, 152, 153, 154, 166 subacute, 83
signs, 13, 74, 75, 76, 80, 82, 142, 182, 183 subcutaneous tissue, 80
silica, 174 substrates, 12, 22, 104, 106
silver, 75, 90, 167, 171 success rate, 54, 56, 158, 165
Singapore, vii, 1, 4, 5, 24, 26, 34, 35, 71, 174, 191, sulfate, 107
203 Sun, 27, 31, 33, 90, 202, 213
sinuses, 80, 81 supplementation, x, 99, 114, 177, 178, 180, 182, 185
sinusitis, 75, 80, 90 suppression, 22, 53, 55, 57, 59, 194
skin, viii, 4, 5, 13, 14, 18, 19, 22, 69, 70, 71, 72, 73, surface component, 11
80, 83, 172, 192 surgical resection, viii, 2
skin diseases, 22 surgical technique, 158
slit lamp, 124, 127 surveillance, 71, 87, 91
smooth muscle, 143, 144 survival, x, 9, 71, 157, 159, 174, 179, 180, 181, 182,
snake venom, 175 183, 188, 189
SNP, 6 susceptibility, vii, 1, 5, 71, 72, 81, 84, 85, 88, 149,
SNS, 102 150, 152
socioeconomic status, 73 suture, 78, 138, 140, 146, 150
sodium, 104, 107 Sweden, 177
software, 194 swelling, 122
solution, 8, 20, 75, 123, 168 Switzerland, 76
Spain, 93, 137 symptoms, 5, 8, 13, 14, 74
spatial frequency, 140 syncytium, 216, 217, 220
species, viii, 13, 69, 78, 85, 100, 104, 140, 146, 154, syndrome, 12, 13, 14, 78, 206, 212, 213
180, 198, 218 synthesis, 19, 24, 70, 139, 140, 141, 142, 143, 145,
specifications, 117 151, 152, 153, 159, 167, 173, 207
spectroscopy, 101, 103, 104, 112 systolic blood pressure, 24
Spring, 29
sprouting, x, 177, 182
T
stability, 12, 53, 54, 128, 155
standard deviation, 121
Taiwan, 6, 7, 19, 26, 71
staphylococci, 72, 86
tamoxifen, 217, 221
Staphylococcus Aureus (SA), viii, 69
target, 19, 23, 48, 49, 52, 53, 54, 55, 56, 84, 106,
starvation, 180
144, 150, 162, 184, 201, 212
state, vii, ix, x, 9, 20, 57, 68, 120, 128, 137, 138,
teams, 81
146, 149, 162, 167, 183, 191

techniques, ix, 53, 85, 93, 95, 102, 116, 122, 124,
stem cells, x, 17, 177, 178, 184, 185, 186
125, 129, 131, 135, 148, 149, 178, 184, 185, 211
steroids, 8, 76, 83
technological advancement, 200
Stevens-Johnson syndrome, 78
technologies, 116, 119, 128
stimulus, 139
technology, 107, 116, 123, 127, 166, 213
storage, 79, 90
teens, 5
strabismus, viii, 37, 39, 41, 44, 51, 52, 56, 57, 61,
tension, 128
65, 66, 67, 68, 77, 78, 90
terminals, 182
testing, ix, 51, 52, 54, 60, 93, 94
PHILIPPINES
Account: ns004409
tetracyclines, 70 transplantation, 78, 98, 178, 184, 187, 188, 189

therapeutic approaches, x, 157, 160 transport, 99, 104, 105, 109, 110, 113
therapeutic targets, 106 transportation, 159
therapeutics, 200 trauma, x, 77, 78, 80, 83, 158, 159, 163, 174, 191
therapy, 8, 11, 25, 84, 91, 99, 102, 110, 115, 174, treatment, viii, ix, x, xi, 2, 8, 13, 14, 20, 37, 65, 74,
180, 210, 220 75, 77, 80, 81, 82, 83, 84, 87, 88, 90, 91, 94, 95,
thinning, 77, 143, 145, 155 98, 107, 132, 148, 157, 158, 164, 165, 166, 167,
threonine, 24 168, 169, 170, 171, 173, 174, 177, 181, 182, 185,
thrombin, 209 186, 189, 191, 192, 199, 201, 212
thrombosis, 101 trial, 53, 63, 65
Tibet, 4 triggers, 19, 180
Tibetans, 30 tuberculosis, 76
time periods, 14 tumor, 6, 9, 14, 102, 166, 171
TIMP, 9, 10, 11, 14, 28, 32, 33, 180 tumor cells, 166
TIMP-1, 9, 10, 11, 14, 32, 33 tumor growth, 171
tissue, vii, xi, 1, 2, 3, 4, 6, 7, 9, 10, 11, 12, 13, 16, tumor invasion, 9
18, 20, 21, 72, 73, 75, 81, 83, 98, 99, 100, 103, tumor necrosis factor (TNF), vii, 1, 14, 23, 28, 102,
104, 108, 115, 119, 122, 123, 130, 158, 162, 178, 112, 183
180, 181, 183, 184, 187, 188, 191, 206, 210, 214, tumors, 7, 16, 19, 21, 22, 172, 184
215 Turkey, 71
tissue degeneration, 183 turnover, 106, 216, 220
TNF-alpha, 102, 112 type 1 diabetes, 94
TNF-α, 14, 23 type 2 diabetes, 94, 107
tocopherols, 110 tyrosine, 9, 162, 165, 173, 200
tonic, 60, 61 tyrosine hydroxylase, 200
tooth, 80
tooth abscess, 80
U
total cholesterol, 24
toxicity, 185, 199, 201
ulcer, 76, 77, 89
toxin, 70, 72, 76, 88, 200
ultrasound, 122, 123, 133, 134, 135, 136
trafficking, 23
underlying mechanisms, 85
training, 72
uniform, xi, 167, 215
transcription, ix, 15, 19, 20, 23, 137, 144, 145, 150,
united, 70, 71, 72, 76, 85, 91, 138, 154, 155, 214
155, 185
United States, 70, 71, 72, 76, 85, 91, 138, 154, 155,
transcription factors, ix, 15, 19, 137, 145, 185
214
transcriptomics, 95
upper respiratory infection, 80
transcripts, viii, 2, 20, 23, 95, 148
urban, 4, 5, 89
transducer, 122, 123
urinary tract, viii, 69, 82
transduction, 170, 179
urine, 102, 103, 112
transferrin, 206
USA, 70, 72, 111, 113, 127, 158, 173, 187, 192, 220
transformation, x, 2, 19, 120, 151, 160, 191
UV light, 14, 18, 113
transforming growth factor (TGF), 3, 14, 35, 142,

UV radiation, vii, 1, 14
143, 144, 145, 148, 150, 151, 153, 154, 162, 164,
uveitis, 83
169, 171, 172, 173, 175, 208
uvula, 60
transgene, 192, 195, 198, 201, 202
translation, 185, 209, 210
translocation, vii, 21, 23, 159, 173, 217, 218 V
transmission, 72, 75, 79, 80, 81, 84, 86, 88, 89, 90,
138, 140, 142, 179 vancomycin, 70, 74, 77, 83, 84, 91
transparency, ix, 115, 130, 216, 220 vapor, 112
PHILIPPINES
Account: ns004409
variables, 103
Z
variations, ix, 123, 137, 194
vascular endothelial growth factor (VEGF), vii, x, 1,
zinc, 9, 111, 144, 148, 206
3, 7, 157
vasculature, viii, 2, 7, 8, 14, 197
vasoactive intestinal peptide, 139, 151, 153
vector, 20
vein, 80, 199
vessels, 74, 101, 102, 203, 210
viral vectors, 98
viscoelastic properties, 95
vision, vii, x, 1, 48, 51, 55, 56, 57, 58, 59, 60, 70, 77,
81, 82, 83, 94, 137, 146, 151, 152, 154, 159, 165,
168, 169, 179
visual acuity, 48, 55, 82, 83, 94, 159, 216
visual area, 60
visual field, 94, 207
visual stimulus, 140
visual system, 140
visualization, 203
vitamin A, 99, 110, 159
vitamin C, 104, 106, 113
vitamin D, 19, 101
vitreous hemorrhage, 95, 162
waking, 74
Wales, 86
war, 73
water, x, 47, 103, 124, 137, 165, 215
weak interaction, xi, 215
wear, 77, 79, 89, 90, 122
Western Australia, 72
western blot, 10, 16, 21, 96
white blood cells, 80
witnesses, 167
workers, 4, 5, 56, 58, 59, 60, 72, 74, 84, 218
World Health Organization, 220
worldwide, 70, 72, 76, 84, 138
wound healing, 9, 22, 130, 136, 158, 159, 162, 172,
174, 208
wound infection, 83
yield, 118, 119
PHILIPPINES
Account: ns004409

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ADVANCES IN EYE RESEARCH

ADVANCES IN EYE RESEARCH

ADVANCES IN EYE RESEARCH

Additional books in this series can be found on Nova‘s website

Additional E-books in this series can be found on Nova‘s website

ADVANCES IN EYE RESEARCH

ADVANCES IN EYE RESEARCH

Nova Science Publishers, Inc.

Copyright © 2012 by Nova Science Publishers, Inc.

NOTICE TO THE READER

Library of Congress Cataloging-in-Publication Data

ISBN: 978-1-62257-129-1 (eBook)

Published by Nova Science Publishers, Inc.  New York

Bose Karthikeyan, Venkataraman Deepak and

Chapter IX Non-Invasive Imaging of Retinal Gliosis for Screening

Advances in Pathogenesis of Pterygium

Free radicals-induced stress generated by UVB results in oxidative damage to DNA,

Keywords: Pterygium, review, pathogenesis, epidemiology, genetics, metalloproteinases,

these nerve endings were found to be increased in pterygium compared to conjunctival

an urban or large metropolitan center is being studied, compared to special occupational

Previous reviews on pterygium have included the discussion of inheritable defects in

Recently, a study in Taiwan has found benzo[a]pyrene 7,8-diol 9,10-epoxide (BPDE), a

Vascular Endothelial Growth Factor and Clinical Trials

Ephrin and Eph Receptors

5. Metalloproteinases and Their Natural

Tsai 2010 IHC, WB √ √

Inflammatory Cells and Pterygium

Several studies have pointed to an immune response-inflammatory component in

immune cells in the conjunctiva, the presence of lymphocytes in pterygium may be a

Ultraviolet Radiation and Inflammation

inflammatory. Hence, upregulation of COX-2 may, at least in theory, contribute to

7. Cell Transformation and Wound Remodeling

In a recent review of pterygium, the role of epithelial-mesenchymal transition (EMT) has

Spatial Differences in the Phenotype of Pterygium Epithelial Cells

the pterygium, which was not investigated in this study.

involvement of epigenetics in pterygium is reported by Young et al in 2010. [157] This paper

Gene Expression Studies in Epithelial-Mesenchymal Transition

Insulin-Like Growth Factor Binding Protein 3

Trefoil Factor (TFF) 1 is a protease-resistant protein commonly found in tumours and

help to stabilize mucin. [11, 128]

9. Stress Related Signaling

The NFκB pathway is an important signaling pathway that controls proliferation,

The phospholipase (PL) D is a family of enzymes that catalyses the metabolism of

Polo-Like Kinase Signaling

Treatment. Curr Opin Ophthalmol. 2007 Jul;18(4):308-13.

An In Vivo Analysis. Ophthalmology. 2010 Sep;117(9):1782-91.

[21] Di Girolamo N, Chui J, Coroneo MT, Wakefield D. Pathogenesis Of Pterygia: Role Of

From An Invasion Of Vimentin-Expressing Altered Limbal Epithelial Basal Cells.

Metalloproteinases During Apoptosis. Apoptosis. 2005 Jan;10(1):19-24.

Lipoprotein Receptors And Hydroxy-Methylglutaryl-Coenzyme A-Reductase In

[104] Perez-Martinez L, Jaworski DM. Tissue Inhibitor Of Metalloproteinase-2 Promotes

[120] Siak J, Ng S, Seet L, Et Al. The Nuclear-Factor Kb Pathway Is Activated In Pterygium.

Locus. Mutat Res. 1995 Jul;329(2):97-105.

Insights And Preliminary Results. Can J Ophthalmol. 2009 Apr;44(2):185-8.

Role of Prisms in the Management

Rehab Rashad Kassem

Optics of Ophthalmic Prisms and Fresnel Optics

Figure 2. Definition of "prism diopter".

even though the eye remains anatomically deviated (1).

Figure 5. Esotropia. Image falls nasal to fovea in esotropic left eye.

Figure 6. Exotropia. Image falls temporal to fovea in exotropic left eye.

Figure 7. Prism placed apex in to neutralize an esotropia.