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Principles of Molecular Neurosurgery
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Progress in Neurological
Surgery
Vol. 18
Series Editor
L. Dade Lunsford Pittsburgh, Pa.

Principles of Molecular
Neurosurgery
Volume Editors
Andrew Freese Minneapolis, Minn.

Frederick A. Simeone Philadelphia, Pa.
Paola Leone Camden, N.J.
Christopher Janson Camden, N.J.
96 figures, 12 in color, and 25 tables, 2005
Basel · Freiburg · Paris · London · New York ·

Bangalore · Bangkok · Singapore · Tokyo · Sydney
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Andrew Freese, MD, PhD Frederick A. Simeone, MD
Department of Neurosurgery, University of Pennsylvania,
University of Minnesota School of Simeone Neuroscience Center,
Medicine MMC 96, Pennsylvania Hospital,
420 Delaware St., S.E., 800 Spruce St.,
Minneapolis, MN 55455 (USA) Philadelphia, PA 19107 (USA)
Paola Leone, PhD Christopher Janson, MD
Cell and Gene Therapy Center, Cell and Gene Therapy Center,
UMDNJ-Robert Wood Johnson Medical UMDNJ-Robert Wood Johnson
School, Division of Neurosurgery, Medical School, Division of
401 Haddon Ave., Rm 390, Neurosurgery, 401 Haddon Ave., Rm 390,
Camden, NJ 08103 (USA) Camden, NJ 08103 (USA)
Library of Congress Cataloging-in-Publication Data
Principles of molecular neurosurgery / volume editors, Andrew Freese ... [et al.].
p. ; cm. – (Progress in neurological surgery, ISSN 0079-6492 ; v. 18)
Includes bibliographical references and indexes.
ISBN 3-8055-7784-2 (hard cover : alk. paper)
1. Nervous system–Diseases–Treatment. 2. Molecular neurobiology. 3.
Nervous system–Surgery.
[DNLM: 1. Nervous System Diseases–therapy. 2. Gene Therapy–methods.
WL 140 P9573 2005] I. Freese, Andrew. II. Series.
RC349.8.P75 2005
616.8–dc22
2004024871
Bibliographic Indices. This publication is listed in bibliographic services, including Current Contents® and
Index Medicus.
Drug Dosage. The authors and the publisher have exerted every effort to ensure that drug selection and
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However, in view of ongoing research, changes in government regulations, and the constant flow of information
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change in indications and dosage and for added warnings and precautions. This is particularly important when the
recommended agent is a new and/or infrequently employed drug.
All rights reserved. No part of this publication may be translated into other languages, reproduced or uti-
lized in any form or by any means electronic or mechanical, including photocopying, recording, microcopying, or
by any information storage and retrieval system, without permission in writing from the publisher.
© Copyright 2005 by S. Karger AG, P.O. Box, CH-4009 Basel (Switzerland)

www.karger.com
Printed in Switzerland on acid-free paper by Reinhardt Druck, Basel
ISSN 0079–6492
ISBN 3–8055–7784–2
Contents
IX Dedication
X Acknowledgements
XI Series Editor’s Note

Lunsford, L.D. (Pittsburgh, Pa.)
XIII Foreword
Anderson, W.F. (Los Angeles, Calif.)
1 Introduction
Freese, A. (Minneapolis, Minn.); Janson, C.; Leone, P. (Camden, N.J.);
Simeone, F.A. (Philadelphia, Pa.)
The Spine and Spinal Cord
5 The Molecular Basis of Intervertebral Disc Degeneration

Leo, B.M.; Walker, M.H. (Charlottesville, Va.); Anderson, D.G.
(Philadelphia, Pa.)
30 Genetics of Degenerative Disc Disease

Kurpad, S.N.; Lifshutz, J. (Milwaukee, Wisc.)
37 Gene Therapy for Degenerative Disc Disease

Kim, J.; Gilbertson, L.G.; Kang, J.D. (Pittsburgh, Pa.)
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52 Bone Morphogenetic Proteins. Spinal Fusion Applications
Bomback, D.A.; Grauer, J.N. (New Haven, Conn.)
65 Cellular and Gene Therapy Approaches to Spinal Cord Injury

Steinmetz, M.P.; Liu, J.K.; Boulis, N.M. (Cleveland, Ohio)
104 Neural Stem Cell Transplantation for Spinal Cord Repair

Iwanami, A.; Ogawa, Y.; Nakamura, M.; Kaneko, S. (Tokyo/Osaka);
Sawamoto, K. (Osaka); Okano, H.J.; Toyama, Y.; Okano, H. (Tokyo/Osaka)
Functional and Restorative Molecular Neurosurgery
124 Contemporary Applications of Functional and Stereotactic Techniques

for Molecular Neurosurgery
House, P.A.; Rao, G.; Couldwell, W. (Salt Lake City, Utah)
146 Xeno-Neurotransplantation
Schumacher, J.M. (Miami, Fla.)
154 Adeno-Associated Viral Vectors for Clinical Gene Therapy

in the Brain
Samulski, R.J.; Giles, J. (Chapel Hill, N.C.)
169 Molecular Mechanisms of Epilepsy and Gene Therapy

Telfeian, A. (Lubbock, Tex.); Celix, J. (Seattle, Wash.);
Dichter, M. (Philadelphia, Pa.)
202 Emerging Treatment of Neurometabolic Disorders

Brady, R.O.; Brady, R.O., Jr. (Bethesda, Md.)
213 Gene Therapy for Parkinson’s Disease

Hadaczek, P.; Daadi, M.; Bankiewicz, K. (San Francisco, Calif.)
246 Simplifying Complex Neurodegenerative Diseases by

Gene Chip Analysis
Scherzer, C.R.; Gullans, S.R. (Cambridge, Mass.); Jensen, R.V.
(Middletown, Conn.)
258 Molecular Pathology of Dementia. Emerging Treatment Strategies

Gouras, G.K. (New York, N.Y.)
Contents VI
270 Expanding the Role of Deep Brain Stimulation from Movement
Disorders to Other Neurological Diseases
Leone, M.; Franzini, A.; Broggi, G.; Bussone, G. (Milano)
284 Molecular Mediators of Pain

Chaudhary, P.; Burchiel, K. (Portland, Oreg.)
322 Gene Transfer in the Treatment of Pain

Fink, D.; Mata, M. (Ann Arbor, Mich.); Glorioso, J.C. (Pittsburgh, Pa.)
Neurovascular Disorders
336 Gene Discovery Underlying Stroke

Barone, F.C. (King of Prussia, Pa.); Read, S.J. (Macclesfield)
377 Molecular Mediators of Hemorrhagic Stroke

Macdonald, R.L. (Chicago, Ill.)
413 Advances towards Cerebrovascular Gene Therapy

Watanabe, Y.; Heistad, D.D. (Iowa City, Iowa)
439 Ex vivo Gene Therapy and Cell Therapy for Stroke

Kondziolka, D. (Pittsburgh, Pa.); Sheehan, J. (Charlottesville, Va.);
Niranjan, A. (Pittsburgh, Pa.)
Neuro-Oncology
458 Neurosurgical Applications for Polymeric Drug Delivery Systems

Wang, P.P.; Brem, H. (Baltimore, Md.)
499 Immunotherapy Strategies for Treatment of Malignant Gliomas

Harshyne, L.; Flomenberg, P.; Andrews, D.W. (Philadelphia, Pa.)
521 Glioma-Genesis. Signaling Pathways for the Development of Molecular

Oncotherapy
Kapoor, G.S.; O’Rourke, D.M. (Philadelphia, Pa.)
557 Oncolytic Viral Therapy for Glioma

Lamfers, M.L.M.; Visted, T. (Charlestown, Mass.); Chiocca, E.A. (Columbus, Ohio)
580 Molecular Neurosurgery in the Pituitary Gland. Gene Transfer as an

Adjunctive Treatment Strategy
Castro, M.G.; Jovel, N.; Goverdhana, S.; Hu, J.; Yu, J.; Ehtesham, M.; Yuan, X.;
Greengold, D.; Xiong, W.; Lowenstein, P.R. (Los Angeles, Calif.).
Contents VII
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624 Stem Cells for Targeting CNS Malignancy
Yip, S. (Vancouver); Sidman, R.L. (Boston, Mass.); Snyder, E.Y.
(Boston, Mass./La Jolla, Calif.)
645 Author Index
646 Subject Index
Contents VIII
Dedication
This book is dedicated to the memory of Drs. Ernst and Elisabeth Freese,
brilliant scientists and wonderful parents who deciphered the chemical basis of
mutagenesis, the engine of evolution and God’s way of making us better.
We also dedicate the section on functional and restorative molecular neu-
rosurgery to the memory of Anne Janson, a victim of Alzheimer’s Disease, and
all the other patients who suffer from this terrible disease which robs the mind
of its memories and dignity, as well as all children affected by neurodegenera-
tive diseases – for those who have died, they are not forgotten; and for those
who are living, may they soon have the promise of a cure.
The section on Oncology is dedicated to the memory of Jack Geary, a vic-
tim of cancer. Finally, we dedicate the section on the spine and spinal cord to
the memory of Anthony Simeone, M.D., whose devotion to his patients and
family will always be remembered.
The Editors
Andrew Freese
Frederick A. Simeone
Paola Leone
Christopher Janson
IX
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Acknowledgements
We gratefully acknowledge the dedicated, diligent work of Jackie Alutis in

helping produce this book, and the valuable contributions of Joanne Coughlin and
Marcia Freese as well. Without them, this book would not have been possible.
In addition, we acknowledge the support of the CNS Gene Therapy Con-
sortium in the production of this book.
Otto Freese produced the front cover illustration, for which we are grateful
as well.
The Editors
Andrew Freese
Frederick A. Simeone
Paola Leone
Christopher Janson
X
Series Editor’s Note
Changing the Paradigm of Neurosurgery
During the last decade of the 20th century and the first years of the 21st cen-
tury, neurosurgery has been part of an enormous paradigm shift. While we previ-
ously concentrated on dealing with the removal or management of structural
masses (blood clots, aneurysms, brain tumors, spinal bone spurs, ruptured discs),
the future of neurosurgery lies in the application of a wide variety of new knowl-
edge. Loosely termed ‘molecular’, this new knowledge can be applied widely to
the diagnosis, management, and possible prevention of serious neurological ill-
ness. As such, we practitioners and surgeons must embrace this new knowledge
and attempt to implement it in the current practice of neurosurgery. The future of
neurosurgery is molecular, minimally invasive, and multidisciplinary. For the first
time, we will be bringing the emerging data from the laboratory and beginning to
apply it to clinical problems, rather than the reverse, the old paradigm of trying
something in the operating room and then going back to the laboratory to see why
it did or didn’t work. Volume 18 of Progress in Neurological Surgery is an elegant
compilation of current and emerging knowledge related to the influence of
molecular neurosurgery on both the present and the future. Drs. Freese, Simeone,
Leone, and Janson have accumulated a wealth of information which can be
applied to the spinal column and spinal cord disorders, functional and restorative
brain surgery, neurovascular disorders and neuro-oncology. The author list is
impressive, and the story that is presented should entice the reader to glimpse the
XI
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future of neurosurgery, which is rapidly descending on us. I congratulate the
current authors for their excellent collaboration, and believe the readership will
enjoy this new volume which does, indeed, represent progress in neurological
surgery.
L. Dade Lunsford, MD
The University of Pittsburgh
School of Medicine,
Pittsburgh, Pa., USA
Series Editor’s Note XII

Foreword
There are not many advantages to becoming ‘senior’, an euphemism for

being an old man. But there are a few, and two of them are exemplified for me
by this volume. The first is in seeing how scientific/medical progress can mush-
room over a span of decades. As I think back on my training, in the 1950s and
early 1960s, and then look through this fascinating book, I am in awe at the
progress. Yes, I have been intimately involved in gene therapy and the genetic
basis of disease my whole career, but the laying out of the application of these
technologies to the single field of neurosurgery leaves me in wonder. Using
gene therapy to treat malignant gliomas is revolutionary enough, but scanning
the chapters herein reveals the use of gene therapy and genetic approaches for
degenerative disc disease, for spinal cord injuries, for epilepsy, for Parkinson’s
and other neurodegenerative diseases, for the treatment of pain, for stroke as
well as for malignancies. And not just genetic approaches: neurosurgery now
embraces protein therapy, cell therapy, oncolytic viral therapy, immunotherapy,
stem cell transplantation, and xeno-neurotransplantation. Truly a glorious story
of scientific/medical progress!
The second advantage of growing old is seeing the successes of the many
young physician investigators that one has trained. Everywhere that I travel,
there seems to be someone who had passed through my laboratory over the past
40 years. The joy of hearing the stories of successful, productive careers is
wonderful. But this book brings an added touch. Andrew Freese is the son of
one of the men that played a significant role in my career. When I interviewed
at NIH in 1963 for a position of Research Associate (following my medical
XIII
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training), I narrowed my choices to two: Marshall Nirenberg (who was in the
middle of deciphering the genetic code) and Ernst Freese, who was doing fas-
cinating work in genetic model systems. This was one of the toughest decisions
of my career. I found Dr. Freese (I still cannot call him by his first name
although he has asked me to for years!) to be a brilliant scientist and marvelous
human being. So much so that, although I joined Marshall Nirenberg and
helped in the final genetic code decipherment, I maintained a long and fruitful
friendship and scientific mentorship with Dr. Freese.
Thus, it was with extraordinary pleasure that I received a letter from Dr.
Freese’s son, Andrew, to write the Foreword for this book. As I think back to all
the advice and knowledge that I received from Dr. Freese over so many years,
I am honored and humbled to add my name to a volume that is dedicated to his
memory by his son.
W. French Anderson, MD
USC Keck School of Medicine,
Los Angeles, Calif., USA
Foreword XIV
Freese A, Simeone FA, Leone P, Janson C (eds): Principles of Molecular Neurosurgery.
Prog Neurol Surg. Basel, Karger, 2005, vol 18, pp 1–4
Introduction
Andrew Freesea, Christopher Jansonb, Paola Leoneb,
Frederick A. Simeonec
a
Department of Neurosurgery, University of Minnesota School of
Medicine MMC 96, Minneapolis, Minn., bCell and Gene Therapy Center,
UMDNJ-Robert Wood Johnson Medical School, Division of Neurosurgery,
Camden, N.J., cDepartment of Neurosurgery, University of Pennsylvania,
Philadelphia, Pa., USA
Until recently, Neurosurgery has been a surgical discipline largely focused

on ablative approaches to diseases affecting the nervous system – clipping
aneurysms, evacuating hematomas, removing disc herniations, decompressing
the stenotic spine, extirpating tumors, lesioning the neostriatum, and others.
However, as physician-scientists have begun to unravel the molecular basis of
many neurological disorders, a paradigm shift has begun to occur – one that
promises to dramatically alter the face and texture of Neurosurgery, and convert
it into a field that not only ablates diseased tissues and structures, but also
restores and improves function within the nervous system. Thus, we believe the
advent of a variety of molecular and cellular technologies will have a marked
impact on what neurosurgeons, neurologists, psychiatrists, neuroradiologists,
and other medical practitioners focused on the nervous system can offer
patients. Indeed, distinctions among these medical disciplines will begin to
fade, as psychiatric diseases increasingly find neurosurgical solutions, and neu-
rosurgical diseases ‘molecularize’ into nonsurgical, neurological approaches.
As the field of medicine in general continues to hone in on molecular inter-
ventions, in parallel, the field of Neurosurgery will convert itself from a macro-
molecular discipline to one that relies increasingly on molecular approaches to
improve the outcomes of patients. To hopefully assist our colleagues in medical
and surgical disciplines dealing with the nervous system in anticipating this
future, we believe that a book focused on the current principles of molecular
Neurosurgery is needed, and we have, therefore, attempted to bring together a
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variety of superb contributors who can further shed light on this topic in this
compilation of chapters.
In this edition, several categories of neurological diseases are examined. It
is important to note that the chapters are not meant to be an exhaustive compi-
lation of facts detailing the entire field of molecular neurosurgery, but instead
are meant to highlight some of the most exciting advances. Thus, this book
attempts to spark the imagination of its readers as much as it tries to recite and
explain exhaustive details of the most recent research work and discoveries. If
there is one fact of which we are certain, it is that this book will be obsolete
within several years of its publication, and a number of promising technologies
outlined therein will have proven to be failures and subsequently will have been
abandoned. Similarly, undoubtedly a number of approaches that are only briefly
skimmed or even inadvertently omitted in this book will prove to be extraordi-
narily successful and important. We cannot predict which will succeed and
which will fail. However, we are certain that Neurosurgery faces an exciting
future as these types of approaches continue to evolve and ultimately allow us
to better care for our patients.
The first section of the book, encompassing the first six chapters, addresses
disorders of the spine and spinal cord. The chapter by Leo et al. focuses on
understanding the molecular and biochemical alterations associated with
degenerative disc disease, a problem that has a huge financial and emotional
impact on a large proportion of our society. Only by understanding the molec-
ular basis of disc disease can we ever hope to meaningfully intervene prior to
the development of symptoms and the progressive cycle of incapacitation that
results from degenerative disease. Kurpad and Lifshutz discuss the genetics
of disc and spinal degeneration, as there is clear evidence of a genetic predis-
position to this disease process, and a variety of gene defects and altered
protein profiles have been associated with it. Once a better understanding of the
genetic alterations, and subsequent molecular and biochemical disruptions that
occur in degenerative spine disease can be garnered, then one can begin to
envisage meaningful interventions. The chapter by Kim et al. outlines one
exciting area of investigation, focused on genetic intervention using different
gene transfer technologies to introduce therapeutic gene products directly into
the disc to retard degeneration or promote restoration of normal function.
Although ideally one would want to intervene in the spine before the emergence
of axial back pain and/or radicular symptoms, surgical intervention will still
be required in a number of patients who need fusion procedures. To optimize
the success of this type of surgery, however, molecular approaches to enhance
fusion need to be developed and promise to significantly improve outcomes
from fusion procedures. The chapter by Bomback and Grauer enumerates these
approaches. Although degenerative spine disorders have an enormous impact
Freese/Janson/Leone/Simeone 2
on our society, it is those patients who have traumatic spinal cord injuries that
frequently suffer the most. Relatively little can be done currently for these
patients, but enormous progress is being made in understanding the molecular
basis of spinal cord injury and hopes for restoration of function. The last two
chapters (Steinmetz et al. and Iwanami et al.) in this section on the spine thus
discuss a variety of molecular and cellular interventions that are being devel-
oped to improve outcomes in patients with spinal cord injury.
The second section of the book, encompassing 11 chapters, addresses func-
tional and metabolic disorders of the nervous system, and restorative approaches
to these diseases. In the chapter by House et al., an overview of the field is
provided with an emphasis on targeting structures within the nervous system
using contemporary neurosurgical techniques. Schumacher as well as Samulski
and Giles identify some exciting technological advances that allow molecular and
functional intervention in the nervous system using cellular transplantation and
gene therapy approaches, selecting two unique and promising systems, one
based on xenotransplantation, and the other, a promising gene transfer system
based on adeno-associated virus, both of which have already been used in
clinical trials for human neurological diseases. Although these two chapters
focus on a specific tissue source and viral vector, respectively, they also discuss
alternatives and principles underlying cell transplantation and viral vector-
mediated gene transfer. Telfeian et al. discuss current concepts regarding
the molecular basis of epilepsy and seizure disorders, identifying promising
research approaches to these diseases. Since pharmacological approaches to
epilepsy are often suboptimal, it is likely that cellular and genetic intervention
will allow more directed and efficacious therapy to the tissue source of the
aberrant electrical activity. Brady and Brady Jr. discuss the advent of molecular
intervention for neurometabolic diseases, starting with the successful develop-
ment of enzyme replacement therapy, and then branching out to exciting new
advances in gene and cell therapy. The following four chapters address a variety
of neurodegenerative diseases, including Parkinson’s disease, Alzheimer’s dis-
ease, and others, with a focus on further elucidating the molecular bases of these
diseases, and then improving neurosurgical approaches to them. Chaudhary and
Burchiel as well as Fink et al. then focus on pain, and include a thorough evalu-
ation of the molecular mediators of pain, and subsequently novel genetic and
surgical interventions for patients with intractable pain disorders.
The third section of the book, encompassing four chapters, focuses on
neurovascular disorders. The first two chapters (Barone/Read and Macdonald)
identify different genes and molecular mediators associated with stroke, an
understanding of which may identify potential genetic and molecular targets for
improved clinical intervention. The chapter by Watanabe and Heistad focuses
on genetic intervention in stroke, including an evaluation of preclinical studies,
Introduction 3
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with the hope of developing neurosurgical or endovascular delivery systems to
minimize damage to neural tissue associated with stroke. Kondziolka et al. then
discuss the advent of cellular transplant therapy for stroke, including some
results of a recent clinical neurosurgical trial.
The fourth and final section, encompassing the last six chapters, addresses
the field of neuro-oncology, with a neurosurgical perspective. The chapter by
Wang and Brem provides a thorough overview of novel polymeric and related
intracranial drug delivery systems for chemotherapeutic agents as adjuvant
therapy following surgical extirpation of brain tumors. Harshyne et al. discuss
immunotherapy strategies for malignant brain tumors, and their likely impact
on developing a more global approach to a disease process that usually extends
beyond the typical resection margins of surgery, virtually assuring recurrence.
The chapter by Kapoor and O’Rourke identifies common molecular signaling
events involved in gliomagenesis, and their relevance for developing targeted
approaches to intervene in these biochemical sequences and their role in pro-
gression to a more malignant tumor phenotype. Lamfers et al. then focus on
viral gene therapy approaches to malignant brain tumors, including an evalua-
tion of failures of clinical trials of gene therapy in the past, and opportunities
for improvement in the future. The chapter by Castro et al. further provides an
overview of the potential for genetic intervention in brain tumors, but in a
particular subset, pituitary adenomas, in which focal gene expression may well
have a significant impact clinically, and may someday be the primary modality
of treatment for these typically benign tumors which nonetheless cause signif-
icant morbidity. The final chapter by Yip et al. gives an exciting glimpse into
the potential for stem cell technology to seek out malignancies in the nervous
system, and selectively destroy them.
It is our hope that by providing an overview of the developing interface
between molecular biology and clinical Neurosurgery, we will further stimulate
the imaginations of clinicians and scientists, and provide additional impetus for
aggressive investigation of these and related technologies.
Andrew Freese, MD, PhD

Professor and Vice Chairman of Neurosurgery
Director of Spinal Neurosurgery
University of Minnesota
420 Delaware Street, S.E.
Suite D429, Mayo Memorial Building
Minneapolis, MN 55455 (USA)
Tel. ⫹1 612 624 2471, Fax ⫹1 612 624 0644, E-Mail afreese@umn.edu
Freese/Janson/Leone/Simeone 4
The Spine and Spinal Cord

The Molecular Basis of Intervertebral

Disc Degeneration
Brian M. Leoa, Matthew H. Walker a, D. Greg Andersonb
a
Department of Orthopaedic Surgery, University of Virginia,
Charlottesville, Va., bDepartment of Orthopaedic Surgery, Thomas Jefferson
University, Philadelphia, Pa., USA
Introduction
Low back pain is an endemic problem in Western societies leading to sig-

nificant morbidity [1–3]. Not only does back pain account for much individual
suffering, but the societal costs of time lost from work, for treatment, and for
compensation of lost wages numbers in the billions of dollars annually. It has
been estimated that up to 80% of the population experiences some form of back
pain over the course of their lives, making this a leading health concern [1–5].
Although the etiologies are many, intervertebral disc degeneration appears to be
the leading cause for chronic axial low back pain [6]. Significant strides have
been made in understanding the molecular basis for disc degeneration. Despite
this, the currently available treatment modalities for disc-related spinal pain
continue to focus on alleviating symptoms rather than addressing the underlying
cause of degeneration. It is likely that clinical outcomes for patients with painful
intervertebral disc degeneration would improve if therapies were developed that
could slow, halt, or reverse this process.
Histopathological changes classified as ‘degeneration’ have been recog-
nized as early as the second decade of life and are known to progress through a
series of stages that have been quantified histologically [7–10] and character-
ized noninvasively using magnetic resonance imaging [7, 9, 11, 12]. Initially,
the degenerative process begins asymptomatically in the nucleus pulposus (NP)
with cell loss and matrix alteration. This leads to an inability to retain water and
results in slight disc space narrowing. As this process progresses, the outer annu-
lus fibrosis (AF) becomes increasingly disorganized, losing its normal lamellar
arrangement and leading to diminished mechanical strength. Tissue fissures
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and clefts begin in the inner AF and progress outwards towards the periphery,
ultimately culminating in a loss of mechanical integrity [13]. Mechanical stresses
are progressively transferred to the surrounding vertebral endplates causing
microfractures and marginal osteophyte formation. Cytokines, produced within
the disc, leach out, leading to the ingrowth of nerve and vascular elements that
may play a role in the etiology of spinal pain [14–17]. The secondary bony
changes serve to stiffen the spinal segment and restabilize the spine. Interestingly,
although this sequence happens throughout life in essentially all humans, there
are significant variations in the degree of symptoms noted by different people.
Equally perplexing is the poor correlation between the degree of degenerative
changes noted on imaging studies and the presence of symptoms of spinal pain
[18]. Clearly, we have much to learn regarding the relationship between spinal
degenerative disease and various pain syndromes.
In recent years, researchers have attempted to understand the genetic and
molecular aspects of disc degeneration in order to determine the etiology of
degeneration and to identify stages in this process where therapeutic intervention
would be beneficial. There has been a growing enthusiasm in the concept of
developing tissue engineering strategies that can alter the course of the degen-
erative process and address the underlying disease process. This chapter will
review the normal disc development and structure as well as the changes that
occur during degeneration on the biomechanical, biochemical and ultrastruc-
tural levels. By gaining a better understanding of the cellular and molecular
biology of the disc, the various strategies that are being discussed or studied to
combat disc degeneration can be evaluated in a rational manner.
Embryological Development of the Disc
Although the embryology of the human disc has not been well studied
directly, animal models have contributed significantly to our understanding of
the development of the intervertebral disc and the axial skeleton.
The development of the spine begins during the third week of gestation in
a phase termed ‘gastrulation.’ It is during this phase that the ectoderm, meso-
derm, and endoderm are formed; embryological layers, which eventually give
rise to all tissues of the body. Differentiation of these tissues begins with the
formation of the primitive streak. Associated with the primitive streak is a prim-
itive node surrounding a small invagination known as the primitive pit, located
slightly caudally from the midline of the embryo. Cells in the primitive pit,
known as prenotochordal cells, migrate cephalad to the prechordal plate and
contribute to the cell layers forming the notochordal plate. As these cells prolif-
erate and detach from the endoderm, they form a solid cord of cells that becomes
Leo/Walker/Anderson 6
The developing embryo
Primitive node
Primitive streak
Prenotochordal
cells migrating
Fig. 1. The developing embryo has a

primitive streak extending caudad from the
primitive node. Arrows approximate the
migration of the prenotochordal cells cepha-
lad to the prechordal plate.
the definitive notochord and forms the basis of the axial skeleton (fig. 1).
Concurrently, the vertebral column and outer portion of the intervertebral discs
form from an aggregation of mesenchyme surrounding the notochord. This
process involves the medial migration of somatic mesoderm from the ventro-
medial portions of the somites, the sclerotomes. In a process called ‘segmentation,’
a pattern of alternating light and dark bands becomes evident in the mesenchy-
mal column by the time the embryo reaches 5 mm in length [19] (fig. 2). The
dark bands contain cells with a greater nuclear density than those of the light
bands and are the precursors of the intervertebral discs, termed the perichordal
discs. The light bands are the precursors of the vertebral bodies [19]. By the
time the embryo reaches 12.5 mm, the perichordal disc becomes trilaminar with
a denser middle region surrounded by lighter regions both cranially and cau-
dally [20]. At this point, the outer mesenchymal cells begin to arrange them-
selves in a lamellar manner with their long axis parallel to the long axis of the
embryo [19]. This outer lamellar zone darkens as it becomes populated with
fibroblasts forming the primitive AF [19]. The lighter inner cell mass is formed
primarily from notochordal cells and embryonic cartilage and can be well seen
by the 40–50 mm stage [19].
Cells in the AF begin to deposit collagen fibers in the outer region of the
perichordal disc by the 20–40 mm stage [19]. With continued growth, the outer
AF becomes progressively more fibrous and less cellular, while the inner AF
becomes fibrocartilaginous and retains a high cell density [21]. Growth of the
disc is hypothesized to occur due to increasing lamellar thickness, as opposed
to an increase in the number of lamellar layers; the number of lamellae remain
Molecular Basis of Intervertebral Disc Degeneration 7
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Precursor of
vertebral body
Notochord
Precursor of
intervertebral
disc –
perichordal
disc
Fig. 2. The mesenchymal column segments into light and dark bands. The dark bands
contain cells with a greater nuclear density and represent the precursors to the intervertebral
discs, the perichordal discs. The light bands represent the precursors to the vertebral bodies.
fairly constant throughout development, and in lumbar discs constitutes twelve

to sixteen layers [19, 21]. The lamellar fibers in these alternating layers of the
AF are oriented at oblique angles to each other, a fashion designed to optimally
dissipate multidirectional stresses on the disc.
The NP continues to develop from both the intervertebral expansion of the
notochord and the growth of primitive cartilage. As the NP region develops, the
ground substance softens leading to a loosely arranged matrix [19, 22]. The
notochordal cells of the NP play a key role in cell division and matrix forma-
tion. As the matrix of the NP becomes looser, the once compact mass of noto-
chordal cells is broken up into cellular clusters in a loose network known as the
‘chorda reticulum’ [23]. The NP continues to expand in size during fetal devel-
opment and early postnatal life with an 18-fold expansion in the number of
notochordal cells before becoming quiescent [19]. Although the appearance of
the NP gradually changes to become more fibrous, resembling the transition zone,
the embryological notochordal cells remain active producers of matrix material
until the end of the first decade of life, at which time a ‘notochordal’ NP can no
longer be defined due to the increased collagen content and loss or metaplasia
of notochordal cells [19, 24].
Insults to the developing fetus can have severe consequences to organ
systems undergoing rapid development at the time of the insult. The time period
for the most rapid development of the lumbar vertebral canal is between 12
and 32 weeks in utero [116]. A recent retrospective study by Jeffrey et al. [25]
Atlas
Axis 7
Cervical curvature Cervical
vertebrae
12
Thoracic curvature Thoracic
vertebrae
5
Lumbar
vertebrae
Lumbar
curvature
Sacrum
(5 fused vertebrae)
Coccyx
(4 fused vertebrae)
Sacral
curvature
Fig. 3. Schematic of the human spine. (Reproduced with permission [117].)
showed that low birth weight, low placental weight, low socioeconomic class,
and smoking during pregnancy can have detrimental effects on the size of
the lumbar vertebral canal and may predispose these people to future spinal
problems.
Structure and Anatomy
The human spine contains twenty-three intervertebral discs (fig. 3). The
size of the discs generally increases in both height and diameter as one moves
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Fig. 4. Micrograph of rabbit intervertebral disc histology. # ⫽ Outer AF; * ⫽ inner AF;
arrowhead ⫽ transition zone; NP ⫽ nucleus pulposus.
more caudally within the spine. The disc itself is a component of a complex
biomechanical system composed of the bony vertebral endplate, the cartilagi-
nous endplate, the AF, and the NP. This complex organ functions to allow
motion and to dissipate stress within the spinal column. Similar to other con-
nective tissues, the disc is composed mostly of extracellular matrix with a
complex array of structural and water-binding proteins and a relative paucity
of cells. Each disc can be further subdivided into four regions: the outer annu-
lus, inner annulus, transition zone, and NP (fig. 4). The outer annulus is com-
posed of highly organized, directionally oriented collagenous lamellae
running at alternating 30-degree angles to the long axis of the spine. The col-
lagen in this region of the disc is mostly type I and includes fibrils that insert
into the vertebral bodies. The cell population in the outer annulus is primarily
fibroblastic. The inner AF is larger and more fibrocartilaginous, containing
less collagen and lacking the lamellar architecture of the outer AF. Collagen
in this region is mostly type II. In addition, the inner AF contains a higher pro-
portion of large proteoglycan aggregates. The cell population in the inner AF
has characteristics of both fibroblasts and chondrocytes. The transition zone
is a distinct, thin acellular fibrous layer that separates the inner AF from the
NP. The NP contains an amorphous matrix of highly hydrated proteoglycans
embedded in a loose network of collagen. Like the inner AF, the collagen in
the NP is mostly type II. The cell population of the NP is sparse and unevenly
distributed with more cells present in the central regions of the NP than at the
periphery. At least two distinct cell populations are recognized in the NP in
early life. The first is a small round cell resembling a chondrocyte. The second
is much larger and has a vacuolated appearance described by Virchow [22] as
Table 1. Collagen composition of the intervertebral disc. (Reproduced
with permission from [116])
Type Predominant Percent of total

location collagen (%)
Fibril-forming collagens
I Annulus 0–50
II Annulus and nucleus 0–50
III Annulus ⬍5
V Annulus and nucleus 1–2
XI Annulus and nucleus 1–2
Short helix collagens
VI Annulus and nucleus 5–20
IX Annulus and nucleus 1–2
XII Annulus ⬍1
‘physoliferous’ (or ‘bubble-bearing’), containing prominent cellular processes

and intracellular glycogen deposits. This cell type is thought to be of noto-
chordal origin. As mentioned, in humans, these large, notochordally derived
cells tend to disappear (or become rare) by adolescence, leaving scattered
chondrocyte-like cells in their place [7, 8, 26, 27]. A recent study by Kim
et al. [28] demonstrated the migration of endplate chondrocytes into the NP
region of the disc, which might account in part for the shift in cell populations
within the NP region.
The disc matrix consists primarily of collagens and proteoglycans, but
varies significantly between regions of the disc. Collagen cross-linking gives
the disc substantial tensile strength, while highly hydrated proteoglycans give
the disc stiffness and resistance to compression [27]. Collagens account for
60% of the dry weight of the AF, with type I collagen being the most abundant
type (80%); the NP, on the other hand, contains up to 20% collagen with a pre-
dominance of type II collagen [7, 29, 30]. Additional collagen types are also
present throughout the disc in much smaller quantities. For example, other fib-
rillar collagens such as type V and type XI are found in greatest concentrations
in areas with high type I collagen and type II collagen, respectively [27]. Non-
fibrillar collagens found in the disc include short, helical collagens, specifically
types VI (up to 10% in the annulus and 15% in the nucleus), IX, and XII [27]
(table 1). Proteoglycans comprise a small proportion of the outer AF, but become
increasingly abundant as one moves toward the more hydrated central regions
of the disc, accounting for 50% of the dry weight of the NP. Large proteoglycan
aggregates consist of central hyaluronan molecules with multiple attached
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Fig. 5. Schematic of a proteoglycan
molecule with a central core of hyaluronic
acid and attached chains of aggrecan.
proteoglycan side chains predominantly of aggrecan. The connection between

the proteoglycan side chains and the hyaluronan backbone is stabilized by small
link proteins. The proteoglycan molecules contain numerous sulfated polysac-
charide molecules leading to a strong net negative charge. It is this net negative
charge that serves to attract water within the disc (fig. 5).
The relative proportion of collagens and proteoglycans changes through-
out life and with disc degeneration. Age-related changes lead to a decline in
the amount of large proteoglycan aggregates in the NP, thus resulting in dimin-
ished water-binding capacity. In addition, there is an increase in the proportion
of nonaggregated proteoglycans and a shift in the composition of sulfated
polysaccaride side chains leading to the diminished structural properties of the
disc [31].
The vertebral endplate is a specialized structure that contains both a bony
and a cartilaginous portion. The bony endplate is made up of cortical bone,
which is thicker around the periphery of the disc and thinner in the central
region. Adjacent to the bony endplate are specialized capillaries, which are the
primary source of nutrient exchange to the disc. The bony endplate is covered
by a layer of hyaline cartilage that forms a barrier between the vertebral body
and the disc and limits solute transport into and out of the disc. With age, the
cartilaginous endplate undergoes progressive calcification, a process which
diminishes its diffusional capacity and may lead to a nutritional crisis within the
disc [32]. In addition to aging, a number of environmental and genetic factors
have been linked to disc degeneration possibly by altering the disc by limiting
endplate diffusion. Smoking, in particular, causes shrinkage of the vascular
buds adjacent to the endplate that undoubtedly has a negative impact on disc
nutrition [33–35].
The intervertebral disc is innervated only in its outermost portion. Small
unmyelinated and encapsulated nerve endings have been found on the surface
of the outer annulus, and small free nerve endings may penetrate the outermost
layers of the annulus. The recurrent nerve of Luschka (sinuvertebral nerve),
formed from small branches of the lumbar ventral ramus, provides this sensory
innervation to the disc and is likely to be responsible for the discogenic pain. In
addition to supplying the outer annulus, these branches also supply the poste-
rior longitudinal ligament and ventral dural tube. Nerve endings have not been
found in the inner annulus or NP region of normal human discs [27].
Disc Nutrition
Most of the normal intervertebral disc is avascular, depending on the dif-

fusion for nutrient and waste exchange. Although small blood vessels can be
found on the surface of the annulus and may penetrate a short distance into the
outer layers of the disc, the central region has cells which can lie 6–8 mm from
the closest blood supply, making the disc the largest avascular organ in the
human body [27]. Unfortunately, the diffusional capacity of the disc is rela-
tively poor even in the nonpathological state, and is further limited by aging and
degenerative changes. Studies comparing the transport of small molecules into
the disc in exercised and anesthetized dogs have shown no differences between
the two groups, suggesting that simple diffusion rather than a physiological
‘pump’ mechanism is responsible for small molecule exchange within the disc
[35]. These results have been confirmed in motionless [36] and mobilized
spinal segments [37]. Recently it has been recognized that the supply of simple
nutrients is a crucial factor regulating the density of disc cells. Diffusion cham-
ber studies have suggested that glucose supply rather than oxygen is the major
factor regulating cell viability within the disc [38]. Smoking probably promotes
disc degeneration via a nutritional mechanism although a direct toxic insult to
disc cells is also possible [39].
In the largely avascular environment of the disc, cells can survive with lit-
tle oxygen [38]. However, under anaerobic conditions disc cells produce lactate
as a by-product of metabolism (fig. 6), thus leading to an acidic pH. Normal
disc tissue has a mildly acidic pH of 6.9–7.2, but under conditions of stress
(degenerative disc disease) the disc pH can be as low as 6.1 [35]. It is known
that an acidic pH leads to the inhibition of proteoglycan and collagen synthesis
and may, therefore, contribute to matrix deterioration [40]. However, matrix
degradation is likely to be dependant on many other factors, as suggested by
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Glucose
ATP
1
ADP
Glucose-6-phosphate
Fructose-6-phosphate
ATP
3
ADP
Fructose-1,6-bisphosphate
4
5
Glyceraldehyde-3-phosphate Dihydroxyacetone-phosphate
6 2 NAD ⫹Pi 2 NADH ⫹2 H
2 1,3-bis-phosphoglycerate
ADP
7
ATP Enzyme key
2 3-Phosphoglycerate
1. Hexokinase
8 2. Phosphoglucoisomerase
3. Phosphofructokinase
4. Aldolase
2 2-Phosphoglycerate
5. Triose-P-isomerase
6. Glyceraldehyde-3-P-
9 dehydrogenase
7. Phosphoglycerate kinase
8. Phosphoglycerate mutase
2 Phosphoenolpyruvate 9. Enolase
10. Pyruvate kinase
ADP
11. Lactate dehydrogenase
10
ATP 11
2 Pyruvate 2 Lactate
2 NADH ⫹ 2 H 2 NAD
Fig. 6. The glycolytic pathway. Anaerobic metabolism leads to lactate as a by-product,

an acid that can lower the pH in the environment of disc cells.
Compressive force
causes disc bulging
Vertebral body
Disc
Vertebral
body
Fig. 7. Disc in compression (intervertebral disc between two vertebral bodies). Com-
pressive forces on one side of the disc lead to disc bulging, with these forces being converted
to tensile hoop stresses by the AF. On the opposite side, the disc fibers stretch.
studies demonstrating a poor correlation between measured oxygen or lactate

levels and the grade of degeneration [41].
Disc Biomechanics
Intervertebral discs in humans have evolved to withstand the significant

forces of an upright posture. When healthy, the normal disc can withstand forces
greater than the surrounding bone, which fractures prior to the disruption of the
disc. By dissipating the large compressive forces in the spine, generated as the
result of musculature activity and vigorous physical situations, the disc serves to
protect the surrounding spine from trauma. Forces up to 17,000 N have been
estimated in lumbar discs during heavy lifting activities [42] (fig. 7). To dissi-
pate these loads, the disc converts compressive forces to tensile stresses in the
outer annulus by exerting a hydrostatic pressure via the interstitial fluid within
the disc. However, the tensile properties vary within different regions of the
annulus leading to a ‘biphasic phenomenon’ during loading. Because fibers in
the anterior/outer regions are stiffer than those in posterolateral/inner regions of
the annulus, the stiffer outer layers convert compressive loads into hoop stresses
while the inner layers act as a ‘shock absorber’ [27]. The high tensile modulus of
the normal annulus helps to prevent disc bulging. During aging and in the
degenerating disc, the swelling pressure of the NP decreases and the stiffness of
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the AF increases [43, 44]. This results in poor load dissipation and increased
stress transfer to the bony elements of the spine [43–46].
Several studies have shown that pathological loading of the spine may play a
role in disc degeneration [47–49]. Hadjipavlou et al. [50] demonstrated disc degen-
eration following a 30-degree torsional injury to the spine in an animal model. This
stress led to early degenerative changes in the disc including an increase in phos-
pholipase A2 and a decrease in NP volume by 60–90 days. Following the onset of
degeneration, increased levels of calcitonin gene-related peptide and vasoactive
intestinal peptide were found within the ganglion, supporting the association of
disc degeneration with spinal pain [51]. Researchers have shown, in animal mod-
els, that discs loaded statically are more prone to degeneration when compared to
those loaded cyclically. However, a recent investigation by MacLean et al. [52]
demonstrated that dynamic loading (12.6 N with a frequency of 0.2 Hz for 2 h) in
rats led to decreased collagen type I and II gene expression in the AF, and an
increase in the catabolic genes collagenase and stromelysin. In the NP, these
mechanically induced changes in gene expression were less significant [52]. In
addition, the magnitude and frequency of loading serve to affect the rate of degen-
eration [53]. For example, Kasra et al. [54] demonstrated that rabbit intervertebral
disc cells in the AF and the NP respond to high-freqenecy (20 Hz) and high-
amplitude (1.7 MPa for annular cells and up to 3.0 MPa for nuclear cells) loading
by increasing collagen synthesis and decreasing collagen degradation when sub-
jected to 3 days of loading. Nuclear cells demonstrated significantly less collagen
degradation as the load increased in amplitude from 1.0 to 3.0 MPa [54]. These
contrasting studies suggest a role in mechanical loading for both degenerative and
regenerative phenomenon in the disc suggesting a complex interaction between
mechanical stress and metabolism within the disc.
Segmental spinal instability associated with disc degeneration has been
quoted as a cause of low back pain. Although there is some controversy in the lit-
erature regarding the relationship between disc degeneration, annular fissures and
nonlinear segmental instability, several researchers have documented an increased
range of axial rotation in degenerative discs. Mimura et al. [55] studied flexion-
extension forces in human lumbar cadaveric spines and found that the range of
motion in flexion-extension decreased while axial rotation increased in degener-
ative spines. Krismer et al. [56] documented similar findings and correlated
increased axial instability with fissure formation in the outer annulus.
Etiology of Disc Degeneration
Disc degeneration is undoubtedly a multifactorial process involving both

environmental and genetic contributions. Some environmental factors thought
Table 2. Factors that have been impli-
cated to cause intervertebral disc degenera- Possible etiologies of disc degeneration
tion.
1. Heavy lifting
2. Vibration
3. Immobilization
4. Trauma (torsion)
5. Smoking
6. Diabetes
7. Vascular disease
8. Genetics
9. Infection
to contribute to disc degeneration include: demanding physical activities, such

as heavy lifting; vibration (experienced while driving or operating machinery);
immobilization, and repetitive torsional loads. Other environmental factors that
affect disc metabolism include smoking, poor glycemic control and vascular
disease [57] (table 2).
In spite of the many environmental factors linked to disc degeneration, it
is felt that genetic influences play the predominant role in early and perhaps
more symptomatic disc disease. The genetic contribution is supported by fam-
ily studies, case-control studies, and twin studies as well as the identification of
certain genetic linkages and single gene defects leading to degenerative disc
disease. Scapinelli [58] identified disc degeneration as a familial trait. In a
case-control study, Matsui et al. [59] documented an increased incidence of disc
degeneration among the family members of patients requiring lumbar surgery.
Disc degeneration has been found to be significantly more prevalent in siblings
of patients with disc degeneration than in random population samples [60].
Twin studies by Sambrook et al. [61] have shown a strong heritable component
to disc degeneration in both the cervical and lumbar regions.
Genetic studies have identified molecular defects contributing to disc
degeneration in certain subgroups of patients. Videman et al. [62] compared
MRI-documented disc degeneration in Finnish twins with alleles of the vita-
min D receptor and found an increased risk of disc degeneration with two
specific vitamin D receptor alleles. In a study of Japanese women, Kawaguchi
et al. [63] found that women with smaller numbers of tandem repeats in the
aggrecan gene had more severe disc degeneration. Annunen et al. [64] iden-
tified mutations in the type IX collagen ␣-2 gene which causes a single codon
substitution (tryptophan for glutamine) leading to disc degeneration in 4% of
the Finnish back pain population. Another defect in the ␣-3 chain of type IX
collagen was shown to be associated with an elevated risk of disc degenera-
tion [65]. Certain alleles of the matrix metalloproteinase-3 (MMP-3) gene
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have been linked with disc degeneration in elderly Japanese patients [69]. The
environmental risk factor obesity has been shown to act synergistically with
an allele of the COL9A3 gene, leading to a high rate of early degenerative
disc disease [70].
Genesis of Back Pain
Exactly how disc degeneration relates to back pain is poorly understood.

Some patients with minimal morphological changes in the disc complain of
chronic back pain while others with significant changes note minimal symp-
toms. Many factors including structural changes in the spine, soluble media-
tors and nerve/vessel ingrowth into the outer annulus have all been
hypothesized to be a cause of chronic spinal pain [57]. Studies have shown
that mononuclear cells infiltrating along the margins of herniated discs
express inflammatory mediators such as interleukin-1 (IL-1), intracellular
adhesion molecule-1, lymphocyte function-associated antigen and basic
fibroblast growth factor [68]. These mediators may contribute to persistent
inflammation and pain and the induction of neovascularization [68]. Sang-Ho
et al. [69] studied the expression of mRNA of various cytokines and
chemokines in herniated lumbar discs and noted an association between the
expression of IL-8 and radicular pain produced by back extension, suggesting
IL-8 as a possible target for symptomatic treatment. Alterations in the disc can
lead to changes in the alignment and the mechanical milieu of the vertebral
bodies, facet joints, spinal ligaments and muscles, producing a complex and
poorly understood biomechanical environment that may contribute to spinal
pain. It is known that the degenerating disc is capable of producing a spectrum
of cytokines and chemical mediators that are capable of stimulating pain in
the surrounding nerve endings and inducing blood vessel and nerve ingrowth
into annular defects in the disc [57]. For example, tumor necrosis factor-␣
(TNF-␣), a proinflammatory cytokine, has been shown to be a key pain medi-
ator in neurogenic pain following disc herniation. Onda et al. [70] demon-
strated, using electrophysiological testing, that an antibody to tumor necrosis
factor-␣ partially blocked the neurogenic response suggesting that tumor
necrosis factor-␣ blockage may have a therapeutic role in treating sciatica. In
addition, Nygaard et al. [71] studied leukotriene and thromboxane levels in
herniated intervertebral discs and found significantly higher levels of
leukotrienes B4 and thromboxanes B2 in noncontained as compared to con-
tained disc herniation; they suggested that these inflammatory mediators play
a role in discogenic pain and sciatica and thus were possible targets for thera-
peutic intervention.
Degenerative Events: Apoptosis, Degradative Enzymes
and Inflammatory Cytokines
In order to develop rational therapies that can slow or reverse the degen-
erative process, it is first necessary to understand the molecular events leading
to disc degeneration. In recent years, investigators have identified candidate
molecules and cellular processes such as apoptosis, or programmed cell death,
which appear to play a prominent role in disc degeneration. Although the current
molecular understanding of disc degeneration is relatively crude, knowledge in
this area is expanding rapidly.
Cell viability has been shown to be affected by age, with a larger proportion
of necrotic cells present in older individuals. In fetal and infantile intervertebral
discs approximately 2% of NP cells are necrotic; this number increases steadily to
50% by adolescence and 80% in elderly humans [57]. In addition, apoptosis, or
programmed cell death, appears to play a prominent role in the disc, with higher
rates of apoptosis present in older individuals [72]. High rates of apoptosis have
also been recognized in herniated disc fragments [73, 74]. This process appears to
be related in part to the expression of Fas and the Fas ligand, which trigger an inter-
cellular cascade leading to programmed cell death when a Fas-bearing cell comes
into contact with a Fas ligand-carrying cell. Normal disc cells do not appear to
express the Fas receptor, but do up-regulate this membrane-bound protein shortly
after the onset of experimental disc degeneration [75]. As mentioned previously,
static compression of intervertebral discs can lead to degeneration. A recent study
by Ariga et al. [76] showed that static loading of mouse intervertebral discs
resulted in higher numbers of apoptotic cells in the cartilaginous endplate; the
number of apoptotic cells increased with the load. Inhibitors of mitogen-activated
protein kinase and p38 significantly increased the number of apoptotic cells in the
loaded discs. Certain growth factors, such as insulin-like growth factor-1 (IGF-1)
and platelet-derived growth factor can exert an anti-apoptotic effect on cultured
disc cells suggesting a possible mechanism for the apoptosis of cells within the
disc [76, 77].
Some researchers have suggested that degeneration represents an alteration
in the disc cellular homeostasis, with the balance tipping away from anabolic
events and towards disc catabolism. Antoniou et al. [78] found decreased levels
of aggrecan and type II collagen production by old and degenerated disc cells
when compared to younger nondegenerated disc cells. Aguiar et al. [79] found
that NP cells were able to up-regulate their production of proteoglycans when
cocultured with notochordal cells, which are found in high concentrations only
in younger humans. The effect appeared to be due to the presence of a soluble
mediator produced by the notochordal NP cells and may explain the onset of
degeneration shortly after these notochordal cells disappear within the disc.
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In addition to soluble mediators, direct cell-to-cell communication probably
contributes to the behavior of the disc cells, as gap junctions and connexin pro-
teins that have been identified within the disc [80].
A host of inflammatory, degradative, and catabolic factors have been iden-
tified that may play a role in disc degeneration. These include proteolytic and
degradative enzymes, oxygen free radicals, nitric oxide, ILs, and prostaglandins.
Proteolytic enzymes involved in disc degeneration include cathepsin, lysozyme,
aggrecanase, and several MMPs [81–86]. A positive correlation between the
level of MMPs-1, -2, -3 and -9 and the grade of disc degeneration has been
documented [81, 83, 86]. Similarly, Melrose et al. [85] found higher levels of
lysozyme in older and degenerative discs.
Membrane-damaging oxygen-derived free radicals and nitric oxide have
been observed in cultured disc cells [87]. Herniated discs have also been shown
to express inducible nitric oxide synthetase and produce nitric oxide [82]. These
molecules have the potential to cause direct chemical injury to cell membranes
and matrix proteins. Collagens and fibronectin, for example, are known to
undergo cleavage or form high-molecular-weight complexes following exposure
to superoxides and other oxygen-derived free radicals [87] and may accumulate
as lipoprotein complexes within the matrix [88, 89]. Other disc macromolecules
undergo complex glycation reactions to form sugar-amino acid by-products that
may interfere with normal cell-matrix interactions [90].
Inflammatory cytokines such as IL-1 have been shown to play a major role
in articular cartilage degeneration and may play a role in disc degeneration as
well. Cell culture experiments have demonstrated that rabbit disc cells increase
their rate of caseinolytic activity in response to IL-1 [91]. IL-1 has been shown
to decrease the rate of proteoglycan synthesis by the disc, an effect that could be
blocked by an IL-1 receptor antagonist [92]. Other effects of IL-1 include induc-
ing increased expression of stromelysin-1, a matrix degradation protease, and an
increased production of prostaglandin E2, an inflammatory mediator [93].
Other mediators produced by disc cells include IL-6, nitric oxide, and
prostaglandin E2 [94, 95]. Herniated disc fragments are capable of producing
very high levels of phospholipase A2, an enzyme critical in the production of
prostaglandins and leukotrienes, which are important mediators of inflammation
and pain [96–98].
As the matrix of the disc undergoes degeneration, many of the macromole-
cules are only partially broken down. These nonfunctional molecules accumu-
late within the disc matrix and may be seen as lipofusion or amyloid by light
microscopy [88] or dense granular material when viewed with the electron
microscope [99–101]. Some by-products, such as fibronectin, appear to build up
within the disc during degeneration. Of interest is the role of bioactive fragments
of fibronectin which may promote tissue degeneration [102–104] (fig. 8).
a b
Fig. 8. a Degenerative disc histology. b Degenerative changes in the AF with chon-

droid nests 16 weeks after injection of FN-f (the amino terminal fragment of fibronectin).
c Higher powered micrograph of NP degeneration with a loss of cells and matrix disarray
4 weeks after FN-f injection.
Growth factor decline has also been implicated in disc degeneration. For
instance, the IGF-1 receptor appears to decrease in older animals leading to a
decrease in the IGF-1-dependent proteoglycan synthesis and perhaps the
expression of an IGF-1-binding protein [105]. Therapeutic interventions with
growth factors remain an active area of interest.
Biological Approaches to Disc Degeneration
A thorough understanding of the molecular aspects of intervertebral disc

degeneration is fundamental to the development of rational biological therapies
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Replication-deficient virus
carrying a therapeutic gene
Growth factor
RNA
Cell
Fig. 9. Schematic of virally mediated gene transfer. A desired gene is inserted into a
viral carrier that is taken up by the cell. Viruses depend on host cells for replication; this
process increases the expression of the desired gene.
aimed at slowing or stopping this process. A large number of factors have been
elucidated that contribute to the process of degeneration and it is likely that
the interplay of many different pathways contribute to the end result, making
therapeutic intervention a challenge. However, by understanding the molecular
basis of the disc degeneration, researchers hope to target specific steps in the
degenerative process, thus producing a therapeutic benefit for patients with
degenerative disc disease.
Because growth factors and stimulatory cytokines can stimulate both cell
division and metabolism, these molecules have been seen as good targets for
therapeutic intervention. Epidermal growth factor and transforming growth
factor-␤ (TGF-␤) were shown to produce a 5-fold increase in the metabolic
activity of cultured NP cells [106], and were more effective than fibroblast
growth factor or IGF-1. Osteogenic protein-1 has been shown to overcome
the degradative effects of IL-1 on cultured disc cells [107]. TGF-␤ also has the
added benefit of promoting the ‘chondrogenic phenotype’ by increasing the
production of type II collagen and proteoglycan, an effect that is desirable in
the inner regions of the disc [108]. Despite the promising results in vitro,
direct injections of recombinant proteins into the disc does not appear to be a
viable solution to disc degeneration due to the limited half-life of these mole-
cules in vivo [109].
Gene therapy has generated a high level of interest for achieving a long-
term solution to disc degeneration. Nishida et al. [108] transduced rabbit
NP cells in vivo with an adenovirus carrying the TGF-␤ gene driven by a
cytomegalovirus promotor and observed a 2-fold increase in proteoglycan
production at the one week time point (fig. 9). Although encouraging, the
authors noted that the long-term expression of a therapeutic gene might be
difficult using the viral vector systems due to the possibility of immuno-
logical activity against viral antigens. Additionally, it is unclear whether a
degenerating disc in metabolic disarray would be able to respond to growth
factors in the same manner as the normal disc cells. Investigations are cur-
rently ongoing to determine the optimal genes and transduction mechanisms
for gene therapy within the disc.
Another attractive therapeutic option being investigated currently is the use
of a cell-based therapy using either mature cells (chondrocytes or disc cells) or
pluripotent cells (stem cells). Gruber et al. [110] successfully studied autologous
disc cells transplanted into the intervertebral disc of the sand rat (Psammomys
obesus). These authors demonstrated that autografted cells were able to exhibit
morphologies similar to native disc cells and were able to survive at least 33
weeks in vivo.
Others have suggested that stem cells, which are able to differentiate
into multiple cell types, including chondrocytes, may be an ideal vehicle for
cell-based therapy. These cells could be treated with therapeutic genes ex vivo
prior to implantation and thus used to deliver therapeutic genes to the disc and
participate in the repair process. Although encouraging, such strategies have
yet to be successfully achieved in a reasonable model of degenerative disc
disease.
Early tissue engineering approaches have evaluated cellular scaffolds for
the delivery of cells to the disc. The advantage of a cell scaffold is that the
therapeutic cells are maintained at the implantation site and are provided
with a three-dimensional environment necessary for division and migration
within the disc [111]. Perka et al. [112] found that NP cells suspended in fib-
rin-alginate beads and fibrin beads were capable of proliferating and produc-
ing extracellular matrix. Lee et al. utilized cells transduced with the TGF-␤
gene in a ‘pellet culture’ system as an alternative to alginate bead micros-
pheres and achieved a native cell phenotype capable of producing type II
collagen and proteoglycan [111]. Some scaffolds may not be as effective as
previously hoped. Alini et al. [113] demonstrated problems with proteogly-
can retention when using scaffolds of type I collagen and hyaluronan seeded
with bovine NP and AF cells. In contrast, Sato et al. [114] found that allo-
grafted AF cells placed in an atelocollagen honeycomb-shaped scaffold with
a membrane seal (ACHMS-scaffold) were able to proliferate, retain type II
collagen mRNA, and actually decrease the narrowing of intervertebral disc
space in Japanese white rabbits [115]. Much more research is needed to
determine the role of cell scaffolds and cellular therapies in treating inter-
vertebral disc degeneration.
medwedi.ru
Conclusion
The intervertebral disc is a part of a complex mechanical system, pivotal

in the dissipation of forces in the spinal column. Aging, environmental and
molecular-genetic factors all contribute to the degeneration of the disc and its
ultimate mechanical failure. Recently, researchers around the world have begun
to understand the molecular basis of intervertebral disc degeneration. This under-
standing forms the basis for the design of biological therapies aimed at slowing
or reversing the degenerative process. With time, these early efforts may lead to
a shift in treatment options from those focusing on symptomatic relief to those
aimed at correcting the underlying disease process. Although promising, these
early attempts are far from achieving the goal of tissue regeneration. Much
work remains to define the ability of gene- and cell-based strategies to produce
a biologically desirable result and to apply these clinically for the benefit of
patients. Fortunately, the underlying molecular basis of this ubiquitous disease
process is rapidly becoming clear.
References
1 McMeeken J, Tully E, Stillman B, Nattrass C, Bygott IL, Story I: The experience of back pain in
young Australians. Man Ther 2001;6:213–220.
2 Waddell G: Low back disability. A syndrome of Western civilization. Neurosurg Clin N Am
1991;2:719–738.
3 Waddell G: Low back pain: A twentieth century health care enigma. Spine 1996;21:2820–2825.
4 Fischgrund JS, Montgomery DM: Diagnosis and treatment of discogenic low back pain. Orthop
Rev 1993;22:311–318.
5 Frymoyer JW, Cats-Baril WL: An overview of the incidences and costs of low back pain. Orthop
Clin North Am 1991;22:263–271.
6 Schwarzer AC, Aprill CN, Derby R, Fortin J, Kine G, Bogduk N: The relative contributions of the
disc and zygapophyseal joint in chronic low back pain. Spine 1994;19:801–806.
7 Buckwalter JA: Aging and degeneration of the human intervertebral disc. Spine 1995;20:
1307–1314.
8 Boos N, Weissbach S, Rohrbach H, Weiler C, Spratt KF, Nerlich AG: Classification of age-related
changes in lumbar intervertebral discs: 2002 Volvo Award in Basic Science. Spine 2002;27:
2631–2644.
9 Miller JA, Schmatz C, Schultz AB: Lumbar disc degeneration: Correlation with age, sex, and
spine level in 600 autopsy specimens. Spine 1988;13:173–178.
10 Boos N, Dreier D, Hilfiker E et al: Tissue characterization of symptomatic and asymptomatic disc
herniations by quantitative magnetic resonance imaging. J Orthop Res 1997;15:141–149.
11 Antoniou J, Pike GB, Steffen T, et al: Quantitative magnetic resonance imaging in the assessment
of degenerative disc disease. Magn Reson Med 1998;40:900–907.
12 Boos N, Wallin A, Schmucker T, Aebi M, Boesch C: Quantitative MR imaging of lumbar inter-
vertebral disc and vertebral bodies: Methodology, reproducibility, and preliminary results. Magn
Reson Imaging 1994;12:577–587.
13 Berlemann U, Gries NC, Moore RJ: The relationship between height, shape and histological
changes in early degeneration of the lower lumbar discs. Eur Spine J 1998;7:212–217.
14 Andersson GB: What are the age-related changes in the spine? Baillieres Clin Rheumatol 1998;
12:161–173.
15 Freemont AJ, Watkins A, Le Maitre C, et al: Nerve growth factor expression and innervation of
the painful intervertebral disc. J Pathol 2002;197:286–292.
16 Jones MD, Pais MJ, Omiya B: Bony overgrowths and abnormal calcifications about the spine.
Radiol Clin North Am 1988;26:1213–1234.
17 O’Neill TW, McCloskey EV, Kanis JA, et al: The distribution, determinants, and clinical correlates
of vertebral osteophytosis: A population based survey. J Rheumatol 1999;26:842–848.
18 Videman T, Battie MC, Gibbons LE, Maravilla K, Manninen H, Kaprio J: Associations between
back pain history and lumbar MRI findings. Spine 2003;28:582–588.
19 Taylor JR, Twomey LT: The development of the human intervertebral disc; in Ghosh P (ed): The
Biology of the Intevertebral Disc. Boca Raton, CRC Press, 1988, pp 39–82.
20 Wyburn GM: Observations on the development of the human intervertebral column. J Anat 1944;78.
21 Walmsley R: The development and growth of the intervertebral disc. Edinburgh Med J 1953;60.
22 Virchow R: Untersuchungen über die Entwicklung des Schädelgrundes. Berlin, Reimer, 1857.
23 Peacock A: Observations on the prenatal development of the intervertebral disc in man. J Anat
1951;85.
24 Peacock A: Observations on the postnatal structure of the intervertebral disc in man. J Anat
1952;86.
25 Jeffrey J, Campbell D, Golden M, et al: Antenatal factors in the development of the lumbar verte-
bral canal. Spine 2003;28:1418–1423.
26 Freemont AJ, Peacock TE, Goupille P, Hoyland JA, O’Brien J, Jayson MI: Nerve ingrowth into
diseased intervertebral disc in chronic back pain. Lancet 1997;350:178–181.
27 Buckwalter JA, Mow VC, Boden SD, Eyre DR, Weidenbaum M: Intervertebral disk structure,
composition, and mechanical function; in Buckwalter JA, Einhorn TA, Simon SR (eds):
Orthopaedic Basic Science – Biology and Biomechanics of the Musculoskeletal System, ed 2.
Rosemont, American Academy of Orthopaedic Surgeons, 2000, pp 548–555.
28 Kim KW, Lim TH, Kim JG, Jeong ST, Masuda K, An HS: The origin of chondrocytes in the
nucleus pulposus and histologic findings associated with the transition of a notochordal nucleus
pulposus to a fibrocartilaginous nucleus pulposus in intact rabbit intervertebral discs. Spine 2003;
28:982–990.
29 Eyre DR, Muir H: Types I and II collagens in intervertebral disc. Interchanging radial distributions
in annulus fibrosus. Biochem J 1976;157:267–270.
30 Eyre DR: Biochemistry of the intervertebral disc. Int Rev Connect Tissue Res 1979;8: 227–291.
31 Buckwalter JA, Martin J: Intervertebral disk degeneration and back pain; in Weinstein JN, Gordon
SL (eds): Low Back Pain: A Scientific and Clinical Overview. Rosemont, American Academy of
Orthopaedic Surgeons, 1996.
32 Roberts S, Urban JP, Evans H, Eisenstein SM: Transport properties of the human cartilage end-
plate in relation to its composition and calcification. Spine 1996;21:415–420.
33 Iwahashi M, Matsuzaki H, Tokuhashi Y, Wakabayashi K, Uematsu Y: Mechanism of intervertebral
disc degeneration caused by nicotine in rabbits to explicate intervertebral disc disorders caused by
smoking. Spine 2002;27:1396–1401.
34 Uematsu Y, Matuzaki H, Iwahashi M: Effects of nicotine on the intervertebral disc: An experi-
mental study in rabbits. J Orthop Sci 2001;6:177–182.
35 Holm S, Maroudas A, Urban JP, Selstam G, Nachemson A: Nutrition of the intervertebral disc:
Solute transport and metabolism. Connect Tissue Res 1981;8:101–119.
36 Urban JP, Holm S, Maroudas A, Nachemson A: Nutrition of the intervertebral disc: Effect of fluid
flow on solute transport. Clin Orthop 1982;296–302.
37 Katz MM, Hargens AR, Garfin SR: Intervertebral disc nutrition. Diffusion versus convection. Clin
Orthop 1986;243–245.
38 Horner HA, Urban JP: 2001 Volvo Award Winner in Basic Science Studies: Effect of nutrient sup-
ply on the viability of cells from the nucleus pulposus of the intervertebral disc. Spine 2001;26:
2543–2549.
39 Holm S, Nachemson A: Nutrition of the intervertebral disc: Acute effects of cigarette smoking.
An experimental animal study. Ups J Med Sci 1988;93:91–99.
medwedi.ru
40 Ohshima H, Urban JP: The effect of lactate and pH on proteoglycan and protein synthesis rates in
the intervertebral disc. Spine 1992;17:1079–1082.
41 Bartels EM, Fairbank JC, Winlove CP, Urban JP: Oxygen and lactate concentrations measured
in vivo in the intervertebral discs of patients with scoliosis and back pain. Spine 1998;23:1–7.
42 Cholewicki J, McGill SM, Norman RW: Lumbar spine loads during the lifting of extremely heavy
weights. Med Sci Sports Exerc 1991;23:1179–1186.
43 Acaroglu ER, Iatridis JC, Setton LA, Foster RJ, Mow VC, Weidenbaum M: Degeneration and
aging affect the tensile behavior of human lumbar anulus fibrosus. Spine 1995;20:2690–2701.
44 Panagiotacopulos ND, Knauss WG, Bloch R: On the mechanical properties of human interverte-
bral disc material. Biorheology 1979;16:317–330.
45 Fujita Y, Duncan NA, Lotz JC: Radial tensile properties of the lumbar annulus fibrosus are site
and degeneration dependent. J Orthop Res 1997;15:814–819.
46 Ishihara H, Urban JP: Effects of low oxygen concentrations and metabolic inhibitors on proteo-
glycan and protein synthesis rates in the intervertebral disc. J Orthop Res 1999;17:829–835.
47 Kroeber MW, Unglaub F, Wang H, et al: New in vivo animal model to create intervertebral disc
degeneration and to investigate the effects of therapeutic strategies to stimulate disc regeneration.
Spine 2002;27:2684–2690.
48 Natarajan RN, Ke JH, Andersson GB: A model to study the disc degeneration process. Spine
1994;19:259–265.
49 Sandover J: Dynamic loading as a possible source of low-back disorders. Spine 1983;8:652–658.
50 Hadjipavlou AG, Simmons JW, Yang JP, et al: Torsional injury resulting in disc degeneration. I. An
in vivo rabbit model. J Spinal Disord 1998;11:312–317.
51 Hadjipavlou AG, Simmons JW, Yang JP, Bi LX, Simmons DJ, Necessary JT: Torsional injury
resulting in disc degeneration in the rabbit. II. Associative changes in dorsal root ganglion and
spinal cord neurotransmitter production. J Spinal Disord 1998;11:318–321.
52 MacLean JJ, Lee CR, Grad S, Ito K, Alini M, Iatridis JC: Effects of immobilization and dynamic
compression on intervertebral disc cell gene expression in vivo. Spine 2003;28:973–981.
53 Ching CT, Chow DH, Yao FY, Holmes AD: The effect of cyclic compression on the mechanical
properties of the intervertebral disc: An in vivo study in a rat tail model. Clin Biomech (Bristol,
Avon) 2003;18:182–189.
54 Kasra M, Goel V, Martin J, et al: Effect of dynamic hydrostatic pressure on rabbit intervertebral
disc cells. J Orthop Res 2003;21:597–603.
55 Mimura M, Panjabi MM, Oxland TR, Crisco JJ, Yamamoto I, Vasavada A: Disc degeneration
affects the multidirectional flexibility of the lumbar spine. Spine 1994;19:1371–1380.
56 Krismer M, Haid C, Behensky H, Kapfinger P, Landauer F, Rachbauer F: Motion in lumbar func-
tional spine units during side bending and axial rotation moments depending on the degree of
degeneration. Spine 2000;25:2020–2027.
57 Buckwalter JA, Boden SD, Erye DR, Mow VC, Weidenbaum M: Intervertebral disk aging, degen-
eration, and herniation; in Buckwalter JA, Einhorn TA, Simon SR (eds): Orthopaedic Basic
Science – Biology and Biomechanics of the Musculoskeletal System, ed 2. Rosemont, American
Academy of Orthopaedic Surgeons, 2000, pp 557–566.
58 Scapinelli R: Lumbar disc herniation in eight siblings with a positive family history for disc dis-
ease. Acta Orthop Belg 1993;59:371–376.
59 Matsui H, Kanamori M, Ishihara H, Yudoh K, Naruse Y, Tsuji H: Familial predisposition for lum-
bar degenerative disc disease. A case-control study. 1998.
60 Bijkerk C, Houwing-Duistermaat JJ, Valkenburg HA, et al: Heritabilities of radiologic osteoarthri-
tis in peripheral joints and of disc degeneration of the spine. Arthritis Rheum 1999;42:1729–1735.
61 Sambrook PN, MacGregor AJ, Spector TD: Genetic influences on cervical and lumbar disc degen-
eration: A magnetic resonance imaging study in twins. Arthritis Rheum 1999;42:366–372.
62 Videman T, Leppavuori J, Kaprio J, et al: Intragenic polymorphisms of the vitamin D receptor
gene associated with intervertebral disc degeneration. Spine 1998;23:2477–2485.
63 Kawaguchi Y, Osada R, Kanamori M, et al: Association between an aggrecan gene polymorphism
and lumbar disc degeneration. Spine 1999;24:2456–2460.
64 Annunen S, Paassilta P, Lohiniva J, et al: An allele of COL9A2 associated with intervertebral disc
disease. Science 1999;285:409–412.
65 Paassilta P, Lohiniva J, Goring HH, et al: Identification of a novel common genetic risk factor for
lumbar disk disease. JAMA 2001;285:1843–1849.
66 Takahashi M, Haro H, Wakabayashi Y, Kawa-uchi T, Komori H, Shinomiya K: The association of
degeneration of the intervertebral disc with 5a/6a polymorphism in the promoter of the human
matrix metalloproteinase-3 gene. J Bone Joint Surg Br 2001;83:491–495.
67 Solovieva S, Lohiniva J, Leino-Arjas P, et al: COL9A3 gene polymorphism and obesity in inter-
vertebral disc degeneration of the lumbar spine: Evidence of gene-environment interaction. Spine
2002;27:2691–2696.
68 Doita M, Kanatani T, Harada T, Mizuno K: Immunohistologic study of the ruptured intervertebral
disc of the lumbar spine. Spine 1996;21:235–241.
69 Sang-Ho A, Yoon-Woo C, et al: mRNA expression of cytokines and chemokines in herniated lum-
bar intervertebral discs. Spine 2002;27:911–917.
70 Onda A, Yabuki S, Kikuchi S: Effects of neutralizing antibodies to tumor necrosis factor-alpha on
nucleus pulposus-induced abnormal nociresponses in rat dorsal horn neurons. Spine 2003;28:
967–972.
71 Nygaard O, Mellgren S, Osterud B: The inflammatory properties of contained and noncontained
lumbar disc herniation. Spine 1997;22:2484–2488.
72 Gruber HE, Hanley EN Jr: Analysis of aging and degeneration of the human intervertebral disc.
Comparison of surgical specimens with normal controls. Spine 1998;23:751–757.
73 Park JB, Chang H, Kim KW: Expression of Fas ligand and apoptosis of disc cells in herniated
lumbar disc tissue. Spine 2001;26:618–621.
74 Park JB, Kim KW, Han CW, Chang H: Expression of Fas receptor on disc cells in herniated lum-
bar disc tissue. Spine 2001;26:142–146.
75 Anderson DG, Izzo MW, Hall DJ, et al: Comparative gene expression profiling of normal and
degenerative discs: Analysis of a rabbit annular laceration model. Spine 2002;27:1291–1296.
76 Ariga K, Yonenobu K, Nakase T, et al: Mechanical stress-induced apoptosis of endplate chondro-
cytes in organ-cultured mouse intervertebral discs. Spine 2003;28:1528–1533.
77 Gruber HE, Norton HJ, Hanley EN Jr: Anti-apoptotic effects of IGF-1 and PDGF on human inter-
vertebral disc cells in vitro. Spine 2000;25:2153–2157.
78 Antoniou J, Steffen T, Nelson F, et al: The human lumbar intervertebral disc: Evidence for changes
in the biosynthesis and denaturation of the extracellular matrix with growth, maturation, aging,
and degeneration. J Clin Invest 1996;98:996–1003.
79 Aguiar DJ, Johnson SL, Oegema TR: Notochordal cells interact with nucleus pulposus cells:
Regulation of proteoglycan synthesis. Exp Cell Res 1999;246:129–137.
80 Gruber HE, Ma D, Hanley EN Jr, Ingram J, Yamaguchi DT: Morphologic and molecular evidence
for gap junctions and connexin 43 and 45 expression in annulus fibrosus cells from the human
intervertebral disc. J Orthop Res 2001;19:985–989.
81 Crean JK, Roberts S, Jaffray DC, Eisenstein SM, Duance VC: Matrix metalloproteinases in the
human intervertebral disc: Role in disc degeneration and scoliosis. Spine 1997;22:2877–2884.
82 Furusawa N, Baba H, Miyoshi N, et al: Herniation of cervical intervertebral disc:
Immunohistochemical examination and measurement of nitric oxide production. Spine 2001;26:
1110–1116.
83 Kanemoto M, Hukuda S, Komiya Y, Katsuura A, Nishioka J: Immunohistochemical study of
matrix metalloproteinase-3 and tissue inhibitor of metalloproteinase-1 human intervertebral discs.
Spine 1996;21:1–8.
84 Konttinen YT, Kaapa E, Hukkanen M, et al: Cathepsin G in degenerating and healthy discal tis-
sue. Clin Exp Rheumatol 1999;17:197–204.
85 Melrose J, Ghosh P, Taylor TK: Lysozyme, a major low-molecular-weight cationic protein of the
intervertebral disc, which increases with ageing and degeneration. Gerontology 1989;35:173–180.
86 Roberts S, Caterson B, Menage J, Evans EH, Jaffray DC, Eisenstein SM: Matrix metallopro-
teinases and aggrecanase: Their role in disorders of the human intervertebral disc. Spine 2000;25:
3005–3013.
87 Oegema T: The role of proteinases and other degradative mechanics in idiopathic low back pain;
in Weinstein JN, Gordon SL (eds): Low Back Pain: A Scientific and Clinical Overview. Rosemont,
American Academy of Orthopaedic Surgeons, 1996.
medwedi.ru
88 Ishii T, Tsuji H, Sano A, Katoh Y, Matsui H, Terahata N: Histochemical and ultrastructural obser-
vations on brown degeneration of human intervertebral disc. J Orthop Res 1991;9:78–90.
89 Yasuma T, Koh S, Okamura T, Yamauchi Y: Histological changes in aging lumbar intervertebral
discs. Their role in protrusions and prolapses. J Bone Joint Surg Am 1990;72:220–229.
90 Hormel SE, Eyre DR: Collagen in the ageing human intervertebral disc: An increase in covalently
bound fluorophores and chromophores. Biochim Biophys Acta 1991;1078:243–250.
91 Shinmei M, Kikuchi T, Yamagishi M, Shimomura Y: The role of interleukin-1 on proteoglycan
metabolism of rabbit annulus fibrosus cells cultured in vitro. Spine 1988;13:1284–1290.
92 Maeda S, Kokubun S: Changes with age in proteoglycan synthesis in cells cultured in vitro from
the inner and outer rabbit annulus fibrosus. Responses to interleukin-1 and interleukin-1 receptor
antagonist protein. Spine 2000;25:166–169.
93 Rannou F, Corvol MT, Hudry C, et al: Sensitivity of anulus fibrosus cells to interleukin 1 beta.
Comparison with articular chondrocytes. Spine 2000;25:17–23.
94 Kang JD, Georgescu HI, McIntyre-Larkin L, Stefanovic-Racic M, Evans CH: Herniated cervical
intervertebral discs spontaneously produce matrix metalloproteinases, nitric oxide, interleukin-6,
and prostaglandin E2. Spine 1995;20:2373–2378.
95 Kang JD, Georgescu HI, McIntyre-Larkin L, Stefanovic-Racic M, Donaldson WF III, Evans CH:
Herniated lumbar intervertebral discs spontaneously produce matrix metalloproteinases, nitric
oxide, interleukin-6, and prostaglandin E2. Spine 1996;21:271–277.
96 Franson RC, Saal JS, Saal JA: Human disc phospholipase A2 is inflammatory. Spine 1992;17:
S129–S132.
97 Gronblad M, Virri J, Tolonen J, et al: A controlled immunohistochemical study of inflammatory
cells in disc herniation tissue. Spine 1994;19:2744–2751.
98 Saal JS, Franson RC, Dobrow R, Saal JA, White AH, Goldthwaite N: High levels of inflammatory
phospholipase A2 activity in lumbar disc herniations. Spine 1990;15:674–678.
99 Trout JJ, Buckwalter JA, Moore KC: Ultrastructure of the human intervertebral disc. II. Cells of
the nucleus pulposus. Anat Rec 1982;204:307–314.
100 Yasuma T, Makino E, Saito S, Inui M: Histological development of intervertebral disc herniation.
J Bone Joint Surg Am 1986;68:1066–1072.
101 Yasuma T, Arai K, Suzuki F: Age-related phenomena in the lumbar intervertebral discs.
Lipofuscin and amyloid deposition. Spine 1992;17:1194–1198.
102 Oegema TR Jr, Johnson SL, Aguiar DJ, Ogilvie JW: Fibronectin and its fragments increase with
degeneration in the human intervertebral disc. Spine 2000;25:2742–2747.
103 Trout JJ, Buckwalter JA, Moore KC, Landas SK: Ultrastructure of the human intervertebral disc.
I. Changes in notochordal cells with age. Tissue Cell 1982;14:359–369.
104 Hollander AP, Heathfield TF, Webber C, et al: Increased damage to type II collagen in osteoarthritic
articular cartilage detected by a new immunoassay. J Clin Invest 1994;93:1722–1732.
105 Okuda S, Myoui A, Ariga K, Nakase T, Yonenobu K, Yoshikawa H: Mechanisms of age-related
decline in insulin-like growth factor-I dependent proteoglycan synthesis in rat intervertebral disc
cells. Spine 2001;26:2421–2426.
106 Thompson JP, Oegema TR Jr, Bradford DS: Stimulation of mature canine intervertebral disc by
growth factors. Spine 1991;16:253–260.
107 Takegami K, Thonar EJ, An HS, Kamada H, Masuda K: Osteogenic protein-1 enhances matrix
replenishment by intervertebral disc cells previously exposed to interleukin-1. Spine
2002;27:1318–1325.
108 Nishida K, Kang JD, Gilbertson LG, et al: Modulation of the biologic activity of the rabbit inter-
vertebral disc by gene therapy: An in vivo study of adenovirus-mediated transfer of the human
transforming growth factor beta 1 encoding gene. Spine 1999;24:2419–2425.
109 Nishida K, Gilbertson LG, Robbins PD, Evans CH, Kang JD: Potential applications of gene ther-
apy to the treatment of intervertebral disc disorders.
110 Gruber HE, Johnson TL, Leslie K, et al: Autologous intervertebral disc cell implantation: A model
using Psammomys obesus, the sand rat. Spine 2002;27:1626–1633.
111 Sato M, Asazuma T, Ishihara M, et al: An experimental study of the regeneration of the interver-
tebral disc with an allograft of cultured annulus fibrosus cells using a tissue-engineering method.
Spine 2003;28:548–553.
112 Perka C, Arnold U, Spitzer RS, Lindenhayn K: The use of fibrin beads for tissue engineering and
subsequential transplantation. Tissue Eng 2001;7:359–361.
113 Alini M, Li W, Markovic P, Aebi M, Spiro RC, Roughley PJ: The potential and limitations of a
cell-seeded collagen/hyaluronan scaffold to engineer an intervertebral disc-like matrix. Spine
2003;28:446–454.
114 Sato M, Asazuma T, Ishihara M, et al: An atelocollagen honeycomb-shaped scaffold with a mem-
brane seal (ACHMS-scaffold) for the culture of annulus fibrosus cells from an intervertebral disc.
J Biomed Mater Res 2003;64A:248–256.
115 Yung LJ, Hall R, Pelinkovic D, et al: New use of a three-dimensional pellet culture system for
human intervertebral disc cells: Initial characterization and potential use for tissue engineering.
Spine 2001;26:2316–2322.
116 Buckwalter JA, Einhorn TA, Simon SR (eds): Orthopaedic Basic Science – Biology and
Biomechanics of the Musculoskeletal System, ed 2. Rosemont, AAOS, 2000, p 550.
117 Ashton-Miller JA, Schultz AB: Biomechanics of the human spine; in Mow BC, Hayes WC (eds):
Basic Orthopaedic Biomechanics. Philadelphia, Lippincott-Raven, 1997, pp 353–393.
D. Greg Anderson, MD
Department of Orthopaedic Surgery
Thomas Jefferson University
925 Chestnut St., 5th Floor
Philadelphia, PA 19107 (USA)
Tel. ⫹1 267 339 3623, E-Mail greg.anderson@rothmaninstitute.com
medwedi.ru
Genetics of Degenerative
Disc Disease
Shekar N. Kurpad, Jason Lifshutz
Department of Neurosurgery, Medical College of Wisconsin,
Milwaukee, Wisc., USA
Introduction
Back and neck pain are some of the most common conditions for which
patients seek medical attention. 80% of the population has reported this com-
plaint at some point during their lifetime, with 5% having chronic pain [8, 14].
Back pain is the most frequent cause of activity limitation in patients under the
age of 45 and is a common cause of disability and medical cost [8, 14]. While
there are several causes of back pain, degenerative and mechanical disorders of
the spine and intervertebral discs encompass the most common reasons.
Traditionally, degeneration has been considered to be a mechanical phen-
omenon; current studies suggest that genetic and biochemical mechanisms may
play a much larger role than is generally appreciated. Topics examined in this
review include a brief description of the normal anatomy and biochemistry of
the intervertebral disc. In addition, the disc complex is examined from a cellular,
biochemical, and genetic basis. We conclude with a look toward the future, and
how these new developments may be used in translational research in going
from bench to bedside.
Biochemistry of the Intervertebral Disc
The adult intervertebral disc is an avascular fibrocartilaginous complex

that links the adjacent vertebrae of the spine. Each disc is made of a gelatinous
nucleus pulposus surrounded by a laminated annulus fibrosus [3, 5–8, 14].
There is no definitive interface between these two regions, and it is referred to
in the literature as the ‘transition zone’ [8, 14]. The adult disc is comprised of
poorly characterized cells surrounded by an exhaustive extracellular matrix.
There are generally two types of cells, fibrocytes in the outer annulus and chon-
drocytes in the remaining layers [3, 5–8, 14], which are thought to be better
equipped to withstand the avascular environment of the disc. The role of these
cell populations is to synthesize, maintain and repair the matrix of the disc.
The matrix of the disc is a framework of polar macromolecules bound with
water, mainly collagen fibrils and proteoglycans, which are glycosaminoglycans
such as chondroitin sulfate and keratin sulfate attached to a protein core.
Collagens are important in conferring tensile strength to the disc, and appear to
play a role in the genetic predisposition toward spinal degeneration. They make
up approximately 70% of the annulus but only 20% of the nucleus pulposus. In
the annulus, collagen is found in tightly packed fibrils arranged in specific lamel-
lae. The majority of collagen found in this region is type I, with smaller amounts
of types II, III, V, and IX also being present. Within the nucleus pulposus, 85%
of the collagen is of type II, with smaller amounts of VI and IX also being found
[3, 7, 8, 11, 14]. Proteoglycans absorb water, conferring both stiffness and
resilience to the disc; they are present within the lamellae of the collagen fibrils
and are found in the nucleus pulposus in their greatest concentrations. The
proteoglycan aggregate is made up of a central glycosaminoglycan hyaluronate
filament that is attached to various proteoglycans via linker proteins [7, 8, 14].
Intervertebral discs are situated between the cartilaginous endplates of adja-
cent vertebrae. The endplates are initially composed of hyaline cartilage, produced
by chondrocytes; later in life, this is replaced in part by calcified cartilage.
Collagen fibers of the annulus attach directly to the endplates. This is a site sus-
ceptible to mechanical failure, especially when exposed to shear forces [8, 11, 14].
At birth the intervertebral discs have an abundant vascular supply. As one
ages, the discs generally lose this vascular supply (usually by 2 years of age), a
process accelerated by the degeneration and calcification of the vertebral end-
plate, and subsequently derive their nutrients and eliminate their waste through
the process of diffusion, which is driven by the high osmotic pressure within the
discs plus the hydrostatic pressure acting on the discs. This process requires a
high intrinsic water content, provided by the proteoglycan attraction.
Molecular Biology of Disc Degeneration
Diffusion
The main characteristic of disc degeneration is the loss of the hydrostatic
properties of the disc. At birth, the nucleus pulposus contains 85–90% water.
With aging, this percentage drops to approximately 70%. This drop in water
content occurs with changes in the proteoglycan extracellular matrix of the disc.
Genetics of Degenerative Disc Disease 31
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A decrease in the turgor of the disc causes the disc to lose its ability to resist
compression and provides resilience to the spine. The diffusion of disc homeo-
static processes may become impaired in this dehydrated state due to quantita-
tive and qualitative changes in the extracellular matrix [8, 14].
Diffusion may become impaired by other means that decrease blood flow
to the spine/disc. Blood flow to the disc space regresses as aging proceeds and
other concomitant factors such as diabetes and vascular disease may impair the
vascular supply to the vertebral endplate [8, 14]. Calcification of the vertebral
endplate may have a detrimental effect on the diffusive processes necessary for
the nutrition of resident cells. Lack of adequate oxygen supply to cellular struc-
tures of the disc may lead to anaerobic glycolysis, lactate production, a decrease
in the pH, and ultimately breakdown and damage to the extracellular matrix.
This pH change causes poor hydrostatic pressure, and hence further deteriora-
tion in the diffusion process, with its related consequences [3, 5, 7, 8, 14].
Cellular Activity
During the early phases of degeneration, there is thought to be a prolifera-
tion of cells within the annulus, with a metaplasia of these cells into chondro-
cytes [7, 8, 14]. As degeneration continues, diffusion through the disc declines,
leading to anaerobic metabolism and cell death. With the loss of support cells,
synthesis and maintenance of the disc matrix disappears with decreased water
content and hence a decrease in the diffusion of essential nutrients.
Biochemical Changes
Proteoglycans
Both quantitative and qualitative changes are seen in the extracellular
matrix of the aging disc as it degenerates. First, the amount of proteoglycans
decreases in the degenerating disc. This decrease in proteoglycans is generally
an early sign of degeneration. The mucopolysaccharide complexes in young
discs are made up primarily of chondroitin sulfate A and C side chains, which
are strongly hydrophilic [8, 14]. As the disc ages, these large molecules break
down into smaller ones, such as chondroitin sulfate B and keratin sulfate, which
do not have the water-storing capacity of types A and C chondroitin sulfate, and
subsequently lead to disc dehydration [8, 14]. Keratin sulfate, in particular, has
been found to be a marker of disc degeneration in surgical and pathological
specimens [5]. Disc dehydration eventually results in impaired diffusion and
further disc degeneration [14].
Collagen
As previously mentioned, the intervertebral disc is composed mostly of
type I and type II collagen [3, 5–8, 14]. Type I collagen is found primarily in
Kurpad/Lifshutz 32
the annulus fibrosus and type II collagen in the nucleus pulposus. In early
degeneration, the location and types of collagen do not change. However,
there is an increase in the amount of collagen found within both the annulus
and nucleus. There also may be an increase in collagen type III, V, and VI
[3, 7, 8, 14].
As degeneration continues, there are qualitative changes seen in the type
of collagen in various portions of the disc. For example, there is an increase in
the amount of collagen type I within the nucleus. In addition, there is a loss of
collagen type II at the endplates. Further changes that are seen include the
formation of collagen types IV and X, and changes in the post-translational
modification of these new fibrils. These larger collagen fibrils are thought to be
weaker than their narrow counterparts, which may lead to tearing and disrup-
tion [8, 14].
Young and healthy discs have been found to have active matrix formation
and denaturation of collagen type II. Studies on the role of collagen in disc
degeneration show a decrease in the levels of aggrecan and type II procolla-
gen formation and a general increase in type II collagen degeneration and
type I synthesis. Type IX collagen has recently been reported to be present in
both the aging and degenerated disc, whose formation may represent a
compensatory repair process. A molecular defect in type IX collagen has been
discovered as a contributing factor toward disc degeneration, specifically a
conversion of the codon for glutamate to tryptophan in the COL9A2 gene.
This genetic polymorphism was found in approximately 10% of patients with
disc disease, and MRI correlated an association of the genetic defect with
radial tears within the disc. In addition, another genetic mutation in collagen
formation, the TRP3 allele, has been found in 12% of patients studied with
disc degeneration.
Inflammation
There are several inflammatory mediators which are present within
the degenerative disc. Some of those that are present include nitric oxide,
interleukin-6 (IL-6), IL-8 [2] (which has been associated with radiculopathy),
prostaglandin E2, and a family of enzymes known as matrix metalloproteinases
(MMPs) [3–13]. IL-6 and Prostaglandin E2 seem to exert their effects by
inhibiting proteoglycan synthesis. They appear to be under the influential con-
trol of IL-1, which is both a central mediator of the inflammatory process, and
a direct toxin to the proteoglycan matrix. The principal cause of the release of
IL-1 is yet to be determined [4, 7, 9].
The role of extracellular matrix degeneration in the disc disease process is
becoming better understood. MMPs are proteinases that degrade at least one
component of the extracellular matrix; they are secreted in a latent form and
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require activation for proteolytic activity. Their activity is inhibited by specific
tissue inhibitors. This family of enzymes can be divided into four groups; col-
lagenases, stromelysins, gelatinases and membrane metalloproteinases [3, 4, 7,
8, 14]. Recent studies suggest that this family of proteases, in particular MMP-1,
MMP-2, MMP-3 and MMP-9, play a significant role in the degradation and
degeneration of the intervertebral disc [3–5, 7–12]. Recent studies showed that
increases in the mRNA in MMP-1 and MMP-3 have been reported in cell
populations of discs cultured with IL-1, IL-12 and tumor necrosis factor-␣.
Further evidence of the role of MMPs is demonstrated in their activity in the
degeneration of aggrecan in degenerated discs. Finally, changes in the role of
cathepsins, fibromodulin, and fibronectin are being described and elucidated in
the degenerative disease process [8, 14]. A therapeutic goal is to find potential
inhibitors of these enzymes. Some experimental work has targeted the enzymes
with specifically designed inhibitors. Other inhibitors being investigated
include hydroxamic acid derivatives, tetracyclines and quinolones [8, 14].
Vertebral Endplates
There are a variety of biological changes that occur in the degeneration of
the vertebral endplates, which contribute, at least in part, to this disease phe-
nomenon. As the endplate ages, it becomes calcified and replaced by bone. This
calcification and bone formation impedes the vascular supply to the disc and
accelerates the negative diffusion process described above. In addition, these
bony changes within the endplate lead to an unequal distribution of load-
sharing forces across the disc complex, which further accelerates the degener-
ative process. There is currently a paucity of biological study on endplate
changes in degenerative disease. However, recent work has described differ-
ences in the proteoglycan composition on degenerated endplates, the contribution
of MMP-3 in endplate degeneration, and a possible role for apoptosis in this
disease process.
Genetics in Disc Disease
The role of genetics in degenerative disc disease is still largely unknown.

Any genetic defect affecting collagen synthesis, proteoglycan synthesis, or
growth and development of resident support cells (e.g., chondrocytes) would be
expected to impact the rate of disc degeneration. As described above, Annunen
et al. reported on the role of the COL9A2 gene in disc degeneration in a cohort
of patients in Finland. In their report, this gene, which codes for a portion of
the type IX collagen of the disc, was screened and found to have common
polymorphisms in patients with intervertebral disc disease, associated with
Kurpad/Lifshutz 34
abnormal formation of the collagen 3 chain. Gruber and Hanley have described
the role of apoptosis in both age and degenerative changes which related to a
decrease in the amount of cells in the disc, and it appears likely that genetic fac-
tors could contribute toward a susceptibility to this process.
Fibroblast-derived growth factors may regulate proteolytic activity in
herniated discs. Nagano et al. studied degenerated disc in the rat model and
showed that that normal structures were replaced with scattered chondrocytes
with fibroblast growth factor-like activity and receptors [7]. New studies of the
canine disc have demonstrated that both epidermal growth factor and trans-
forming growth factor are associated with matrix synthesis and cell prolifera-
tion within the disc, and could represent another genetic risk factor if such
factors are deficient.
Conclusion
Degenerative spine disease is a common problem for which neurosurgical

patients seek treatment. New technology and advances in basic science has led
to a better and more thorough understanding of the molecular biology of this
disease process. With this new information, we may be able to develop novel
and minimally invasive therapeutics for this very common disease entity. The
role of genetics has until recently been underemphasized in the etiology of disc
disease, but as more genetic influences are recognized, the number of targets for
in vivo and ex vivo gene transfer will increase and new therapies will become
available.
References
1 Ahn S-H, et al: mRNA expression of cytokines and chemokines in herniated lumbar interverte-
bral discs. Spine 2002;27:911–917.
2 Burke JG, et al: Spontaneous production of monocyte chemoattractant protein-1 and interleukin-8
by the human lumbar intervertebral disc. Spine 2002;27:1402–1407.
3 Crean JKG, et al: Matrix metalloproteinases in the human intervertebral disc: Role in disc degen-
eration and scoliosis. Spine 1997;22:2877–2884.
4 Doita M, et al: Influence of macrophage infiltration of herniated disc tissue on the production of
matrix metalloproteinases leading to disc resorption. Spine 2001;26:1522–1527.
5 Fujita K, et al: Neutral proteinases in human intervertebral disc – Role in degeneration and prob-
able origin. Spine 1993;18:1766–1773.
6 Furusawa N, Baba H, et al: Herniation of cervical intervertebral disc – Immunohistochemical
examination and measurement of nitric oxide production. Spine 2001;26:1110–1116.
7 Goupille P, Jayson M, et al: Matrix metalloproteinases: The clue to intervertebral disc degenera-
tion? Spine 1998;23:1612–1626.
8 Guiot B, Fessler R: Molecular biology of degenerative disc disease. Neurosurgery 2000;47:
1034–1040.
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9 Kang J, et al: Toward a biochemical understanding of human intervertebral disc degeneration and
herniation – Contributions of nitric oxide, interleukins, prostaglandin E2 and matrix metallopro-
teinases. Spine 1997;22:1065–1073.
10 Matsui Y, et al: The involvement of matrix metalloproteinases and inflammation in lumbar disc
herniation. Spine 1998;23:863–869.
11 Nemoto O, et al: Matrix metalloproteinase-3 production by human degenerated intervertebral
disc. J Spinal Disord 1997;10:493–498.
12 Roberts S, Caterson B, et al: Matrix metalloproteinases and aggrecanase – Their role in disorders
of the human intervertebral disc. Spine 2001;25:3005–3013.
13 Takao T, Iwaki T: A comparative study of localization of heat shock protein 27 and heat shock
protein 72 in the developmental and degenerative intervertebral discs. Spine 2002;27:361–368.
14 Vaccaro A, Betz R, Zeidman S (eds): Principles and Practice of Spine Surgery. Mosby Publisher,
2003, chaps 6, 28.
Shekar N. Kurpad, MD, PhD

Department of Neurosurgery, Medical College of Wisconsin
9200 West Wisconsin Avenue, Milwaukee, WI 53226 (USA)
Tel. ⫹1 414 805 3666, E-Mail skurpad@neuroscience.mcw.edu
Kurpad/Lifshutz 36
Gene Therapy for Degenerative

Disc Disease
Joseph Kim, Lars G. Gilbertson, James D. Kang
Department of Orthopaedic Surgery, University of Pittsburgh Medical Center,
Pittsburgh, Pa., USA
Introduction
Degenerative disc disease (DDD) is a chronic process that can clinically

manifest in multiple disorders, such as idiopathic back pain, disc herniation,
radiculopathy, myelopathy, and spinal stenosis. It is a significant source of
patient pain and morbidity, utilizing a large portion of health care resources
[2, 3, 5, 15, 34]. The available treatment options for the clinical manifestations
of DDD include conservative measures such as bed rest, anti-inflammatory
drugs, analgesia, and physical therapy. This approach is usually effective in
alleviating symptoms within 2 months in the majority of cases. However, when
conservative methods fail, invasive surgical procedures such as discectomy,
instrumentation, or fusion, with their inherent complication risks and expense,
may be required. These treatment modalities focus on the clinical symptoms of
intervertebral disc (IVD) degeneration without addressing the underlying
pathological processes occurring early in the course of degeneration. However,
recent advances in molecular biology may result in the development of novel
therapies that target the ongoing physiological changes that occur in this
disease.
Although the pathophysiology of DDD is not completely understood, an
insult to the disc or its supporting structures initially leads to a cascade of
cellular changes that may promote either healing or further disc degeneration.
One major contributing factor includes the progressive decline in aggrecan, the
primary proteoglycan of the nucleus pulposus [1, 7, 24]. At the biochemical
level, aggrecan homeostasis is altered by various combinations of decreased
synthesis and increased breakdown. Kang et al. [13] demonstrated increased
levels of matrix metalloproteinases in degenerated human discs compared to
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normal, nondegenerated controls. These enzymes are known to contribute to
net proteoglycan loss by increasing its degradation.
With reductions in proteoglycan content of the intervertebral matrix, the
nucleus pulposus dehydrates, decreasing both disc height and its load-bearing
capacity [8, 32, 33]. This may directly affect biomechanical function by alter-
ing the loads experienced by the facet joints, leading to degenerative changes.
Although disc degeneration most probably evolves in response to a complex
interplay of multiple biochemical and biomechanical factors [10], the ability to
restore proteoglycan content may have therapeutic benefit by increasing disc
hydration and potentially improving biomechanics.
The ability to increase proteoglycan synthesis in the IVD was demon-
strated by Thompson et al. [30] who showed that the exogenous application of
human transforming growth factor (TGF)-1 to canine disc tissue in culture
stimulated in vitro proteoglycan synthesis. The authors suggested that growth
factors might be useful for the treatment of disc degeneration. Subsequent stud-
ies with other growth factors such as insulin-like growth factor-1 (IGF-1), bone
morphogenic protein-2 (BMP-2), and osteogenic protein-1 also exhibited the
ability to up-regulate proteoglycan content in IVD cells [23, 29]. However, due
to the relatively brief half-life of these factors, practical application of growth
factor therapy to chronic conditions such as DDD would necessitate repeated
administrations. Consequently, efforts were directed at developing approaches
to induce endogenous synthesis of growth factors via gene therapy such that
genetically modified disc cells manufacture the desired growth factors on a
continuous basis.
Overview of Gene Therapy
The definition of gene therapy has become quite broad. The term was
previously used to describe replacement of a defective gene with a functional
copy by means of gene transfer. The diseases originally targeted for gene ther-
apy were classic, heritable genetic disorders. The term now defines therapy
involving the transfer of genes encoding therapeutic proteins into cells to treat
any disease [26]. Genetically altered cells are made into factories producing
disease-altering proteins. These proteins affect not only the metabolism of the
cells from which they were made, but they can also affect the metabolism of
adjacent nongenetically altered cells via paracrine mechanisms (fig. 1).
Successful gene therapy for DDD will depend on efficient transfer of
genes to target cells with sustained expression. With few exceptions, naked
DNA is not taken up and expressed by cells and consequently, vectors are nec-
essary to package and insert genes into cells in such a way that the genetic
Kim/Gilbertson/Kang 38
DNA coding for Growth factor released
growth factor from cell
Viral vector carrying

growth factor gene
Ribosomes making
growth factors
Cell Nucleus
Fig. 1. The DNA encoding the growth factor of interest is constructed into a viral
vector that is rendered incapable of replication. The vector is then exposed to host cells,
attaches to their surface, and is then internalized. The released genetic information can then
either travel to the nucleus, where it may become integrated into the host genome or remain
episomal. It then commandeers the normal protein-making machinery of the cell and
produces large quantities of the transgene.
information can be expressed. The two broad categories of vectors are viral and
nonviral. The most commonly used nonviral vectors are liposomes. These phos-
pholipid vesicles deliver genetic material into a cell by fusing with the cell’s
phospholipid membrane. Liposome vectors are simple, inexpensive, and safe.
Their drawbacks are transient expression of the transgene, cytotoxicity at
higher concentrations, and low efficiency of transfection. Other nonviral meth-
ods of gene delivery include DNA-ligand complexes and the biolistics or pen-
etration of cells with a ‘gene gun.’ These vectors are nonpathogenic and
relatively inexpensive to construct. However, the overall transfection efficiency
of nonviral vectors is generally inferior to that of viral-mediated gene transfer.
Thus, most current studies involving gene therapy employ viral vectors.
The most commonly used viral vectors are retroviruses, herpes simplex
viruses, adeno-associated viruses, and adenoviruses; although, there are likely
to be many other naturally occurring viruses which could be adapted for gene
transfer. Viruses are frequently rendered incapable of replication prior to gene
therapy application in an effort to make them less pathogenic. The various viral
vectors and their advantages and disadvantages are discussed elsewhere in this
volume, and will not be discussed further.
Gene Therapy for Degenerative Disc Disease 39
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There are two fundamental approaches to delivering exogenous genes with
vectors to target cells within the body. The first is the direct, in vivo method in
which the gene-carrying vector is directly injected into the patient. The second
approach, known as ex vivo gene therapy, involves removing target cells from
the body, genetically altering them in vitro, and then reimplanting them in the
body. There are advantages and disadvantages to both of these approaches that
depend on the anatomy and physiology of the target organs, the pathophysiology
of the disease being treated, the vector of choice, and safety considerations [9].
Biology of the IVD
The IVD is an avascular organ consisting of nucleus pulposus cells scat-

tered within an extracellular matrix. This gel-like inner core is encircled by an
outer annulus fibrosis. The highly differentiated, nondividing cells of the
nucleus pulposus are responsible for matrix synthesis. The matrix of a healthy
nucleus pulposus is normally rich in proteoglycans and type II collagen, with a
water content of over 85% by volume in juveniles, decreasing to approximately
70–75% in adults, and decreasing even further with aging and degeneration [11,
12]. This progressive loss of matrix and water content reflects the inability of
the IVD for self-repair, as demonstrated by Bradford et al. [6]. The avascular
disc receives the majority of its nutrition via passive diffusion through the car-
tilaginous endplates. The lack of a direct vascular supply results in low oxygen
tension within the disc and causes the cells of the nucleus pulposus to undergo
anaerobic metabolism. The ensuing high lactate concentration and subsequent
low environmental pH most likely inhibit matrix repair.
The avascularity of the IVD, though limiting its potential for repair and
regeneration, does confer a distinct advantage in the context of gene therapy
application. Early research attempting to characterize the IVD demonstrated an
autoimmune response when subjects were exposed to their own nucleus pulpo-
sus tissue, suggesting that the IVD is an immune-privileged site exempt from
prior exposure to the host immune system [4].
Adenoviral (Ad) Vectors for Gene Therapy to the IVD
Vectors based on adenoviruses have been frequently used in gene therapy

studies for the IVD due to their ability to efficiently transduce highly differen-
tiated, nondividing cells such as the cells of the nucleus pulposus. However,
successful gene therapy depends not only on efficient gene transfer, but also on
the expression of transgene for sufficiently long periods of time. The duration
of gene expression following adenoviral (Ad) transfer to an immunocompetent
animal is limited in most organs and tissues by immune reactions to viral pro-
teins and to foreign proteins encoded by the transgene [17, 31, 36]. Specifically,
expression for longer than 12 weeks has been difficult to achieve in muscu-
loskeletal tissues following Ad delivery due to brisk immune responses.
On the other hand, sustained gene expression can occur for longer periods
of time in an immune-privileged site such as the IVD. Kang et al. [14] demon-
strated positive expression of the marker genes lacZ and luciferase in the rabbit
lumbar disc one year after adenovirus-mediated transduction. A corollary
experiment attempting to characterize the immune response in the same study
showed no production of neutralizing antibody to viral proteins in 3 of 6 rabbits
after intradiscal injections of Ad vectors carrying the luciferase marker gene.
The positive immune response in the other 3 rabbits was presumably due to the
leakage of virus from an injected disc. Importantly, all 6 rabbits, including the
3 with circulating antibodies, had positive transgene expression several weeks
after injections. On histological review, there was no evidence of cellular
infiltration, increased vascularity, fibrosis, or other hallmarks of an immune
response. Furthermore, intradiscal expression of luciferase was apparent for up
to at least 42 days in rabbits whose immune system had been deliberately
primed by subcutaneous inoculations with Ad proteins 2 weeks prior to
intradiscal vector-gene injections (fig. 2). This implied that circulating anti-
bodies do not reach the IVD in significant quantities to exert an immune
response against the transduced disc cells. These findings further confirmed the
IVD as being an immune-privileged organ and demonstrated the feasibility of
using Ad vectors to achieve efficient, sustained expression of foreign genes.
Comparative Studies of Intradiscal Gene Therapy
With the success of in vitro studies demonstrating an increase in proteo-

glycan synthesis in IVD cells treated with the exogenous administration of
growth factors, efforts were directed towards stimulating endogenous synthesis
of these proteins by IVD cells with the use of gene therapy. Nishida et al. [22]
reported the first successful in vivo gene transfer to the IVD in 1998, using an
Ad vector to deliver the lacZ marker gene to the rabbit lumbar disc. The authors
were able to demonstrate sustained transgene production with no significant
reduction in the expression for up to 3 months after transduction (fig. 3). Follow-
up studies revealed evidence of continued foreign gene expression even at one
year [14]. Notably, the rabbits used in these studies showed no signs of systemic
illness in response to the Ad vector and its transgene synthesis. In addition, no
histological changes suggesting a cellular immune response were observed.
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Group A 0.4 Group B
0.4
0.3 0.3
O.D. 405
O.D. 405
0.2 0.2
0.1 0.1
0 0
0 7 14 21 28 35 42 0 2 4 6 8 0 7 14 21 28 35 42 0 2 4 6 8
Intradiscal Time (days) (log10 RLU) @42 days Intradiscal and subcutaneous (log10 RLU) @42 days
a injection b injection
0.4 Group C
0.3
O.D. 405
0.2
0.1
0
14 7 0 7 14 21 28 35 42 0 2 4 6 8
Subcutaneous Intradiscal (log10 RLU) @42 days

c injection injection
Fig. 2. a–c Plots on the left show sequential production of specific antibodies for Ad
proteins in peripheral blood. Bar charts on the right show luciferase activity at 42 days
postintradiscal injection of Ad-luciferase. a In Group A, 3 rabbits produced little or no anti-
body to Ad proteins in the peripheral blood after injection of Ad-luciferase into the lumbar
IVD. In the remaining 3 rabbits, antibody was produced within 3 weeks, presumably due to
the leakage of virus from injected discs. b In Group B, all rabbits exhibited significantly
increased production of antibody within 2 weeks after simultaneous subcutaneous and
intradiscal injection of Ad-luciferase. c In Group C, the rabbits were immunized by subcuta-
neous injections of Ad-luciferase 2 weeks prior to the intradiscal gene therapy. All rabbits
exhibited increased production of antibody in the peripheral blood by the time of the intradis-
cal injection. a–c As shown in the bar charts (right), all rabbits from the three groups exhib-
ited significant amounts of intradiscal transgene expression. There was no correlation
between the neutralizing antibody titer and intradiscal transgene expression at 6 weeks
postintradiscal injection by Pearson correlation analysis (p 0.395).
Encouraged by these results with marker proteins, successful in vivo trans-

duction of the IVD with a putative therapeutic gene was soon accomplished
[21]. Using Ad vector, the gene for human TGF-1 was delivered. This study
demonstrated a 30-fold increase in active TGF-1 synthesis and a 5-fold
increase in total TGF-1 production in discs injected with the Ad-growth fac-
tor construct (fig. 4). Biological modulation was also documented by a 100%
increase in proteoglycan synthesis (fig. 5). Assays for TGF-1 production and
proteoglycan synthesis were performed with experimental discs that had been
injected with Ad vector carrying only the luciferase marker gene. These viral
control discs demonstrated no increase in production of TGF-1 or proteogly-
can, indicating that the increases in the TGF-1 experimental group were a
result of transgene expression and not a nonspecific response to the Ad vector.
As in the previous studies, no signs of local or systemic immune response were
noted.
Additional in vitro studies with cultured human nucleus pulposus cells
yielded similar results. Successful transduction of the lacZ marker gene deliv-
ered via Ad vectors was achieved with human cells from degenerated discs [19].
Similar experiments with retroviral delivery of marker genes resulted in a
smaller percentage of transduction [25], perhaps due to the minimal mitotic
activity of the IVD cells. The response of human cells from degenerated discs
to Ad-mediated delivery of TGF-1 was assessed; increased expression of
TGF-1, as well as increased proteoglycan and collagen synthesis, was demon-
strated in cells receiving gene therapy as compared to controls [20]. Of note,
cells receiving the Ad-TGF-1 construct showed increased proteoglycan and
collagen synthesis when compared to cells receiving exogenous TGF-1 pro-
tein, presumably in response to the sustained expression of this growth factor.
Interestingly, the viral dose required to increase proteoglycan synthesis was
significantly less than that required for 100% transduction of the cells, perhaps
highlighting the ability of a transduced cell to influence the biological activity
of nongenetically altered neighboring cells. The concept that successfully trans-
duced cells appear to exert a paracrine-like effect on their nontransduced neigh-
boring cells implies that significant alteration in protein synthesis can be
achieved with a small number of transduced cells [9]. A better understanding of
this paracrine effect with TGF-1 gene transfer may enable the use of decreased
viral loads to achieve a therapeutic effect, thereby minimizing potential viral
toxicity. These experiments were also performed with a viral control, which fur-
ther established that the increase in biological activity was the result of the
delivered genetic material and not of the Ad vector.
Subsequent in vitro studies with other growth factors such as BMP-2 and
IGF-1 documented the potential of Ad delivery of these factors to increase
the proteoglycan synthesis in a viral dose-dependent manner [18, 35]. Tissue
inhibitor of metalloproteinase-1 also demonstrated the same ability [34]
following Ad vector delivery. Tissue inhibitor of metalloproteinase-1 is an
endogenous inhibitor of matrix metalloproteinases, which are enzymes capable of
degrading the extracellular matrix of the IVD [27]. This finding established a
second gene therapy strategy to modify the disrupted balance of synthesis
and catabolism occurring in the degenerated IVD, namely, inhibition of
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a b
c d
e f
140 N.S. 350 N.S.
1 week 1 week
120 6 weeks 6 weeks
300 *
*
pg/ml/mg (wet weight)
pg/ml/mg (wet weight)

100 250
*
80 * 200
60 150
40 100
20 50
0 0
a Intact Saline Ad/luciferase Ad/TGF-1 b Intact Saline Ad/luciferase Ad/TGF-1
Fig. 4. a Active TGF-1 production in rabbit nucleus pulposus tissue one and 6 weeks
after in vivo injection of Ad-TGF-1, as compared to intact control discs and discs injected
with saline and Ad-luciferase. b Total (active and latent) TGF-1 production in rabbit disc
tissue one and 6 weeks after in vivo injection. There were no significant differences in either
active or total TGF-1 production at one week and 6 weeks, indicating that the therapeutic
gene expression was sustained. * A significant increase over corresponding intact, saline,
and viral (Ad-luciferase) control groups (p 0.05).
matrix degradation with ensuing net increases or stabilization of proteoglycan

content.
Considering the potential adverse effects of viral vectors, studies have
been undertaken to develop strategies to minimize viral loads while maintain-
ing the same biological effects. Experiments with combination gene therapy
involving TGF-1, IGF-1, and BMP-2 suggested that these growth factors are
synergistic in amplifying matrix synthesis [18]. Ad delivery of a single growth
factor increased proteoglycan synthesis by a range of 180–295%, whereas
combination gene therapy with two agents resulted in increases of 322–398%.
When all three growth factors were combined, proteoglycan synthesis was
increased by 471% (fig. 6). It remains to be determined if combination gene
Fig. 3. a–g Qualitative analysis of intradiscal lacZ transgene expression up to and includ-
ing one year after injection of Ad-lacZ into lumbar intervertebral discs of adult New Zealand
white rabbits. Serial histological sections were stained with X-Gal and counter-stained with
eosin. Representative sections of lumbar discs at 3 weeks (a, b), 6 weeks (c, d), and 24 weeks
(e, f ) postinjection are shown. All of the discs injected with Ad-LacZ exhibited positive X-Gal
staining. [Original magnifications: a, c, e. 40; b, d, f. 200.] At 52 weeks postinjection,
positive X-Gal staining was observed in the discs from two of three rabbits. However, the
intensity of positive staining was less than in discs from the other time periods (g). [Original
magnification: g. 600].
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N.S.
3.5
1 week
6 weeks *
3.0 *
Ratio (experimental/intact)
2.5
2.0
1.5
0.5
0
Intact Saline Ad/luciferase Ad/TGF-1
Fig. 5. Proteoglycan synthesis in rabbit nucleus pulposus tissue one and 6 weeks after
in vivo injection of Ad-TGF-1, as compared to intact control discs and discs injected with
saline and Ad-luciferase. There were no significant differences in proteoglycan synthesis at
one week and 6 weeks, indicating that the biologic effects of transgene synthesis was sus-
tained. * A significant increase over corresponding intact, saline, and viral (Ad-luciferase)
control groups (p 0.05).
therapy with both an anabolic growth factor and a catabolic inhibitor such as
tissue inhibitor of metalloproteinase-1 will have a similar synergistic effect.
Areas of Ongoing Research
For the continued progress of gene therapy for DDD toward successful
human clinical trials, it is critical to rigorously test the proposed gene therapy
strategies in animal models of disc degeneration that closely simulate the
human condition. A number of models have been proposed. Disc degeneration
occurs spontaneously in some species, such as the nonchondrodystrophic bea-
gle and the sand rat [28]. Other species require artificial interventions to bring
about degenerative changes within a reasonable time frame. The annular stab
model of degeneration in the New Zealand white rabbit has been well described
in the literature by Lipson and Muir [16]. In previous gene therapy studies with
this model, our group found that a 3 mm incision of the anterior annulus
allowed escape of nuclear material from the disc, with subsequent loss of viral
700
Triple
600 Double *
Single gene
500
*
*
Percent control
400 *
*
300
* *
200
100
0
Saline Ad/luciferase Ad/TGF-1 Ad/IGF-1 Ad/BMP-2 Ad/TGF1-1 Ad/TGF-1 Ad/IGF-1 Ad/TGF-1

IGF-1 Ad/BMP-2 Ad/BMP-2 Ad/IGF-1

Ad/BMP-2
Fig. 6. Proteoglycan synthesis in human intervertebral disc cells treated with different
combinations of therapeutic Ad vectors (Ad-TGF-1, Ad-IGF-1, Ad-BMP-2). All groups
showed significant increase in synthesis compared to saline and viral (Ad-luciferase) control
groups (* p 0.05).
injections directed at the nucleus pulposus. There was also concern that degen-
erative changes induced by the full thickness 3 mm annular incision were too
abrupt, in contrast to the gradual changes that occur in the human condition. For
these reasons, we modified this technique to produce a puncture injury using a
16-gauge hypodermic needle.
Extensive MRI and histological data have shown that the needle puncture
model produces gradual and consistent degenerative changes that closely parallel
human disc degeneration. The MRI analysis revealed progressive loss of mean
nucleus pulposus signal intensity of stabbed lumbar discs as a function of time
from puncture surgery (fig. 7). Importantly, there was no MRI evidence of spon-
taneous recovery of any of the degenerated discs. Histological examinations of
punctured discs revealed cracks and clefts within the nucleus as well as delami-
nation and infolding of the annulus. In addition, clusters of notochordal cells
were readily apparent in healthy discs but were sparse in discs that had been punc-
tured (fig. 8). Further validation of the puncture model was achieved by the
demonstrated loss of mean water content from 85 to 70% twenty-four weeks after
needle injury.
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NP signal intensity
L1-L2 20
(control) * *
15 L2-L3
10
5
L2-L3
Gray value on NP * Area of NP

0
(stab) Pre-op 12 weeks 24 weeks
* *
15
10 L3-L4
L3-L4 5
(stab)
0
Pre-op 12 weeks 24 weeks
20
L4-L5 15 L4-L5
(stab) 10
5
0
Pre-op 1 wk 2 wks 3 wks 6 wks 12 wks 24 wks Pre-op 12 weeks 24 weeks
Fig. 7. Representative T2-weighted sagittal MRI of a New Zealand white rabbit lum-
bar spine: immediately before and 1, 2, 3, 6, 12 and 24 weeks after puncture surgery (left).
Signal intensity of nucleus pulposus decreased with time after surgery (right). n 8 rabbits.
a b
Pre-operative 24 weeks after stab
Fig. 8. a Healthy L5–6 rabbit disc. Clusters of notochordal cells are apparent.
b Degenerated L4–5 rabbit disc 24 weeks after puncture surgery. Nuclear displacement
occurred, accompanied by infolding of the contralateral inner annulus towards the direction
of nuclear displacement. Clusters of notochordal cells are sparse.
Future Directions
The potential of gene therapy to alter the biological processes occurring in

the degenerated IVD disc has been clearly established. The next step in this devel-
opment will be to assess the feasibility of transducing degenerated rabbit discs,
using needle-stab modeling as described above, with marker and therapeutic
genes. Additional in vivo studies in this model will help to clarify the potential
benefits and toxicity of gene therapy. Because other vectors are now available
which can transduce cartilagenous cells, most notably adeno-associated vector,
expanding this experimental methodology to other vector systems will be impor-
tant. The basic science of the effects of growth factors and catabolic inhibitors in
the biological processes and mechanical functioning of the spine also needs to be
further elucidated, to determine which factors are best to promote growth of col-
lagen and proteoglycans. Biochemical studies are necessary to delineate the rela-
tionship between viral concentration, transgene synthesis, and protein expression
in the disc space. Despite the hurdles that remain, gene therapy to alter the course
of IVD degeneration holds much clinical promise, and will continue to stimulate
future investigations.
References
1 Adler JH, Schoenbaum M, Silberberg R: Early onset of disk degeneration and spondylosis in sand
rats (Psammomys obesus). Vet Pathol 1983;20:13–22.
2 Anderson J: Back pain and occupation; in Jayson MIV (ed): The Lumbar Spine and Back Pain.
London, Churchill Livingstone, 1987, pp 2–36.
3 Anderson JA: Epidemiological aspects of back pain. J Soc Occup Med 1986;36:90–94.
4 Bobechko: Auto-immune response to nucleus pulposus in the rabbit. J Bone Joint Surg Br 1965;
47:574–580.
5 Borenstein D: Epidemiology, etiology, diagnostic evaluation, and treatment of low back pain. Curr
Opin Rheumatol 1992;4:226–232.
6 Bradford DS, Cooper KM, Oegema TR Jr: Chymopapain, chemonucleolysis, and nucleus pulpo-
sus regeneration. J Bone Joint Surg Am 1983;65:1220–1231.
7 Buckwalter JA: Aging and degeneration of the human intervertebral disc. Spine 1995;20:
1307–1314.
8 Butler D, Trafinow JH, Andersson GB, McNeil TW: Discs degenerate before facets. Spine 1990;
15:111–113.
9 Evans CH, Robbins P: Possible orthopaedic applications of gene therapy. J Bone Joint Surg Am
1995;77:1103–1113.
10 Garfin SR: The intervertebral disc: Disc disease – Does it exist? in Weinstein JN (ed): The Lumbar
Spine. Philadelphia, W.B. Saunders, 1990, pp 369–380.
11 Hallen A: Hexosamine and ester suphate content of the human nucleus pulposus at different ages.
Acta Chem Scand 1958;12:1869–1872.
12 Hallen A: The collagen and ground substance of the human nucleus pulposus at different ages.
Acta Chem Scand 1962;16:705–709.
13 Kang JD, Georgescu HI, McIntyre-Larkin L, Stefanovic-Racic M, Donaldson WF 3rd, Evans CH:
Herniated lumbar intervertebral discs spontaneously produce matrix metalloproteinases, nitric
oxide, interleukin-6, and prostaglandin E2. Spine 1996;21:271–277.
medwedi.ru
14 Kang JD, Boden SD: Orthopaedic gene therapy. Spine. Clin Orthop 2000;379S:256–259.
15 Kraemer J: Natural course and prognosis of intervertebral disc diseases. International Society for
the Study of the Lumbar Spine, Seattle, Washington, June 1994. Spine 1995;20:635–639.
16 Lipson SJ, Muir H: 1980 Volvo Award in Basic Science: Proteoglycans in experimental interver-
tebral disc degeneration. Spine 1981;6:194–210.
17 McCoy RD, Davidson BL, Roessler BJ: Expression of human interleukin-1 receptor antagonist in
mouse lungs using recombinant adenovirus: Effects on vector induced inflammation. Gene Ther
1995;2:437–442.
18 Moon S, Nishida K, Gilbertson LG, Hall RA, Robbins PD, Kang JD: Biologic response of human
intervertebral disc cell to gene therapy cocktail. San Francisco, Orthopaedic Research Society,
2001.
19 Moon SH, Gilbertson LG, Nishida K, Knaub M, Muzzingro T, Robbins PD, Evans CH, Kang JD:
Human intervertebral disc cells are genetically modifiable by adenovirus-mediated gene transfer.
Spine 2000;25:2573–2579.
20 Moon SH, Nishida K, et al: Proteoglycan synthesis in human intervertebral disc cells cultured in
alginate beads; exogenous TGF-1 vs adenovirus-mediated gene transfer of TGF 1 cDNA.
Orlando, Florida Orthopaedic Research Society 2000; (Abstr 1061).
21 Nishida K, Kang JD, Gilbertson LG, Moon SH, Suh JK, Vogt MT, Robbins PD, Evans CH:
Modulation of the biologic activity of the rabbit intervertebral disc by gene therapy: An in vivo
study of adenovirus-mediated transfer of the human transforming growth factor beta 1 encoding
gene. Spine 1999;24:2419–2425.
22 Nishida K, Kang JD, Suh JK, Robbins PD, Evans CH, Gilbertson LG: Adenovirus-mediated gene
transfer to nucleus pulposus cells. Implications for the treatment of intervertebral disc degenera-
tion. Spine 1998;23:2437–2442; discussion 2443.
23 Osada R, Oshima H, Ishihara H: Autocrine/paracrine mechanism of insulin-like growth factor-1
secretion, and the effect of insulin-like growth factors-1 on proteoglycan synthesis in bovine inter-
vertebral discs. J Orthop Res 1996;14:690–699.
24 Pearce RH, Grimmer BJ, Adams ME: Degeneration and the chemical composition of the human
lumbar intervertebral disc. J Orthop Res 1987;5:198–205.
25 Reinke J, et al: Transfer of therapeutic genes to human chondrocytes-like cells of lumbar disc
prolapse (abstract 56). Annual Meeting of International Society for the Study of the Lumbar
Spinel, Singapore, 1997.
26 Robbins PD, Ghivizzani SC: Viral vectors for gene therapy. Pharmacol Ther 1998;80:35–47.
27 Roberts S, Caterson B, Menage J, Evans EH, Jaffray DC, Eisenstein SM: Matrix metallopro-
teinases and aggrecanase: Their role in disorders of the human intervertebral disc. Spine 2000;
25:3005–3013.
28 Silberberg R, Aufdermaur M, Adler JH: Degeneration of the intervertebral disks and spondylosis
in aging sand rats. Arch Pathol Lab Med 1979;103:231–235.
29 Takegami K, Thonar EJ, An HS, Kamada H, Masuda K: Osteogenic protein-1 enhances matrix
replenishment by intervertebral disc cells previously exposed to interleukin-1. Spine 2002;27:
1318–1325.
30 Thompson JP, Oegema TR Jr, Bradford DS: Stimulation of mature canine intervertebral disc by
growth factors. Spine 1991;16:253–260.
31 Tripathy SK, et al: Immune responses to transgene-encoded proteins limit the stability of
gene expression after injection of replication-defective adenovirus vectors. Nat Med 1996;2:
545–550.
32 Urban JP, McMullin JF: Swelling pressure of the intervertebral disc: Influence of proteoglycan
and collagen contents. Biorheology 1985;22:145–157.
33 Urban JP, McMullin JF: Swelling pressure of the lumbar intervertebral discs: influence of age,
spinal level, composition, and degeneration. Spine 1988;13:179–187.
34 Waddell G: Low back pain: A twentieth century health care enigma. Spine 1996;21:
2820–2825.
35 Wallach CJ, Sobajima S, Watanabe Y, Gilbertson LG, Kang JD: Gene transfer of the catabolic
inhibitor TIMP-1 increases measured proteoglycans in human intervertebral disc cells.
International Society for the Study of the Lumbar Spine, Cleveland, Ohio, 2002.
36 Yang Y, et al: Cellular immunity to viral antigens limits E1-deleted adenoviruses for gene therapy.
Proceedings of the National Academy of Sciences of the United States of America 1994;
91:4407–4411.
James D. Kang, MD
Assistant Professor of Orthopaedic Surgery and Neurological Surgery
Division of Spinal Surgery, University of Pittsburgh Medical Center
Department of Orthopaedic Surgery, Liliane Kaufmann Building
3471 Fifth Avenue, Suite 1010, Pittsburgh, PA 15213 (USA)
Tel. 1 412 605 3241, Fax 1 412 687 3724, E-Mail jkangjd@msx.upmc.edu
medwedi.ru
Bone Morphogenetic Proteins

Spinal Fusion Applications
David A. Bomback, Jonathan N. Grauer

Department of Orthopaedics and Rehabilitation, Yale University
School of Medicine, New Haven, Conn., USA
Overview of Bone Morphogenetic Proteins (BMPs)
The clinical track record of autogenous iliac crest bone graft makes it the
current ‘gold standard’ for spinal arthrodesis. However, autograft utilization is
accompanied by a number of limitations. For example, pseudoarthrosis rates
vary from 5 to 35% [1], which prolongs recovery from surgery and leads to
potential complications such as graft migration, instability, or even spinal cord
impingement. Moreover, chronic donor site pain has been reported in up to
25% of patients who undergo removal of iliac crest material for spinal auto-
grafting. The availability of donor bone from a given patient may also be lim-
ited secondary to prior graft harvest or poor bone quality. Finally, an additional
operative site increases blood loss, operative time, and cost [1].
Such limitations have prompted investigations into a variety of bone graft
alternatives. The goals of such efforts are aimed at eliminating donor-site pain
and increasing union rate with a product that is virtually limitless in supply.
Such alternatives can be classified as either bone graft extenders or substitutes.
Graft extenders, when added to autogenous bone, allow for arthrodesis of a
greater number of levels or the use of less autograft and yield a fusion rate equal
to or superior to that of autograft alone. Graft substitutes completely replace
autogenous bone yet allow for comparable or increased fusion rates compared
with the autograft [2].
In order to understand the biological application of bone graft alternatives,
one must be familiar with the basic terminology. Osteoconduction is the ability
of a material to behave as a scaffold for the ingrowth of new host bone.
Osteoinduction is defined as the capability of initiating de novo bone formation
by inducing osteoblastic precursor stem cells to differentiate into mature bone-
forming cells. An ideal bone graft substitute must possess both of these char-
acteristics. Osteogenesis simply refers to the ability of graft cells to directly
form bone. Only autogenous bone graft and bone marrow aspirates possess
osteogenic properties [3].
In 1965, Urist [4] made the observation that implanted devitalized bone
was capable of inducing a cellular response resulting in new bone formation.
His laboratory subsequently demonstrated that proteins extracted from the
organic component of bone were responsible for such a behavior [5, 6].
Implantation of this bone matrix protein mixture into animals resulted in a
multitude of cellular events including mesenchymal cell infiltration, carti-
lage formation, vascular ingrowth, bone formation, and bony remodeling [7].
Urist thus coined the term ‘bone morphogenetic protein’ (BMP). Over time,
such extracts and proteins have become exploited and modified to induce
fusions.
Biology of Spinal Fusion
The goal of spinal arthrodesis using decortication and autogenous bone

graft is the development of a well-formed fusion mass bridging one bony sur-
face to another. In order to achieve such an endpoint, a specific set of events
needs to occur. First, osteoprogenitor cells must enter the fusion bed.
Decortication of host bone enables cells to exit the bone marrow and enter the
fusion environment. Next, osteoprogenitor cells differentiate into osteoblast
precursors and ultimately mature osteoblasts, depositing new bone matrix.
Finally, bony remodeling of the fusion mass occurs according to Wolff’s law
(i.e., remodeling occurs in response to physical stresses; bone is deposited in
sites subjected to stress and resorbed from sites of little stress), resulting in a
stable fusion mass able to withstand physiological stress [3].
To study the many variables which affect bone formation and fusion,
animal models have been developed. One such approach is the New Zealand
white rabbit posterolateral lumbar fusion model, which has been validated
and extensively studied [8]. Histological analysis reveals that maturation of
the spine fusion mass occurs first in the ‘outer zone’ (adjacent to the trans-
verse processes) followed by the ‘central zone’ (between transverse
processes). This temporal and spatial sequence would be expected postdecor-
tication because osteoprogenitor cells from the marrow must travel a longer
distance to reach the central zone. In each region, inflammatory, reparative,
and remodeling histological phases of bone healing can be observed [3]. In
further evaluating the process, mRNA expression of various BMPs has been
Bone Morphogenetic Proteins 53
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shown to occur at different times and at different locations during fusion.
Such findings suggest unique roles for specific BMPs during spine fusion
and indicate the potential for clinical applications of these proteins. A full
understanding of the process of BMP expression during fusion has not yet
been achieved, although limited gene expression studies following induction
with BMP have been performed.
Molecular and Cellular Mechanisms of Action of BMPs
The BMPs are dimeric molecules belonging to the transforming growth

factor-␤ superfamily based on amino acid homology [9, 10]. BMP molecules
are multifunctional proteins that exhibit both autocrine and paracrine effects.
They act by binding to specific serine-threonine kinase receptors present on the
surface of undifferentiated mesenchymal stem cells. The receptors then trans-
duce a signal via a group of G-proteins known as Smads, which in turn activate
genes in the nucleus of the cell related to the osteoblast phenotype [11]. When
applied in vivo, BMPs induce undifferentiated mesenchymal stem cells to
switch from a fibrogenic to an osteogenic pathway of development, culminating
in mature bone with normal marrow cavities [12].
The activity of BMPs is tightly controlled and self-limiting. Outside the
cell, inhibitory proteins (e.g., noggin, chordin, follistatin) can bind specific
BMPs, thus preventing their binding to cell surface receptors [11, 13, 14].
Furthermore, intracellular BMP transcription and translation is regulated by a
combination of signal-transducing and inhibitory Smad proteins. BMPs can
themselves up-regulate the expression of these extracellular antagonists and
intracellular inhibitors, suggesting a negative feedback autoregulation cycle. As
a result of all of these regulatory mechanisms, bone induction is tightly limited
and bone overgrowth is avoided [11].
Research and Clinical Use of BMPs
Several BMP preparations have been, and are currently being investigated
preclinically and clinically for use in spinal arthrodesis. These BMP products
include recombinant human BMPs (rhBMPs) and demineralized bone matrices
(DBMs). The two rhBMPs which have been most investigated are rhBMP-2
(Medtronic Sofamore Danek, Memphis, Tenn., USA) and rhBMP-7 also
known as osteogenic protein-1 or OP-1 (Stryker Biotech, Hopkinton, Mass.,
USA). They are highly purified single proteins produced by recombinant DNA
biotechnology. Others include BMP-9 [9, 15] and growth and differentiation
Bomback/Grauer 54
factor-5 [16]. Small amounts of BMP may also be present in some DBM prepa-
rations. Examples of these formulations, which are derived from human allo-
graft bone, include, but are not limited to, Grafton (Osteotech, Eatontown, N.J.,
USA), Dynagraft (Gen-Sci Regeneration Laboratories, Calif., USA), and
Osteofil (Regeneration Technologies, Fla., USA). However, concentration of
BMPs in DBM is not thought to be sufficient for it to be a complete bone graft
substitute in spinal applications [17, 18]. In addition, a more concentrated
DBM preparation, bovine bone-derived BMP extract or bBMPx (Sulzer
Orthopedics Biologics, Denver, Colo., USA), has been investigated [10, 19].
Theoretically, this may offer more osteogenic potential than the standard
human DBM preparations.
Preclinical Studies with Specific Classes of BMP
rhBMP-2
The first preclinical interbody cage study using rhBMP-2 was by Sandhu
et al. [20]. L4-L5 retroperitoneal anterior lumbar interbody fusions were per-
formed in a sheep model. Cylindrical, threaded titanium fusion cages were filled
with either iliac crest autograft or rhBMP-2. The 6-month follow-up results
demonstrated a 100% fusion rate for the rhBMP-2 group compared with a 37%
fusion rate for the autograft controls. A similar study in a goat model using tita-
nium BAK fusion cages (Spinetech, Minneapolis, Minn., USA) packed with
either autograft or rhBMP-2 yielded a 95% fusion rate in the rhBMP-2 and a
48% fusion rate in the autograft group [21]. Finally, another study utilizing allo-
graft bone dowels for anterior interbody fusion in nonhuman primates showed
that dowels filled with rhBMP-2 resulted in a 100% fusion rate at 6 months, as
opposed to the 33% fusion rate seen in the autograft-filled dowels [22].
Numerous posterolateral fusion studies have been reported in the past
decade, with similar conclusions. Schimandle et al. [23] noted a 100% fusion
rate for rhBMP-2 in a rabbit posterolateral fusion model, while autograft con-
trols fused only 42% of the time. In addition, fusions in the rhBMP-2 animals
were biomechanically stronger and stiffer than autograft fusions. A canine
model demonstrated 100% rhBMP-2 fusions and 0% autograft fusions at
12 weeks [24]. Use of rhBMP-2 as an autograft enhancer also has been studied
in a canine model; in that model, gross specimens and CT scans demonstrated
significantly increased fusion mass volume 6 months after surgery in rhBMP-2
autograft dogs when compared with autograft alone dogs [25, 26].
Martin et al. [27] established the systemic effects of ketorolac on postero-
lateral spine fusion and then tested rhBMP-2’s ability to overcome such inhibi-
tion. First, the investigators demonstrated an autograft fusion rate of 35% with
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IV ketorolac pump infusion as compared to an autograft fusion rate of 75%
with IV saline infusion. Fusion rates subsequently increased to 100% in an
autograft/rhBMP-2 group with IV ketorolac infusion. In a related study, Silcox
et al. [28] demonstrated that the inhibitory effect on fusion of systemic nicotine
could be overcome with rhBMP-2. These investigators administered nicotine to
rabbits via mini-osmotic pumps. They subsequently performed single-level
posterolateral fusions on these rabbits comparing autograft alone to autograft
mixed with rhBMP-2 to allogeneic DBM mixed with BMP-2. They achieved a
100% fusion rate in the autograft/rhBMP-2 group, a 64% fusion rate in the
DBM/rhBMP-2 group and a 0% fusion rate in the autograft alone group.
rhBMP-7 (OP-1)
Cook et al. [29] reported the first spinal application of OP-1 using a canine
posterolateral fusion model. Radiographical and histological examination
revealed solid fusion for the OP-1 group 6 weeks after surgery. The autograft
group attained comparable fusion rates but not until 26 weeks postsurgery. No
fusions were observed for negative controls (i.e., no implant material or carrier
alone). Grauer et al. [30] have studied comparative intertransverse lumbar
fusion in the New Zealand white rabbit model. Study groups were autograft
alone, carrier alone, or OP-1 with carrier. Manual palpation and biomechanical
testing at 5 weeks confirmed a 0% fusion rate in the carrier group, a 63% fusion
rate in the autograft group, and a 100% fusion rate in the OP-1 group. At
5 weeks, histology revealed more mature bone in the OP-1 group. Cunningham
et al. [31] studied a skip-level posterolateral canine model using autograft alone,
autograft and OP-1, or OP-1 alone. Statistically significant differences in the
rate of fusion between the autograft alone and the OP-1-containing specimens
were noted at all timepoints studied (4, 8 and 12 weeks postoperatively).
Only a few other studies reporting the use of OP-1 in interbody fusion have
been reported. Magin and Delling [32] compared OP-1, autograft alone, and an
osteoconductive hydroxyapatite bone graft alternative using a posterior lumbar
interbody fusion sheep model. At 4 months time, OP-1 animals had an 80%
fusion rate with a 60% increase in bone formation compared to the other groups.
Cunningham et al. [33] studied a sheep thoracic spine model using threaded
fusion cages (BAK devices) placed thoracoscopically. BAK cages packed with
OP-1 had fusion rates equivalent to those packed with autograft and to autograft
bone dowel alone, suggesting that OP-1 is as effective as autograft in obtaining
interbody fusion.
Similar to the rhBMP-2 nicotine study above, Patel et al. [34] tested the
ability of OP-1 to overcome the inhibitory effects of nicotine in a rabbit pos-
terolateral fusion model. L5-L6 fusions were performed using either iliac crest
autograft or OP-1. Nicotine was administered to all animals via a subcutaneous
Bomback/Grauer 56
mini-osmotic pump. The fusion rate was 25% for the nicotine-exposed autograft
group and 100% for the nicotine-exposed OP-1 group at 5 weeks, demonstrat-
ing the latter’s ability to overcome the negative effects of nicotine in this model.
DBM
Grafton DBM was shown to be effective as a graft extender with autograft

in a rabbit posterolateral spine fusion model. It demonstrated equal effective-
ness with autograft in a 1:1 or 3:1 dosing ratio. However, it never demonstrated
fusion rates greater than the autograft alone [1]. In a canine posterior fusion
model, Cook et al. [35] evaluated fusion in 9 adult mongrel dogs at 6, 12, and
26 weeks. Four sites on each animal received implants consisting of DBM gel,
DBM gel with allograft, allograft alone, or autograft alone. Radiographical
studies demonstrated that the autograft sites had achieved fusion by 26 weeks
postoperatively. Conversely, the DBM gel alone and with the allograft demon-
strated some new bone formation but did not achieve fusion by 26 weeks.
Mechanically, the autograft sites demonstrated torsional stability significantly
greater than all other fusion sites. Histological analysis confirmed the radio-
graphical and mechanical findings. The results indicate that the DBM gel alone
or with the allograft is inferior to the autograft.
Only sparse reports are available regarding utility of bBMPx in spinal
arthrodesis, but the limited information appears encouraging. Boden et al. [36]
demonstrated a dose response with bBMPx in the rabbit intertransverse process
fusion model. bBMPx mixed with collagen and DBM achieved fusion rates of
50–100% depending on dose. Autograft fusion rates were 62%, and DBM with
collagen alone were only 17%. More recently, a posterolateral fusion model was
studied in nonhuman primates comparing bBMPx delivered in DBM to the
autograft alone. Efficacy data demonstrated an autograft fusion rate of 21%; the
bBMPx displayed a dose response in which 3 mg per side gave twice the fusion
rate as that of the autograft [19].
Clinical Studies Using BMPs
The preclinical data cited above paved the way for completed, ongoing,
and future human trials with these three differentiation factors.
rhBMP-2
In 1996, 14 patients with single-level lumbar degenerative disc disease
were enrolled in a prospective randomized nonblinded controlled trial to test
interbody cage with BMP treatment versus bone autograft. All patients received
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Bridging bone
Fig. 1. Patient one-year status postinterbody fusion with Infuse (BMP-2 on absorbable
collagen sponge) in an LT Cage (image courtesy of Medtronic Sofamor Danek, Memphis,
Tenn., USA).
a tapered titanium interbody fusion device (LT Cage, Medtronic Sofamor

Danek, Memphis, Tenn., USA) filled with either rhBMP-2 or iliac crest auto-
graft. All 11 patients randomized to the rhBMP-2 group were fused radiograph-
ically at 6 months, while one of the 3 patients in the autograft group had a
nonunion at one-year follow-up. Given the small numbers, the differences were
not statistically significant [37], although the clinical results were considered
excellent with rhBMP-2 compared to other interventions.
A variety of clinical trials followed this pilot study, which have demon-
strated efficacy in varied clinical applications of BMPs. Anterior lumbar inter-
body fusion rates performed either open or laparoscopically using
rhBMP-2-filled interbody fusion cages have been shown to be equivalent to
those with autograft-filled cages [37] (fig. 1). When rhBMP-2 was used with
machined allografts (bone dowels) for anterior lumbar interbody fusion, it
yielded higher fusion rates, superior improvement in pain and function, and
a greater likelihood of returning to work compared with autograft-filled dowel
controls [38]. It should be noted, however, that heterotopic bone within the
spinal canal was noted in patients enrolled in an rhBMP-2 posterior lumbar
interbody fusion or PLIF trial. There were no neurological sequelae reported,
but the study was halted prior to completion [39]. Further investigation is war-
ranted to define appropriate safety parameters, given the concern about bony
overgrowth. Finally, the first human trial investigating rhBMP-2 as an adjunct
to posterolateral intertransverse arthrodesis has recently been reported. Twenty-
five patients undergoing lumbar arthrodesis were randomized based on the
arthrodesis technique: autograft/Texas Scottish Rite Hospital (TSRH) pedicle
Bomback/Grauer 58
screw instrumentation (n ⫽ 5), rhBMP-2/TSRH (n ⫽ 11), and rhBMP-2 only
without internal fixation (n ⫽ 9). On each side, 20 mg of rhBMP-2 were
implanted. The patients had single-level disc degeneration, grade 1 or less
spondylolisthesis, mechanical low back pain with or without leg pain, and fail-
ure of nonoperative treatment for at least 6 months. The radiographical fusion
rate was 40% (2/5) in the autograft/TSRH group and 100% (20/20) with
rhBMP-2 group with or without TSRH internal fixation (p ⫽ 0.004). In addi-
tion, statistically greater and quicker improvement in patient-derived clinical
outcome was measured in the rhBMP-2 groups [40]. These results strongly sug-
gest that BMP treatment augments the efficacy of spinal fusion in the setting of
autographs, with or without internal fixation. All interbody clinical trials with
rhBMP-2 demonstrated less blood loss compared to autograft controls. In addi-
tion, the incidence of donor site pain in those patients who underwent bone
graft harvest was 30–40% at 2 years [37].
rhBMP-7 (OP-1)
All OP-1 human trials have involved uninstrumented posterolateral inter-
transverse process fusion in the setting of degenerative spondylolisthesis. An
early Australian study [41] placed autograft on one side and OP-1 on the con-
tralateral side. The 6-month follow-up noted bone formation to be equal or
greater on the OP-1 side (assessed by CT scan) as compared with the autograft
side. Although this was encouraging, it is difficult to interpret the results of
such studies with different bone graft materials on different sides, as one side
may affect the other.
An initial safety and efficacy study in the United States compared auto-
graft alone to autograft augmented with OP-1 for posterolateral arthrodesis.
Sixteen patients with degenerative lumbar spondylolisthesis and spinal steno-
sis were randomized to each treatment arm. At 6-month follow-up, 75% of
patients in the OP-1/autograft group were radiographically fused, whereas
only 50% in the autograft only group were fused [42] (fig. 2). A subsequent
study of similar design is underway, in which patients receive either iliac crest
autograft alone or OP-1 alone [43]. The 6-month results for 36 enrolled
patients have shown a clinical success rate 32% higher in the OP-1 group
than in the autograft group [44]. No OP-1-related adverse events have been
observed to date.
DBM
At the present time, there have been no prospective clinical trials with
human DBM products in the spine literature. A clinical trial is underway in
Switzerland testing bBMPx in a posterolateral lumbar fusion but results are not
yet available [19].
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Bridging bone
Fig. 2. Patient one-year status postdecompression and posterolateral fusion with OP-1
alone for degenerative spondylolisthesis.
Future Directions in Spinal Fusion Research
Gene therapy may play an active role in future preclinical and clinical trials
with various BMPs. Gene-based therapies attempt to deliver specific genes,
known as transgenes, to target cells to change the existing physiological state or
disease process [45]. Genes encoding for factors in the osteogenic cascade are
either inserted into the patient’s own cells that exist at the fusion site (in vivo)
or into cells that have been removed and will be reimplanted at the site of fusion
(ex vivo) [3]. Once these cells are in place, the transgene produces a protein that
initiates the bone-formation cascade. Hence, it is the activity and half-life of the
transgene itself that is the limiting temporal factor for the presence of osteoin-
ductive stimulatory signals at the fusion site [3]. This therapy might allow for
potentially longer expression of BMP activity and thus perhaps an increased
window of time for bone formation.
Boden et al. [46] have reported successful use of gene therapy techniques in
an athymic rat posterolateral spine fusion model. The gene encoding for anos-
teoinductive intracellular signaling protein named LIM mineralization protein-1
was identified and cloned [47]. It appears to be regulated by BMP-6 and to func-
tion very early in the cascade of events leading to de novo bone formation [46].
Bomback/Grauer 60
Using an ex vivo gene therapy strategy, the LIM mineralization protein-1 gene
was transfected into the harvested bone marrow cells of athymic rats and then
reimplanted at appropriate posterolateral fusion sites. Successful arthrodesis was
achieved in 100% of the sites receiving cells containing the LIM mineralization
protein-1 gene and 0% of the sites receiving control cells [46]. In vivo gene ther-
apy methodology has also been reported. In two separate studies, an adenovirus
vector containing either rhBMP-2 or rhBMP-9 was injected percutaneously into
the lumbar paraspinal musculature of athymic rats. Both studies reported suc-
cessful arthrodesis at the experimental sites without any evidence of canal or
neuroforaminal compression [9, 48].
Future techniques may include delivery of genetic material via nonviral
means (e.g., liposomes, gene gun therapy) or with newer viral vectors (e.g.,
adeno-associated virus) demonstrating less immunogenicity. All of these
promising techniques, however, are certainly associated with greater expense
and there are concerns of oncogenesis and bony overgrowth. Although gene
therapy may allow for higher levels of osteoinductive proteins to be expressed
for longer time periods, it is unknown if high fusion rates can be achieved with
one-time applications. In addition, potentially increased bone production might
compromise the safety of these implants. Vector design must incorporate gene
regulation techniques and spine-specific targeting strategies before human
clinical trials can be safely conducted [45].
In terms of local application of recombinant BMP products at the time of
surgery, the use of carrier systems with BMP recombinant proteins is still in
evolution. Carriers for BMP in spine fusion are used to increase the retention of
these differentiation factors at the fusion site while at the same time providing
an osteoconductive matrix on which bone formation can occur. Four major
categories of carriers are used for BMP delivery: inorganic materials (e.g.,
hydroxyapatite, tricalcium phosphate), synthetic polymers (e.g., polylactide,
polyglycolide), natural polymers (e.g., collagen formulations), and composites of
the above three materials [49]. Carrier efficacy is both site specific and species
specific. The dosing of BMP products with their associated carriers is currently
under investigation. At the present time, it is unclear if the optimal dose or the
optimal delivery system has been established, and if delayed-release products can
successfully compete with the theoretical advantages of gene transfer.
Conclusions
The ability of BMPs to promote, extend, or enhance spinal fusion is attract-

ing interest in both the basic science and clinical settings. Although autograft
currently remains the ‘gold standard’ for initiating spine fusion in the clinical
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arena, osteoinductive differentiation factors combined with osteoconductive
matrices are being investigated in preclinical and clinical trials. Results of these
early clinical investigations indicate that rhBMP may be an acceptable, safe bone
graft alternative. However, current numbers are small and follow-up is still of
relatively short duration. Longer follow-up and additional studies are, therefore,
needed to test acute application and long-term application of osteoinductive fac-
tors. Newer gene therapy techniques have not yet been introduced into clinical
trials, but preliminary animal study results are promising.
References
1 Morone MA, Boden SD: Experimental posterolateral lumbar spinal fusion with a demineralized
bone matrix gel. Spine 1998;23:159–167.
2 Martin GJ Jr, Boden SD, Titus L, Scarborough NL: New formulations of demineralized bone
matrix as a more effective graft alternative in experimental posterolateral lumbar spine arthrode-
sis. Spine 1999;24:637–645.
3 Boatright KC, Boden SD: Biologic enhancement of spinal arthrodesis: Past, present, and future; in
Fardon DF, Garfin SR, North American Spine Society, Abitbol J-J (eds): Orthopaedic Knowledge
Update: Spine 2, ed 2. Am Acad Orthopaedic Surgeons, 2002, pp 459–468.
4 Urist MR: Bone formation by autoinduction. Science 1965;150:893.
5 Urist MR, Iwata H, Ceccotti PL, Dorfman RL, Boyd SD, McDowell RM, Chien C: Bone mor-
phogenesis in implants of insoluble bone gelatin. Proc Natl Acad Sci USA 1973;70:3511–3515.
6 Urist MR, Mikulski A, Leitze A: A solubilized and insolubilized bone morphogenetic protein.
Proc Natl Acad Sci USA 1979;76:1828.
7 Reddi AH: Cell biology and biochemistry of endochondral bone development. Coll Relat Res
1981;1:209–226.
8 Boden SD, Schimandle JH, Hutton WC: An experimental lumbar intertransverse process spinal fusion
model: Radiographic, histologic, and biomechanical healing characteristics. Spine 1995;20:412.
9 Helm GA, Alden TD, Beres EJ, Hudson SB, Das S, Engh JA, Pittman DD, Kerns KM, Kallmes DF:
Use of bone morphogenetic protein-9 gene therapy to induce spinal arthrodesis in the rodent.
J Neurosurg (Spine 2) 2000;92:191–196.
10 Wozney JM: Overview of bone morphogenetic proteins. Spine 2002;27:S2–S8.
11 Ebara S, Nakayama K: Mechanism for the action of bone morphogenetic proteins and regulation
of their activity. Spine 2002;27:S10–S15.
12 Urist MR, DeLange RJ, Finerman GAM: Bone cell differentiation and growth factors: Induced
activity of chondro-osteogenetic DNA. Science 1983;220:680.
13 Piccolo S, Sasai Y, Lu B, DeRobertis EM: Dorsoventral patterning in Xenopus: Inhibition of ven-
tral signals by direct binding of chordin to BMP-4. Cell 1996;86:589–598.
14 Zimmerman LB, DeJesus-Escobar JM, Harland RM: The Spemann organizer signal noggin binds
and inactivates bone morphogenetic protein 4. Cell 1996;86:599–606.
15 Varady P, Li JZ, Cunningham M, Beres EJ, Das D, Engh J, Alden TD, Pittman DD, Kerns KM,
Kallmes DF, Helm GA: Morphologic analysis of BMP-9 gene therapy-induced osteogenesis.
Hum Gene Ther 2001;12:697–710.
16 Spiro RC, Liu L, Heidaran MA, Thompson AY, Ng CK, Pohl J, Poser JW: Inductive activity of
recombinant human growth and differentiation factor-5. Biochem Soc Trans 2000;28:362–368.
17 Boden SD, Andersson GBJ, Anderson DG, Damien C, Ebara S, Helm G, Lane JM, McKay B,
Sandhu HS, Seeherman H, Wozney J: Summary statement: Overview of bone morphogenetic
proteins for spine fusion. Spine 2002;27:S1.
18 Bomback DA, Grauer JN, Lugo R, Troiano N, Patel TC, Friedlaender GE: Comparison of
posterolateral lumbar fusion rates of Grafton Putty and OP-1 Putty in an athymic rat model. Spine
2004;29:1612–1617.
Bomback/Grauer 62
19 Damien CJ, Grob D, Boden SD, Benedict JJ: Purified bovine BMP extract and collagen for spine
arthrodesis. Spine 2002;27:S50–S58.
20 Sandhu HS, Turner S, Kanim LEA, Kabo JM, Toth JM: Augmentation of threaded titanium fusion
cages with rhBMP-2 for experimental anterior lumbar fusion. Presented at the 11th Annual Meeting
of the North American Spine Society, October 23–26, Vancouver, Canada, 1996.
21 Zdeblick TA, Ghanayem AJ, Rapoff AJ, Swain C, Bassett T, Cooke ME, Markel M: Cervical inter-
body fusion cages: An animal model with and without bone morphogenetic protein. Spine 1998;
23:758–765.
22 Hecht BP, Fischgrund JS, Herkowitz HN, Penman L, Toth JM, Shirkhoda A: The use of recombi-
nant human bone morphogenetic protein 2 (rhBMP-2) to promote spinal fusion in a nonhuman
primate anterior interbody fusion model. Spine 1999;24:629.
23 Schimandle JH, Boden SD, Hutton WC: Experimental spinal fusion with recombinant human
bone morphogenetic protein-2. Spine 1995;20:1326.
24 Sandhu HS, Kanim LE, Kabo JM, Toth JM, Zeegan EN, Liu D, Seeger LL, Dawson EG:
Evaluation of rhBMP-2 with an OPLA carrier in a canine posterolateral spinal fusion model.
Spine 1995;20:2669.
25 Fischgrund JS, James SB, Chabot MC, Hankin R, Herkowitz HN, Wozney JM, Shirkhoda A:
Augmentation of autograft using rhBMP-2 and different carrier media in the canine spinal fusion
model. J Spinal Disord 1997;10:467.
26 Helm GA, Sheehan JM, Sheehan JP, Jane JA Jr, diPierro CG, Simmons NE, Gillies GT,
Kallmes DF, Sweeney TM: Utilization of type I collagen gel, demineralized bone matrix and
bone morphogenetic protein-2 to enhance autologous bone lumbar spinal fusion. J Neurosurg
1997;86:93–100.
27 Martin GJ, Boden SD, Titus L: Recombinant human bone morphogenetic protein-2 overcomes the
inhibitory effect of ketorolac, a nonsteroidal anti-inflammatory drug (NSAID), on posterolateral
lumber intertransverse process spine fusion. Spine 1999;24:2188–2193.
28 Silcox DH, Boden SD, Schimandle JH, Johnson P, Whitesides TE, Hutton WC: Reversing the
inhibitory effect of nicotine on spinal fusion using an osteoinductive protein extract. Spine 1998;
23:291–296.
29 Cook SD, Dalton JE, Tan EH, Whitecloud TS III, Rueger DC: In vivo evaluation of recombinant
human osteogenic protein (rhOP-1) implants as a bone graft substitute for spinal fusions. Spine
1994;19:1655.
30 Grauer JN, Patel TC, Erulkar JS, Troiano NW, Panjabi MM, Friedlaender GE: Evaluation of
OP-1 as a graft substitute for intertransverse process lumbar fusion. Spine 2001;26:127–133.
31 Cunningham BW, Shimanoto N, Sefter JC, McAfee A: Posterolateral spinal arthrodesis using
osteogenic protein-1: An in vivo time course study using a canine model. Presented at the 15th
Annual Meeting of the North American Spine Society, New Orleans, LA, 2000.
32 Magin MN, Delling G: Improved lumbar vertebral interbody fusion using rhOP-1: A comparison
of autogenous bone graft, bovine hydroxylapatite (Bio-Oss), and BMP-7 (rhOP-1) in sheep. Spine
2001;26:469–478.
33 Cunningham BW, Kanayama M, Parker LM, Weis JC, Sefter JC, Fedder IL, McAfee PC:
Osteogenic protein versus autologous interbody arthrodesis in the sheep thoracic spine: A compar-
ative endoscopic study using the Bagby and Kuslich interbody fusion device. Spine 1999;24:509.
34 Patel TC, Erulkar JS, Grauer JN, Troiano NW, Panjabi MM, Friedlaender GE: Osteogenic protein-1
overcomes the inhibitory effect of nicotine on posterolateral lumbar fusion. Spine 2001;26:1656.
35 Cook SD, Dalton JE, Prewett AB, Whitecloud TS III: In vivo evaluation of demineralized bone
matrix as a bone graft substitute for posterior spinal fusion. Spine 1995;20:877–886.
36 Boden SD, Schimandle JH, Hutton WC: Lumbar intertransverse-process spinal arthrodesis with
use of a bovine bone-derived osteoinductive protein. A preliminary report. J Bone Joint Surg Am
1995;77:1404–1417.
37 McKay B, Sandhu HS: Use of recombinant human bone morphogenetic protein-2 in spinal fusion
applications. Spine 2002;27:S66–S85.
38 Burkus JK, Transfeldt EE, Kitchel SH, Watkins RG, Balderston RA: Clinical and radiographic
outcomes of anterior lumbar interbody fusion using recombinant human bone morphogenetic
protein-2. Spine 2002;27:2396–2408.
medwedi.ru
39 Poynton AR, Lane JM: Safety profile for the clinical use of bone morphogenetic proteins in the
spine. Spine 2002;27:S40–S48.
40 Boden SD, Kang J, Sandhu H, Heller JG: Use of recombinant human bone morphogenetic protein-2
to achieve posterolateral lumbar spine fusion in humans: A prospective, randomized clinical pilot
trial: 2002 Volvo Award in Clinical Studies. Spine 2002;27:2662–2673.
41 Speck G: Posterolateral fusion using OP-1: A model using degenerative spondylolisthesis.
Australian Spine Meeting, Adelaide, Australia, 2000.
42 Patel TC, McCullough JA, Vaccaro AR: A pilot safety and efficacy study of OP-1 (rhBMP-7) in
posterolateral lumbar fusion as a replacement for iliac crest autograft. Seattle, North Am Spine
Society, 2001.
43 Vaccaro AR, Anderson G, Toth CA: Recombinant human osteogenic protein-1 (bone morpho-
genetic protein-7) as an osteoinductive agent in spinal fusion. Spine 2002;27:S59–S65.
44 Patel TC, Vacarro AR, Truumees E: A safety and efficacy study of OP-1 (rhBMP-7) as an adjunct
to posterolateral lumbar fusion. Seattle, North Am Spine Society 2001.
45 Alden TD, Varady P, Kallmes DF, Jane JA, Helm GA: Bone morphogenetic protein gene therapy.
Spine 2002;27:S87–S93.
46 Boden SD, Titus L, Hair G, Liu Y, Viggeswarapu M, Nanes MS, Baranowski C: Lumbar spine
fusion by local gene therapy with cDNA encoding a novel osteoinductive protein (LMP-1). Spine
1998;23:2486–2492.
47 Liu Y, Hair G, Titus L: BMP-6 induces a novel LIM protein involved in bone mineralization and
osteocalcin secretion. J Bone Min Res 1997;12:S115.
48 Alden TD, Pittman DD, Berer EJ, Hankins GR, Kallmes DF, Wisotsky BM, Kerns KM, Helm GA:
Percutaneous spinal fusion using bone morphogenetic protein-2 gene therapy. J Neurosurg (Spine 1)
1999;90:109–114.
49 Seeherman H, Wozney J, Li R: Bone morphogenetic delivery systems. Spine 2002;27:S16–S23.
David Bomback, MD
400E 71st Street
Apt. #51
New York, NY 10021 (USA)
Tel. ⫹1 212 472 2143, Fax ⫹1 212 774 2779, E-Mail dbomback@yahoo.com
Bomback/Grauer 64
Cellular and Gene Therapy

Approaches to Spinal Cord Injury
Michael P. Steinmetz, James K. Liu, Nicholas M. Boulis
Department of Neurosurgery S31, Cleveland Clinic Foundation,
Cleveland, Ohio, USA
Introduction
Acute spinal cord injury (SCI) is relatively uncommon, affecting about

one in 40 patients who present to a major trauma center [1]. It is estimated
that there are 11,000 new cases per year or 40 cases per million population
(National Spinal Cord Injury Databank, 2001). Despite this relatively low
incidence, these injuries pose serious problems for the patients, their families,
and society in general [2]. Mortality from SCI is estimated to range from
4.4 to 16.7% for those that survive the initial injury and receive treatment [3].
Aside from the obvious physical damage, there may be serious psychological
effects on both the patients and their families. The financial burden to the
patient, the health care system, and society is great in terms of both direct and
indirect costs (i.e., lost income and productivity) [4]. In 1990, it was esti-
mated that the cost to the United States of caring for all SCI patients was USD
4 billion annually [5].
Clinical therapy for acute SCI is sparse and often disappointing. The clini-
cian is limited to surgical decompression (if appropriate), IV methylprednisolone,
and acute and long-term SCI rehabilitation. Various research protocols are also in
progress. Despite these therapies, the prognosis for SCI remains dismal.
The failure of functional recovery following SCI is multifactorial. Axonal
regeneration requires neuroprotection, neuronal cell-body stimulation, the need
to overcome local inhibitors at the injury site, and finally reconnection of neu-
ronal pathways (both ascending and descending) essential for functional recov-
ery. Current research is focused on each of these areas. Both genetic and
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cellular therapies are emerging as strategies to overcome barriers to neural
regeneration. This chapter will review past and current research with genetic
and cellular therapeutic options.
Cellular Therapies
There are many evolving cellular therapeutic strategies for SCI. The
focus of this therapy is on neuroprotection, remyelination, and regeneration.
Cellular therapies include endogenous and transplanted stem cells, fetal tissue
transplants, allo- and xenografted Schwann cells and olfactory ensheathing
cells (OECs) as well as autologous macrophages. Each area of research has
uncovered difficulties unique to individual cellular approaches. These include
the ethical and moral concerns raised by embryonic stem (ES) cells and fetal
grafts, as well as the potential need for immunosuppression after cellular
transplants.
Models of SCI
There are many animal models of SCI available to the researcher. The most
popular animals for these injury paradigms include the rat and mouse. Both are
fairly inexpensive and are readily available. The mouse model has the further
advantage of being used for transgenic experiments.
Methods of experimental SCI entail complete spinal cord transection,
partial transection, contusion, and compression [6]. Complete transection
involves the complete disruption of the spinal cord. The main advantage of this
model is that all tracts are transected; therefore, any axons demonstrated on ret-
rograde labeling (see below) are due to regeneration and not from sparing (not
destroyed during the experimental injury). Partial transection models utilize
animals in which only certain tracts are cut (e.g., the rubrospinal tract). This
leaves the contralateral tract available for comparison [6]. The absence of com-
plete paraparesis and urinary retention renders these animals easier to care for
(i.e., one does not need to manually express the bladder in those rats who have
only a unilateral rubrospinal transection). A serious disadvantage of these mod-
els is the potential for confusing spared and regenerating axons. Contusion and
compression models reflect more accurately the SCIs that generally occur in
humans [6]. Methods available for contusion and compression models include
weight-drop and clip-compression strategies. Because the lesions in these mod-
els are even less discrete, they have an even greater potential for confusion
between axon sparing and true regeneration.
Steinmetz/Liu/Boulis 66
Recovery Assays
Immunohistochemical, electrophysiological, and behavioral assays can all

be used to measure spinal cord recovery [6]. Axons tracers are used to follow
tracts in the spinal cord (both anterograde and retrograde). Anterograde tracers
include biotinylated dextran amine and the cholera toxin B subunit. These trac-
ers are applied to the cell body and then transported anterograde and may be
used to identify axons regenerating at the injury site. Retrograde tracers are
taken up by axons and transported back to the cell body. These may then be
placed distal to the injury site to assess regeneration at a site of injury.
Examples include Flouro-Gold and the cholera toxin B subunit.
Electrophysiological tests may assess axon integrity, both in vivo and in
vitro. Examples of in vivo tests include somatosensory evoked and motor
potentials. Isolated spinal cords (in vitro) may also be assessed neurophysio-
logically [6].
Finally, behavioral tests are available to evaluate the neurological recovery
of the injured animal. These include open field test of locomotion, such as the
BBB; sensory tests, such as sensing and removing a piece of tape from a paw;
and other tests or motor and skilled behavior, such as walking across a wire grid
or narrow beam.
Cellular Therapies for SCI
Stem Cells
Neural stem cells (NSCs) are defined by their ability to generate neural tis-
sue (both neuronal and glial), their ability to self-renew, and their pluripotency.
Pluripotency refers to a cell’s ability to generate a variety of lineages through cell
division [7]. Progenitor cells have a more restricted fate (fig. 1). For example,
neural progenitors differentiate into all the neuronal cells of the central nervous
system (CNS), but not the glia.
NSCs may be isolated from adult and fetal brain and also embryonic
tissue. Cells may be harvested from the adult subventricular zone, hippocampus
[7], the fetal telencephalon, or the inner cell mass of blastocyst-stage embryos
[8]. These cells are then grown in cell culture in the presence of a high concen-
tration of mitogens such as fibroblast growth factor (FGF) or epidermal growth
factor [7]. After several rounds of division, the cells are exposed to either media
with the mitogens withdrawn or to a new substrate. Different substrates can
drive stem cell differentiation into specific lineages (e.g., oligodendrocyte).
Immunostaining for specific marker antigens can be used to identify these line-
ages. Cells may be infected with a replication-incompetent retroviral vector
Cellular and Gene Therapy Approaches 67
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Cell type
Pluripotent embryonic stem cell

self-renewing
Multipotent stem cells

self-renewing
Neural progenitor cells

limited self-renewal
Committed progenitor cells

no self-renewal
Neuronal Glial
Differentiated cells
no self-renewal
Neuron Glial
Fig. 1. The progression of cell development from a pluripotent, self-renewing,

embryonic stem cell to differentiated neurons and glia.
encoding lacZ in order to assess clonal relationships of progeny and identify

them in situ following transplantation [9].
NSCs
The adult CNS has a limited capacity to repair itself after injury. This is due
in part to the inability to generate new neurons and the inability to initiate
Exogenous stem cells
1. 2.
Syrinx Syrinx
a
Exogenous factor
1. 2.
Central canal Syrinx Potential stem cells Central canal Syrinx

b
Fig. 2.a Exogenous stem cell transplantation. 1. Exogenous stem cells have been trans-
planted into a syrinx cavity following a chronic SCI. 2. Following transplantation, the stem
cells have populated the cavity and migrated into the spinal cord parenchyma. b Endogenous
stem cells. 1. The endogenous stem cells of the spinal cord probably reside in the region of
the central canal. 2. Following injury or factor injection, these cells are stimulated to divide
and migrate into the area of injury (in this case a syrinx cavity). Some cells also migrate into
the cord parenchyma.
functional axonal regrowth. The ability to transplant multipotent NSCs may

overcome the former. As such, significant enthusiasm has focused on the appli-
cation of NSCs for the repair of focal neural tissue destruction, including that
which is seen with SCI. These stem cells may be isolated from embryonic or
adult brain tissue of a variety of species, including mouse, rat, and human [7, 10].
They may also be derived from the mouse and human ES cells [8, 11–13] derived
from nonneural embryonic tissue. These cells are stable through multiple pas-
sages in vitro without loss of their multipotentiality [14]. Multipotentiality or
‘pluripotency’ refers to the NSC’s ability to differentiate into a variety of line-
ages, including neuronal, oligodendrocytic, or astrocytic phenotypes. These stem
cells have been shown to survive transplantation into the CNS and also have the
ability to migrate. Thus, these cells may be transplanted into the injured CNS
with the potential to repair specific regions. In addition to the ability to transplant
exogenous stem cells, it may be possible to induce endogenous multipotential
cells to ‘self-repair’ after injury or disease [14] (fig. 2).
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Recent evidence also suggest that NSCs may be derived from bone marrow
and umbilical cord blood, thus serving as another source of both exogenous and
endogenous stem cells, but residing outside the CNS [15]. Both in vivo and
in vitro studies have demonstrated neuronal and glial differentiation; further-
more, these cells (i.e., bone marrow or umbilical cord blood) have the potential
to be delivered systemically for the treatment of CNS pathology as opposed to
direct transplantation into the CNS [15]. The use of marrow-derived stem cells
may permit the clinician to harvest autologous stem cells, amplify them in vitro
and then transplant them back into the patient. This may obviate the need for
chronic immunosuppression and its inherent morbidity. Experimentally, auto-
logous bone marrow-derived stem cells have been used to regenerate infarcted
myocardium [16]. Further progress may provide the same opportunity for the
nervous system.
Exogenous NSCs
Experiments have demonstrated that NSCs demonstrate significant sur-
vival, migration, and differentiation. NSCs undergo area-specific differentiation
following transplantation into the CNS [9, 17, 18]. It appears that these cells
have the capacity to respond appropriately to local signals in the developing
CNS [14]. It may be that the local environment is the predominant determinant
of the differentiated fate of the engrafted cells. When pluripotent NSCs are
transplanted into the injured spinal cord, the engrafted cells differentiate only
into astrocytes, and the temporal progression of that differentiation is markedly
retarded [19, 20]. The mechanism regulating this transformation is unknown.
Successful neuronal replacement may, therefore, require transplanting NSCs
already committed to a neuronal lineage to avoid local environmental cues
defining a glial lineage. Neural restricted precursors (NRPs) are an exciting
alternative to multipotent NSCs. These cells are committed to a neuronal lineage
at the time of isolation, and have been isolated from embryonic CNS tissue,
ES cells, and multipotential NSCs [11] (fig. 1). These cells have been trans-
planted into the adult rat spinal cord. Neuronal maturation was observed, but
was significantly retarded. It appears that additional modification of the grafts
and/or the host environment will be needed for mature neuronal differentiation
[14]. The intrinsic state of NSCs at the time of transplant, like the host environ-
ment, may also be important [21]. There appear to be differences between neural
progenitor cells isolated from different brain regions [22, 23]. When NRPs
derived from embryonic spinal cord are grafted into the subventricular zone
(SVZ), the cells were observed to migrate extensively and generate mature neu-
rons of various neurochemical and morphological phenotypes [24]. When NRPs
isolated from the SVZ are engrafted back to the SVZ, they demonstrate less
migratory and differentiation potential [25]. Spinal cord-derived NRPs were
also observed to differentiate into a neuronal phenotype expressing choline
acetyltransferase in brain areas where endogenous choline acetyltransferase-
positive neurons have not been found. In this instance, therefore, intrinsic char-
acteristics of the transplanted NRPs committed them to a restricted phenotype
even after ectopic engraftment [14]. This finding suggests that one may need to
isolate precursors from specific CNS regions for proper functional neural
replacement [14].
Many studies have demonstrated successful transplantation of NSCs and
NRPs into the CNS, but the physiological function of these grafts has not been
completely elucidated. McDonald et al. [26] demonstrated functional improve-
ment (locomotor) after transplantation of NSCs derived from ES cells into a rat
spinal cord in a contusion injury model. ES cell embryoid bodies were derived
from the D3 cell line (mouse) [27] at the 4⫺/4⫹ stage (i.e., 4 days without, then
4 days with retinoic acid) for transplantation. These cells were transplanted as
cell aggregates directly into the syrinx (fig. 2a) 9 days after the experimental
SCI. Two weeks after transplantation, labeled ES cells [Bromodeoxyuridine
(BrdU), or mouse-specific antibodies M2, EMA, or Thy 1.1/1.2] were identi-
fied in situ. Cells were found to be filling the syrinx cavity, but also as far as
8 mm away from the syrinx edge in either the rostral or caudal direction. 43%
of these cells were found by immunohistochemistry to be oligodendrocytic and
19% were found to be astrocytic. Many of the ES-derived oligodendrocytes
were immunoreactive for myelin basic protein. 8% demonstrated neuronal
staining (neuron-specific nuclear protein, NeuN).
One month following transplantation, a difference of 2 points on the BBB
scale was observed (7.9 vs. 10) between treated animals and sham controls. The
difference in the BBB score reflected the ability to mobilize with partial hind
limb weight-bearing and coordination as opposed to no hind limb weight-bearing
or coordination. It is unclear what factors were responsible for the improved func-
tional score following transplantation. The ES cells may have remyelinated the
injured axons or provided neurotrophic or tissue-sparing effects. Furthermore,
ES-derived neurons may have matured and made functional connections with
injured spinal tracts. The rapidity of locomotor improvement and the observation
that most ES cell-derived cells were oligodendrocytes positive for myelin basic
protein make remyelination the most probable cause of recovery. The fact that
oligodendrocyte precursors transplanted into chemical lesions have previously
been associated with remyelination and improved axonal conduction creates
further precedence for this explanation [28].
Various goals underlie the rationale for NSCs or NRPs spinal cord trans-
plantation. As previously discussed, it is unlikely that transplanted stem cells
will be able to completely recapitulate the injured ascending and descending
tracts of the spinal cord (fig. 3e). Although stem cell-derived neurons have been
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NSC Trophic NSC Native glial Trophic
factors cell factors
a b
Ex vivo Trophic NSC precursor Glia
Fibroblast
gene transfer factors
c d
NSC
e
Fig. 3. Various mechanisms of NSC graft-induced recovery following spinal cord

injury. a NSCs may secrete various trophic factors that induce regrowth of spinal cord axons.
b NSCs may induce native CNS support cells (e.g., glial cells) to secrete trophic factors that
lead to axonal regrowth. c Fibroblasts implanted following ex vivo gene transfer may secrete
trophic factors, leading to axonal regrowth. d NSCs that have been stimulated to become
oligodendrocyte or glial precursors are transplanted into the spinal cord. Following further
differentiation, remyelination of damaged axons is initiated. e A common misconception of
NSC transplantation is that NSCs completely recapitulate a new axon, replacing the dam-
aged axon. It is highly unlikely that this occurs following NSC transplantation.
shown to possess ion channels similar to native neurons and are capable of gen-
erating action potentials, no study has clearly shown that these cells integrate
into host circuitry and create functional synapses. It is more likely that these
grafts provide neuroprotection (tissue sparing), trophic support, or remyelina-
tion, although direct evidence is speculative (fig. 3a). Furthermore, stem cell
grafts may provide effective tissue bridges that permit or promote the passage
of endogenous regenerating axons.
Endogenous Stem Cells

The use of exogenous stem cells necessitates a grafting procedure and
possibly immunosuppression. The use and manipulation of endogenous stem
cells may obviate these cumbersome and potentially hazardous interventions. It
is now widely accepted that neurogenesis occurs in the adult CNS. This process
has been demonstrated in the hippocampus and the SVZ [29–32]. Lois and
Alvarez-Buylla [31] labeled the brains of adult male mice with [3H]thymidine.
Proliferating, hence, dividing cells were localized almost exclusively to the SVZ.
To test the fate of these cells, the SVZ of labeled brains were isolated and grown
in culture. By six cell divisions, the explants had generated an outgrowth of flat
glial cells (GFAP positive) and cells containing processes growing on the glial
monolayer. These cells were determined to be neurons due to their immunoreac-
tivity to neuronal markers (MAP-2, NF, and NSE) and absence of staining for
glial markers (GFAP). Explants stained with neuron-specific antibodies were
processed for autoradiography to detect the presence of [3H]thymidine in the
cell nuclei. It was found that approximately 84% of neurons were labeled with
[3H] thymidine. These results indicate that proliferating cells do exist in the adult
SVZ in vivo and these cells possess the ability to generate neurons and glia.
Eriksson et al. [29] demonstrated similar results in the adult human dentate
gyrus. They examined the hippocampus and SVZ of patients who had suc-
cumbed to cancer and had received an intravenous infusion of BrdU before they
died. The results of these experiments demonstrated that in all BrdU-treated
patients, the granule cell layer contained BrdU-positive cells, which also double-
stained with neuron-specific markers (e.g., NeuN). Furthermore, the SVZ also
contained BrdU-positive cells, but these did not colabel with neuron-specific
markers. It is believed that these too are progenitor cells, but they must first
migrate from the SVZ before they differentiate. It is believed that the cellular
substrate for this neurogenesis is the endogenous stem cell [33]. The mechanisms
that induce and control this process are unknown.
It may be that one can manipulate these endogenous cells to replace
damaged neural tissue following injury. Indeed some important observations
have been made. Johansson et al. [34] demonstrated that multipotential NSCs
migrate to the area of injury after dorsal funiculus sectioning. However, similar
to cellular transplants, most differentiated into astrocytes. After prolonged
administration of BdrU to the spinal cord of rats, a substantial number of
ependymal cells lining the central canal were labeled and few were seen outside
the spinal cord central canal ependyma (due to the lack of an SVZ in the spinal
cord). After an incision (one day following injury) was made in the dorsal
funiculus at T2, there was a 50-fold increase in the proliferating ependymal
cells. Electron microscopy demonstrated the cell division to be asymmetric. To
demonstrate the fate of these ependymal cells, lesions were made in animals
that had received an injection with a fluorescent marker called Dil that labels
the ependymal cells prior to the lesion. Dil-labeled cells were abundantly seen
in the injury site within one week after the lesion. These cells demonstrated
immunoreactivity to GFAP, but neither ␤-tubulin nor O4, confirming them to
be astrocytes, and not neurons or oligodendrocytes. Therefore, local cues will
have to be overcome so that neurons are formed rather than glia, which may
exacerbate scarring. It is also postulated that since there is little neurogenesis
occurring in the mature spinal cord, the number of endogenous stem cells may
be inappropriate for the replacement of tissue following injury.
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Remyelination of the Spinal Cord
Glial precursors have been found throughout the developing and adult
CNS. A large proportion of these are oligodendrocyte precursors. When adult
rats are pulse-labeled with BrdU, 70–75% of BrdU-labeled cells found in the
spinal cord and cortex express NG2, a marker for oligodendrocyte precursors
[35, 36]. The exact function of these cells is unknown, but one may speculate
that these cells remyelinate the CNS following injury. It has been demonstrated
that the numbers of these oligodendrocyte precursors are markedly increased
following SCI and demyelination [37–39]. It is hypothesized that these glial
precursor cells may be transplanted into the injured spinal cord to remyelinate
axons and promote functional recovery.
As previously mentioned, when multipotential NSCs are grafted into the
injured CNS, only a fraction differentiates into oligodendrocytes. Therefore,
stimulating oligodendrocyte lineage commitment prior to transplantation may
be necessary to achieve effective remyelination. Grafts of this type are called
oligospheres. When oligospheres are transplanted into the myelin-deficient
spinal cord, significantly larger areas of myelination were demonstrated com-
pared to neurosphere transplantation [13, 40]. It appears that astrocytes also
play an important role during myelination. Therefore, glial restricted precursors
may prove more effective for remyelination than oligospheres due to their abil-
ity to differentiate into both astrocytes and oligodendrocytes after transplanta-
tion [41]. The efficacy of these glial restricted precursors for functional
myelination is still in question.
Although the use of endogenous and transplanted stem cells has demon-
strated some remyelination of axons, no study has clearly demonstrated ‘func-
tional’ myelination after this cellular therapy. As with other NSC grafting
strategies, the therapeutic mechanism of oligospheres remains unknown. As with
the other grafts, neuroprotective or trophic mechanisms may contribute (fig. 3b).
In vitro studies have demonstrated partial electrophysiological recovery of
remyelinated axons [42]. However, there has not been electrophysiological
evidence of recovery in the live animal [14].
Although few studies have demonstrated functional recovery following
stem cell therapy, the field is advancing rapidly. The ability to replace lost
neural elements (i.e., neurons and glia) is paramount to neural regeneration
following injury.
Non-Stem Cell Strategies

Fetal Tissue
After SCI, a gap often exists at the site of injury. There is often a cyst or
syrinx cavity in the spinal cord. Therefore, a bridge may be necessary to permit
adequate neural regeneration for both spinal and supraspinal projections. Fetal
tissue transplants have been demonstrated to provide a permissive condition for
axonal regrowth and provide such a bridge.
After complete spinal cord transection in the newborn rat or kitten, fetal
spinal cord tissue transplanted into the site of injury has allowed some restora-
tion of supraspinal projections and improvement in the locomotor function
[43, 44]. In a transection model of adult SCI, transplantation of fetal tissue at the
lesion permitted axonal regeneration into the graft, but not beyond the graft-host
interface [45–48]. The failure to achieve significant numbers of graft-spanning
axons has remained an obstacle to most studies involving tissue grafts for the
treatment of SCI. Some have demonstrated that the exogenous administration of
brain-derived neurotrophic factor (BDNF) or neurotrophin-3 (NT-3) increases
supraspinal axonal growth into the transplant fetal tissue grafts and prevents the
atrophy of axotomized supraspinal neurons [49].
It is also problematic that most studies utilizing tissue bridges employ acute
models of SCI. These models may not be clinically relevant. Often a syrinx cav-
ity does not develop until the subacute or chronic phase of SCI. Coumans et al.
[50] demonstrated that if a fetal tissue transplant and neurotrophin administra-
tion is delayed 2–4 weeks after a complete SCI in the rat, axonal regrowth from
both propriospinal and supraspinal neurons is increased within the transplant and
the host cord caudal to the lesion. These animals also demonstrated significant
improvement in locomotion, including recovery of weight-supported plantar
stepping on both treadmill and over-ground tasks such as stair climbing.
In summary, although fetal tissue transplants have shown some success as
tissue bridges, experiments remain hampered with the distal host-graft inter-
face. Some studies have demonstrated axonal presence across the host-graft
interface. The numbers of these axons is sparse and may actually represent
‘axonal sparing’ and not regeneration.
OECs
Axonal regrowth into a site of injury following cellular grafting is plagued
by the inability of those axons, which have entered the graft, to cross the host-
graft interface. Therefore, a cell, which may enable axons to re-enter the CNS,
may be useful to overcome this barrier to regeneration. Olfactory axons con-
tinue to re-enter the olfactory bulb throughout adult life. The entry point is asso-
ciated with special glial cells known as OECs [51–54]. Investigators have
demonstrated that OECs transplanted into the spinal cord mediate the re-entry
of regenerating dorsal root axons into the spinal dorsal horn and the injections
also increased axon growth into Schwann cell-filled guidance channels [55, 56].
As opposed to other cellular grafts, transplantation of OECs facilitated axonal
growth past the host-graft interface. This may be due to the migratory capacity
of these cells. In a study by Li and Raisman [57], regenerating axons were
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demonstrated to re-enter the distal host corticospinal tract up to 10 mm caudal
to the injection site. These regenerating axons are covered by peripheral myelin
formed by the OEC cell.
Schwann Cells
Because the environment of the peripheral nervous system (PNS) is
permissive for regeneration, Schwann cell transplants have also been used as a
strategy for the treatment of experimental SCI. These cells may be neuropro-
tective and have been demonstrated to secrete various growth factors.
Theoretically, these cells are also able to form myelin around spared and regen-
erating axons. As with other cellular grafts, investigators have found that fol-
lowing the transplantation of cultured Schwann cells, the cells integrate into
the host tract glial structure [57, 58]. These cellular grafts greatly increase
axon sprouting in lesions of the corticospinal tract, but few axons were found
to re-enter the distal tract [59].
In contrast, other investigators have demonstrated axonal growth beyond
the graft. Schwann cells transplanted after a moderate contusion of the rat tho-
racic spinal cord permitted propriospinal and supraspinal axons reaching
5–6 mm beyond the graft. A modest improvement in hind limb locomotor per-
formance was detected at 8–11 weeks after injury [60]. Nonetheless, the lim-
ited growth beyond the graft, even in this experiment, suggests that recovery
was likely to be due to neuroprotection or remyelination of spared axons rather
than axonal regeneration.
Schwann cells have also been seeded into mini-channels that have been
used as bridges. When this transplantation technique is combined with exog-
enous neurotrophin administration, axonal growth was demonstrated into the
graft and into the distal spinal cord, albeit for a limited distance [61].
In summary, these studies demonstrate that both OECs and Schwann cell
transplants may be useful to induce axonal regeneration and remyelination
after SCI. Although some studies have demonstrated some functional improve-
ment following Schwann cell or OEC transplants, it is unclear if the improve-
ment is due to neuroprotection, trophic factor secretion, or remyelination (see
above). The myelination that has been observed has not been demonstrated to
be functional nor has it been quantified. While some studies have demonstrated
axonal growth into the distal spinal cord, the amount of regrowth is not highly
significant, and is unlikely to account for a significant amount of functional
improvement following SCI.
Macrophages
Macrophage recruitment and stimulation are among the earliest events in
the multifactorial process of tissue healing. This observation has led to the
hypothesis that stimulating an appropriate inflammatory response could
encourage a cascade of events necessary for regeneration and repair [62].
Because of the immune-privileged status of the CNS, a restricted inflammatory
response is seen following injury. This restriction may contribute to the poor
regenerative capacity of the CNS compared to the PNS [63–65]. Macrophages,
previously exposed in vitro to regenerating peripheral nerve segments, have
been shown to induce axonal regrowth in completely transected rat optic nerves
[64]. This observation drew attention to the potential for stimulated
macrophages to play a role in spinal cord repair.
Macrophages stimulated via exposure to peripheral nerve segments in vitro
and then re-implanted at the site of a complete SCI in the rat induce partial
recovery of motor function [62]. The fact that this function was lost after retran-
section proved that the recovery was not due to intrinsic spinal cord reflex path-
ways. Anterograde labeling demonstrated continuity of nerve fibers across the
transection site. The authors of this study hypothesize that activated
macrophages may provide cytokines, growth factors, and other wound-healing
factors [41, 66, 67]. These factors may control the astrocytic response seen after
injury, thus reducing the glial scar known to inhibit axonal regeneration [68].
Stimulated macrophages may accelerate processes that normally occur relatively
slowly in the injured CNS [62].
Because the activated macrophage strategy is aimed at upstream processes
in the injury cascade, this one intervention may then affect numerous down-
stream events. Given the complexity of SCI pathophysiology, multifactorial
therapies of this kind may ultimately prove the most effective. Furthermore,
because autologous cells are utilized, many of the ethical and immunological
difficulties inherent in other cellular therapies are absent.
Molecular Therapies for SCI
Concepts of Gene Transfer

A variety of investigators have pursued gene transfer as a means of inducing
neuroprotection and axonal regeneration in the injured spinal cord. As with
cellular therapies, a variety of potential strategies exist for spinal cord gene trans-
fer. Transfer can be affected with viral and nonviral vectors. In vivo strategies,
which entail gene transfer directly into injured cord parenchyma, and ex vivo
strategies, which entail gene transfer into cells that are subsequently grafted, have
both been proposed.
The best method for gene delivery remains debated. An effective method
must accomplish four basic steps. Gene delivery is initiated when a vector binds
to the host cell. The cell membrane constitutes the first barrier to gene delivery.
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Efforts are ongoing in our laboratory to create neurotrophic vectors that will
target specific cell types. Once past the cell membrane, a vector must provide
protection from degradation in the cytoplasm. If the vector enters through endo-
cytosis, lysosomal fusion may result in enzymatic degradation of the transgene.
An effective delivery method must either avoid entry into the cell via endocy-
tosis thus preventing lysosomal degradation, or allow entry into the cell via an
alternative pathway.
The next barrier to gene delivery is the nuclear membrane. In order for
most transgenes to be expressed, they must enter the nucleus. The nuclear
membrane serves as a relatively effective barrier against foreign entry into the
nucleus [69]. Early in the development of gene therapy strategies, entry into
the nucleus was limited to mitotically active host cells, and thus naturally
occurring breakdown of the nuclear membrane [70–72]. Recent advances have
involved the use of nuclear localization signals and viral vectors to overcome
this barrier.
The final step of gene delivery is expression of the transgene. Because
recovery from SCI will require significant amounts of time, gene-based
approaches to SCI require long-term gene expression. The duration of expres-
sion can vary depending on the vector being used, from transient to extended
expression. Long-term expression is usually accomplished through integration
of the transgene into the host DNA. Integration may be accomplished through
a variety of methods depending on the vector being used.
While an effective gene therapy delivery system is able to accomplish
these four basic steps, an ideal vector should not be a source of pathogenicity
to the host cell. Thus an ideal vector must be nontoxic and elicit little, if any,
immune response in the host. Vectors for gene therapy can be divided into two
main groups: viral and nonviral gene therapy. Here we will discuss the advances
that have been made in each category of gene therapy delivery.
Non-Viral Gene Therapy

Nonviral gene therapy poses several advantages over viral gene therapy.
The main advantage is the lack of pathogenicity of nonviral vectors. The sim-
plest method to provide a transgene for a host is the delivery of naked DNA.
In 1980, Capecchi [69] was able to successfully microinject DNA via glass
micropipettes directly into the nuclei of host cells in vitro, although DNA
expression could not be detected when DNA was injected into the cytoplasm.
These results emphasize the importance of overcoming the nuclear mem-
brane as a barrier to transgene delivery. In 1990, Wolff et al. [73] showed that
both DNA and RNA transgenes could be effectively expressed when injected
into mouse skeletal muscle. However, injection into other major organs, such
as the liver, spleen and brain, resulted in relatively inefficient transgene
expression. In order to increase the efficiency of gene delivery while retain-
ing the simplicity of naked DNA delivery, researchers have developed meth-
ods to augment transgene uptake into the host cell. One such method is
utilizing electropermeabilization to enhance the uptake of plasmid DNA.
Electrically mediated gene transfer has proven effective for gene delivery to
murine melanoma cells [74]. Lin et al. [75] designed an intrathecal electro-
poration probe to be used following intrathecal injection of plasmid DNA.
This device greatly enhanced transgene uptake into the spinal cord.
Unfortunately, expression of the transgene was transient, greatly diminishing
after 2 weeks.
To further increase the uptake of DNA in the host cell, researchers have
combined plasmid DNA with nonviral carriers. One method utilized by Yang
et al. [76] involved coating the transgene onto fine gold particles. In vivo par-
ticle bombardment was shown to be effective in a variety of major organs in
both rats and mice. Another method of delivery involved combining plasmid
DNA with cationic lipid-forming lipoplexes [77]. Variant forms of lipoplexes
can improve the efficiency of transfer. The addition of cationic polymers,
such as poly-L-lysine or protamine, to the DNA/liposome complex can
greatly enhance transgene delivery through a number of methods. The poly-
mers enhance lipoplex endocytosis, provide protection from nuclease activ-
ity and enhance transgene entry into the host cell nucleus [78]. Another
common addition to lipoplexes is dioleoylphosphatidylethanolamine.
Dioleoylphosphatidylethanolamine is a neutral lipid that is capable of desta-
bilizing the lysosomal membrane, permitting the release of the plasmid into
the host cell cytoplasm, thus reducing lysosomal degradation of the transgene
[79, 80].
An alternative to combining plasmid DNA with cationic lipids is the use
of cationic polymers. Cationic polymers have been found to be far more effec-
tive than their lipid counterparts at condensing DNA. One such polymer being
used is polyethylenimine. Polyethylenimine also acts as a proton sponge, which
causes osmotic disruption of the lysosome, rescuing the transgene from enzy-
matic degradation. Protection of the delivered gene allows for the greater trans-
fection efficiency, which has allowed for polyethylenimine to become one of
the most efficient synthetic delivery systems available [81].
Transposons have also emerged as an effective method of delivering plasmid
DNA into the host cell. Transposons are naturally occurring elements capable of
integrating foreign plasmids into the host cell DNA with the help of two
enzymes, integrase and transposase. Transposons have proven to be an extremely
effective transgene delivery system for their ability to integrate into the host
genome and allow for long-term expression. Transposons can also specifically
direct the site of transgene integration [82].
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Viral Vectors
Given the limitations present in using nonviral vectors, viral vectors have
emerged as the most efficient form of gene therapy delivery. Viral vectors pre-
sent a variety of advantages over nonviral vectors, including increased efficiency
of transfection as well as extended transgene expression. The drawbacks of using
viral vectors include the immune response induced as well as constraints on
transgene size. Nonetheless, the overall transfection efficiency of viral over non-
viral vectors has led researchers to develop novel viral vectors that minimize
their limitations while maintaining their effectiveness. Here, we will discuss the
four main types of viral vectors being used for gene therapy delivery.
Retroviral Vectors
Researchers have used retroviral vectors for the purpose of gene delivery
for a relatively long time. Retroviruses are advantageous for gene delivery
because they allow integration of the transgene into the host genome. This
allows for the expression of the transgene for the life of the host cell. Early retro-
viral studies were successful in using oncoretroviruses such as the Moloney
murine leukemia virus for in vivo transfection [83, 84]. The problem posed by
these early retroviral vectors was the inability to infect nondividing cells; a prob-
lem also faced when using nonviral vector delivery [85, 86]. Thus, focus has
turned to lentiviruses, a form of retroviruses that are able to infect nondividing
cells. Retroviruses replicate with the help of a preintegration complex that repli-
cates viral RNA through a DNA intermediate, which allows for integration into
the host genome. The preintegration complex in oncoretroviruses is believed to
be excluded by the nuclear membrane while the matrix protein in lentiviruses
contains a nuclear-targeting component which allows for transport of the trans-
gene into the host nucleus, explaining the ability of lentiviruses to infect nondi-
viding cells [87–89]. Human immunodeficiency virus type 1 (HIV-1) is a
well-known member of the lentivirus family, which was found to be able to
infect nondividing macrophages [90, 91]. HIV-based lentivirus vectors are capa-
ble of transfecting liver and muscle tissue, sustaining expression of the trans-
gene for over 6 months [92]. In order to increase the range of transfection, the
membranes of lentivirus vectors were modified to contain envelop proteins from
different viruses. One common virus whose envelope was used for this purpose
was the vesicular stomatitis virus G (VSV-G) [93].
An obvious concern in the use of HIV-1 as vehicle for gene therapy delivery
is the inadvertent infection of the host. This has led to the development of atten-
uated forms of the virus. Attenuation of lentiviruses can be accomplished by
eliminating accessory viral genes without hindering the transfection efficiency of
the vector. HIV requires several basic genes for function. In addition to structural
genes gag, pol, and env, HIV-1 genome contains two regulatory genes, tat and
rev, and four accessory genes. nef, vif, vpr, vpu [94]. Researchers have attempted
to create attenuated HIV vectors that can eliminate as many regulatory and acces-
sory genes as possible to allow for minimal pathogenicity while maintaining
transfection efficiency. Zufferey et al. [95] first introduced the attenuated HIV
vector by deleting accessory genes vif, vpr, vpu, and nef as well as the structural
gene env. They showed that this first generation-attenuated vector was able to
retain its transfection ability in nondividing cells. Further studies by Kim et al.
[96] demonstrated that the tat gene is not necessary for HIV-1 transfection in
nondividing cells in vitro. Tat is a strong transcriptional activator of HIV-1, but
Kim showed that this function might be replaced by inserting a human
cytomegalovirus promoter into the HIV genome. A third generation lentivirus
vector was created which, containing only the gag, pol, and env genes, was shown
to be successful in transfecting neurons in vivo [97].
In addition to these attenuated viruses, Zufferey et al. [98] also developed
self-inactivating lentiviruses through deletions in the 3⬘ long terminal repeats
of the HIV genome. Using self-inactivating viruses decreases the possibility of
recombination with wild-type virus, further rendering the vector safe for gene
delivery. An alternative to attenuation is the use of nonprimate lentiviruses
such as feline immunodeficiency virus. Feline immunodeficiency viruses are
unable to infect human cells, but when pseudotyped with VSV-G, transfection
of nondividing human cells is possible [99].
Herpes simplex Virus Vectors

Herpes simplex virus 1 (HSV-1) is a member of the human herpes viruses.
HSV-1 became an attractive vector for the delivery of therapeutic transgenes
for several reasons. Like lentivirus, HSV-1 is able to infect nondividing cells.
The HSV-1 genome is composed of 152-kb double-stranded DNA, which
allows the insertion of large transgenes and general ease of genetic manipula-
tion. Lastly, HSV-1’s most distinguishing characteristic as a viral vector is its
ability to establish latent infection in neurons [100].
Like lentivirus, HSV-1 must be attenuated to prevent viral replication in
the host. One method to accomplish this was the creation of defective viral vec-
tors. One example is amplicons, which contain the transgene to be delivered
flanked by viral recognition sequences. The absence of any genes encoding
viral proteins reduces the potential for an inflammatory response to these pro-
teins and prevents replication. However, in order for the transgene to be pack-
aged in an HSV-1 coat, the viral genes for replication and packaging must be
provided in trans through a helper virus. Thus HSV-1 vectors may be produced
by transfecting cells with amplicon and either cotransfecting with helper virus
DNA or superinfecting with HSV [101]. Geller and Breakefield [102] demon-
strated the use of such a defective HSV-1 vector to deliver the Escherichia coli
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lacZ gene to neurons in vitro. Originally, Geller used an HSV-1 temperature-
sensitive mutant, ts K, as the helper virus in HSV-1 vector production to avoid
cell damage. However, because ts K was later found to revert to wild type, an
HSV-1 deletion mutant which also effectively packages the amplicon was
substituted [103].
An alternative to amplicons is the creation of recombinant viruses. The
HSV-1 genome contains three main classes of genes; immediate early genes
(IE), early (E) and late (L) genes. Mutational analysis revealed that most of these
genes were nonessential for viral replication in cell cultures. The development of
HSV-1 deletion mutants has allowed the effective delivery of reporter genes into
postmitotic cells [104]. Research is continuing to define deletions capable of
further reducing the potential for wild-type reversion of HSV recombinants.
Adenovirus
Adenovirus holds several advantages over its viral counterparts as a trans-
gene delivery vehicle. Adenovirus is comprised of a 36-kb double-stranded
DNA genome, allowing for a large area of transgene manipulation [105].
Several generations of attenuated adenovirus have been created in an attempt to
decrease viral toxicity while maintaining efficiency of infectivity. First genera-
tion adenovirus was created by deleting the E1 gene, which is necessary for viral
gene expression and replication. These viruses were used to successfully deliver
the cystic fibrosis transmembrane conductance regulator gene into the lungs of
nonhuman primates [106]. The attenuated virus was able to induce transgene
expression, though only for a limited time. Transient gene expression is one of
the major obstacles to the application of adenovirus. The lack of extended
expression may be secondary to the immune response initiated by the low level
expression of the remaining viral genes. Such an immune response may precip-
itate the destruction of transfected cells eliminating transgene expression
[107–109]. Immunosuppression in parallel with adenoviral administration pro-
longs transgene expression [110]. An alternative approach was to completely
eliminate viral gene expression. Thus, subsequent generations of adenoviruses
were developed each with a larger deletion of viral genes. Second and third gen-
eration viruses include deletions in the E1, E2a, as well as the E4 genes. These
further-attenuated forms of adenovirus caused less inflammation and allow for
longer transgene expression [111–116].
The most advanced generation of adenoviral vectors involve removal of all
viral genes. These ‘gutless’ vectors lack all viral genes with the exception of the
inverted terminal repeats and packaging sequences required for inclusion into
the vector. The lack of viral genes allows insertion of transgenes up to 28 kb in
size. In order for this vector to be packaged, a helper virus is needed to provide
viral genes in trans. This helper virus lacks the inverted terminal repeats and
packaging sequences, thus inhibiting it from being packaged into the virus
particle [117, 118]. These gutless vectors have allowed for high levels of gene
expression with little immune response.
Adeno-Associated Virus
Adeno-associated virus (AAV) is a 4.7-kb single-stranded DNA virus.
Unlike the other systems discussed, AAV is not infectious to humans in its wild-
type form. In fact, wild-type AAV is naturally attenuated requiring a helper
virus to provide the necessary genes before initiating replication [119]. The
AAV vector itself contains no viral genes with the exception of the 125-bp AAV
terminal repeats flanking the transgene in question. In order for the vector to be
packaged into the AAV vector, viral genes rep and cap are provided in trans
through the use of a helper plasmid. This helper plasmid, much like the helper
plasmid used in gutted adenoviral production, contains the necessary viral
genes for replication and encapsidation, but lacks the terminal repeats neces-
sary to package the helper plasmid itself into the viral vector. AAV is capable
of gene delivery to terminally differentiated cells, with minimal inflammatory
response and resulting long-term gene expression.
More recent studies have been able to further reduce the potential for tox-
icity of AAV by applying the use of a truncated adenoviral genome rather than
a virus to supply the necessary helper genes. This eliminates the risk of contam-
ination of viral preparations by infectious helper virus [120]. Another advan-
tage of AAV is the ability to integrate into the host genome. AAV has been
shown to be capable of specific integration into chromosome 19. This capacity
may be responsible for the vector’s prolonged transgene expression [121].
Integration along with the lack of a significant immune response has allowed
for AAV-delivered transgene expression for up to 18 months [122]. Kaplitt et al.
[123] were the first to demonstrate the use of AAV for delivery of transgenes
into postmitotic cells in vivo. Much recent work in viral gene therapy has
focused on the use of AAV due to its nonpathogenic properties.
One of the major drawbacks of AAV is the limited genome size. The AAV
genome is not able to accommodate transgenes greater than 4.7 kb in size [124].
Recent work by Sun et al. [125] has provided a strategy to overcome this hand-
icap by utilizing the ability of AAV to heterodimerize. A large single transgene
is split and packaged into two separate AAV vectors and coinfected into the host
cell. Once inside the cell, heterodimerization occurs which allows for expression
of the original transgene.
Therapeutic Transgenes
The application of gene therapy to SCI depends on the existence of genes
capable of stimulating neuroprotection, remyelination, or regeneration. Because
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neurotrophic factors possess these properties, they have attracted the most
enthusiasm for application to SCI. A great deal of focus has surrounded mem-
bers of the ‘classic’ neurotrophin family, including nerve growth factor (NGF),
BDNF, and NT-3. Different neurotrophic factors show varying degrees of effec-
tiveness in promoting regeneration of the spinal cord. The difference in effec-
tiveness of these neurotrophic factors can be accounted for by the presence or
absence of receptors for these factors on varying types of neurons. For exam-
ple, in the adult rat lumbar dorsal root ganglia (DRG), receptors for NGF,
known as TrkA, are found predominantly in small, unmyelinated neurons, which
enter into the dorsal horn from the DRG. Oudega and Hagg [126] have shown
that continuous infusion of NGF into the spinal cord following peripheral nerve
transection and insertion of a peripheral nerve graft promoted re-entry of sen-
sory axons into the denervated dorsal columns. Further studies by Ramer et al.
[127] confirmed NGF’s ability to promote sensory axon growth into the spinal
cord. The axons responding to NGF are positive for calcitonin gene-related
peptide, a marker for small, umyelinated peptidergic axons. In contrast, TrkB
and TrkC receptors, which bind BDNF and NT-3 respectively, are found mainly
in DRG neurons possessing thick, myelinated axons [128], which form most of
the ascending fibers of the dorsal columns. The scarcity of TrkB receptors
within the dorsal horn may explain the failure of spinal BDNF administration
to promoted sensory axon growth into the spinal cord [127]. In contrast, BDNF
has been shown to promote motor axon growth, illustrating that different neu-
rotrophic factors exert their effects on different neuronal subtypes [129]. Unlike
BDNF, NT-3 has been shown to be able to promote sensory axon growth into
the spinal cord. Oudega and Hagg [130] showed that continuous infusion of
NT-3 into the spinal cord promotes regeneration of dorsal column sensory
axons into the spinal cord. In a separate experiment, NT-3 was infused into the
spinal cord at the site of a crush lesion. NT-3 was shown to stimulate axonal
regrowth in the region of the lesion and distally, without the use of a peripheral
nerve graft. NT-3 is also the only neurotrophic factor capable of promoting the
growth of corticospinal axons. Axonal sprouting has been observed following
a single injection [131].
Glia cell line-derived neurotrophic factor (GDNF) is a neurotrophic factor,
which belongs to the cytokine rather than the neurotrophin family. Once
thought to be specific for the protection of dopaminergic neurons, GDNF has
been proven effective in protecting from motor neuron death following axotomy
[132]. Although GDNF was not shown to have an effect on the regrowth of
lesioned dorsal column axons, it is more effective than NGF on stimulating
axonal growth into the spinal cord [127, 133].
In contrast to the growth-inducing abilities of neurotrophic factors,
anti-apoptotic proteins have been utilized in an effort to protect neuronal
degeneration following injury. The Bcl-2 family of proto-oncogenes is a group
of apoptosis-regulating proteins. Two proteins from this family, Bcl-2 and
Bcl-XL, play an anti-apoptotic role and are believed to exert their actions by
preventing the release of cytochrome C from the mitochondria, an important
step in the apoptosis pathway [134]. Overexpression of Bcl-2 into embryonic
sensory neurons was able to prevent death following deprivation of certain
growth factors, while Bcl-XL has been shown to prevent death in primary neu-
ronal cultures when delivered via adenoviral gene transfer [135, 136]. In addi-
tion, Bcl-xL has also been shown in vivo to prevent apoptotic death of
cholinergic neurons following axotomy [137]. Thus anti-apoptotic proteins have
become a reasonable addition to the library of therapeutic transgenes capable of
protecting neuron survival.
Viral Gene Delivery to the Spinal Cord

While direct spinal cord injection of viral vectors carrying therapeutic
transgenes is the most efficient method of introducing transgenes into the spinal
cord, several complications have arisen from this method of injection. Liu et al.
[138] attempted to inject recombinant first generation adenovirus expressing
the lacZ gene under control of the cytomegalovirus promoter directly into the
T7–8 levels of spinal cord in adult Sprague-Dawley rats. Transgene expression
was effective after one week, but quickly diminished thereafter, almost com-
pletely disappearing by 2 months postinjection. This down-regulation is most
likely due to the intense immune response elicited with the injection of adeno-
viral vectors. Our laboratory observed a cellular infiltrate in spinal cords 7 days
after adenoviral injection (fig. 4). Immunohistochemistry suggested that this
response was predominantly gliotic although a variety of mononuclear cells
were also observed. In animals immunsuppressed with cyclosporin A, ␤-gal
transgene staining remained robust up to 2 months postinjection. The immune
response can be partly attributed to a specific reaction in response to the early
generation adenovirus and partly attributed to the nonspecific immune reaction
in response to the trauma induced by spinal cord injection. The former can be
partly solved using the now available ‘gutless’ adenoviral vectors. Because
these vectors lack viral genes, the host cannot present the viral gene products
on major histocompatibility complexes. However, an immune response can still
be mounted against the viral capsid, which is itself immunogenic. In addition,
the application of gutless vectors does not eliminate the problem of the trauma
of direct injection.
The presence of a significant immune response to direct spinal cord injec-
tion and the potential trauma of this approach has spurred the search for alter-
natives. Direct intraparenchymal spinal cord and brain injection of early
generation adenoviral vectors results in a mononuclear inflammatory infiltrate,
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a b c
Fig. 4. Photomicrographs demonstrating local inflammatory response following ade-

noviral injection into the T-8 spinal cord. a At 7 days post-PBS (vehicle) injection, no infil-
trate surrounding the cannula tract (arrow) is seen. b At 7 days post-lacZ adenovirus
injection, inflammatory infiltrate is revealed (arrow). c At 7 days post-NGF adenovirus
injection, a mild inflammatory infiltrate (arrow) is found surrounding the cannula tract.
eliminating transgene expression [139, 140]. Researchers, including us, have

thus turned to peripheral injections as an alternative. In theory, this approach
should remove the viral capsid from the spinal cord, hence eliminating the
potential for damage through an immune response to the viral coat proteins.
This approach also avoids direct trauma to the spinal cord. Earlier studies have
shown that replication-defective adenoviruses have the ability to undergo retro-
grade transport following injection into the CNS [141, 142]. Kuo et al. [142]
used adenoviral vectors containing the lacZ gene under the control of the Rous
sarcoma virus promoter for retrograde axonal tracing studies. Kuo demon-
strated staining at the site of injection as well as several sites distal to the injec-
tion. These studies led our laboratory to evaluate the retrograde transport of
vectors injected into the PNS and its projection areas. Attenuated adenoviral
vectors with deletions in the E1a, E1b, and E3 viral genes expressing lacZ
under the Rous sarcoma virus promoter were injected into the sciatic nerve,
foot pad, and anterior tibialis muscle of adult rats. Histological examination of
the spinal cord revealed ␤-gal staining (transgene expression) occurring pre-
dominantly in neuronal cells with large cell bodies (fig. 5). Staining was also
present in the DRG. This phenomenon, which we call ‘remote delivery’, was
significantly greater in spinal cords following injection into the sciatic nerve in
comparison with foot pad and intramuscular injection [140]. These studies
demonstrated that vectors based on viruses that had not previously been con-
sidered neurotrophic were capable of penetrating the CNS from peripheral
inoculation sites.
Fig. 5. ␤-Galactosidase staining of motor neuron cell bodies from rat spinal cord.
Animals were injected into the sciatic nerve with adenovirus carrying a lacZ reporter gene
7 days prior to histology.
In order to confirm that retrograde axonal transport is responsible for the

remote delivery phenomenon, we studied the effects of intraneural colchicine
on adenoviral vector transport. Colchicine inhibits tubulin polymerization and
hence disrupts axonal transport. Intrasciatic colchicine injection inhibits
remote adenoviral and AAV gene delivery following sciatic injection in a
dose-dependant fashion implicating retrograde axonal transport in this
process [143].
Spinal cord gene expression following peripheral injections did not trigger
the inflammatory response observed following direct injections. Because the
termination of gene expression is linked to the inflammatory response, this dis-
covery led to the hope that remote delivery might prolong transgene expression.
Nonetheless, spinal cord transgene expression following remote delivery fol-
lowed the same time course as direct injection deteriorating within 3 weeks of
injection [140]. Both chronic dexamethasone and cyclosporine treatment stim-
ulated higher levels of gene expression in the lumbar spinal cord and DRG and
prolonged gene expression following remote adenoviral injection [144].
Nonetheless, no sign of cell death could be detected in parallel with the disap-
pearance of transgene expression. Together, these findings suggested that an
inflammatory response to the vector at the site of injection was shutting off
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gene expression through a promoter level noncytolytic mechanism. Because the
neurons of interest appeared to remain healthy after the disappearance of trans-
gene expression, we conducted repeated sciatic nerve injection of adenoviral
vectors in the hope of boosting gene expression. While repeated injection
resulted in prolonged gene expression at the site of injection, spinal cord gene
delivery was not boosted. Following initial injection, gene expression in the
nerve was found predominantly in the perineurium without a significant
inflammatory response at early timepoints. In contrast, gene expression at the
repeat-injection sites occurs within phagocytic infiltrative cells noted shortly
after injection. This immune response is likely to reduce the available titer of
vector for remote delivery and may inhibit axonal uptake.
Glatzel et al. [145] were also able to use adenovirus for the delivery of
transgenes into the spinal cord. Though they were unable to show spinal cord
expression of the transgene following intramuscular injection, they were able to
demonstrate staining following adenoviral sciatic nerve injection. In their exper-
iment, injection directly into the DRG resulted in a much higher efficiency of
gene transfer compared to sciatic nerve injections, without an increase in neuro-
logical side effects. In addition, they evaluated the role that the immune response
had on eliminating gene expression. Rag-1⫺/⫺ mice, which lack differentiated
B and T cells, were transfected with adenoviral vectors containing the lacZ trans-
gene. ␤-Gal expression could be seen for up to 102 days without any signs of
deterioration, which further confirms the role of the immune response as the
rate-limiting step in the duration of transgene expression.
Because AAV vectors induce prolonged gene expression with a minimal
inflammatory response, attention has turned to their application to spinal cord
gene transfer. AAV vectors were demonstrated to successfully allow for trans-
gene expression following direct injection into the cervical enlargement of
adult rat spinal cord [146]. Thus, attention turned to using AAV as a vector for
delivery given the lack of an immune response generated by administration of
AAV [124]. Glatzel injected recombinant AAV delivering enhanced green fluo-
rescent protein (EGFP) into the DRG of L4/L5. Expression of EGFP was
detectable up to 52 days postinjection without any signs of deterioration [145].
Our laboratory has also observed excellent transduction of cervical spinal cord
neurons following direct spinal cord injection (fig. 6). The next step with AAV,
as with adenovirus, was to utilize AAV’s property of retrograde transport to
allow for the remote delivery of transgene into the spinal cord following indi-
rect injection in the PNS. Kaspar et al. [147] have demonstrated retrograde
transport of AAV-delivered GFP from the hippocampus and striatum to the
entorhinal cortex and substantia nigra. Our laboratory has observed retrograde
delivery of AAV following peripheral nerve injection in mice at a variety of
locations [143].
Fig. 6. Motor neuron GFP expression 3 weeks after AAV.GFP injection of the cervical
spinal cord in SOD1 mutant mice.
Lentiviral vectors have also proven useful for transgene delivery to the
spinal cord. Mazarakis et al. [148] was able to demonstrate that equine infec-
tious anemia virus pseudotyped either with rabies-G envelope or VSV-G can
effectively deliver transgenes into the rat spinal cord following intraspinal
injection. Following injection, the spinal cord failed to show any significant cell
damage and only a mild degree of inflammation, confirming the low toxicity of
lentiviral vector delivery. Mazarakis also showed that rabies-G pseudotyped
lentivirus, but not VSV-G, is able to undergo retrograde transport following
injection into rat gastrocnemius muscle. Transgene expression was detected at
3 weeks following lentiviral delivery, but is believed to persist for much longer
time periods based on observations in other parts of the CNS. Prior to this
study, lentiviral vectors have not been shown to undergo retrograde transport.
This new capacity for retrograde transport is likely to be secondary to innate
properties of the rabies-G protein to convey axonal uptake and transport [149].
Our laboratory has achieved retrograde delivery to cervical spinal cord motor
neurons through injection into the brachial plexus using the rabies-G pseudo-
typed equine infectious anemia virus carrying a lacZ reporter gene. Spinal cord
stained 3 weeks following brachial plexus injection of the virus showed
transgene expression, confirming retrograde transport (fig. 7). The properties
of the rabies-G envelope combined with a low immune toxicity should provide
several new possibilities for using lentiviruses as potential vectors for transgene
delivery into the spinal cord. In addition to making ‘remote delivery’ possible,
direct injection of this vector into the spinal cord should make gene delivery to
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Fig. 7. ␤-Galactosidase staining of motor neuron cell bodies in the ventral horn of
spinal cord from mice injected with lacZ expressing rabies-G pseudotyped equine infectious
anemia virus. The mice were injected with 4 ⫻ 108 PFU titer of EIAV.RabG.lacZ into the
brachial plexus. The spinal cord was stained 3 weeks postinjection.
both the site of injection as well as upstream projection areas possible. This may
prove useful for trophic factor delivery to upper motor neurons projecting into
the site of injury.
Therapeutic Animal Models

Several animal models have been used to test the therapeutic potential of
the viral vectors discussed above. One animal model is the delivery of GDNF
into transgenic superoxide dismutase 1 (SOD1) mice. Transgenic SOD1 mice
contain a mutation in the Cu/Zn SOD1 gene on chromosome 21, mimicking a
form of amyotrophic lateral sclerosis (ALS) found in 20% of all ALS cases.
GDNF has been shown to demonstrate an overwhelmingly potent ability to pro-
tect motor neuron survival compared to other neurotrophic factors, and is thus
an ideal candidate for use in ALS animal models [132].
Acsadi et al. [150] delivered GDNF via intramuscular adenoviral vector
injection into SOD1 mice. This adenoviral vector contained the rat GDNF
cDNA under a cytomegalovirus promoter followed by an internal ribosomal
entry signal and an EGFP cDNA (AVR-GDNF). The virus was injected at a titer
of 5 ⫻ 109 plaque forming units (PFUs) into the anterior tibialis, gastrocnemius,
quadriceps, and paraspinal muscles of 5- to 7-day-old SOD1 mice. Control ani-
mals were injected with virus containing lacZ as the reported gene in order to
confirm uptake of the virus into the muscle and retrograde transport into the
spinal cord via ␤-gal staining. ELISA analysis was used to confirm the level of
GDNF in the muscles, measuring the GDNF expression at 3 months postinjec-
tion to be 454 ⫾ 268 pg/mg (mean ⫾ SE), and at 4 months postinjection to be
180 ⫾ 106 pg/mg. GDNF levels in untreated mice were at undetectable levels.
Importantly, injections in neonatal animals appeared to induce a longer lasting
period of gene expression. This effect may be secondary to limited immune
recognition in the neonate. SOD1 mice treated with AVR-GDNF showed a clear
delay in the development of ALS symptoms. Untreated mice developed symp-
toms (e.g., hind limb tremor, slowing of movements) at 106.2 ⫾ 2.71
(mean ⫾ SD) days of age compared to treated mice, which developed symptoms
at 116.1 ⫾ 8.6 days of age. Injection with AVR-GDNF also increased the life-
span of SOD1 mice following onset of symptoms by 8 days compared to
untreated SOD1 mice, increasing lifespan overall by an average of 14 days.
To quantitatively measure the effect of the disease on mice, the ability of
the mice to maintain their balance on a rotating rod (RotaRod) was measured.
RotaRod performance started to decline in SOD1 mice compared to wild-type
mice following 8 weeks of age. The study showed that SOD1 mice treated with
AVR-GDNF showed a significantly slower decline in performance compared to
untreated SOD1 mice. Finally, the effect of GDNF was demonstrated in motor
neuron counts of the spinal cord anterior horn 2, 3, and 4 months postinjection
compared to untreated SOD1 mice. AVR-GDNF demonstrated an ability to pro-
long large motor neuron (⬎20 ␮m) survival for up to 2 months, after which
motor neuron survival declined in a similar fashion to that found in untreated
SOD1 mice.
A similar study conducted by Manabe et al. [151] also delivered an adeno-
viral vector-containing GDNF into SOD1 mice. In this study, adenovirus at 108
PFU was injected into the gastrocnemius muscle of SOD1 mice, once a week
starting at 35 weeks of age. Quantitative measurements of disease included
evaluation of clinical grade, unilateral movement in a circular cage, and
RotaRod performance at 35, 40, 42, and 46 weeks of age. Although there was
not a significant difference between adenoviral vector-containing GDNF-
treated mice compared to untreated mice, there was a tendency of improvement
in the treated animals. In addition, large motor neuron survival was evaluated
via hematoxylin and eosin staining as well as immunohistochemistry for p-Akt
positive large motor neurons indicative of apoptotic death. Both means of
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evaluation showed significant preservation of motor neurons in adenoviral
vector-containing GDNF-treated SOD1 mice. The difference in the survival
outcome between these two studies may be derived from the age of the animal
at the time of treatment.
Wang et al. [152] were able to demonstrate the neuroprotective effects of
AAV vector GDNF gene delivery. Once again, SOD1 mice were used as animal
models. In order to differentiate GDNF that is transgenically expressed from
those being endogenously expressed, an AAV vector containing a transgene
expressing a GDNF-FLAG fusion protein was developed. GDNF-FLAG can be
easily determined via immunofluorescence staining with polyclonal rabbit
anti-FLAG antibodies. The AAV-GDNF was injected into the gastrocnemius
and triceps brachii muscles of all the four limbs of SOD1 mice at 9 weeks of
age. At 110 days of age (7 weeks postinjection), GDNF levels in AAV-GDNF-
treated mice was 7,985.0 ⫾ 874.0 pg/mg, an increase of ⬎120-fold compared to
untreated mice. AAV-GDNF-treated mice also showed considerable preservation
of the gastrocnemius muscle, showing little evidence of neutrogenic atrophy and
weighing nearly one and a half times more than untreated mice. Retrograde
transport of GDNF was demonstrated via FLAG staining of the spinal cord.
Nissl staining of the motor neurons in the lumbar spinal cord showed significant
protective effects of AAV-GDNF in the side of the cord ipsilateral to AAV-GDNF
injection. Finally, AAV-GDNF demonstrated similar effects as adenovirus-
delivered GDNF on RotaRod testing. Performance on the RotaRod deteriorated
after 12 weeks of age in SOD1 mice compared to wild-type mice. AAV-GDNF
was able to delay the onset of motor deficits as well as slow the deterioration of
performance on the RotaRod. The onset of motor deficit in AAV-GDNF treated
mice was 114.0 ⫾ 4.0 days compared to 101.3 ⫾ 5.4 days in untreated mice.
Lifespan in treated mice was increased by a mean of 16.6 ⫾ 4.1 days; an
improvement remarkably similar to the one found in AVR-GDNF-treated SOD1
mice in the study conducted by Acsadi et al. [150]. Despite the delay in the onset
of symptoms and increase in lifespan, AAV-GDNF-treated mice showed no
difference in the duration of disease when compared to untreated mice. The sig-
nificantly higher level of expression of GDNF in these experiments compared to
those found in the AVR-GDNF experiments lends a great deal of promise to the
use of AAV as an ideal vector for delivery of therapeutic transgenes. Further
experiments are necessary to test whether administering the transgene at an
earlier age can affect the duration of disease [152].
GDNF’s ability to protect motor neuron following axotomy was also illus-
trated by Baumgartner and Shine [153]. Adenoviral vectors were created con-
taining expression cassettes for BDNF, GDNF, NGF, or ciliary neurotrophic
factor. Adenoviral vectors were injected into the gastrocnemius, flexor longus
digitorum, and tibialis PFU titer. Adenoviruses carrying a lacZ transgene and
adenovirus void of the transgene, as well as a virus-free vehicle, were injected
into the same hind limb muscles as controls. Retrograde transport of virally
delivered transgenes was confirmed with ␤-gal staining in the lumbar spinal cord
motor neurons. In order to study the neuroprotective effects of the growth factors,
2 days following injection the sciatic nerve was severed. Seven days postaxo-
tomy, the lumbar spinal cord segments were removed and it was found that only
animals treated with GNDF-containing adenovirus showed a significant differ-
ence in preserving neuron survival when compared to empty adenovirus or
vehicle-treated animals. At 2 days postaxotomy, animals treated with GDNF
showed preservation of 70% of its motor neurons compared to 44% seen in vehi-
cle controls. The neuroprotective effect of GDNF proved to be transient, show-
ing no difference from control animals at 42 days postaxotomy. These results
further demonstrate the potent ability of GDNF in neuroprotection as well as
setting the stage for the use of neurotrophic factors for protection following SCI.
Smith and his colleagues [154] have been able to utilize adenoviral vectors
for the delivery of neurotrophic factors to induce functional recovery of axons
into the dorsal root entry zone. Recombinant adenoviral vectors were created
containing transgenes encoding for FGF2, NGF, L1 cell adhesion molecule, or
␤-galactosidase (LacZ). Sprague-Dawley rats were treated with triple crush
lesions at the L4 and L5 dorsal roots. Under natural conditions, peripheral
nerve regeneration is halted at the dorsal root entry zone, the CNS border. In
rats injected with a 7.5 ⫻ 106 PFU/␮l titre of adenovirus carrying either NGF
or FGF2 16 days following rhizotomy, large numbers of calcitonin gene-related
peptide-positive axon fibers can be seen growing into the dorsal horn compared
to uninfected rats. In addition to histological analysis, Smith was able to
demonstrate functional recovery following NGF or FGF2 administration. Rats
treated with NGF or FGF2 showed recovery of nociception as evaluated using
a plantar heat test. Furthermore, recovery of proprioception was evaluated
using a grid runway test. None of the neurotrophic factors administered was
able to induce any recovery of proprioception, indicating specific targeting by
NGF and FGF2.
Ex vivo Gene Transfer

The use of cell grafts that express a therapeutic transgene is an alternative
to in vivo gene transfer. This method, known as ex vivo gene transfer, involves
genetically modifying cells in vitro to express a gene of interest, and then trans-
planting the cell graft into the host. One advantage of this method is the poten-
tial to verify transgene expression in the desired cells before transplantation
into the host. Another major advantage is the ability of the transplanted cell to
produce a long-term steady-state therapeutic level of transgene, a problem
commonly encountered with the use of viral or nonviral vectors (fig. 8).
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Retroviral vector Retroviral vector
Fibroblast
Spinal cord
NGF NGF
Spinal cord
NGF
Spinal cord
Fig. 8.a Ex vivo gene transfer. Following in vitro infection with a retroviral (or an
alternative virus) vector carrying a therapeutic transgene, the fibroblasts are grown in cul-
ture. Verification of the gene expression can be confirmed before transplantation of the
fibroblast into animal spinal cords. b In vivo gene transfer. Retroviral vectors carrying the
therapeutic transgene are directly injected into the spinal cord of animal models.
The first consideration in ex vivo gene transfer is the type of cell to be used
for grafting. Ease of infection in vitro and the viability of the cells themselves, as
well as their effects on the viability of the cells around them once grafted into the
host, are all factors that can mitigate the choice of cell type. For these reasons,
primary fibroblasts have been a popular choice for ex vivo experiments. Not only
are fibroblasts easily sustained in vitro and well accepted into the CNS, fibroblasts
naturally secrete collagen and fibronectin, which provide a conducive environment
for neurite growth. Also, since fibroblasts are nonneuronal and nonglial in origin,
they are void of any growth-inhibiting molecules that may prohibit neuronal
growth. The earliest studies using fibroblasts to provide therapeutic transgenes to
the CNS came from Rosenberg et al. [155], who were able to modify fibroblasts
by infecting them with mouse NGF cDNA via a retroviral vector. The cells were
then grafted into the brain following surgical lesion between the fimbria and
the fornix. Two weeks after graft implantation, the animals were sacrificed and
stained for choline acetyltransferase, an indication of survival of cholinergic cell
bodies. It was found that animals receiving the NGF-secreting cell graft preserved
92% of their cholinergic cells compared to only 49% in control animals. These
experiments set the stage for using fibroblasts for ex vivo gene transfer.
Fibroblasts producing NGF have been transplanted into the spinal cord in
an attempt to induce neuronal growth. Such cell grafts were found to be viable
and producing NGF for up to one year following transplantation [156]. The
grafts heavily induced growth of sensory axons, verified by calcitonin gene-
related peptide staining, proving the grafts’ ability to induce growth of a spe-
cific axonal type. The ability of fibroblast grafts to induce axonal growth
following acute SCI was also observed. NGF-producing fibroblasts were
implanted into the spinal cord following spinal dorsal hemisection lesions. The
cell grafts were able to induce growth of not only sensory axons, but also of
motor axons, albeit to a lesser extent [157]. The injury-evoked responsiveness
to NGF provides important insight into the selective use of NGF as a neu-
rotrophic factor in SCI. Similar results were obtained in studies in which NGF
grafts were implanted 1–3 months following spinal cord lesion to study the
effects on the chronically injured spinal cord. While sensory fibers were noted
to regenerate, no motor neuron response was observed in the chronically
injured spinal cord. Following SCI neurotrophin receptors are hypothesized to
increase [158]. This upregulation is most likely transient, explaining the lack of
motor neuron response following chronic injury [159].
The above experiments were carried out using early generation retrovirus
to genetically modify the cell grafts. Retroviral vectors can be effective because
they integrate into the host genome and allow for long-term gene expression.
Alternative vectors have also been utilized and proved to be successful.
Lentiviral vectors, retroviral vectors capable of gene transfer to terminally dif-
ferentiated cells, have been successfully used to modify fibroblasts [160, 161].
Liu et al. [162] were also able to use adenoviral vectors to modify fibroblasts.
Though ex vivo neurotrophic gene transfer to fibroblasts has shown
promise, the use of alternative cell lines as well as other neurotrophins are also
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capable of inducing axonal growth in the CNS. Because of their possible advan-
tage for autologous grafting, skin fibroblasts have been attempted for use as cell
grafts. Early attempts at using skin fibroblasts were unsuccessful in producing
prolonged transgene expression [163]. OECs have also been used as implants
for ex vivo gene transfer. Primary OECs have been modified with both adeno-
viral and lentiviral vectors, with grafts allowing for transgene expression for up
to one and 4 months respectively [164]. Cell grafts that have been modified to
produce NT-3 have also been shown to promote corticospinal axon growth
when grafted into the spinal cord following hemisection lesion [165].
Conclusion
Despite many years of research in the field of SCI and regeneration, the
prognosis for those who have sustained an SCI remains dismal. Few therapeutic
strategies are available to the clinician. The barriers to effective neural regener-
ation, and hence functional recovery, are multifactorial. These barriers include
the lack of an intrinsic cellular response to divide and regenerate, the need to
overcome inhibitory barriers at the injury site, and the need to recapitulate the
native circuitry following injury.
Advances in both genetic and cellular therapy for SCI have begun to
unravel some of the difficulties with functional neuronal recovery. Stem cells,
both exogenous and endogenous, have the capability to differentiate into vari-
ous CNS lineages. These cells may, therefore, provide neurogenesis, neuropro-
tection, trophic support, and/or remyelination. Furthermore, they may provide
effective tissue bridges for regenerating axons. Stem cell therapy may supplant
existing tissue or cell transplant paradigms and their inherent shortcomings. The
fields of molecular neurobiology and genetic therapy are rapidly advancing.
This therapeutic strategy will grant clinicians the ability to alter the function of
intrinsic or extrinsically placed cells. These cells may then produce growth fac-
tors or other growth-permissive and neural-protective proteins capable of sup-
porting the injured spinal cord. These therapies may be delivered systemically
without the need to access the CNS surgically.
Despite the grim prognosis of SCI, cellular and genetic therapies continue
to provide the hope of recovery following an SCI. In experimental animal mod-
els of SCI, both are providing evidence of functional recovery. This work lays
the groundwork for human clinical trials.
References
1 Burney R, Maio R, Maynard F: Incidence, characteristics, and outcome of spinal cord injury at
trauma centers in North America. Arch Surg 1992;128:569–599.
2 Tator C: Epidemiology and general characteristics of the spinal cord injury patient; in Contempory
Management of Spinal Cord Injury. Illinois, American Association of Neurological Surgeons,
1995, pp 9–13.
3 Kraus J: Injury to the head and spinal cord: The epidemiological relevance of the medical litera-
ture published from 1960 to 1978. J Neurosurg 1980;53(suppl):S3–S10.
4 Tator C, Duncan E, Edmonds V: Complications and costs of management of acute spinal cord
injury. Paraplegia 1993;31:700–714.
5 Stripling T: The cost of economic consequences of traumatic spinal cord injury. Paraplegia News
1990;August:50–54.
6 Kwon D, Oxland T, Tetzlaff W: Animal models used in spinal cord regeneration research. Spine
2002;27:1504–1510.
7 Gage F: Mammalian neural stem cells. Science 2000;287:1433–1438.
8 Brustle I, Jones K, Learisk R, Karram K, Choudhary K, Wiestler O, Duncan I, McKay R:
Embryonic stem cell-derived glial precursors: A source of myelinating transplants. Science 1999;
285:754–756.
9 Flax J, Aurora S, Yang C, Simonin C, Wills A, Billinghurst L, Jendoubi M, Sidman R, Wolfe J,
Kim S, Snyder E: Engraftable human neural stem cells respond to developmental cues, replace
neurons, and express foreign genes. Nat Biotechnol 1998;16:1033–1039.
10 McKay R: Stem cells in the central nervous system. Science 1997;276:66–71.
11 Mujtaba T, Piper D, Kalyani A, Groves A, Lucero M, Rao M: Lineage-restricted neural precursors
can be isolated from both the mouse neural tube and cultured ES cells. Dev Biol 1999;214:113–127.
12 Reubinoff B, Itsykson P, Turetsky T, Pera M, Reinhartz E, Itzik A, Ben-Hur T: Progenitors from
human embryonic stem cells. Nat Biotechnol 2001;19: 1134–1140.
13 Zhang S, Wernig M, Duncan I, Brustle O, Thomson J: In vitro differentiation of transplantable
neural precursors from human embryonic stem cells. Nat Biotechnol 2002;19:1129–1133.
14 Cao Q, Benton R, Whittemore S: Stem cell repair of central nervous system injury. J Neurosci Res
2002;68:501–510.
15 Sanchez-Ramos J: Neural cells derived from adult bone marrow and umbilical cord blood.
J Neurosci Res 2002;69:880–893.
16 Penn M, Francis G, Ellis S, Young J, McCarthy P, Topol E: Autologous cell transplantation for the
treatment of damaged myocardium. Prog Cardiovasc Dis 2002;45:21–32.
17 Brustle I, Choudhary K, Karrma K, Huttner A, Murray K, Dubois-Dalcq M: Chimeric brains
generated by intraventricular transplantation of fetal human brain cells into embryonic rats. Nat
Biotechnol 1998;16:1040–1044.
18 Rosser A, Tyerrs P, Dunnett S: The morphological development of neurons derived from EGF and
FGF-2 driven human CNS precursors depends on their site of integration in the neonatal rat brain.
Eur J Neurosci 2000;12:2405–2413.
19 Cao Q, Zhang Y, Howard R, Walters W, Tsoulfas P, Whittemore S: Pluripotent stem cells engrafted
into the normal or lesioned adult rat spinal cord are restricted to a glial lineage. Exp Neurol
2001;167:48–58.
20 Chow S, Moul J, Tobias C, Himes B, Liu Y, Obrocka M, Hodge L, Tessler A, Gischer I:
Characterization and intraspinal grafting of EGF/bFGF-dependent neurospheres derived from
embryonic rat spinal cord. Brain Res 2000;874:87–106.
21 White P, Morrison S, Orimoto K, Kubu C, Verdi J, Anderson D: Neural crest stem cells undergo
cell-intrinsic developmental changes in sensitivity to instructive differentiation signals. Neuron
2001;29:57–71.
22 Rao M, Mayer-Proschel M: Precursor cells for transplantation. Prog Brain Res 2000;128:273–292.
23 Anderson D: Stem cells and pattern formation in the nervous system: The possible versus the
actual. Neuron 2001;30:19–35.
24 Yang H, Mtjtaba T, Benkatraman G, Wu Y, Rao M, Luskin M: Region-specific differentiation of
neural tube derived neuronal restricted progenitor cells after heterotopic transplantation. Proc Natl
Acad Sci USA 2000;97:13366–13371.
25 Zigova T, Pencea V, Betarbert R, Wiegand S, Alexander C, Bakay RA, Luskin MB: Neuronal prog-
enitor cells of the neonatal subventricular zone differentiate and disperse following transplanation
into the adult rat striatum. Cell Transplant 1998;7:137–156.
medwedi.ru
26 McDonald J, Liu X, Qu Y, Liu S, Mickley S, Turetsky D, Gottlieb D: Transplanted embryonic stem
cells survive, differentiate and promote recovery in injured rat spinal cord. Nat Med 1999;
5:1410–1412.
27 Bain G, Kitchen D, Yao M, Huettner J, Gottlieb D: Embryonic stem cells express neuronal
properties in vitro. Dev Biol 1995;168: 342–357.
28 Gimenez Y, Ribotta M, Feraboli-Lohnherr O, Privat A: Recovery of locomotion following
transplantation of monoaminergic neurons in the spinal cord of paraplegic rats. Ann NY Acad Sci
1998;860:393–411.
29 Eriksson P, Perfilieva E, Bjork-Eriksson T, Alborn A, Nordborg C, Peterson D, Gage F: Neurogenesis
in the adult human hippocampus. Nat Med 1998;4:1313–1317.
30 Gould E, Tanapat P: Stress and hippocampal neurogenesis. Biol Psychiatry 1999;46:1472–1479.
31 Lois C, Alvarez-Buylla A: Proliferating subventricular zone cells in the adult mammalian fore-
brain can differentiate into neurons and glia. Proc Natl Acad Sci USA 1993;90:2074–2077.
32 Rietze R, Poulin P, Weiss S: Mitotically active cells that generate neurons and astrocytes are
present in multiple regions of adult mouse hippocampus. J Comp Neurol 2000;424:397–408.
33 Temple S, Alvarez-Buylla A: Stem cells in the adult mammalian central nervous system. Curr
Opin Neurobiol 1999;9:135–141.
34 Johansson C, Momma S, Clarke K, Risling M, Lendahl U, Friesen J: Identification of a neural
stem cell in the adult mammalian central nervous system. Cell 1999;96:25–34.
35 Horner P, Power AE, Kempermann G, Kuhn H, Palmer T, Winkler J, Thal L, Gage F: Proliferation
and differentiation of progenitor cells throughout the intact adult rat spinal cord. J Neurosci 2000;20:
2218–2228.
36 Levine J, Reynolds R, Fawcett J: The oligodendrocyte precursor cell in health and disease. Trends
Neurosci 2001;24:39–47.
37 Keirstead H, Levine J, Blakemore W: Response of the oligodendrocyte progenitor cell population
(defined by NG2 labelling) to demyelination of the adult spinal cord. Glia 1998;22:161–170.
38 Levine J, Reynolds R: Activation and proliferation of endogenous oligodendrocyte precursor cells
during ethidium bromide-induced demyelination. Exp Neurol 1999;160:333–347.
39 McTigue D, Wei P, Stokes B: Proliferation of NG2-positive cells and altered oligodendrocyte
numbers in the contused rat spinal cord. J Neurosci 2001;21:3392–3400.
40 Zhang S, Lipsitz D, Duncan I: Self-renewing canine oligodendroglial progenitor expanded as
oligospheres. J Neurosci Res 1998;54:181–190.
41 Herrera J, Yang H, Zang S, Proschel C, Tresco P, Duncan I, Luskin M, Mayer-Proschel M:
Embryonic-derived glial-restricted precursor cells (GRP cells) can differentiate into astrocytes
and oligodendrocytes in vivo. Exp Neurol 2001;171:11–21.
42 Akiyama Y, Honmou O, Kato T, Uede T, Hashi K, Kocsis J: Transplantation of clonal neural pre-
cursor cells derived from adult human brain establishes functional peripheral myelin in the rat
spinal cord. Exp Neurol 2001;167:27–39.
43 Howland D, Bregman B, Tessler A, Goldberger M: Transplants enhance locomotion in neonatal
kittens whose spinal cords are transected: A behavioral and anatomical study. Exp Neurol 1995;
135:123–145.
44 Miya D, Giszter S, Mori F, Adipudi V, Tessler A, Murrary M: Fetal transplants alter the develop-
ment of function after spinal cord transection in newborn rats. J Neurosci 1997;17:4865–4872.
45 Bregman B, Diener P, McAtee M, Dai H, James C: Intervention strategies to enhance anatomical
plasticity and recovery of function after spinal cord injury. Adv Neurol 1997;72:257–275.
46 Bregman B, Kunkel-Bagden E, McAtee M, O’Neill A: Extension of the critical period for devel-
opmental plasticity of the corticospinal pathway. J Comp Neurol 1989;282:355–370.
47 Bregman B, McAtee M, Dai H, Kuhn P: Neurotrophic factors increase axonal growth after
spinal cord injury and transplantation in the adult rat. Exp Neurol 1997;148:475–494.
48 Jakeman L, Reier P: Axonal projections between fetal spinal cord transplants and the adult rat spinal
cord: A neuroanatomical tracing study of local interactions. J Comp Neurol 1991;307:311–334.
49 Bregman B, Broude E, McAtee M, Kelly M: Transplants and neurotrophic factors prevent atrophy
of mature CNS neurons after spinal cord injury. Exp Neurol 1998;149:13–27.
50 Coumans J, Lin T, Dai H, MacArthur L, McAtee M, Nash C, Bregman B: Axonal regeneration and
functional recovery after complete spinal cord transection in rats by delayed treatment with trans-
plants and neurotrophins. J Neurosci 2001;21:9334–9344.
51 Barnett S, Hutchins A, Noble M: Purification of olfactory nerve ensheathing cells from olfactory
bulb. Dev Biol 1993;155:337–350.
52 Raisman G: Specialized neuroglial arrangement may explain the capacity of vomeronasal axons
to reinnervate central neurons. Neuroscience 1995;14:237–254.
53 Ramon-Cueto A, Nieto-Sampedro M: Glial cells from adult rat olfactory bulb: Immunocytochemical
properties of pure cultures of ensheathing cells. Neuroscience 1992;47:213–220.
54 Valverde F, Lopez-Mascarauque L: Neuroglial arrangements in the olfactory glomeruli of the
hedgehog. J Comp Neurol 1991;307:658–674.
55 Ramon-Cueto A, Nieto-Sampedro M: Regeneration into the spinal cord of transected dorsal root
axons is promoted by enhancing glia transplants. Exp Neurol 1994;127:232–244.
56 Ramon-Cueto A, Plant G, Avila J, Bunge M: Long-distance axonal regeneration in the transected adult
rat spinal cord is promoted by olfactory ensheathing glia transplants. J Neurosci 1998;18:3803–3815.
57 Li Y, Raisman G: Integration of transplanted cultured Schwann cells into the long myelinated fiber
tracts of the adult spinal cord. Exp Neurol 1997;145:397–411.
58 Brook G, Lawrence J, Raisman G: Morphology and migration of cultured Schwann cells trans-
planted into the fimbria and hippocampus in adult rats. Glia 1993;9:292–304.
59 Li Y, Raisman G: Schwann cells induce sprouting in motor and sensory axons in the adult rat
spinal cord. J Neurosci 1994;14:4050–4063.
60 Takami T, Oudega M, Bates M, Wood P, Kleitman N, Bunge M: Schwann cell but not olfactory
ensheathing glia transplants improve hindlimb locomotor performance in the moderately contused
adult rat thoracic spinal cord. J Neurosci 2002;22:6670–6681.
61 Bamber N, Li H, Lu X, Oudega M, Aebisher P, Xu X: Neurotrophins BDNF and NT-3 promote
axonal re-entry into the distal host spinal cord through Schwann cell-seeded mini-channels. Eur J
Neurosci 2001;13:257–268.
62 Rapalino O, Lazarov-Spiegler O, Agranov E, Velan G, Yoles E, Fraidakis M, Solomon A,
Gepstein R, Katz A, Belkin M, Hadani M, Schwarta M: Implantation of stimulated homologous
macrophages results in partial recovery of paraplegic rats. Nat Med 1998;4:814–821.
63 Lazarov-Spiegler O, Rapalino O, Agranov G, Schwartz M: Restricted inflammatory response in
the CNS: A key impediment to axonal regeneration? Mol Med Today 1998;4:337–342.
64 Lazarov-Spiegler O, Solomon A, Hirschberg D, Levine V, Schwartz M: Transplantation of activated
macrophages overcomes central nervous system regrowth failure. FASEB J 1996;10:1296–1302.
65 Schwartz M, Hirschberg D, Beserman P: Central nervous system regeneration and the immune
system. Mol Med Today 1995;1:60–61.
66 Faber-Elman A, Levine V, Schvartz I, Shaltiel S, Schwartz M: Vitronectin overrides a negative
effect of TNF-alpha on astrocytes migration. FASEB J 1995;9:1605–1613.
67 Faber-Elman A, Solomon A, Abraham J, Marikovsky M, Schwartz M: Involvement of wound-
associated factors in rat brain astrocytes migratory response taxonal injury: In vitro stimulation.
J Clin Invest 1996;97:162–171.
68 Mckeon R, Hoke A, Silver J: Injury-induced proteoglycans inhibit the potential for laminin-mediated
axon growth on astrocytic scars. Exp Neurol 1995;136:32–43.
69 Capecchi M: High efficiency transformation by direct microinjection of DNA into cultured
mammalian cells. Cell 1980;22:479–488.
70 Cartier R, Reszka R: Utilization of synthetic peptides containing nuclear localization signals for
nonviral gene transfer systems. Gene Ther 2002;9:157–167.
71 Tseng W, Haselton F, Giorgio T: Mitosis enhances transgene expression of plasmid delivered by
cationic liposomes. Biochim Biophys Acta 1999;1445:53–64.
72 Wilke M, Fortunati E, van den Broek M, Hoogeveen A, Scholte B: Efficacy of a peptide-based
gene delivery system depends on mitotic activity. Gene Ther 1996;3:1133–1142.
73 Wolff J, Malone R, Williams P, Chong W, Acsadi G, Jani A, Felgner P: Direct gene transfer into
mouse muscle in vivo. Science 1990;247:1465–1468.
74 Rols M, Teissie J: Electropermeabilization of mammalian cells to macromolecules: Control by
pulse duration. Biophys J 1998;75:1415–1423.
75 Lin C, Tai M, Cheng J, Chou A, Wang J, Tan P, Marsala M, Yang L: Electroporation for direct
spinal gene transfer in rats. Neurosci Lett 2002;317:1–4.
76 Yang N, Burkholder J, Roberts B, Martinell B, McCabe D: In vivo and in vitro gene transfer to
mammalian somatic cells by particle bombardment. Proc Natl Acad Sci USA 1990;87:9568–9572.
medwedi.ru
77 Felgner P, Gadek T, Holm M, Roman R, Chan H, Wenz M, Northrop J, Ringold G, Danielson M:
Lipofection: A highly efficient, lipid-mediated DNA-transfection procedure. Proc Natl Acad Sci
USA 1987;84:7413–7417.
78 Gao X, Huang L: Potentiation of cationic liposome-mediated gene delivery by polycations.
Biochemistry 1996;35:1027–1036.
79 Farhood H, Gao X, Son K, Yang Y, Lazo J, Huang L, Barsoum J, Bottega R, Epand R: Cationic
liposomes for direct gene transfer in therapy of cancer and other diseases. Ann NY Acad Sci 1994;
716:23–34.
80 Hui S, Langner M, Zhao Y, Ross P, Hurley E, Chan K: The role of helper lipids in cationic
liposome-mediated gene transfer. Biophys J 1996; 71:590–599.
81 Boussif O, Lezoualc’h F, Zanta M, Mergny M, Scherman D, Demeneix B, Behr J: A versatile
vector for gene and oligonucleotide transfer into cells in culture and in vivo: Polyethylenimine.
Proc Natl Acad Sci USA 1995;92:7297–7301.
82 Kaminski J, Huber M, Summers J, Ward M: Design of a nonviral vector for site-selective, efficient
integration into the human genome. FASEB J 2002;16:1242–1247.
83 Guild B, Finer M, Housman D, Mulligan R: Development of retrovirus vectors useful for expressing
genes in cultured murine embryonal cells and hematopoietic cells in vivo. J Virol 1988;62:3795–3801.
84 Miller A, Skotzko M, Rhoades K, Belldegrun A, Tso C, Kaboo R, McBride W, Jacobs E, Kohn D,
Moen R: Simultaneous use of two retroviral vectors in human gene marking trials: Feasibility and
potential applications. Hum Gene Ther 1992;3:619–624.
85 Miller D, Adam M, Miller A: Gene transfer by retrovirus vectors occurs only in cells that are
actively replicating at the time of infection. Mol Cell Biol 1990;10:4239–4242.
86 Roe T, Reynolds T, Yu G: Integration of murine leukemia virus DNA depends on mitosis. EMBO J
1993;12:2099–2108.
87 Bukrinsky M, Sharova N, Dempsey M, Stanwick T, Bukrinskaya A, Haggerty S, Stevenson M:
Active nuclear import of human immunodeficiency virus type 1 preintegration complexes. Proc
Natl Acad Sci USA 1992;89:6580–6584.
88 Bukrinsky M, Haggerty S, Dempsey M, Sharova N, Adzhubel A, Spitz L, Lewis P, Goldfarb D,
Emerma M, Stevenson M: A nuclear localization signal within HIV-1 matrix protein that governs
infection of non-dividing cells. Nature 1994;369:107–108.
89 Gallay P, Stitt V, Mundy C, Oettinger M, Trono D: Role of the karyopherin pathway in human
immunodeficiency virus type 1 nuclear import. J Virol 1996;70:1027–1032.
90 Lewis P, Hensel M, Emerma M: Human immunodeficiency virus infection of cells arrested in the
cell cycle. EMBO J 1992;11:3053–3058.
91 Lewis P, Emerman M: Passage through mitosis is required for oncoretroviruses but not for the
human immunodeficieny virus. J Virol 1994;68:510–516.
92 Kafri T, Blomer U, Peterson D, Gage F, Verma I: Sustained expression of genes delivered directly
into liver and muscle by lentiviral vectors. Nat Genet 1997;17:314–317.
93 Naldini L, Blomer U, Gallay P, Ory D, Mulligan R, Gage F, Verma I, Trono D: In vivo gene delivery
and stable transduction of nondividing cells by a lentiviral vector. Science 1996;272:263–267.
94 Trono D: HIV accessory proteins: Leading roles for the supporting cast. J Virol 1995;82:189–192.
95 Zufferey R, Nagy D, Mandel R, Naldini L, Trono D: Multiply attenuated lentiviral vector achieves
efficient gene delivery in vivo. Nat Biotechnol 1997;15:871–875.
96 Kim V, Mitrophanous K, Kingsman S, Kingsman A: Minimal requirement for a lentivirus vector
based on human immunodeficiency virus type 1. J Virol 1998;72:811–816.
97 Dull T, Zufferey R, Kelly M, Mandel R, Nguyen M, Trono D, Naldini, L: A third-generation
lentivirus vector with a conditional packaging system. J Virol 1998;72:8463–8471.
98 Zufferey R, Dull T, Mandel R, Bukovsky A, Quiroz D, Naldini L, Trono D: Self-inactivating
lentivirus vector for safe and efficient in vivo gene delivery. J Virol 1998;72:9873–9880.
99 Poeschla E, Won-Staal F, Looney D: Efficient transduction of nondividing human cells by feline
immunodeficieny virus lentiviral vectors. Nat Med 1998;4:354–357.
100 Stevens J: Latent herpes simplex virus and the nervous system. Curr Top Microbiol Immunol
1975;70:31–50.
101 Kaplitt M, Pfaff D: Viral vectors for gene delivery and expression in the CNS. Methods 1996;
10:343–350.
102 Geller A, Breakefield X: A defective HSV-1 vector expresses Escherichia coli beta-galactosidase
in cultured peripheral neurons. Science 1988;241:1667–1669.
103 Geller A, Keyomarsi K, Bryan J, Pardee A: An efficient deletion mutant packaging system for
defective herpes simplex virus vectors: Potential applications to human gene therapy and neuronal
physiology. Procl Natl Acad Sci USA 1990;87:8950–8954.
104 Glorioso J, Goins W, Meaney C, Fink D, DeLuca N: Gene transfer to brain using herpes simplex
virus vectors. Ann Neurol 1994; 35(suppl):S28–S34.
105 Shenk T: Group C adenoviruses as vectors for gene therapy; in Kaplitt M, Loewy A (eds): Viral
Vectors: Gene Therapy and Neuroscience Applications. San Diego, Academic Press, 1995, pp 43–54.
106 Engelhardt J, Simon R, Yang Y, Zepeda M, Weber-Pendleton S, Doranz B, Grossman M, Wilson J:
Adenovirus-mediated transfer of the CFTR gene to lung of nonhuman primates: Biological efficacy
study. Hum Gene Ther 1993;4:759–769.
107 Byrnes A, Wood M, Charlton H: Role of T cells in inflammation caused by adenovirus vectors in
the brain. Gene Ther 1996;3:644–651.
108 Yang Y, Li Q, Ertl HC, Wilson JM: Cellular and humoral immune responses to viral antigens create
barriers to lung-directed gene therapy with recombinant adenoviruses. J Virol 1995;69:2004–2015.
109 Yang Y, Nunes F, Berencsi K, Furth E, Gonczol E, Wilson J: Cellular immunity to viral antigens
limits E1-deleted adenoviruses for gene therapy. Proc Natl Acad Sci USA 1994;91:4407–4411.
110 Romero M, Smith G: Adenoviral gene transfer into the normal and injured spinal cord: Enhanced
transgene stability by combined administration of temperature-sensitive virus and transient
immune blockage. Gene Ther 1998;5:1612–1621.
111 Engelhardt J, Litzky L, Wilson J: Prolonged transgene expression in cotton rat lung with recom-
binant adenoviruses defective in E2a. Hum Gene Ther 1994;5:1217–1229.
112 Armentano D, Sookdeo C, Hehir K, Gregory R, St George J, Prince G, Wadsworth S, Smith A:
Characterization of an adenovirus gene transfer vector containing an E4 deletion. Hum Gene
Ther 1995;6:1343–1353.
113 Gorziglia M, Kadan M, Yei S, Lim J, Lee G, Luthra R, Trapnell B: Elimination of both E1 and E2a
from adenovirus vectors further improve prospects for in vivo human gene therapy. J Virol 1996;
70:4173–4178.
114 Pao C, Lin K, Zhu J, Wu G, Farmer P, Phillips L: In vitro transcription of the rat insulin-like
growth factor-I gene. J Biol Chem 1996; 271:8667–8674.
115 Goldman M, Litzky L, Engelhardt J, Wilson J: Transfer of the CFTR gene to the lung of nonhu-
man primates with E1-deleted, E2a-defective recombinant adenoviruses: A preclinical toxicology
study. Hum Gene Ther 1995;6: 839–851.
116 Yang Y, Nunes F, Berencsi K, Gonczol E, Engelhardt J, Wilson J: Inactivation of E2a in recombi-
nant adenoviruses limits cellular immunity and improves the prospect for gene therapy of cystic
fibrosis. Nat Genet 1994;7:362–369.
117 Parks R, Chen L, Anton M, Sankar U, Rudnicki M, Graham F: A helper-dependent adenovirus
vector system: Removal of helper virus by Cre-mediated excision of the viral packaging signal.
118 Parks R, Graham F: A helper-dependent system for adenovirus vector production helps define a
lower limit for efficient DNA packaging. J Virol 1997;71:3293–3298.
119 Berns K, Bohenzky R: Adeno-associated viruses: An update. Adv Virus Res 1987;32:243–306.
120 Xiao X, Li J, Samulski R: Production of high-titer recombinant adeno-associated virus vectors in
the absence of helper adenovirus. J Virol 1998;72:2224–2232.
121 Kotin R, Siniscalco M, Samulski R, Zhu X, Hunter L, Laughlin C, McLaughlin S, Muzycka N,
Rocchi M, Berns K: Site-specific integration by adeno-associated virus. Proc Natl Acad Sci USA
1990;87:2211–2215.
122 Xiao X, Li J, Samulski RJ: Efficient long-term gene transfer into muscle tissue of immunocom-
petent mice by adeno-associated virus vector. J Virol 1996;70:8098–8108.
123 Kaplitt M, Leone P, Samulski R, Xiao X, Pfaff D, O’Malley K: Long-term gene expression and
phenotypic correction using adeno-associated virus vectors in the mammalian brain. Nat Genet
1994;8:148–154.
124 Xiao X, Li J, McCown TJ, Samulski RJ: Gene transfer by adeno-asociated virus vectors into the
central nervous system. Exp Neurol 1997;144:113–124.
medwedi.ru
125 Sun L, Li J, Xiao X: Overcoming adeno-associated virus vector size limitations through viral
DNA heterodimerization. Nat Med 2000;6:599–602.
126 Oudega M, Hagg T: Nerve growth factor promotes regeneration of sensory axons into adult rat
spinal cord. Exp Neurol 1996;140:218–229.
127 Ramer M, Priestley J, McMahon S: Functional regeneration of sensory axons into the adult spinal
cord. Nature 2000;403:312–316.
128 McMahon S, Armanini M, Ling L, Phillips H: Expression and coexpression of Trk receptors in
subpopulations of adult primary sensory neurons projecting to identified peripheral targets. Neuron
1994;12:1161–1171.
129 Jakeman L, Wei P, Guan Z, Stokes B: Brain-derived neurotrophic factor stimulates hindlimb step-
ping and sprouting of cholinergic fibers after spinal cord injury. Exp Neurol 1998;154:170–184.
130 Oudega M, Hagg T: Neurotrophins promote regeneration of sensory axons in the adult rat spinal
cord. Brain Res 1999;818:431–438.
131 Schnell L, Schneider R, Kolbeck R, Barde Y, Schwab M: Neurotrophin-3 enhances sprouting of cor-
ticospinal tract during development and after adult spinal cord lesions. Nature 1994;367:170–173.
132 Henderson C, Phillips H, Pollock R, Davies A, Lemeulle C, Armanini M, Simpson L, Moffet B,
Vandlen R, Koliatsos V, Rosenthal A: GDNF: A potent survival factor for motor neurons present
in peripheral nerve and muscle. Science 1994;266:1062–1064.
133 Bradbury E, Khemani S, King V, Priestley J, McMahon S: NT-3 promotes growth of lesioned adult rat
sensory axons ascending in the dorsal columns of the spinal cord. Eur J Neurosci 1999;11:3873–3883.
134 Kluck R, Bossy-Wetzel E, Green D, Newmeyer D: The release of cytochrome c from mitochon-
dria: A primary site for Bcl-2 regulation of apoptosis. Science 1997;275:1132–1136.
135 Allsopp T, Wyatt S, Paterson H, Davies A: The proto-oncogene bcl-2 can selectively rescue
neurotrophic factor-dependent neurons from apoptosis. Cell 1993;73:295–307.
136 Matsuoka N, Yukawa H, Ishii K, Hamada H, Akimoto M, Hashimoto N, Miyatake S: Adenovirus-
mediated gene transfer of Bcl-xL prevents cell death in primary neuronal culture of the rat.
Neurosci Lett 1999;270:177–180.
137 Blomer U, Kafri T, Randolph-Moore L, Verma I, Gage F: Bcl-xL protects adult septal cholinergic
neurons from axotomized cell death. Proc Natl Acad Sci USA 1998;95:2603–2608.
138 Liu Y, Himes B, Moul J, Huang W, Chow S, Tessler A, Fischer I: Application of recombinant
adenovirus for in vivo gene delivery to spinal cord. Brain Res 1997;768:19–29.
139 Byrnes A, Rusby J, Wood M, Charlton H: Adenovirus gene transfer causes inflammation in the
brain. Neuroscience 1995; 66:1015–1024.
140 Boulis N, Turner DE, Dice J, Bhatia V, Feldman E: Characterization of adenoviral gene expession
in spinal cord after remote vector delivery. Neurosurgery 1999;45:131–137.
141 Ghadge G, Roos R, Kang U, Wollmann R, Fishman P, Kalynych A, Barr E, Leiden J: CNS gene
delivery by retrograde transport of recombinant replication-defective adenoviruses. Gene Ther
1995;2:132–137.
142 Kuo H, Ingram D, Crystal R, Mastrangeli A: Retrograde transfer of replication deficient recombinant
adenovirus vector in the central nervous system for tracing studies. Brain Res 1995;705:31–38.
143 Boulis NM, Willmarth N, Song D, Feldman E, Imperiale M: Intraneural colchicine inhibition
of adenoviral and adeno-associated viral vector remote spinal cord gene delivery. Neurosurgery
2003;52:381–387.
144 Turner D, Noordmans A, Feldman E, Boulis N: Remote adenoviral gene delivery to the spinal
cord: Contralateral delivery and reinjection. Neurosurgery 2001;48:1309–1316.
145 Glatzel M, Flechsig E, Navarro B, Klein M, Paterna J, Bueler H, Aguzzi A: Adenoviral and adeno-
associated viral transfer of genes to the peripheral nervous system. Proc Natl Acad Sci USA 2000;
97:442–447.
146 Peel A, Zolotukhin S, Schrimsher G, Muzyczka N, Reier P: Efficient transduction of green
fluorescent protein in spinal cord neurons using adeno-associated virus vectors containing cell
type-specific promoters. Gene Ther 1997;4: 16–24.
147 Kaspar B, Erickson D, Schaffer D, Hinh L, Gage F, Peterson D: Targeted retrograde gene delivery
for neuronal protection. Mol Ther 2002;5:50–56.
148 Mazarakis N, Azzouz M, Rohll F, Ellard F, Wilkes F, Olsen A, Carter E, Barber R, Baban D,
Kingsman S, Kingsman A, O’Malley K, Mitrophanous K: Rabies virus glycoprotein pseudotyping
of lentiviral vectors enables retrograde axonal transport and access to the nervous system after
peripheral delivery. Hum Mol Genet 2001;10:2109–2121.
149 Tsiang H: Evidence for an intraaxonal transport of fixed and street rabies virus. J Neuropathol
Exp Neurol 1979;38:286–299.
150 Acsadi G, Anguelov R, Yang H, Toth G, Thomas R, Jani A, Wang Y, Ianakova E, Mohammad S,
Lewis R, Shy M: Increased survival and function of SOD1 mice after glial cell-derived neuro-
trophic factor gene therapy. Hum Gene Ther 2002;13:1047–1059.
151 Manabe Y, Nagano I, Gazi M, Murakami T, Shiote M, Shoji M, Kitagawa H, Setoguchi Y, Abe K:
Adenovirus-mediated gene transfer of glial cell line-derived neurotrophic factor prevents motor
neuron loss of transgenic model mice for amyotrophic lateral sclerosis. Apoptosis 2002;7:329–334.
152 Wang L, Lu Y, Muramatsu S, Ikeguchi K, Fujimoto K, Okada T, Mizukami H, Matsushita T,
Hanazono Y, Kume A, Nagatsu T, Ozawa K, Nakano I: Neuroprotective effects of glial cell line-
derived neurotrophic factor mediated by an adeno-associated virus vector in a transgenic animal
model of amyotrophic lateral sclerosis. J Neurosci 2002;22:6920–6928.
153 Baumgartner B, Shine H: Neuroprotection of spinal motor neurons following targeted transduc-
tion with an adenoviral vector carrying the gene for glial cell line-derived neurotrophic factor. Exp
Neurol 1998;153:102–112.
154 Romero M, Rangappa N, Garry M, Smith G: Functional regeneration of chronically injured sensory
afferents into adult spinal cord after neurotrophin gene therapy. J Neurosci 2001;21:8408–8416.
155 Rosenberg M, Friedmann T, Robertson R, Tuszynski M, Wolff J, Breakefield X, Gage F: Grafting
genetically modified cells to the damaged brain: Restorative effects of NGF expression. Science
1988;242:1575–1578.
156 Tuszynski M, Peterson D, Ray J, Baird A, Nakahara Y, Gage F: Fibroblasts genetically modified
to produce nerve growth factor induce robust neuritic ingrowth after grafting to the spinal cord.
Exp Neurol 1994;128:1–14.
157 Tuszynski M, Gabriel K, Gage F, Suhr S, Meyer S, Rosetti A: Nerve growth factor delivery by
gene transfer induces differential outgrowth of sensory, motor, and noradrenergic neurites after
adult spinal cord injury. Exp Neurol 1996; 137:157–173.
158 Ernfors P, Henschen A, Olson L, Persson H: Expression of nerve growth factor receptor mRNA is
developmentally regulated and increased after axotomy in rat spinal cord motor neurons. Neuron
1989;2:1605–1613.
159 Grill R, Blesch A, Tuszynski M: Robust growth of chronically injured spinal cord axons induced
by grafts of genetically modified NGF-secreting cells. Exp Neurol 1997;148:444–452.
160 Englund U, Ericson C, Rosenblad C, Mandel R, Trono D, Wictorin K, Lundberg C: The use of a
recombinant lentiviral vector for ex vivo gene transfer into the rat CNS. Neuroreport 2000;11:
3973–3977.
161 Ericson C, Wictorin K, Lundberg C: Ex vivo and in vitro studies of transgene expression in rat
astrocytes transduced with lentiviral vectors. Exp Neurol 2002;173:22–30.
162 Liu Y, Himes B, Tyron B, Moul J, Chow S, Jin H, Murray M, Tessler A, Fischer I: Intraspinal grafting
of fibroblasts genetically modified by recombinant adenoviruses. Neuroreport 1998;9:1075–1079.
163 Palmer TD, Rosman G, Osborne W, Miller A: Genetically modified skin fibroblasts persist long
after transplantation but gradually inactivate introduced genes. Proc Natl Acad Sci USA 1991;
88:1330–1334.
164 Ruitenberg M, Plant G, Christensen C, Blits B, Niclou S, Harvey A, Boer G, Verhaagen J: Viral
vector-mediated gene expression in olfactory ensheathing glia implants in the lesioned rat spinal
cord. Gene Ther 2002;9:135–146.
165 Grill R, Murai K, Blesch A, Gage F, Tuszynski M: Cellular delivery of neurotrophin-3 promotes
corticospinal axonal growth and partial functional recovery after spinal cord injury. J Neurosci
1997;17:5560–5572.
Nicholas M. Boulis, MD
Department of Neurosurgery
S31, Cleveland Clinic Foundation
9500 Euclid Ave, Cleveland, OH 44195 (USA)
Tel. ⫹1 216 444 5188, Fax ⫹1 216 445 1466, E-Mail boulisn@ccf.org
medwedi.ru
Neural Stem Cell Transplantation

for Spinal Cord Repair
Akio Iwanami a–c, Yuto Ogawa a–c, Masaya Nakamura b,c,
Shinjiro Kaneko a–c, Kazunobu Sawamoto c,d, Hirotaka James Okano a,c,
Yoshiaki Toyama b,c, Hideyuki Okano a,c,d
Departments of aPhysiology and bOrthopaedic Surgery, Keio University
School of Medicine, Shinjuku, Tokyo, cCore Research for Evolutional
Science and Technology, Japan Science and Technology Corporation,
Osaka and dDepartment of Neuroanatomy, Osaka University Graduate School
of Medicine, Suita, Osaka, Japan
Introduction
Ever since the neuroanatomist Cajal [1] reported in the 1920s that the
mature central nervous system (CNS) cannot generate new neurons, it was
believed that the mammalian CNS does not have the capacity for repair after
injury. Nevertheless, in the 1970s, preliminary work in neural transplantation
was done which indicated that the neural tissue obtained from fetal rat brain
survived, reconstructed neuronal networks, and reversed motor abnormalities
when grafted into the animal model of Parkinson’s disease.
With the discovery of the potential for functional brain transplantation,
interest in neural transplantation escalated sharply and the field of
‘Functional Neurosurgery’ was born. During the 1980s, clinical transplanta-
tion trials for Parkinson’s disease attempted to replace dopaminergic neurons
with transplants of dopamine-producing cells of various derivations. Clinical
application of neural tissue transplantation is still practically limited by the
lack of availability of donor fetal brain or spinal cord tissue. In recent years,
however, it has become evident that the developing and even the adult mam-
malian CNS contains self-renewing, multipotent neural stem cells (NSCs),
which can be harvested as a source of material for grafting. Recent techno-
logical advances developed for the identification, isolation, and expansion of
NSCs raises the enormous potential of therapeutic applications for nervous
system disorders [2].
Studies of neural progenitor cells or NSCs are broadly divided into stud-
ies on the activation of endogenous NSCs in situ, or studies involving trans-
plantation of NSCs isolated from the brain or spinal cord. At least within the
spinal cord, therapeutic strategies using activation of endogenous NSCs are not
expected to be practical, because endogenous NSCs appear to proliferate but
differentiate only into astrocytes after spinal cord injury (SCI) [3–5]. On the
other hand, there are many reports demonstrating the transplantation potential
of neural progenitor cells for various CNS disorders or trauma. These cells
have not only shown the reconstruction of neuronal networks, but have also
rendered functional recovery in animal models. In this chapter, we will intro-
duce recent progress in transplantation, especially as it pertains to practical
issues of timing. In addition, we will discuss the future prospects for their
clinical application.
Definition and Selective Culture of NSCs
NSCs have been defined as neural cells with the potential to self-renew and
generate all three major cell types of the CNS: neurons, astrocytes, and oligo-
dendrocytes. The existence of mammalian NSCs was first suggested by
researchers such as Altman, Bayer, Kaplan and others starting in the 1960s, but
it was not until the 1990s that these cells were demonstrated in humans and
were isolated in culture. Enormous progress has been made in studies on the
biological properties of NSCs and their localization in the body over the last
decade since Reynolds and Weiss and coworkers [6, 7] developed the neu-
rosphere technique, a selective culture technique for NSCs in 1992. They
cultured CNS cell populations including NSCs derived from the mouse embry-
onic striate body and spinal cord in serum-free culture medium containing
insulin, transferrin, selenium, progesterone, and cell division-promoting epi-
dermal growth factor or fibroblast growth factor 2. Although many of these
cells could not survive in serum-free medium, the cells surviving in this unusual
environment could be grown as floating cell aggregates or ‘neurospheres’.
When the neurosphere was dissociated into single cells and cultured in the same
medium, they formed the neurospheres again, indicating a self-renewing ability.
Moreover, the differentiation into the three neural cell types was driven by
growth factor withdrawal, demonstrating multipotency. Thus, the neurosphere
technique was shown to expand multipotent, self-renewing NSCs for many pas-
sages without apparent phenotypic change (fig. 1) [8]. Subsequently, other cul-
ture techniques, such as a low-density monolayer technique, were developed by
Neural Stem Cell Transplantation 105
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Neural stem cell Other cells
Growth factors
(EGF, FGF2)
Removal of Neuron
growth factors
1. Self-renewal Astrocyte
Oligodendrocyte
2. Multipotency
Repeat
Fig. 1. Neurosphere method. The major breakthrough for research on stem cell biol-
ogy of the CNS was the development of the clonogenic expansion of NSCs in floating cul-
ture, called neurosphere culture, within a serum-free medium containing epidermal growth
factor EGF and/or fibroblast growth factor 2 or FGF2. A neurosphere derived from a single
cell is capable of generating the major three lineages of the CNS, i.e., neurons, astrocytes,
and oligodendrocytes, indicating the multipotency of the neurosphere-initiating cell, upon
the differentiation assay. If the neurosphere is dissociated into single cells, each cell starts to
form a secondary neurosphere again with high frequency. From [6].
Davis and Temple [9] and a high-density monolayer technique was developed
by Gage and his coworkers [10].
Selective Markers of NSCs
Even though selective culture methods for NSCs have been established,
specific markers for NSCs have not been identified. Instead, highly selective
markers of NSCs are known. These include the intermediate filament Nestin
[11], the RNA-binding protein Musashi1 [12] identified by our group, and RC2
(i.e., marker of radial glia). These markers are strongly expressed in NSCs;
however, they are also expressed in intermediate progenitor cells such as neu-
ronal and glial progenitor cells. Therefore, they are not 100% specific for NSCs.
Since cell populations expanded by the neurosphere technique include neural
progenitor cells that have differentiated to some degree, it is currently impossi-
ble to completely discriminate NSCs from partially differentiated progenitor
cells using a positive marker.
Iwanami/Ogawa/Nakamura/Kaneko/Sawamoto/Okano/Toyama/Okano 106
Existence and Localization of NSCs in Adult Mice and Humans
Reynolds and Weiss and coworkers [6, 7, 13] demonstrated the existence
of NSCs in the embryonic mouse striate body and spinal cord and successfully
cultured them. In 1996, Gritti et al. [14] showed that multipotent, self-renew-
ing NSCs also exist in the adult mouse striatum, indicating that NSCs exist not
only in the embryonic but also in the adult mouse brain. Furthermore,
Eriksson et al. [15] demonstrated that neurogenesis also occurred in the adult
human brain. That study stained postmortem brain samples from cancer
patients with 5-bromodeoxyuridine (BrdU) treatment and demonstrated that
neurons incorporating BrdU were present in the hippocampal dentate gyrus.
Using Nestin and Musashi1 as markers, the collaborative team of Goldman’s
group [16] at Cornell University and our own research group showed that
NSC-like cells were present around the lateral ventricles of intractable epilep-
tics who had undergone temporal lobectomy. In support of the studies by
Eriksson et al. [15], these observations also indicate the existence of NSCs in
the adult brain. Their locations correspond to sites of neurogenesis in the
granule cell layer of the hippocampal dentate gyrus, the subventricular zone
facing the lateral ventricles, and/or the ependymal layer. Recent studies have
also suggested the existence of NSCs in the parenchyma of the adult cerebral
cortex and spinal cord [10, 17]. Although CNS injury leads to the prolifera-
tion of endogenous NSCs, these cells are not usually capable of self-repair.
While recent results suggest that forebrain damage due to ischemia could be
recovered by activating endogenous NSCs to induce de novo neurogenesis
[18, 19], such a strategy has not yet been successful in the injured spinal cord.
This failure is presumably because there are few endogenous NSCs in the
adult spinal cord or because their differentiation into neuronal cells is inhib-
ited by some mechanism, which remains to be elucidated. Studies are in
progress throughout the world in two major areas of research to develop ther-
apeutic strategies for CNS injuries and disorders: first, the activation of
endogenous NSCs and second, the transplantation of harvested NSCs. This
chapter primarily addresses transplantation therapy using neuronal precursor
cells or NSCs.
In situ Identification and Effective Isolation of NSCs
The effective isolation, culture, and expansion of NSCs are essential in

considering the clinical application of transplantation therapy, and it is neces-
sary to develop appropriate methods to achieve these purposes. As described
above, it is feasible to expand NSCs by the methods represented by the
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neurosphere technique. However, we can define the neurosphere-initiating
cells as NSCs retrospectively only after the formation of the neurosphere. It
has been difficult to prospectively identify them in the early stages of culture
and impossible to do so in situ. Thus, for experimental purposes we have made
transgenic mice that express enhanced green fluorescent protein (EGFP) under
the control of the Nestin gene promoter or enhancer to isolate Nestin-positive
cells by a fluorescence-activated cell sorter according to the intensity of EGFP
expression and fluorescence [20]. We found that the activity of the isolated
cells as NSCs correlates well with the intensity of fluorescence; a more
intensely fluorescent group of cells had a higher formation rate of neu-
rospheres, showing self-renewing ability and multipotency even in low-density
culture. This finding is significant not only because it became possible to
prospectively identify NSCs in a ‘living state’ by GFP fluorescence using
fluorescence-activated cell sorter, but also because NSCs can be concentrated
by this method without using growth factors as in conventional methods [8].
Recently, it is feasible to perform transplantation therapy with a new source of
NSCs in place of embryonic tissue transplantation.
Shifting from Neural Tissue Transplantation to Neural

Precursor Cell Transplantation
Studies on Transplantation for Parkinson’s Disease

Studies on transplantation therapy for CNS disorders have been more
advanced for Parkinson’s disease than for other diseases because of the earlier
establishment of animal models. In 1979, Björkund and Stenevi [21] reported
that rats with experimental Parkinson’s disease recovered from symptoms after
transplantation of embryonic rat midbrain tissue into their striata. Later,
numerous studies reported results including symptomatic recovery following
transplantation of fetal cells of different derivations, and clinical trials also
started. In fact, some patients transplanted with fetal tissue have achieved
symptomatic relief for more than 10 years and have been demonstrated by PET
to have cell transplants functioning effectively [22]. However, fetal tissue
transplantation posed many problems such as low engrafting rates and the need
for as many as five to ten fetal midbrains for a unilateral striatum transplant.
Because of these practical and ethical problems, it was hoped that new donor
cells would be developed. Against this background, in 1996, Svendsen et al.
[23, 24] transplanted rat neural progenitor cells and human-derived cells
shortly afterwards into the striata of model rats with Parkinson’s disease
(6-OH-dopamine-administered rats), and reported successful engrafting of
the transplants. They reported that although many of the transplanted cells
differentiated into glia, a few tyrosine hydroxylase-positive cells were observed,
with modest functional improvement. By taking advantage of the Nestin-
EGFP system, we tried to isolate neural progenitor cells from the fetal ventral
mesencephalic region. In fact, we obtained a strongly GFP-positive undiffer-
entiated cell population from the fetal ventral mesencephalon of the Nestin-
EGFP mouse by fluorescence-activated cell sorter, which was transplanted
into the striatum body of rat models of Parkinson’s disease. We demonstrated
the differentiation of the cell transplants into dopaminergic neurons, with
recovery from symptoms of Parkinson’s disease [25].
Studies on Transplantation for SCI

Experiments on transplantation of nervous system cells for SCI started in
1980 with peripheral nerve transplantation by Aguayo and his coworkers [26].
Then in 1993, Bregman et al. [27] reported that both immature and adult rats
in which the thoracic spinal cords had been partially transected and which
were transplanted with a fetal spinal cord showed elongation of injured axons
with functional recovery, which was predominant in the immature rats. These
studies indicated that the introduction of an appropriate environment into the
injured site could cause injured axons to regenerate. In addition, other reports
described limited spinal cord regeneration including the promotion of the
regeneration of injured axons by neurotrophic factors [28] and the identifica-
tion of axonal growth inhibitors [29]. These studies indicated that regenera-
tion of the injured spinal cord might really be possible. Although researchers
first focused on the effectiveness of fetal spinal cord transplantation for SCI
[30–32], as with Parkinson’s disease, donor tissue shortage and ethical prob-
lems precluded the practical clinical application of this approach. As a result
of remarkable advances in neuroscience in recent years, NSCs also have
stepped into the limelight as a new transplant material in the field of the spinal
cord repair. In 1999, McDonald et al. [33] developed elaborate sequential
culture conditions that differentiated mouse ES cells into NSCs in vitro, and
transplanted them into the traumatic cavity of rat models of thoracic spinal
cord contusion injury. They reported that the engrafted cells could differenti-
ate into neurons, astrocytes, and oligodendrocytes, and that the model rats
improved in lower limb motor function to a greater degree than the control
group. More recently, Vacanti et al. [34] transplanted gels packed with adult
rat-derived NSCs into rat models of thoracic spinal cord transection, with
similar results. Our group has looked specifically at time-dependent changes
in the cavity microenvironment after SCI and achieved excellent results. On
the ninth post-traumatic day during the subacute stage between the immedi-
ate post-traumatic stage and the chronic stage during which glial scarring of
the injured site progressed, we transplanted fetal rat spinal cord-derived
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NSCs into the cavity region of rat models of cervical spinal cord contusion
injury and observed differentiation of the transplanted cells into neurons, form-
ing synapses with host axons, with recovery of motor function [35, 36] (see
Strategies of Transplantation of Fetal Spinal Cord-Derived NSCs for SCI).
Other Reports on NSC Transplantation

Other studies reported that the transplantation of NSCs into the cerebral
ventricle of myelin basic protein-deficient dysmyelinated shiverer mice caused
engrafting of myelin basic protein-positive cells [37]. Moreover, transplanta-
tion of rat hippocampus-derived NSCs into the growing retina of rats resulted
in the appearance of cells expressing molecular markers and having the mor-
phology of Mueller, amacrine, bipolar, horizontal, and photoreceptor cells and
astrocytes [38]. These experiments, unlike the above-described ones, have a
drawback in that there was no testing of functional improvement. However,
based on these results, hopes are mounting for future experiments on their
clinical application.
Aims of Neural Progenitor Cell Transplantation

We consider that the above-described studies on neural precursor cell
transplantation had two broad aims: first, to allow NSC transplants to appro-
priately proliferate and differentiate, replace lost neurons, reform synapses, and
induce remyelination; second, to activate endogenous NSCs by the trophic
effects of the grafted cell, to induce the differentiation in the desired direction
and repair injured neural tissue. As described above, NSCs have now been
shown to exist in a number of separate locations, such the fetal and adult brain,
spinal cord, and retina [39]. As reported by Kempermann et al. [40], some stim-
uli appear to activate endogenous NSCs to increase the generation of neurons
and glia. Unfortunately, the manner and mechanism of this activation remain to
be elucidated. In contrast, the transplantation of exogenous NSCs is aimed at
activating endogenous NSCs through neurotrophic factors or some signal to
participate in the mechanism of repair and regeneration of lost tissue. In line
with these aims, we describe below the present status and future prospects of
NSC transplant-based regenerative medicine for the injured spinal cord.
Strategies of Transplantation of Fetal Spinal

Cord-Derived NSCs for SCI
Properties of Endogenous NSCs of the Spinal Cord

When the spinal cord is injured, Nestin-positive cells, derived from vigor-
ously proliferating cells near the central canal adjacent to the injured site,
Spinal cord injury Reactive astrocytes Glial scar
formation
Cystic
formation
Endogenous neural stem cells
Fig. 2. Properties of endogenous NSCs of the spinal cord. Recent studies have shown
that there are endogenous NSCs in the adult spinal cord near the central canal. However,
these cells differentiate into astrocytes, but not into neurons or oligodendrocytes after SCI.
migrate to the area of the injured site and differentiate into astrocytes [3]. The
study of Johansson et al. [4] in 1999 showed the existence of NSCs in the adult
spinal cord near the central canal. After SCI, these endogenous NSCs prolifer-
ate [5] and differentiate mostly into astrocytes, but not neurons or oligodendro-
cytes. Since endogenous astrocytes eventually form a glial scar around the
wound cavity in a time-dependent manner after injury, the regeneration, elon-
gation and remyelination of damaged axons is entirely disturbed (fig. 2). For
repairing injured spinal cord, therefore, neuronal replacement therapy remains
the most promising therapeutic strategy.
Optimal Timing of NSPCs Transplantation

For the purpose of transplantation into the injured spinal cord, we cultured
NSCs obtained from 14-day gestational age rat spinal cord, using the neu-
rosphere technique. When these cells were induced to differentiate in vitro,
they differentiated into neurons, astrocytes, and oligodendrocytes. About 50%
of the cells formed astrocytes and 5% of the total into neurons [Nakamura,
pers. commun.]. Thus, it seems highly unlikely that the transplantation of NSCs
without any contrivance results in the repair and regeneration of neurons and
oligodendrocytes that have been lost through axonotemesis or apoptosis.
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NSCs obtained from adult CNS showed extensive differentiation poten-
tial when transplanted into an adult neurogenic site (i.e., the hippocampal
dentate gyrus) [41]. It is well known that the surrounding microenvironment
greatly influences the survival and differentiation of engrafted neural progen-
itor cells. To address the optimal time of transplantation, we investigated the
post-traumatic changes of the microenvironment within the injured spinal
cord. In the injured spinal cord, the expression of mRNA for various proin-
flammatory cytokines [e.g., tumor necrosis factor-, interleukin (IL)-1,
IL-1, IL-6] peaked 6–12 h after injury and remained elevated until the fourth
day [42]. Hart and coworkers [43] have reported the same results. Since these
proinflammatory cytokines are known to have biphasic actions, both neuro-
toxic and neurotrophic, their action within the injured spinal cord requires
careful interpretation. Extremely high expression at least within 7 days after
injury is thought to be neurotoxic, representing a microenvironment unfit for
the survival of NSC transplants.
Johe and coworkers [44] have reported that platelet-derived growth factor,
ciliary neurotrophic factor, and thyroid hormone (T3) instructively induced the
fetal rat hippocampus-derived NSCs to differentiate into neurons, astrocytes,
and oligodendrocytes, respectively. Taga and coworkers [45] reported that
leukemia inhibitory factor and bone morphogenic protein-2 promote the differ-
entiation of fetal mouse neuroepithelium-derived NSCs into astrocytes. These
reports both described members of the IL-6 superfamily (e.g., ciliary neu-
rotrophic factor, leukemia inhibitory factor) and suggest that a signal mediated
by the gp130 subunit of the cytokine receptors induces the differentiation of
NSCs into astrocytes. During the acute inflammatory phase immediately after
SCI, under the condition in which there are high levels of IL-6, NSC transplants
are difficult to engraft, but also easy to differentiate into astrocytes if they
engraft.
We have found that the expression of the anti-inflammatory cytokine
transforming growth factor -1 (TGF-1) did not increase immediately after
injury, but gradually increased with a peak on the fourth day after injury [42],
suggesting that TGF-1 acts to alleviate the inflammatory situation. These
observations on the survival and differentiation of NSC transplants indicate
that the optimal time of the transplantation is probably not immediately after
injury. However, if too much time passes after injury, a glial scar forms around
the injured site and inhibits the regeneration of axons; therefore, we consid-
ered the optimal time of transplantation to be 7–14 days after trauma (fig. 3).
In addition, the benefits of NSC transplantation at this timepoint could also
result from microvascular regeneration in the host, considering previous
findings from fetal neural tissues transplanted into the cerebral cortex [46,
47]. Correspondingly, a recent report indicates that the formation of new
Acute phase
Subacute – chronic phase

Inflammation
SCI Glial scar formation

Hemorrhage
BBB destruction
Infiltration of inflammatory cells
Glial scar formation
0 1
4
7 Cystic
Pro-inflammatory cytokine cavity
Free radical
NO Time 14 (Posti
windo njury
Excitatory amino acid w for days)
trans
plant
ation Anti-inflammatory cytokine
?
Fig. 3. Optimal timing of neural stem/progenitor cells (NSPCs) transplantation based

on changes in the microenvironment within the injured spinal cord. We consider the optimal
timing of NSPCs transplantation to be 7–14 days after trauma, when the lesion site is neither
inflammatory and neurotoxic, nor surrounded by glial scar.
vessels occurs most actively 7–14 days after a contusion injury to the rat spinal
cord [48].
Fetal Rat Spinal Cord-Derived Cells for Rat SCI: Delayed

Transplantation
Based on the above considerations, we made models of quantitative cervi-
cal cord contusion injury by performing C4–5 laminectomy on adult rats and
allowing a 35-gram weight to stand still on the exposed dura for 15 min. On the
ninth day after injury, we transplanted fetal rat spinal cord-derived neural prog-
enitor cells that had been cultured and expanded by the neurosphere technique
and labeled with BrdU in and around the injured site (fig. 4). The transplanted
cells survived in the host spinal cord and differentiated into neurons, astrocytes,
and oligodendrocytes at the 5-week timepoint after transplantation (fig. 5a–c). To
investigate the properties of new neurons derived from donors in more detail, we
took advantage of the fact that the 1.1-kb promoter element of the T-1 tubulin
gene is only active in cells of the neuronal lineage (including neuronal progeni-
tors and postmitotic neurons), and not those of the glial lineage (fig. 6a, b)
[49–52]. Here, we used rats that had been treated with transplanted neurospheres
derived from the fetal spinal cords (E14.5) of T-1-EYFP transgenic rats. By
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9 days after injury
Fetal spinal
cord-derived
NSPCs
transplantation
C4 C5
Cervical cord of an adult rat
Fig. 4. Delayed transplantation of NSPCs after SCI. We made models of quantitative

cervical cord contusion injury by performing C4–5 laminectomy on adult rats and allowing
a 35-gram weight to stand still on the exposed dura for 15 min. On the ninth day after injury,
we transplanted fetal rat spinal cord-derived NSPCs that had been cultured and expanded by
the neurosphere technique and labeled with BrdU, in and around the injured site.
a b
HU
CNP GFAP
Fig. 5. Survival and differentiation of the engrafted NSPCs. The NSPC transplants
had survived in the host spinal cord and differentiated into neurons, astrocytes, and oligo-
dendrocytes at the 5-week timepoint after transplantation. a Hu (neuronal marker) and
5-bromodeoxyuridine (BrdU) double-positive cells (brown: Hu; blue: BrdU). b GFAP
(marker of astrocytes) and BrdU double-positive cells (brown: GFAP; blue: BrdU). c CNPase
(marker of oligodendrocytes) and BrdU double-positive cells (brown: CNPase; blue: BrdU).
Scale bar 5 m.
injecting BrdU intraperitoneally, we could label cells that had divided after the
BrdU injection. The presence of postmitotic neurons that were double positive
for BrdU-labeling and EYFP expression demonstrated that donor-derived pro-
genitor cells underwent mitotic neurogenesis within the host spinal cord (fig. 6c).
T-1-EYFP transgene Neural progenitor cells
T-1 promoter EYFP
SV40 derived from T-1-EYFP Tg rats
polyA
Transplantation
1 kb of the 5 flanking region
a of the rat T-1 tubulin gene
SCI rats
Transplantation
Intraperitoneal injection of
BrdU (50g/g B.W.) once a day
from 3 to 14 days after transplantation
-III tubulin EYFP
b c
Fig. 6. The advantage of using T-1-EYFP Tg rats. a k-1 transgene. b The expres-
sion of EYFP in the fetal T-1-EYFP Tg rat brain. As the promoter element of T-1 tubulin
gene is only active in cells of the neuronal lineage (including neuronal progenitors and post-
mitotic neurons), and not those of the glial lineage, the EYFP-positive region is coincident
with the region stained with -III tubulin (neuronal marker). c The proof of neurogenesis by
the transplanted NSPCs. By injecting BrdU intraperitoneally, we could label cells that had
divided after the BrdU injection. The presence of postmitotic neurons that were double pos-
itive for BrdU labeling and EYFP expression demonstrated that donor-derived progenitor
cells underwent mitotic neurogenesis within the host spinal cord.
Five weeks after transplanting neurospheres derived from the fetal spinal
cords of T-1-EYFP transgenic rats, donor-derived EYFP-positive neurons
extended their axons within the host spinal cord (fig. 7a). We found that
T-1-EYFP-positive neurons were surrounded by synaptophysin-immunopos-
itive sites, a presynaptic marker (fig. 7b). Furthermore, we observed EYFP-
positive presynaptic structures with presynaptic vesicles that were connected
with EYFP-negative postsynaptic structures with postsynaptic densities by
immnoelectron microscopic studies (fig. 7c). We also found EYFP-negative
presynaptic structures that were connected with EYFP-positive postsynaptic
structures. Interestingly, we found some cases in which EYFP-positive neu-
rons had formed a synapse with host motor neurons at the injury site. In addi-
tion, compared with the control group (which received an injection of only
culture fluid on the ninth day after injury), the transplantation group showed a
greater degree of functional recovery as demonstrated by the pellet retrieval
test (fig. 8) [32]. These results indicate that if NSCs are transplanted in the
subacute phase, neither in the acute phase after SCI nor in the chronic phase
characterized by marked glial scarring, they can engraft and contribute to
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T-1-EYFP
Synaptophysin
T-1-EYFP
a b
Presynapse
Presynapse PSD
PSD
Fig. 7. Neurons derived from transplanted cells extend their axon into the host spinal
cord. a Five weeks after transplanting neurospheres derived from the fetal spinal cords of
T-1-EYFP transgenic rats, donor-derived EYFP-positive neurons extended their axons
within host spinal cord. Scale bar 50 m. b T-1-EYFP-positive neurons were surrounded
by synaptophysin-immunopositive sites that are well-characterized pre-synaptic markers.
Scale bar 5 m. c EYFP-positive presynaptic structures with presynaptic vesicles that
were connected with EYFP-negative postsynaptic structures with postsynaptic densities.
Immunoelectron microscopic studies. Scale bar 0.2 m.
some degree to the repair of the injured site. It is important to consider the fol-
lowing three possibilities to explain these data: (1) the transplanted cells may
have differentiated into neurons, which formed synapses with neurons above
and below the injured site; (2) the transplanted cells may have differentiated
into oligodendrocytes, which might have remyelinated the axons that had been
demyelinated by the injury; (3) the transplanted cells may have released some
neurotrophic factors, which inhibited neuronal death, induced neuronal pro-
tection, or activated endogenous NSCs to repair the injured site (fig. 9). The
actual functions of the transplanted NSCs are yet to be elucidated and require
further study.
Bregman and coworkers [53] transplanted fetal rat spinal cord tissue into
two groups of rats with SCI immediately after and 2 weeks after injury, and
compared the two groups in terms of their anatomical features and the degree
of functional recovery. They found that the group receiving transplants 2 weeks
after injury showed better regeneration of the injured axons and better lower
limb functional recovery compared with the group receiving transplants
Number of pellets
90
*
80 *
*
70
60
50
40
30
20
10
0
ope () SCI SCImed SCITP
a b
Fig. 8. Transplantation of NSPCs improved functional recovery. a Pellet retrieval test.

Rats could obtain pellets only with their forelimbs (arrows). b Results of pellet retrieval test.
*p 0.01. The p value was determined using a Mann-Whitney U-test.
Synapse formation Remyelination of Trophic support for

a b growing axons c axonal regeneration
Fig. 9. Mechanism of functional recovery by the grafts. a The transplanted NSPCs dif-
ferentiated into neurons, which formed synapses with neurons above and below the injured
site. b The transplanted NSPCs differentiated into oligodendrocytes, which might have
remyelinated the axons that had been demyelinated by the injury. c The transplanted NSPCs
released some neurotrophic factors, which inhibited neuronal death, induced neuronal
protection, or activated endogenous NSCs to repair the injured site.
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immediately after injury. These results also strongly support the effectiveness of
the delayed transplantation paradigm.
Clinical Applications of Human NSC Transplantation
As described above, transplant experiments with NSCs have been con-

ducted throughout the world. Needless to say, the ultimate goal of these exper-
iments is the human clinical application of NSC transplantation. As the culture
techniques for human NSCs become established, transplant experiments in the
clinic are under consideration. After the first demonstration by Svendsen et al.
of transplantation into Parkinson’s models (see Studies on Transplantation
for Parkinson’s Disease), Flax et al. [54] expanded NSCs from the human
fetal telencephalon by the neurosphere method, transplanted them into the
brains of newborn mice, and reported that they differentiated into neurons,
astrocytes, and oligodendrocytes. Brüstle et al. [55] similarly transplanted
NSCs cultured from the human fetal brain into the fetal rat cerebral ventricles
and reported that differentiation and engrafting occurred in the rat forebrain,
midbrain, and hindbrain. In 2001, Ourednik et al. [56] transplanted human fetal
NSCs into the fetal monkey cerebral ventricles, and reported that some of the
engrafted cells differentiated into neurons and glia, while the remaining cells
engrafted and remained undifferentiated. This report holds promise in the sense
that the transplantation of human NSCs into primates is essential as a prelimi-
nary step toward clinical application. It is hoped that, for SCI, experiments in
transplantation of human NSCs in primate models will also yield good results.
Future Prospects
There are still many problems to solve before NSC transplantation finds
clinical application. For further improvement of the transplantation therapy,
more efficient techniques to isolate NSCs are needed. In contrast to the exper-
iments with mice, for the purpose of clinical application, the collaborative team
of Goldman’s group [51, 57] and our group introduced the gene for a fluores-
cent protein into adult human hippocampal cells by the lipofection method, and
successfully isolated human NSCs. We also prepared adenoviruses expressing
EGFP under the control of the Nestin enhancer or the Musashi1 promoter for
gene transfer, and succeeded in effectively isolating NSCs from human fetal
brain tissue [58]. Using the same strategy as for hematopoietic stem cells, an
effective method was developed for effectively isolating NSPCs using antibod-
ies to cell surface antigens [59].
Another problem is how to further examine the mechanisms of the autoreg-
ulation of NSC differentiation. If a favorable environment were created for
endogenous NSCs, they might migrate rapidly to injured or degenerated sites to
self-repair these sites, and the need for heterologous neural cell transplantation
would be reduced. The key to solving this problem may be neurotrophic factors
or activation of the immune system.
Recently, new concepts on the plasticity of NSCs have emerged. For
example, Kondo and Raff [60] have shown that oligodendrocyte precursor
cells acquire multipotency similar to NSCs after the manipulation of culture
conditions. Clarke et al. [61] have shown that adult ROSA26 mouse-derived
NSCs, which have been transplanted into the embryonic chicken amniotic
cavity and the mouse blastocyst, differentiate into ectodermal, mesodermal,
and endodermal tissues and cells. These observations suggest that NSCs have
the potential for differentiation similar to ES cells, and depending on their
environment, sometimes differentiate into non-neural cells. On the other hand,
other studies reported that non-neural bone marrow stromal cells, which are
mesenchymal stem cells, differentiated into neurons in vitro [62], or migrated
to the cerebrum and cerebellum to differentiate into astrocytes after transplan-
tation into the neonatal mouse cerebral ventricle [63]. Furthermore, a study
has reported that bone marrow stromal cells, which have been transplanted
into the injured rat spinal cord one week after injury, bridge the epicenter of
the injury in association with immature astrocytes, thus serving as guiding
strands for regenerating axons, causing significant functional recovery [64].
More intriguingly, marrow stromal cells, previously thought to differentiate
into mesenchymal lineages such as osteocytes, chondrocytes, and adipocytes,
could be induced to generate ectoderm-derived CNS cells. However, it remains
in doubt whether this so-called transdifferentiation actually occurs constantly
in vivo.
Many other related problems with SCI remain unsolved. In addition to
considering NSC transplantation, it is necessary to create a permissive
microenvironment within the site of SCI. This may entail making a biological
or other physical scaffold to facilitate the elongation of regenerating axons into
the traumatic cavity [65], eliminating axonal growth inhibitors (e.g., Nogo
[26], myelin-associated glycoprotein [66], semaphorin [67], chondroitin sul-
fate) that continue to be released from post-traumatic glial scar tissue, or
concomitantly using neurotrophic factors (e.g., neurotrophin-3, brain-derived
neurotrophic factor) [68–70] to create a permissive environment for grafting.
The most important fundamental problem continues to be how to regenerate
the chronically injured spinal cord, in terms of neuronal cell body and axonal
growth in patients with existing or long-standing injuries. More than 99% of
patients with SCI, numbering more than 100,000 in Japan and almost 250,000
medwedi.ru
in the USA, are patients with long-standing injuries. Without their recovery,
there can be no success in the treatment of SCI.
References
1 Ramon y Cajal S: Degeneration and Regeneration of the Nervous System (Translated by RM Day
from the 1913 Spanish edition). Oxford, Oxford University Press, 1928.
2 Okano H: Stem cell biology of the central nervous system. J Neurosci Res 2002;69:698–707.
3 Frisen J, Johansson CB, Torok C, Risling M, Lendahl U: Rapid, widespread, and long-lasting
induction of nestin contributes to the generation of glial scar tissue after CNS injury. J Cell Biol
1995;131:453–464.
4 Johansson CB, Momma S, Clarke DL, Risling M, Lendahl U, Frisen J: Identification of a neural
stem cell in the adult mammalian central nervous system. Cell 1999;96:26–34.
5 Namiki J, Tator CH: Cell proliferation and nestin expression in the ependyma of the adult rat
spinal cord after injury. J Neuropathol Exp Neurol 1999;58:489–498.
6 Reynolds BA, Weiss S: Generation of neurons and astrocytes from isolated cells of the adult mam-
malian central nervous system. Science 1992;255:1707–1710.
7 Reynolds BA, Tetzlaff W, Weiss S: A multipotent EGF-responsive striatal embryonic progenitor
cell produces neurons and astrocytes. J Neurosci 1992;12:4565–4574.
8 Okano H: Neural stem cells: Progression of basic research and perspective for clinical application.
Keio J Med 2002;51:115–128.
9 Davis AA, Temple S: A self-renewing multipotential stem cell in embryonic rat cerebral cortex.
Nature 1994;372:263–266.
10 Palmer TD, Markakis EA, Willhoite AR, Safar F, Gage FH: Fibroblast growth factor-2 activates a
latent neurogenic program in neural stem cells from diverse regions of the adult CNS. J Neurosci
1999;19:8487–8497.
11 Lendahl U, Zimmerman LB, McKay RD: CNS stem cells express a new class of intermediate
filament protein. Cell 1990;60:585–595.
12 Sakakibara S, Imai T, Hamaguchi K, Okabe M, Aruga J, Nakajima K, Yasutomi D, Nagata T,
KuriharaY, Uesugi S, Miyata T, Ogawa M, Mikoshiba K, Okano H: Mouse-Musashi-1, a neural RNA-
binding protein highly enriched in the mammalian CNS stem cell. Dev Biol 1996;176:230–242.
13 Weiss S, Dunne C, Hewson J, Wohl C, Wheatley M, Peterson AC, Reynolds BA: Multipotent CNS
stem cells are present in the adult mammalian spinal cord and ventricular neuroaxis. J Neurosci
1996;16:7599–7609.
14 Gritti A, Parati EA, Cova L, Frolichsthal P, Galli R, Wanke E, Faravelli L, Morassutti DJ, Roisen F,
Nickel DD, Vescovi AL: Multipotential stem cells from the adult mouse brain proliferate and self-
renew in response to basic fibroblast growth factor. J Neurosci 1996;16:1091–1100.
15 Eriksson PS, Perfilieva E, Bjork-Eriksson T, Alborn AM, Nordborg C, Peterson DA, Gage FH:
Neurogenesis in the adult human hippocampus. Nat Med 1998;4:1313–1317.
16 Pincus DW, Keyoung HM, Harrison-Restelli C, Goodman RR, Fraser RA, Edgar M, Sakakibara S,
Okano H, Nedergaard M, Goldman SA: Fibroblast growth factor-2/brain-derived neurotrophic
factor-associated maturation of new neurons generated from adult human subependymal cells. Ann
Neurol 1998;43:576–585.
17 Yamamoto S, Yamamoto N, Kitamura T, Nakamura K, Nakafuku M: Proliferation of parenchymal
neural progenitors in response to injury in the adult rat spinal cord. Exp Neurol 2001;172:115–127.
18 Nakatomi H, Kuriu T, Okabe S, Yamamoto S, Hatano O, Kawahara N, Tamura A, Kirino T,
Nakafuku M: Regeneration of hippocampal pyramidal neurons after ischemic brain injury by
recruitment of endogenous neural progenitors. Cell 2002;110:429–441.
19 Arvidsson A, Collin T, Kirik D, Kokaia Z, Lindvall O: Neuronal replacement from endogenous
precursors in the adult brain after stroke. Nat Med 2002;8:963–970.
20 Kawaguchi A, Miyata T, Sawamoto K, Takashita N, Murayama A, Akamatsu W, Ogawa M,
Okabe M, Tano Y, Goldman SA, Okano H: Nestin-EGFP transgenic mice: Visualization of the
self-renewal and multipotency of CNS stem cells. Mol Cell Neurosci 2001;17:259–273.
21 Björkund A, Stenevi U: Reconstruction of the nigrostriatal dopamine pathway by intracerebral
nigral transplants. Brain Res 1979;177:555–560.
22 Piccini P, Brooks DJ, Bjorklund A, Gunn RN, Grasby PM, Rimoldi O, Brundin P, Hagell P,
Rehncrona S, Widner H, Lindvall O: Dopamine release from nigral transplants visualized in vivo
in a Parkinson’s patient. Nat Neurosci 1999;2:1137–1140.
23 Svendsen CN, Clarke DJ, Rosser AE, Dunnett SB: Survival and differentiation of rat and human
epidermal growth factor-responsive precursor cells following grafting into the lesioned adult cen-
tral nervous system. Exp Neurol 1996;137:376–388.
24 Svendsen CN, Caldwell MA, Shen J, ter Borg MG, Rosser AE, Tyers P, Karmiol S, Dunnett SB:
Long-term survival of human central nervous system progenitor cells transplanted into a rat model
of Parkinson’s disease. Exp Neurol 1997;148:135–146.
25 Sawamoto K, Nakao N, Kakishita K, Ogawa Y, Toyama Y, Yamamoto A, Yamaguchi M, Mori K,
Goldman SA, Itakura T, Okano H: Generation of dopaminergic neurons in the adult brain
from mesencephalic precursor cells labeled with a nestin-GFP transgene. J Neurosci 2001;21:
3895–3903.
26 Richardson PM, McGuinness UM, Aguayo AJ: Axons from CNS neurons regenerate into PNS
grafts. Nature 1980;284:264–265.
27 Bregman BS, Kunkel-Bagden E, Reier PJ, Dai HN, McAtee M, Gao D: Recovery of function after
spinal cord injury: Mechanisms underlying transplant-mediated recovery of function differ after
spinal cord injury in newborn and adult rats. Exp Neurol 1993;123:3–16.
28 Cai D, Shen Y, De Bellard M, Tang S, Filbin MT: Prior exposure to neurotrophins blocks inhibi-
tion of axonal regeneration by MAG and myelin via a cAMP-dependent mechanism. Neuron
1999;22:89–101.
29 Chen MS, Huber AB, van der Haar ME, Frank M, Schnell L, Spillmann AA, Christ F, Schwab ME:
Nogo-A is a myelin-associated neurite outgrowth inhibitor and an antigen for monoclonal antibody
IN-1. Nature 2000;403:434–439.
30 Miya D, Giszter S, Mori F, Adipudi V, Tessler A, Murray M: Fetal transplants after the develop-
ment of function after spinal cord transection in newborn rats. J Neurosci 1997;17:4856–4872.
31 Diener PS, Bregman BS: Fetal spinal cord transplants support the development of target reaching
and coordinated postural adjustments after neonatal cervical spinal cord injury. J Neurosci 1998;
18:763–778.
32 Diener PS, Bregman BS: Fetal spinal cord transplants support growth of supraspinal and segmental
projections after cervical spinal cord hemisection in the neonatal rat. J Neurosci 1998;18:779–793.
33 McDonald JW, Liu XZ, Qu Y, Liu S, Mickey SK, Turetsky D, Gottlieb DI, Choi DW: Transplanted
embryonic stem cells survive, differentiate and promote recovery in injured rat spinal cord. Nat
Med 1999;5:1410–1412.
34 Vacanti MP, Leonard JL, Dore B, Bonassar LJ, Cao Y, Stachelek SJ, Vacanti JP, O’Connell F,
Yu CS, Farwell AP, Vacanti CA: Tissue-engineered spinal cord. Transplant Proc 2001;33:592–598.
35 Ogawa Y, Sawamoto K, Miyata T, Miyao S, Watanabe M, Toyama Y, Nakamura M, Bregman BS,
Koike M, Uchiyama Y, Toyama Y, Okano H: Transplantation of in vitro expanded fetal neural
progenitor cells results in neurogenesis and functional recovery after spinal cord contusion injury
in rats. J Neurosci Res 2002;69:925–933.
36 Okano H, Ogawa Y, Nakamura M, Kaneko S, Iwanami A, Toyama Y: Transplantation of neural
stem cells into the spinal cord after injury. Semin Cell Dev Biol 2003;14:191–198.
37 Yandava BD, Billinghurst LL, Snyder EY: ‘Global’ cell replacement is feasible via neural stem cell
transplantation: Evidence from the dysmyelinated shiverer mouse brain. Proc Natl Acad Sci USA
1999;96:7029–7034.
38 Takahashi M, Palmer TD, Takahashi J, Gage FH: Widespread integration and survival of adult-
derived neural progenitor cells in the developing optic retina. Mol Cell Neurosci 1998;12:340–348.
39 Tropepe V, Coles BL, Chiasson BJ, Horsford DJ, Elia AJ, McInnes RR, van der Kooy D: Retinal
stem cells in the adult mammalian eye. Science 2000;287:2032–2036.
40 Kempermann G, Kuhn HG, Gage FH: More hippocampal neurons in adult mice living in an
enriched environment. Nature 1997;386:493–495.
41 Shihabuddin LS, Horner PJ, Ray J, Gage FH: Adult spinal cord stem cells generate neurons after
transplantation in the adult dentate gyrus. J Neurosci 2000;20:8727–8735.
medwedi.ru
42 Nakamura M, Houghtling RA, MacArthur L, Bayer BM, Bregman BS: Differences in cytokine
gene expression profile between acute and secondary injury in adult rat spinal cord. Exp Neurol
2003;184:313–325.
43 Pan JZ, Ni L, Sodhi A, Aguanno A, Young W, Hart RP: Cytokine activity contributes to induction
of inflammatory cytokine mRNAs in spinal cord following contusion. J Neurosci Res 2002;68:
315–322.
44 Johe KK, Hazel TG, Muller T, Dugich-Djordjevic MM, McKay RD: Single factors direct the
differentiation of stem cells from the fetal and adult central nervous system. Genes Dev 1996;10:
3129–3140.
45 Nakashima K, Yanagisawa M, Arakawa H, Kimura N, Hisatsune T, Kawabata M, Miyazono K,
Taga T: Synergistic signaling in fetal brain by STAT3-Smad1 complex bridged by p300. Science
1999;284:479–482.
46 Miyoshi Y, Date I, Ohmoto T: Three-dimensional morphological study of microvascular regener-
ation in cavity wall of the rat cerebral cortex using the scanning electron microscope: Implications
for delayed neural grafting into brain cavities. Exp Neurol 1995;131:69–82.
47 Miyoshi Y, Date I, Ohmoto T: Neovascularization of rat fetal neocortical grafts transplanted into
a previously prepared cavity in the cerebral cortex: A three-dimensional morphological study
using the scanning electron microscope. Brain Res 1995;681:131–140.
48 Casella GT, Marcillo A, Bunge MB, Wood PM: New vascular tissue rapidly replaces neural
parenchyma and vessels destroyed by a contusion injury to the rat spinal cord. Exp Neurol 2002;
173:63–76.
49 Gloster A, Wu W, Speelman A, Weiss S, Causing C, Pozniak C, Reynolds B, Chang E, Toma JG,
Miller FD: The T-1-tubulin promoter specifies gene expression as a function of neuronal growth
and regeneration in transgenic mice. J Neurosci 1994;14:7319–7330.
50 Wang S, Wu H, Jiang J, Delohery TM, Isdell F, Goldman SA: Isolation of neuronal precursors by
sorting embryonic forebrain transfected with GFP regulated by the T-1 tubulin promoter. Nat
Biotechnol 1998;16:196–201.
51 Roy NS, Wang S, Jiang L, Kang J, Restelli C, Fraser RAR, Couldwell WT, Kawaguchi A, Okano H,
Nedergaard M, Goldman S: In vitro neurogenesis by neural progenitor cells isolated from the adult
human hippocampus. Nat Med 2000;6:271–278.
52 Sawamoto K, Yamamoto A, Kawaguchi A, Yamaguchi M, Mori K, Goldman SA, Okano H:
Visualization and direct isolation of neuronal progenitor cells by dual-color flow cytometric detec-
tion of fluorescent proteins. J Neurosci Res 2001;65:220–227.
53 Coumans JV, Lin TT, Dai HN, MacArthur L, McAtee M, Nash C, Bregman BS: Axonal regener-
ation and functional recovery after complete spinal cord transection in rats by delayed treatment
with transplants and neurotrophins. J Neurosci 2001;21:9334–9344.
54 Flax JD, Aurora S, Yang C, Simonin C, Wills AM, Billinghurst LL, Jendoubi M, Sidman RL,
Wolfe JH, Kim SU, Snyder EY: Engraftable human neural stem cells respond to developmental
cues, replace neurons, and express foreign genes. Nat Biotechnol 1998;16:1033–1039.
55 Brustle O, Choudhary K, Karram K, Huttner A, Murray K, Dubois-Dalcq M, McKay RD:
Chimeric brains generated by intraventricular transplantation of fetal human brain cells into
embryonic rats. Nat Biotechnol 1998;16:1040–1044.
56 Ourednik V, Ourednik J, Flax JD, Zawada WM, Hutt C, Yang C, Park KI, Kim SU, Sidman RL,
Freed CR, Snyder EY: Segregation of human neural stem cells in the developing primate fore-
brain. Science 2001;293:1820–1824.
57 Roy NS, Benraiss A, Wang S, Fraser RA, Goodman R, Couldwell WT, Nedergaard M,
Kawaguchi A, Okano H, Goldman SA: Promoter-targeted selection and isolation of neural
progenitor cells from the adult human ventricular zone. J Neurosci Res 2000;59:321–331.
58 Keyoung HM, Roy NS, Benraiss A, Louissaint A Jr, Suzuki A, Hashimoto M, Rashbaum WK,
Okano H, Goldman SA: High-yield selection and extraction of two promoter-defined phenotypes
of neural stem cells from the fetal human brain. Nat Biotechnol 2001;19:843–850.
59 Uchida N, Buck DW, He D, Reitsma MJ, Masek M, Phan TV, Tsukamoto AS, Gage FH,
Weissman IL: Direct isolation of human central nervous system stem cells. Proc Natl Acad Sci
USA 2000;97:14720–14725.
60 Kondo T, Raff M: Oligodendrocyte precursor cells reprogrammed to become multipotential CNS
stem cells. Science 2000;289:1754–1757.
61 Clarke DL, Johansson CB, Wilbertz J, Veress B, Nilsson E, Karlstrom H, Lendahl U, Frisen J:
Generalized potential of adult neural stem cells. Science 2000;288:1660–1663.
62 Woodbury D, Schwarz EJ, Prockop DJ, Black IB: Adult rat and human bone marrow stromal cells
differentiate into neurons. J Neurosci Res 2000;61:364–370.
63 Kopen GC, Prockop DJ, Phinney DG: Marrow stromal cells migrate throughout forebrain and
cerebellum, and they differentiate into astrocytes after injection into neonatal mouse brains. Proc
Natl Acad Sci USA 1999;96:10711–10716.
64 Hofstetter CP, Schwarz EJ, Hess D, Widenfalk J, El Manira A, Prockop DJ, Olson L: Marrow stro-
mal cells form guiding strands in the injured spinal cord and promote recovery. Proc Natl Acad
Sci USA 2002;99:2199–2204.
65 Teng YD, Lavik EB, Qu X, Park KI, Ourednik J, Zurakowski D, Langer R, Snyder EY: Functional
recovery following traumatic spinal cord injury mediated by a unique polymer scaffold seeded
with neural stem cells. Proc Natl Acad Sci USA 2002;99:3024–3029.
66 Cai D, Deng K, Mellado W, Lee J, Ratan R, Filbin M: Arginase I and polyamines act downstream
from cyclic AMP in overcoming inhibition of axonal growth MAG and myelin in vitro. Neuron
2002;35:711–719.
67 De Winter F, Oudega M, Lankhorst AJ, Hamers FP, Blits B, Ruitenberg MJ, Pasterkamp RJ,
Gispen WH, Verhaagen J: Injury-induced class 3 semaphorin expression in the rat spinal cord. Exp
Neurol 2002;175:61–75.
68 Olson L, Widenfalk J, Josephson A, Greitz D, Klason T, Kiyotani T, Lipson A, Ebendal T,
Cao Y, Hostetter C, Schwartz E, Prockop D, Manson S, Jurban M, Lindqvist E, Lundströmer K,
Nosrat C, Brene S, Spenger C: Experimental spinal cord injury models: Prospective and repair
strategies; in Ikada Y, Oshima N (eds): Tissue Engineering for Therapeutic Use, ed 5. Amsterdam,
Elsevier Science BV, 2001, pp 21–36.
69 Grill R, Murai K, Blesch A, Gage FH, Tuszynski MH: Cellular delivery of neurotrophin-3
promotes corticospinal axonal growth and partial functional recovery after spinal cord injury.
J Neurosci 1997;17:5560–5572.
70 Liu Y, Kim D, Himes BT, Chow SY, Schallert T, Murray M, Tessler A, Fischer I: Transplants of
fibroblasts genetically modified to express BDNF promote regeneration of adult rat rubrospinal
axons and recovery of forelimb function. J Neurosci 1999;19:4370–4387.
Dr. Hideyuki Okano

Department of Physiology, Keio University School of Medicine, Shinjuku
Tokyo 160-8582 (Japan)
Tel. 81 3 5363 3747, Fax 81 3 3357 5445, E-Mail hidokano@sc.itc.keio.ac.jp
medwedi.ru
Functional and Restorative Molecular Neurosurgery

Contemporary Applications of
Functional and Stereotactic
Techniques for Molecular Neurosurgery
Paul A. House, Ganesh Rao, William Couldwell
Department of Neurological Surgery, University of Utah Medical Center,
Salt Lake City, Utah, USA
Introduction
Tremendous advances have been made in understanding the molecular basis

of many neurological diseases. Although the molecular biology of brain tumors
and neurodegenerative diseases has become better understood, utilizing this
information to achieve improved therapeutic results remains a challenge. In
some neurological diseases, the dysfunction of specific neuroanatomic sites is
primarily responsible for a disease process, for example, degeneration of
dopaminergic neurons of the substantia nigra pars compacta in Parkinson’s dis-
ease. Other diseases are more diffuse; for example, a glioblastoma multiforme
(GBM) may have tumor cells several centimeters away from the primary tumor
focus. Each of these scenarios calls for a different type of treatment approach,
either delivering therapy locally or diffusely. In this chapter, we discuss strategies
that are currently under investigation for delivery of drug or molecular-cellular
treatments.
Basic Treatment Approaches to Neurological Disease
Neuro-Oncology
Treatment of brain tumors typically involves some combination of surgi-
cal resection, radiotherapy, and chemotherapy. Surgical resection has been
shown to improve survival in certain tumors such as GBM, whereas others are
definitively treated by radiation, such as germinoma. Still others, such as
oligodendroglioma, respond very favorably to chemotherapy. Despite
advances in these classes of treatment, the survival for patients with most pri-
mary brain tumors remains poor. The survival rate for GBM, the most com-
mon primary brain tumor, has not improved in over a decade. Improved
strategies for treating GBM must address the diffuse nature of intrinsic brain
tumors. Tumor cells spread widely along white matter tracts and can be found
on the contralateral hemisphere from a primary tumor focus. Definitive ther-
apy for primary brain tumors will require treatments such as well-targeted
inactivation of aberrantly expressed oncogenes or re-establishment of the
activity of lost or nonfunctioning tumor suppressor genes.
Neurodegenerative Diseases
Neurodegenerative diseases such as Alzheimer’s disease involve a contin-
uous loss of neurons. In Alzheimer’s disease, diffuse loss of neurons in the cor-
tex as well as basal structures such as the locus ceruleus and nucleus basalis is
characteristic. The same widespread loss of neurons holds true for Parkinson’s
disease, in which dopaminergic neurons are depleted, and Huntington’s disease
in which striatal neurons are lost. In the case of these disorders, dysfunction of
specific neuroanatomic structures must be addressed. Other neurodegenerative
diseases such as amyotrophic lateral sclerosis involve degeneration of anterior
horn cells throughout the spinal cord, providing a unique therapeutic challenge.
Definitive therapy for all of these disorders is likely to involve grafting of cells
to restore function, along with approaches to deliver local trophic and growth
factors.
Spinal Cord Injury

Molecular underpinnings of the normal healing process following spinal
cord injury suggest that a variety of steps in the healing cascade may be
amenable to intervention. While some forms of intervention for spinal cord
injury, such as corticosteroids, can be delivered systemically, future therapies
will involve proteins and small molecules that need to be delivered locally.
Although not classically considered in the realm of functional neurosurgery, the
need for targeted local therapy in the spinal cord may expand the role of the
functional neurosurgeon.
At least three types of axon-inhibiting molecules present in the myelin of
the injured spinal cord have now been characterized. Two separate types of
myelin-associated glycoproteins named Nogo and MAG, along with oligoden-
drocyte-myelin glycoprotein, have been shown to inhibit growth cones [1–3]. It
has been demonstrated that blocking Nogo-A with a monoclonal antibody
(IN-1) leads to enhanced long fiber tract regeneration in spinal cord injury
models [4]. Because diffusely blocking Nogo-A leads to sprouting of uninjured
Techniques for Molecular Neurosurgery 125
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axons in the intact central nervous system (CNS) [5], it is likely that blocking
antibodies will need to be delivered in a focused manner to patients with injured
spinal cord to avoid diffuse axonal sprouting. Similarly, blocking the receptors
to these glycoproteins or blocking the signaling cascade they induce will likely
be treated best in a focused manner. Neurosurgeons will be required to develop
and implement these technologies in the clinic.
New Approaches to Drug Delivery in the Brain
Gene Therapy
Advances in understanding the genetic aberrations leading to GBM have
provided new therapeutic targets. For example, loss of the tumor suppressor
phosphatase and tensin homolog has been shown to be an important event in the
development of GBM [6–9]. Restoration of the function of such tumor sup-
pressor genes has shown promise in vitro. Typically, these therapeutic genes are
delivered via adenoviral or retroviral vectors. Transduction of functional phos-
phatase and tensin homolog into glioma cell lines has been shown to reduce the
proliferation of tumor cells [10]. Other successes have been obtained by intro-
ducing herpes simplex virus type 1 thymidine kinase into glioma cell lines and
inducing cytotoxicity with gancyclovir or other prodrugs [10–15]. Although
many promising therapies have been successful in the laboratory, improvement
in patients has been significantly less dramatic. Translating in vitro successes
to human patients remains a challenge and there are risks inherent to this type
of treatment [16–18].
Antisense gene therapy is being developed for therapy in which overex-
pression of cancer-promoting genes (e.g., oncogenes) plays a role in tumor pro-
gression. The simplest antisense constructs utilize an oligonucleotide sequence
in complementary orientation to a target gene, and the antisense cDNA binds
to a target DNA or mRNA and prevents transcription or translation. Antisense
therapy has been used on glioma targets with some success. For example,
matrix metalloproteinase-9 (MMP-9) has been shown to be important for
cell migration and invasion of gliomas. Antisense constructs targeted against
MMP-9 in both in vitro and in vivo models have shown regression of tumor
growth [19]. Similar preclinical results have been obtained with the delivery
of antisense constructs directed against epidermal growth factor receptor gene,
which is upregulated in gliomas. Another new antisense therapy involves the
use of short interfering RNA, which can bind to mRNA and cause degradation,
known as RNA interference [20].
The major obstacle to gene therapy relates to inefficient delivery to the
CNS. While many gene therapy techniques utilizing adenoviral or retroviral
House/Rao/Couldwell 126
Fig. 1. Localization of GPi with CED.
vectors have been successful in cell lines, targeted delivery in the clinical set-
ting remains quite difficult. The past failures of cancer gene therapy are mainly
due to the poor delivery of the gene to tumor cells, and the method of manual
injection of vector-producing cells limits the distribution of these cells [14].
The development of newer vectors such as recombinant adeno-associated virus
(AAV) has provided hope for in vivo delivery. AAV is based on a nonpatho-
genic, replication-defective virus and has been used successfully for efficient
and sustained gene transfer to proliferating and differentiated cells without a
detectable immune response or toxicity [21–23]. AAV has been shown to be
effective for long-term delivery of genes at biologically relevant levels in both
the CNS and intramuscularly [22, 24–27]. There may be limitations to this vec-
tor, although it has safety advantages over other adenoviral or retroviral vectors
[22, 23, 28].
Convection-Enhanced Delivery
One of the more promising techniques for clinical drug delivery to the
brain is convection-enhanced delivery (CED) or high-flow microinfusion. CED
involves placement of an infusion catheter directly into the brain parenchyma
and relies on bulk flow (as opposed to passive diffusion) through the CNS
parenchyma, thus bypassing the blood brain barrier. Although technical issues
remain (e.g., cannula size, location of the infusion pump, cellular damage
caused by high flow rates), there are distinct advantages over conventional
medwedi.ru
Fig. 2. Targeted brainstem delivery with CED.
intravenous chemotherapy, intraventricular delivery, or drug-impregnated poly-

mer-based therapy. CED allows for significantly higher concentrations of phar-
macotherapy to be delivered to a larger volume of brain tissue, with important
applications in neurodegenerative and neuro-oncological diseases. Stereo-
tactically implanted catheters may be targeted at specific structures. For exam-
ple, high-flow microinfusion of the caudate with biotinylated dextran has been
performed successfully with very little spillover into the adjacent structures
[29]. Similar perfusion of the globus pallidus has also been achieved (fig. 1)
[30]. Indirect targeting through axonal tracts also has been shown by infusion
of the striatum in rats, with subsequent identification of the infusate in the sub-
stantia nigra pars compacta [29]. These findings have therapeutic implications
for the treatment of neurodegenerative diseases such as Parkinson’s or
Huntington’s.
CED also shows promise for diffuse neoplastic disease as there is spread
of the infusate along white matter tracts. It can be used relatively safely in the
cerebral hemispheres, brainstem, and spinal cord (fig. 2) [31]. Preclinical test-
ing has demonstrated a survival advantage in C6 glioma-bearing rats treated
with BCNU or toptecan delivered via CED [32, 33]. This observation has been
extended to human trials, which take advantage of the overexpression of trans-
ferrin, interleukin-13, or interleukin-14 receptors on glioma cells. By linking a
toxic compound (such as Pseudomonas exotoxin) to a ligand specific for these
receptors, glioma cells can be targeted for specific destruction [34, 35]. CED
lends itself nicely to this technique, and recent trials have shown some promise
for this route of delivery.
Stem Cell Therapy
Stem cell therapy has been touted as a potential treatment for neurode-
generative diseases such as Parkinson’s disease, Huntington’s disease, and
Alzheimer’s disease. It is now well established that neural stem cells possess
the ability to differentiate into any of the various cell types in the CNS.
Transplantation of fetal dopamine cells into rat models of Parkinson’s disease
was successfully started decades ago [36–39]. These experiments showed that
transplanted dopaminergic cells could survive and function in vivo. Fetal cell
transplantation has been performed in humans with some success, and in some
cases caused a huge improvement in Parkinsonian symptoms [40]. There are,
however, serious difficulties with current fetal dopamine cell transplants. First,
recovery of these cells from aborted fetuses is expensive and although it is
legal, there are ethical considerations in ramping up production of these cells
from primary sources. Further, the survival rate of these transplants can be
quite poor with the majority of cells undergoing apoptosis in the absence of
immunosuppression.
Embryonic stem cells (ES cells) offer a more attractive source of
dopaminergic cells, as these are available from any number of established
ES cell lines and could be genetically engineered to match a host through new
techniques of somatic cell nuclear transfer. Transplantation of ES cells is still
problematic regarding control of cell growth and differentiation, as well as hav-
ing a sufficient quantity of cells to transplant. In rat models of Parkinson’s dis-
ease many ES cells will not survive in situ, and up to 20% will differentiate into
lethal teratomas [41]. These problems are being addressed by allowing some
cellular differentiation to occur in vitro prior to transplantation. Investigators
are currently attempting to develop cell lines of dopamine-producing ES cells
using gene transfer with prodopaminergic genes (e.g., Nurr1) or treatment with
soluble signaling factors (e.g., epidermal growth factor, insulin-like growth
factor). Also, it may be possible to develop specific neuronal cells from other
stem cells located elsewhere in the body (e.g., blood, bone marrow, skin) for
purposes of autotransplantation.
Growth Factors as a Therapeutic Strategy

Growth factors used to promote functional restoration of neurons that are
affected in neurodegenerative diseases include glial-derived neurotrophic factor
(GDNF), ciliary neurotrophic factor, brain-derived growth factor, and insulin-
like growth factor-1. GDNF in particular has shown clinical promise for neural
repair. Described as the most potent neurotrophic factor for motoneurons, it was
tested heavily beginning in the mid 1990s. In vitro studies showed that GDNF
promoted the survival of motoneurons [42–44]. However, because of difficulties
with delivery, short half-life of the recombinant protein, and various inflammatory
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effects, clinical trials met with poor success and side effects [22, 45]. The com-
bination of growth factor genes and viral vectors such as AAV has restored hope
for its use in neurodegenerative disorders. For example, an AAV-GDNF con-
struct injected intramuscularly into mice has been shown to result in sustained
expression of transgenic GDNF and is delivered via retrograde transport to
spinal motoneurons [22]. This type of therapy holds promise for the treatment of
motor neuron diseases such as amyotrophic lateral sclerosis, because GDNF
expression in the muscles of transgenic amyotrophic lateral sclerosis mice has
improved their survival [24].
Molecular Therapies for Spinal Cord Injury

Neurotrophins have been investigated for their ability to allow regenerating
axons to cross the area of an injured spinal cord. For example, neurotrophin-3
promotes axon sprouting through the gray matter in lesioned spinal cords when
delivered continuously via a fibroblast-producing cell line [46]. Functional
neurosurgeons will need to become involved in the development of new drug
delivery systems to provide such sustained levels of neurotrophins to the sites
of injury. As with the blocking of inhibiting epitopes in the injured spinal cord,
delivery of neurotrophins to the injured spinal cord could possibly utilize CED
[47]. Osmotic pumps have been used in experimental animals to deliver some
of these small molecules and could perhaps be adapted for the purpose in
human subjects. A variety of biomaterials are being developed that might not
only deliver the needed concentration gradients but also provide a permissive
substrate for axon regrowth [48]. The transplantation of stem cells of multiple
lineages also holds promise in providing permissive microenvironments for
spinal cord regeneration.
Novel Surgical Techniques for Spinal Cord Injury
While a progressive understanding of the molecular biology of spinal cord

injury will provide new avenues to aid recently injured patients, those with pre-
existing spinal cord deterioration suffer from a host of secondary complications
for which neuro-augmentative surgery could provide functional improvement.
Many augmentative technologies and techniques would also benefit patients
with progressive degenerative disease. For example, loss of bowel and bladder
control is often cited as one of the most disabling complications of diplegia or
tetraplegia. Indeed, chronic hydronephrosis and secondary infections are often
the ultimate cause of death for those who are disabled. Anterior sacral root stim-
ulation combined with dorsal rhizotomy to treat the neurogenic bladder is the
most widely used neurosurgical method employing a neuroprosthetic device to
augment bladder function. This implantation technique yields full continence in
the majority of patients [49] and has been found to be cost effective [50]. This
system has limitations, however, including side effects on sexual function,
prompting further investigations into mechanisms that allow specific stimula-
tion of coordinated bladder emptying as well as continence. In the decerebrate
cat, stimulation of the dorsolateral funiculus within the lower thoracic spinal
cord (T9–T13) has been shown to produce coordinated bladder contraction with
decreased urethral sphincter tone [51]. If a coordinated control center can be
found in the human spinal cord, it may be possible to produce a microelectrode
system for bladder control. The functional neurosurgeon, already adept at
microelectrode recording and targeting, will be needed to help overcome tech-
nical hurdles and make such a system possible. Although therapy to treat seque-
lae of spinal cord injury may seem like a small goal compared to the ultimate
goal of restoring total spinal cord function, it may be a more achievable goal
and would definitely enhance quality of life until true spinal cord repair is pos-
sible, which may take decades of further work and refinement.
Advancing bionic technologies also present an area where neurosurgical
expertise could lead to the development of new human-prosthetic interfaces,
which would greatly augment the functional capacities of those with disabili-
ties. Commercial examples of advancements in this area include the ‘iBot
Mobility System’ (Independence Technology, LLC) which incorporates gyro-
scopic guidance into a wheelchair design, allowing the system to climb stairs
and balance on two wheels. A similar capability-expanding peripheral device in
development is called ‘Robowalker’ (Yobotics, Inc.). This exoskeleton-type
device could greatly enhance the mobility of those with lower extremity weak-
ness or limited leg control. These technologies demonstrate how sophisticated
movements can be controlled using only a limited amount of input information,
namely, the leaning body weight of a patient lacking full mobility. Other
devices, which rely on tracking eye movement to initate and control move-
ments, are in development for the patient with spinal cord injury.
Neuro-Prosthetic Therapies
For patients with severe neurodegenerative disorders or severe CNS

injury, one factor limiting the utilization of new forms of augmentative tech-
nology is the difficulty in providing communication between the device and
the injured CNS. Providing simple two-dimensional directional control has
been achieved by direct implantation of a microelectrode into the human motor
cortex (fig. 3) [52]. This system provides a brain-computer interface by hav-
ing the computer learn to recognize firing patterns of motor cortex associated
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I
II
III
IVA
IVB
IVC ␣
␤
VI
2 mm
Fig. 3. One device which has been designed to serve as a brain-machine interface is
the Utah Microelectrode Array. A 100 electrode array is pictured above next to a penny for
size comparison. The electrode tips have been machined to penetrate neocortex to layer IV.
An electron micrograph of the 4 ⫻ 4 mm array is below.
with direction-specific movement. A similar system utilizing a vastly
increased number of microelectrodes allowed a monkey to remotely control a
robot arm in a three-dimensional reaching task [53]. These experiments out-
line a strategy that may be employed in the future to extend cognitive control
of artificial limbs or mobility-extending devices to patients with very high cer-
vical cord injuries. A host of technical difficulties must still be overcome to
allow even rudimentary cognitive control of artificial limbs, but there is cer-
tainly reason for hope [54]. Current technology allows the simultaneous
recording of approximately one hundred neurons through implanted micro-
electrodes or microelectrode arrays [55]. Perhaps surprisingly, real-time analy-
sis of only fifty to one hundred motoneurons was sufficient to reproduce the
three-dimensional arm movements previously discussed.
The first devices capable of directly recording electrical information from
the brain were developed in the 1950s as external electroencephalography
recorders. These devices provide limited spatial resolution but are noninvasive.
Through ‘bio-feedback’, patients can operate simple one-variable devices in
controlled situations. The signal resolution from surface recordings, however, is
too limited to provide driving information for even the simplest artificial limb
system. Subdural electrode grids, already widely used as monitoring devices in
the evaluation of epilepsy, deliver finer spatial resolution than superficial
devices. The possibility of implanting subdural grids was a strategy utilized to
provide the first cortical stimulation systems designed to deliver visual infor-
mation to the blind [56]. Such systems are able to provide enhanced communi-
cation with the CNS compared with surface EEG. Yet, each subdural electrode
is affected by many thousands of neurons and spatial resolution is still too
limited to drive most useful artificial limb systems. One benefit of limited reso-
lution is the need to transfer only a limited amount of information to a record-
ing/interpreting computer. Progress on telemetry systems now allows
continuous neuronal recordings to be obtained from an entirely implanted sys-
tem, although limited to only two electrodes [57]. Specially designed integrated
circuits and telemetry devices continue to be developed, but so far, such devices
are not able to provide continuous telemetry of implanted multielectrode
subdural systems.
To provide the cortical spatial resolution needed to drive an artificial limb
or replace sensory information in a detailed manner, several varieties of high-
density microelectrode systems are in development. The Utah microelectrode
array provides 100 electrode contacts at 400-micron spacing [58]. A micro-
electrode system developed at the University of Michigan also provides multi-
ple sites of electrode contacts along each electrode [59]. Each system utilizes
silicon semiconductor fabrication processes producing a robust interface with
high biocompatibility.
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As the ability to communicate with the CNS becomes progressively refined,
a host of prosthetic devices will be introduced for clinical application. The
cochlear implant system is a good example of a device that has been developed
in the past to restore a sense in a functional way [60]. Prosthetics designed to
restore vision to the blind are in development and often receive much attention
from the popular press [61]. It may also become possible to restore somatosen-
sory function to those utilizing artificial limbs. Interpreting neuronal coding sig-
nals offers hope to those with little ability to control their bodies or outside
situations. Another application of these devices is implantable stimulators, which
have been designed to abort epileptic activity [62]. It may even become possible
to artificially replicate portions of the CNS function, such as hippocampal input
[63], through implantable/programmable neurostimulator devices, or to simulate
axonal connections between brain regions with coordinated stimulators in more
than one brain region.
Intraoperative Navigation and Imaging
The application of intraoperative navigation has continued to evolve with

new technology. Routine intraoperative navigation is now employed at most
surgical centers, and many systems are commercially available. Most of these
systems utilize archived data sets (CT/MRI/angiography) to provide target
localization. The most important advances made over the past decade have been
with the application of intraoperative imaging to provide real-time feedback to
the operating surgeon. This is especially important for those instances in which
shifts of important structures occur which render archived data inaccurate, such
as in the resection of large intra-axial tumors.
Refinements in real-time imaging of intracranial tumors are valuable to
neurosurgeons in maximizing resections in a safe manner. The most contem-
porary method of imaging refinement is in the application of CT [64] and MRI
to the operating room environment. The superior soft-tissue resolution of MRI
over CT has made it the preferred intraoperative imaging machine in most
institutions. The advantages of intraoperative MRI (iMRI) will be likely to
make this an ubiquitous feature in neurosurgical operating rooms. Since their
introduction into surgical practice in the mid 1990s, iMRI systems have
allowed the delineation of the lesion, including ‘under the surface’ vision, and
obtained real-time feedback of the extent of resection and the position of any
residual tumor tissue. High-performance computing has extended the capabil-
ities of iMRI with multimodal information and three-dimensional reconstruc-
tions [65]. One of the major issues surrounding the use of intraoperative
magnets is the safety and ease-of-use considerations for the surgeon, nurses,
anesthesiologist, and patient. Safe working environment demands the use of
MR-compatible instruments, head holders, and anesthesia equipment with
most machines. These safety issues have been well reviewed recently by
Russell [66].
Improvements in Surgical Outcomes

In contemporary series, both low- and high-field iMRI have had a positive
impact on patient care, maximizing tumor resection, and shortening length of
stay. In a report by Schulder and Carmel [67], iMRI-guided resection of tumors
in 112 patients resulted in additional tumor removal in 36%. In another 31%,
imaging confirmed that the goals of surgery had been attained, so potentially
harmful further dissection in and around the brain was avoided. iMRI offers the
possibility of further tumor removal during the same surgical procedure in case
of tumor remnants, increasing the rate of complete tumor removal. The effects
of brain shift can be compensated by using intraoperative imaging data for
updating. This capability is especially important in cases involving intrinsic
tumor surgery (especially low-grade tumors), and in skull-base tumors in which
direct surgical view is not possible (e.g., large pituitary tumors with suprasellar
extension).
Most current systems combine the advantages of intracranial computer-
assisted cranial navigation with real-time or intermittent intraoperative imaging
to verify location and tumor resection status. Various other indications for the
use of iMRI as a surgical adjunct include iMRI-guided instillation of phospho-
rous-32 for cystic craniopharyngiomas [68], monitoring resection of epilepsy
foci, and resection of vascular lesions (AVM and cavernous malformations).
Combining iMRI with a comprehensive neuronavigation environment with the
use of ultrasound, cortical stimulation, and navigation system-guidance of
biopsy probes, instruments, and endoscopy has been described [69]. iMRI has
been used with planned adjuvant radiosurgical treatment [70]. The emerging
use of combining functional MRI with diffusion-weighted imaging to provide
the anatomical detail of cortical and subcortical white matter tracts will
enhance safe and complete resections of tumors adjacent to eloquent regions of
the brain.
Costs of Imaging Technology

A concern of many centers is the cost involved with the establishment
of an iMRI program. The Department of Neurosurgery at the University
of Minnesota recently published a retrospective cost comparison of the costs
and benefits of brain tumor resection in a conventional operating room and
those associated with the iMRI suite [71]. A comparison of the length of stay,
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Fig. 4. The low-field Odin 0.12 T Polestar system. This unit may be swung into the
operative field at any time imaging is required. Photo courtesy of Odin company.
hospital charges and payments, hospital direct and indirect costs, readmission
rates, repeat resection interval, and net health outcome was performed between
the patients cared for in the two operating environments. The authors noted a
reduced length of stay, reduced repeat-resection interval, and reduced hospital
charges and costs. Other centers, such as the University of Cincinnati College
of Medicine in Ohio, have utilized a shared-resource MRI, in which the suite
functions to provide both neurosurgical and diagnostic procedures in a single
unit [72]. The open low-field (0.3 T) Hitachi unit is used for diagnostic studies
when not being used for neurosurgical cases. The ability to perform diagnostic
procedures in a shared unit has been a cost-effective solution for this particular
institution.
iMRI System Options

There are several different options for iMRI application in the contemporary
operating room environment. These include the use of low- or high-magnetic
field strength units. There are also different solutions to the layout of the operat-
ing room and the concessions made to be able to image in the OR environment.
The most common iMRI systems are designated low-field systems. Systems
include a Siemens (Erlangen, Germany) 0.2-Tesla Magnetom Open unit [73, 74]
or Odin 0.12-Tesla Polestar system [67, 69, 75, 76] (fig. 4). These systems have
Fig. 5. High-field intraoperative MRI machine in use at the University of Calgary is a
ceiling-mounted system on rails. Photo courtesy of University of Calgary, Department of
Neurosurgery.
gained widespread use and experience in multiple centers, largely for reasons
of reduced cost and ease of implementation with minimal operating room
renovations necessary. Room shielding requirements are minimized, and some
systems require no modification with the use of a portable shielding apparatus
that may be brought over the patient and machine when in use. One of the early
iterations of a low-field designated intraoperative unit was the GE Signa SP sys-
tem, which enables operating within the open magnet [77]. Such a system has the
advantage of performing continuous real-time or periodic imaging. The open-
configuration MRI installed at the Brigham and Women’s Hospital in Boston has
been in use since the mid 1990s [78]. Since that time neurosurgeons at that center
have gained experience with over 500 craniotomies and 100 biopsies. The advan-
tage of such a system is that it allows real-time imaging; disadvantages are
somewhat restricted surgeon and patient positioning and the necessity to utilize
MRI-compatible instruments.
Higher field strength magnets are increasing in popularity, to enable
acquisition of improved quality images. They also enable the use of expanded
MR capabilities such as MR spectroscopy and functional MRI. There are sev-
eral systems available. One well-designed system is the moveable high-field
(1.5 T) magnet that is located on a roller system fixed to the ceiling as
employed at the University of Calgary, Canada [79] (fig. 5). This configura-
tion is similar to the operating microscope and other surgical adjuncts, with
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MR technology moved to and from the patient as needed. Their system has
been used to monitor a variety of neurosurgical procedures, including tumors,
epilepsy, AVMs and other vascular malformations, and some cervical spine
disorders.
Another clever adaptation for the use of a high-field unit is the recent
introduction of the Siemens 1.5 T intraoperative magnet (fig. 6). The
machine is a standard 1.5 T MRI with functional MRI and MR spectroscopy
capability. The room is designed to accommodate the 1.5 T machine with
minimal disruption to the standard neurosurgical operating environment,
including the use of standard operating instruments and microscope, which
are located outside the 5 Gauss line. This enables the use of standard neuro-
surgical instrumentation, microscope, and image guidance systems. The
patient is placed on a mobile operating table which is then rotated to fit on to
the gantry of the MRI when imaging is desired. The machine has capabilities
for intraoperative MR spectroscopy and functional MRI. There is no imped-
iment to the operating surgeon, and operative positioning is independent of
the scanning.
Future Considerations
Future developments in imaging will include more use of advanced MRI
capabilities such as spectroscopy and functional MRI for intraoperative deci-
sion-making [80]. Also several centers are now planning for the adaptation of
higher field strength magnets (e.g., 3 T) to the operating room environment.
The introduction of MR-compatible robotic surgery with integration of robotic
technology to the MR environment is an area that will help to revolutionize
the future of neurosurgery, including the ability to locate and target a variety of
deep structures in the brain. When combined with advances in viral gene
Fig. 6.a The current high-field MRI Siemens system, demonstrating the ability of the
surgeons to operate outside the 5 G line and use standard surgical instruments and microscope.
Photo courtesy of Christopher Nimsky, MD, Department of Neurosurgery, Erlangen,
Germany. b Schematic representation of the operating room layout for use of the Siemens
1.5 T machine with surgeons, nurses, and anesthetists positioned outside the 5 G line.
c Pictures of the operating table rotating into position for intraoperative image acquisition.
d(i) An example of image quality. Pre- and postresection T1-weighted images of a patient with
a pituitary macroadenoma. d(ii) The use of iMRI facilitates glioma resection. Shown are com-
parative pre- and intraoperative images demonstrating the use of both T1-enhanced and T2
images to assess extent resection. e Standard image-guidance systems may also be employed
in conjunction with intraoperative imaging. In this picture, the registration fiducials for a stan-
dard image guidance system are pictured.
6a
Anesthesia
Anesthesia
preparation
5G
-lin
e
Cam
era
NC
MR-
4
RF-cabin console
6b
6c
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6d(i)
6d(ii)
6e
transfer, stem cell engineering, drug delivery devices, and neuroprosthetics,

these complimentary technologies will allow precise targeting and delivery of
molecular neurosurgical drugs to the brain and spinal cord.
References
1 Wang KC, Koprivica V, Kim JA, Sivasankaran R, Guo Y, Neve RL, He Z: Oligodendrocyte-myelin
glycoprotein is a Nogo receptor ligand that inhibits neurite outgrowth. Nature 2002;417:941–944.
2 Mukhopadhyay G, Doherty P, Walsh FS, Crocker PR, Filbin MT: A novel role for myelin-associated
glycoprotein as an inhibitor of axonal regeneration. Neuron 1994;13:757–767.
3 Caroni P, Schwab ME: Antibody against myelin-associated inhibitor of neurite growth neutralizes
nonpermissive substrate properties of CNS white matter. Neuron 1988;1:85–96.
4 Schnell L, Schwab ME: Axonal regeneration in the rat spinal cord produced by an antibody
against myelin-associated neurite growth inhibitors. Nature 1990;343:269–272.
5 Buffo A, Zagrebelsky M, Huber AB, Skerra A, Schwab ME, Strata P, Rossi F: Application of
neutralizing antibodies against NI-35/250 myelin-associated neurite growth inhibitory proteins to
the adult rat cerebellum induces sprouting of uninjured purkinje cell axons. J Neurosci 2000;20:
2275–2286.
6 Smith JS, Tachibana I, Passe SM, Huntley BK, Borell TJ, Iturria N, O’Fallon JR, Schaefer PL,
Scheithauer BW, James CD, Buckner JC, Jenkins RB: PTEN mutation, EGFR amplification, and
outcome in patients with anaplastic astrocytoma and glioblastoma multiforme. J Natl Cancer Inst
2001;93:1246–1256.
7 Fan X, Aalto Y, Sanko SG, Knuutila S, Klatzmann D, Castresana JS: Genetic profile, PTEN muta-
tion and therapeutic role of PTEN in glioblastomas. Int J Oncol 2002;21:1141–1150.
8 Frisk T, Foukakis T, Dwight T, Lundberg J, Hoog A, Wallin G, Eng C, Zendenius J, Larsson C:
Silencing of the PTEN tumor-suppressor gene in anaplastic thyroid cancer. Genes Chromosomes
Cancer 2002;35:74–80.
medwedi.ru
9 Fults D, Pedone C: Immunocytochemical mapping of the phosphatase and tensin homolog
(PTEN/MMAC1) tumor suppressor protein in human gliomas. Neuro-oncol 2000;2:71–79.
10 Cheney IW, Johnson DE, Vaillancourt MT, Avanzini J, Morimoto A, Demers GW, Wills KN,
Shabram PW, Bolen JB, Tavitgian SV, Bookstein R: Suppression of tumorigenicity of glioblas-
toma cells by adenovirus-mediated MMAC1/PTEN gene transfer. Cancer Res 1998;58:
2331–2334.
11 Ikenaka K, Sasaki M, Tamura K, Tamura M, Miyao Y, Nanmoku K, Kawano Y, Nakahira K,
Yoshimine T, Shimizu K: Treatment of glioblastoma by direct inoculation of concentrated high
titer-recombinant retrovirus carrying the herpes simplex virus thymidine kinase gene. Hum Cell
2001;14:49–58.
12 Glenn GM, Chatterjee S: Generation of adenoviruses encoding the herpes simplex virus vhs gene:
A novel strategy to generate adenoviruses expressing genes toxic to producer cells. Cancer Gene
Ther 2001;8:566–572.
13 Maleniak TC, Darling JL, Lowenstein PR, Castro MG: Adenovirus-mediated expression of
HSV1-TK or Fas ligand induces cell death in primary human glioma-derived cell cultures that are
resistant to the chemotherapeutic agent CCNU. Cancer Gene Ther 2001;8:589–598.
14 Rainov NG: A phase III clinical evaluation of herpes simplex virus type 1 thymidine kinase and
ganciclovir gene therapy as an adjuvant to surgical resection and radiation in adults with previ-
ously untreated glioblastoma multiforme. Hum Gene Ther 2000;11:2389–2401.
15 Voges J, Weber F, Reszka R, Sturm V, Jacobs A, Heiss WD, Wiestler O, Kapp JF: Clinical protocol.
Liposomal gene therapy with the herpes simplex thymidine kinase gene/ganciclovir system for the
treatment of glioblastoma multiforme. Hum Gene Ther 2002;13:675–685.
16 Wade N: Death leads to concerns for future of gene therapy. NY Times (Print) 1999:A22.
17 Smith L, Byers JF: Gene therapy in the post-Gelsinger era. JONAS Health Law Ethics Regul
2002;4:104–110.
18 Dettweiler U, Simon P: Points to consider for ethics committees in human gene therapy trials.
Bioethics 2001;15:491–500.
19 Lakka SS, Rajan M, Gondi C, Yanamandra N, Chandrasekar N, Jasti SL, Adachi Y, Siddique K,
Gujrati M, Olivero W, Dinh DH, Kouraklis G, Kyritsis AP, Rao JS: Adenovirus-mediated expres-
sion of antisense MMP-9 in glioma cells inhibits tumor growth and invasion. Oncogene 2002;21:
8011–8019.
20 Hemmings-Mieszczak M, Dorn G, Natt FJ, Hall J, Wishart WL: Independent combinatorial effect
of antisense oligonucleotides and RNAi-mediated specific inhibition of the recombinant rat
P2X(3) receptor. Nucleic Acids Res 2003;31:2117–2126.
21 Bueler H: Adeno-associated viral vectors for gene transfer and gene therapy. Biol Chem 1999;
380:613–622.
22 Lu YY, Wang LJ, Muramatsu S, Ikeguchi K, Fujimoto K, Okada T, Mizukami H, Matsushita T,
Hanazono Y, Kume A, Nagatsu T, Ozawa K, Nakano I: Intramuscular injection of AAV-GDNF
results in sustained expression of transgenic GDNF, and its delivery to spinal motoneurons by
retrograde transport. Neurosci Res 2003;45:33–40.
23 Okada T, Nomoto T, Shimazaki K, Lijun W, Lu Y, Matsushita T, Mizukami H, Urabe M, Hanazono
Y, Kume A, Muramatsu S, Nakano I, Ozawa K: Adeno-associated virus vectors for gene transfer
to the brain. Methods 2002;28:237–247.
24 Wang L, Muramatsu S, Lu Y, Ikeguchi K, Fujimoto K, Okada T, Mizukami H, Hanazono Y, Kume
A, Urano F, Ichinose H, Nagatsu T, Nakano I, Ozawa K: Delayed delivery of AAV-GDNF prevents
nigral neurodegeneration and promotes functional recovery in a rat model of Parkinson’s disease.
Gene Ther 2002;9:381–389.
25 Kirik D, Rosenblad C, Bjorklund A, Mandel RJ: Long-term rAAV-mediated gene transfer of
GDNF in the rat Parkinson’s model: Intrastriatal but not intranigral transduction promotes func-
tional regeneration in the lesioned nigrostriatal system. J Neurosci 2000;20:4686–4700.
26 Kirik D, Georgievska B, Rosenblad C, Bjorklund A: Delayed infusion of GDNF promotes recov-
ery of motor function in the partial lesion model of Parkinson’s disease. Eur J Neurosci 2001;
13:1589–1599.
27 Mandel RJ, Snyder RO, Leff SE: Recombinant adeno-associated viral vector-mediated glial
cell line-derived neurotrophic factor gene transfer protects nigral dopamine neurons after
onset of progressive degeneration in a rat model of Parkinson’s disease. Exp Neurol 1999;160:
205–214.
28 Wright JF, Qu G, Tang C, Sommer JM: Recombinant adeno-associated virus: Formulation chal-
lenges and strategies for a gene therapy vector. Curr Opin Drug Discov Devel 2003;6:174–178.
29 Lieberman DM, Laske DW, Morrison PF, Bankiewicz KS, Oldfield EH: Convection-enhanced
distribution of large molecules in gray matter during interstitial drug infusion. J Neurosurg 1995;
82:1021–1029.
30 Lonser RR, Corthesy ME, Morrison PF, Gogate N, Oldfield EH: Convection-enhanced selective
excitotoxic ablation of the neurons of the globus pallidus internus for treatment of parkinsonism
in nonhuman primates. J Neurosurg 1999;91:294–302.
31 Laske DW, Youle RJ, Oldfield EH: Tumor regression with regional distribution of the targeted
toxin TF-CRM107 in patients with malignant brain tumors. Nat Med 1997;3:1362–1368.
32 Bruce JN, Falavigna A, Johnson JP, Hall JS, Birch BD, Yoon JT, Wu EX, Fine RL, Parsa AT:
Intracerebral clysis in a rat glioma model. Neurosurgery 2000;46:683–691.
33 Kaiser MG, Parsa AT, Fine RL, Hall JS, Chakrabarti I, Bruce JN: Tissue distribution and antitu-
mor activity of topotecan delivered by intracerebral clysis in a rat glioma model. Neurosurgery
2000;47:1391–1398; discussion 1398–1399.
34 Recht L, Torres CO, Smith TW, Raso V, Griffin TW: Transferrin receptor in normal and neoplas-
tic brain tissue: Implications for brain-tumor immunotherapy. J Neurosurg 1990;72:941–945.
35 Joshi BH, Leland P, Asher A, Prayson RA, Varricchio F, Puri RK: In situ expression of interleukin-4
(IL-4) receptors in human brain tumors and cytotoxicity of a recombinant IL-4 cytotoxin in primary
glioblastoma cell cultures. Cancer Res 2001;61:8058–8061.
36 Gage FH, Brundin P, Strecker R, Dunnett SB, Isacson O, Bjorklund A: Intracerebral neuronal
grafting in experimental animal models of age-related motor dysfunction. Ann NY Acad Sci
1988;515:383–394.
37 Perlow MJ, Freed WJ, Hoffer BJ, Seiger A, Olson L, Wyatt RJ: Brain grafts reduce motor
abnormalities produced by destruction of nigrostriatal dopamine system. Science 1979;204:
643–647.
38 Bjorklund A, Stenevi U: Reconstruction of the nigrostriatal dopamine pathway by intracerebral
39 Freed WJ, Perlow MJ, Karoum F, Seiger A, Olson L, Hoffer BJ, Wyatt RJ: Restoration of dopamin-
ergic function by grafting of fetal rat substantia nigra to the caudate nucleus: Long-term behav-
ioral, biochemical, and histochemical studies. Ann Neurol 1980;8:510–519.
40 Freed CR, Greene PE, Breeze RE, Tsai WY, DuMouchel W, Kao R, Dillon S, Winfield H, Culver
S, Trojanowski JO, Eidelberg D, Fahn S: Transplantation of embryonic dopamine neurons for
severe Parkinson’s disease. N Engl J Med 2001;344:710–719.
41 Freed CR: Will embryonic stem cells be a useful source of dopamine neurons for transplant into
patients with Parkinson’s disease? Proc Natl Acad Sci USA 2002;99:1755–1757.
42 Yan Q, Matheson C, Lopez OT: In vivo neurotrophic effects of GDNF on neonatal and adult facial
motor neurons. Nature 1995;373:341–344.
43 Oppenheim RW, Houenou LJ, Johnson JE, Lin LF, Li L, Lo AC, Newsome AL, Prevette DM,
Wang S: Developing motor neurons rescued from programmed and axotomy-induced cell death
by GDNF. Nature 1995;373:344–346.
44 Henderson CE, Phillips HS, Pollock RA, Davies AM, Lemeulle C, Armanini M, et al: GDNF: A
potent survival factor for motoneurons present in peripheral nerve and muscle. Science 1994;266:
1062–1064.
45 Yuen EC: The role of neurotrophic factors in disorders of peripheral nerves and motor neurons.
Phys Med Rehabil Clin N Am 2001;12:293–306, viii.
46 Grill RJ, Blesch A, Tuszynski MH: Robust growth of chronically injured spinal cord axons
induced by grafts of genetically modified NGF-secreting cells. Exp Neurol 1997;148:444–452.
47 Lonser RR, Gogate N, Morrison PF, Wood JD, Oldfield EH: Direct convective delivery of macro-
molecules to the spinal cord. J Neurosurg 1998;89:616–622.
48 Hench LL, Polak JM: Third-generation biomedical materials. Science 2002;295:1014–1017.
49 Brindley GS, Polkey CE, Rushton DN, Cardozo L: Sacral anterior root stimulators for bladder
control in paraplegia: The first 50 cases. J Neurol Neurosurg Psychiatry 1986;49:1104–1114.
medwedi.ru
50 Wielink G, Essink-Bot ML, van Kerrebroeck PE, Rutten FF: Sacral rhizotomies and electrical
bladder stimulation in spinal cord injury. 2. Cost-effectiveness and quality of life analysis. Dutch
Study Group on Sacral Anterior Root Stimulation. Eur Urol 1997;31:441–446.
51 Fedirchuk B, Shefchyk SJ: Effects of electrical stimulation of the thoracic spinal cord on blad-
der and external urethral sphincter activity in the decerebrate cat. Exp Brain Res 1991;84:
635–642.
52 Kennedy PR, Bakay RA, Moore MM, Adams K, Goldwaithe J: Direct control of a computer from
the human central nervous system. IEEE Trans Rehabil Eng 2000;8:198–202.
53 Wessberg J, Stambaugh CR, Kralik JD, Beck PD, Laubach M, Chapin JK, Kim J, Biggs SJ,
Srinivasan MA, Nicolelis MA: Real-time prediction of hand trajectory by ensembles of cortical
neurons in primates. Nature 2000;408: 361–365.
54 Nicolelis MA: Actions from thoughts. Nature 2001;409(suppl):403–407.
55 Guillory KS, Normann RA: A 100-channel system for real time detection and storage of extra-
cellular spike waveforms. J Neurosci Methods 1999;91:21–29.
56 Dobelle WH, Quest DO, Antunes JL, Roberts TS, Girvin JP: Artificial vision for the blind by elec-
trical stimulation of the visual cortex. Neurosurgery 1979;5:521–527.
57 Nieder A: Miniature stereo radio transmitter for simultaneous recording of multiple single-neuron
signals from behaving owls. J Neurosci Methods 2000;101:157–164.
58 Nordhausen CT, Rousche PJ, Normann RA: Optimizing recording capabilities of the Utah
Intracortical Electrode Array. Brain Res 1994;637:27–36.
59 Hoogerwerf AC, Wise KD: A three-dimensional microelectrode array for chronic neural record-
ing. IEEE Trans Biomed Eng 1994;41:1136–1146.
60 Rauschecker JP, Shannon RV: Sending sound to the brain. Science 2002;295:1025–1029.
61 Maynard EM: Visual prostheses. Annu Rev Biomed Eng 2001;3:145–168.
62 Fanselow EE, Reid AP, Nicolelis MA: Reduction of pentylenetetrazole-induced seizure activity in
awake rats by seizure-triggered trigeminal nerve stimulation. J Neurosci 2000;20:8160–8168.
63 Berger T: World’s first brain prosthesis revealed. New Scientist 2003, March 12.
64 Broggi G, Ferroli P, Franzini A, Dones L, Marras C, Marchetti M, Maccagnano E: CT-guided neu-
rosurgery: Preliminary experience. Acta Neurochir Suppl 2003;85:101–104.
65 Jolesz FA, Talos IF, Schwartz RB, Mamata H, Kacher DF, Hynynen K, McDannold N,
Saivironporn P, Zao L: Intraoperative magnetic resonance imaging and magnetic resonance
imaging-guided therapy for brain tumors. Neuroimaging Clin N Am 2002;12:665–683.
66 Russell L: Intraoperative magnetic resonance imaging safety considerations. Norton Healthcare,
Louisville, KY.
67 Schulder M, Carmel PW: Intraoperative magnetic resonance imaging: Impact on brain tumor
surgery. Cancer Control 2003;10:115–124.
68 Hall WA, Liu H, Truwit CL: Intraoperative MR-guided instillation of phosphorus-32 for cystic
craniopharyngiomas: Case report. Technol Cancer Res Treat 2003;2:19–24.
69 Tuominen J, Yrjana SK, Katisko JP, Heikkila J, Koivukangas J: Intraoperative imaging in a com-
prehensive neuronavigation environment for minimally invasive brain tumour surgery. Acta
Neurochir Suppl 2003;85:115–120.
70 Schulder M, Jacobs A, Carmel PW: Intraoperative MRI and adjuvant radiosurgery. Stereotact
Funct Neurosurg 2001;76:151–158.
71 Hall WA, Kowalik K, Liu H, Truwit CL, Kucharezyk J: Costs and benefits of intraoperative
MR-guided brain tumor resection. Acta Neurochir Suppl 2003;85:137–142.
72 McPherson CM, Bohinski RJ, Dagnew E, Warnick RE, Tew JM: Tumor resection in a shared-
resource magnetic resonance operating room: Experience at the University of Cincinnati. Acta
Neurochir Suppl 2003;85:39–44.
73 Nimsky C, Ganslandt O, Gralla J, Buchfelder M, Fahlbusch R: Intraoperative low-field magnetic
resonance imaging in pediatric neurosurgery. Pediatr Neurosurg 2003;38:83–89.
74 Nimsky C, Ganslandt O, Tomandl B, Buchfelder M, Fahlbusch R: Low-field magnetic resonance
imaging for intraoperative use in neurosurgery: A 5-year experience. Eur Radiol 2002;12:2690–2703.
75 Kanner AA, Vogelbaum MA, Mayberg MR, Weisenberger JP, Barnett GH: Intracranial navigation by
using low-field intraoperative magnetic resonance imaging: Preliminary experience. J Neurosurg
2002;97:1115–1124.
76 Schulder M, Sernas TJ, Carmel PW: Cranial surgery and navigation with a compact intraoperative
MRI system. Acta Neurochir Suppl 2003;85:79–86.
77 Vitaz TW, Hushek S, Shields CB, Moriarty T: Intraoperative MRI for pediatric tumor manage-
ment. Acta Neurochir Suppl 2003;85:73–78.
78 Nabavi A, Gering DT, Kacher DF, Talos IF, Wells WM, Kikinis R, Black PM, Jolesz FA: Surgical
navigation in the open MRI. Acta Neurochir Suppl 2003;85:121–125.
79 Sutherland GR, Kaibara T, Louw DF: Intraoperative MR at 1.5 Tesla – Experience and future
directions. Acta Neurochir Suppl 2003;85:21–28.
80 Liu H, Hall WA, Truwit CL: The roles of functional MRI in MR-guided neurosurgery in a com-
bined 1.5 Tesla MR-operating room. Acta Neurochir Suppl 2003;85:127–135.
William T. Couldwell, MD, PhD

Department of Neurological Surgery, University of Utah Medical Center
Suite 3B409, 30 North 1900 East
Salt Lake City, UT 84132-2303 (USA)
Tel. ⫹1 801 581 6908, Fax ⫹1 801 581 4385, E-Mail william.couldwell@hsc.utah.edu
medwedi.ru
Xeno-Neurotransplantation
James M. Schumacher
Center for Movement Disorders, University of Miami School of Medicine,
Miami, Fla., USA
Introduction
Though limited, the human nervous system has some capability to regen-
erate or repair damaged or degenerated cells. This inherent biological mecha-
nism is not sufficient to reverse the devastating effects of neurodegenerative
conditions such as Parkinson’s, Alzheimer’s, and Huntington’s disease.
Strategies to replace degenerative neuronal systems have included transplanta-
tion of human embryonic fetal cells, embryonic stem cells, adult ‘stem’ cells,
genetically modified somatic cells, viral-assisted gene transfer, and cross-
species cell transplants (i.e., xenotransplantation). Cell replacement therapies for
neurodegenerative diseases were considered for human application after
Parkinson’s disease (PD)-like motor deficits in animal models of the disease
were ameliorated using transplanted embryonic dopamine cells. Fetal allogenic
neuronal transplants have been shown to effect functional and behavioral recov-
ery in a variety of animal models of neurodegenerative disease [1, 12, 15].
Using PD as a target syndrome, several investigations have been performed
in humans. The first human neurotransplantation for PD was performed in 1988
[17]. Since then, over 300 patients worldwide have been transplanted with
human tissue. Open-label studies suggested efficacy of transplantation and
resulted in many cases in graft survival and increased dopamine utilization in the
striatum [16]. Until recently, however, none of these surgical studies were done
with adequate controls. Recently, two controlled studies have been performed
using solid grafts in patients with advanced PD. Neither study demonstrated sta-
tistically powered efficacy [6, 7]. In the first study the endpoint was the Global
Rating Scale. This scale is a subjective account of how the patient feels after
surgery. The second study looked at the Unified Parkinson’s Disease Rating
Scale (UPDRS), Part III motor ‘off scores.’
Table 1. Milestones of xeno-neurotransplantation
1890 Thompson. Cat cerebral cortex into brains of

adult dogs. (No survival.)
1917 Dunn. Rat neonatal cerebral cortex into adult
rat brain.
1921 Shirai. First description of brain
immunoprivilege and xenografts.
1979 Bjorklund. Fetal rat brain to adult rat brain.
1985 Isacson. Fetal rat brain into adult rat model of
Huntington’s and Parkinson’s.
1995 Schumacher. Fetal pig dopaminergic cells into
a Parkinson’s patient.
Although results were uneven, there were patients within these studies
and in previous open-label studies who have shown remarkable improvement
in their condition and decreased need for pharmacological dopamine replace-
ment. PET fluorodopa studies have also confirmed restoration of dopamine in
the striatum of previously depleted areas. Patients who improved the most
from transplantation were those who had a large difference between their ‘on’
and ‘off ’ UPDRS scores. Meta-analyses of the published open-label studies
demonstrate that neurotransplantation is a very promising work in progress
[12–14].
In order to demonstrate benefit and adequate dopamine cell survival,
nearly 10 fetuses (3–5 human fetuses per putamen of embryonic age 8–10
weeks) are required for transplantation in any given patient. The logistics of
obtaining this quantity of human tissue are prohibitive, notwithstanding the
ethical considerations involved. Hence, the search for alternate cell sources
has led investigators to cross-species transplants (xenotransplantation) and
embryonic stem cells. In the case of xenotransplantation, neuroblasts from
other species such as pigs could provide unlimited, screenable, precisely incu-
bated cells for transplantation.
Early attempts at cross-species neurotransplantation were unsuccessful
due to immune rejection. With the introduction of cyclosporine and other
immunosuppressive agents, however, successful xenotransplantation has
become possible (table 1). Xenotransplantation is a beneficial laboratory tool in
animal models of neurodegenerative disease. When host animals undergo
immunosuppression, cross-species transplantation of embryonic dopaminergic
cells has shown similar results to allografts [11, 19]. In addition, the unique
antigenicity of the graft and host allows specific antibody cell labeling. In this
Xeno-Neurotransplantation 147
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manner, graft survival, outgrowth and target specificity can be clearly defined.
Several studies using embryonic porcine dopaminergic transplants into animal
models of PD have been successful. Porcine tissue has been chosen for most
studies because it is plentiful (up to 20 fetuses/liter), similar phenotypically to
human, and has been used extensively in human medicine (i.e., cardiac valves,
insulin). Human trials using porcine tissue have now been performed to treat
PD, Huntington’s disease and epilepsy. Recent trials of neurotransplantation
and the safety and immunological concerns of xeno-neurotransplantation are
discussed below.
Immunology of Neural Xenografts
Although the central nervous system has a higher degree of immunoprivi-

lege than other systems such as the heart, lung, liver and kidney, immunologi-
cal reactions are a concern in allografts and especially xenografts. Factors that
determine successful graft-host integration include the donor tissue embryonic
stage, phylogenetic distance between donor and host, method of transplanta-
tion, preparation of graft (i.e., solid vs. suspension), host site, and method of
immunosuppression.
Several factors contribute to the immunological privilege in the host. In
xenotransplantation, the lack of major histocompatibility complex (MHC)
class I and II antigens is probably most important in graft survival. Cytokines
are an important factor in graft cell death. These antigens can be induced in
either system by influx of cytokines in the face of the inflammatory response
of transplantation trauma [22]. Subsequent T-cell and macrophage activation
and cell death is deactivated to a major extent by treatment with cyclosporine
immunosuppression. Continued immunosuppression is important in that sev-
eral other cytokines such as interleukin-2, -4, and -10 are induced for up to
30 days after transplantation [5, 18]. Anti-C5 (complement) antibody treat-
ment has been found to inhibit cell death in xenografts. Further graft survival
is seen with the combination of C5 inhibitor, cyclosporine methylprednisolone
and azathioprine [2].
In the adult brain, MHC antigens are restricted to endothelial cells. Solid
grafts contain intact endothelial cells and supporting cells and for this reason cell
suspension grafts are favored over solid tissue pieces. The role of MHC 1 in tol-
erance induction has been shown to be an effective strategy in animals of xeno-
transplantation [18]. The antibody against the graft cell surface antigen is
thought to promote tolerance by inhibiting T-cell induction. This technique is not
as effective as cyclosporine but may provide an adjunct to immunosuppression
and graft protection.
Schumacher 148
Safety Issues in Xenotransplantation
Acute, type I graft rejection is generally not seen in neurotransplantation

or xeno-neurotransplantation. Patterns of rejection are mild when compared
with that seen in major organ transplantation. In neurotransplantation, T-cell
and macrophage-mediated rejection is seen over days and weeks. This response
is greatly reduced with cyclosporine and other modifiers of immune response.
In experimental animal models and human studies, no adverse side effects in
the host have been seen. Either in animals or in human safety studies, no sys-
temic immune effect has been documented. Local pathological effects of the
inflammatory cell response have been observed. Immunosuppressants carry
their own hazards of use including increased risk of infection and renal dam-
age. Of special concern in cross-species transplantation is the risk of transmis-
sion of viral nucleic acid sequences (i.e., porcine endogenous retrovirus). To
date, no transmission of porcine endogenous retrovirus from animal to man has
been observed. Risk of transmission of virus or bacteria between species (xeno-
zooinosis) is possible. Animals and their tissues must be carefully screened and
monitored to avoid this problem. Prophylactic antibiotics are given before and
after transplantation.
Xeno-Neurograft Procedures in Humans
Clinical trials of xenotransplantation in humans have been quite limited. Our

study in transplanting porcine dopaminergic mesencephalic cells into patients
with PD was the first human study of xeno-neurotransplantation [20], and will be
discussed below. Subsequently, other studies for Huntington’s disease and
epilepsy have been performed and these also will be briefly discussed.
Patient Selection
Patients selected for xenotransplantation were affected with advanced PD,
were failing medical treatment with L-dopa, and were having ‘on-off’ motor
fluctuations but were still responsive to L-dopa. Patients were screened using the
Core Assessment Protocol in Intracerebral Transplantation (CAPIT protocol;
UPDRS ⫹ time testing). Patients with dementia, poor medical condition, or seri-
ous comorbidity were excluded. Fluorodopa PET scans and cranial MRIs were
performed before surgery and at 6 months and one year after transplantation.
Preparation of Embryonic Porcine Ventral Mesencephalon Tissue

Donor animals from a Yorkshire porcine herd were screened by serology
for pathogen exposure, tested for parasites, and isolated. Embryonic tissue was
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prepared by dissection of the ventral mesencephalon region from embryonic
day 25–28 fetuses. Cells were then trypsonized to prepare a cell suspension at
50,000 cells/␮l. In our initial study, some cells were treated with an F(ab⬘)2
fragment of a monoclonal antibody directed against MHC I. This technique has
been shown to promote xenograft survival without pharmacological immuno-
suppression [8, 18]. Viability prior to transplantation was assessed by acridine
orange staining and screen by gram stain for bacteria. Aliquots of cell suspen-
sions were cultured for dopaminergic cells using antibody to tyrosine hydoxy-
lase. Patient’s blood mononuclear cells were obtained, archived and tested for
porcine endogenous retrovirus.
Preoperative Preparation
In our study, 6 patients were loaded preoperatively with cyclosporine
(15 mg/kg) 12 h prior to surgery. Six other patients were transplanted with cells
that had been treated with the monoclonal antibody. All patients received peri-
operative antibiotics.
Surgical Procedure
Patients underwent the procedure with local anesthesia and MRI/CT-guided
stereotaxy. Eighty microliter volumes of suspension were transplanted unilater-
ally in the striatum along three separate 5 mm tracts. One tract was placed in the
caudate head and two in the mid and posterior putamen.
Postoperative Evaluation
MRI after one week showed evidence of the tracts in the striatum. Standard
adverse event reporting, chemistry and blood testing was done per protocol.
Cyclosporin levels were followed in those immunosuppressed patients. CAPIT
testing was performed at 6 months and one year after surgery as was PET scan-
ning and MRI.
Clinical Results in Xenotransplantation

In the original human study of CNS xenotransplantation of porcine cells,
no adverse effects were seen in the 10 patients evaluated. None of the patients’
disease worsened in the year following surgery. In the medication ‘off’ state,
3 patients improved by ⬃30%. As a group the CAPIT scores improved by 19%.
No significant change was seen in PET scans [20].
Another study with PD patients has been recently reported where bilateral
‘solid piece’ grafts were placed. Half the patients were given sham burr holes
as a control. The patients that received xenografts improved 28% and the sham
patients 23%. This study failed to show significant group improvement in
CAPIT scores and showed a remarkable sham placebo effect [10]. Phase I
Schumacher 150
safety trials also have been performed using porcine fetal neural cells in
Huntington’s disease. On evaluation after one year following transplantation, no
adverse events were seen. No significant deterioration and no improvement in
functional capacity were seen [9]. A small group of patients with frontal lobe
epilepsy had GABAergic porcine grafts transplanted in the seizure focus, and
this study is still ongoing.
Future Direction in Xeno-Neurotransplantation
Studies in both animal models and in humans have shown that transplanted
neurons across species barriers can survive and establish functional axonal and
synaptic contact with the immunosuppressed host. Neurotransmitters can be
replenished and neuronal circuitry re-established. Preclinical studies in animals
have clearly shown that pathological and behavioral deficits can be reversed
using xenografts.
The value of having an unlimited supply of selected neuronal cells for
transplantation cannot be underestimated. Xenografts can be carefully screened
for the disease and selected for the function and precise embryonic age. The
greatest obstacle in xenotransplantation is still graft rejection. This obstacle is
somewhat offset by modern methods of immunosuppression, but it is not yet
optimized. Novel strategies are underway to improve cell survival. Transgenic
pigs have been genetically engineered to express human cell surface markers.
These cells are less immunogenic and promote graft survival [3, 4].
Critics of neurotransplantation have cited problems or adverse events in
recent controlled human studies using human fetal material. As a group, these
studies failed to meet their endpoint of statistical significance in improvement.
Of particular concern, dyskinesia was observed during defined ‘off’ periods in
some patients that were transplanted [6]. This phenomenon may represent unreg-
ulated dopamine production by the graft or may reflect more ‘on’ time with
dyskinesia. Those patients did have ‘on’ dyskinesia prior to transplantation and
were successfully treated with medication and in 2 patients with globus pallidus
stimulation.
Although xenografts are not as viable as human allografts, we believe that
studies should continue, in particular to engineer hybrid human-porcine cell
lines that may show less immunogenicity and greater in vivo activity. The util-
ity of the unlimited supply and possibility of tissue screening make xenografts
an important resource. Our current level of understanding of the immune sys-
tem limits the use of xenografts as a treatment in human disease. These studies
also provide the information that will make transplantation with allografts or
embryonic stem cells more successful.
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Ultimately, the answer to curing neurodegenerative disease is not only in the
protection of native cells but in reconstruction of damaged and lost neuronal cir-
cuitry. Pharmacological therapy cannot provide the neurotransmitter and signal
regulation needed at the cellular level. This regulation can only be provided by cell
configurations at the synaptic level. Until we can determine how to promote native
regeneration and regrowth of neural elements, our best strategy for neurodegener-
ation will incorporate aspects of cellular replacement through transplantation.
References
1 Bjorklund A, Stenevi U, et al: Cross species neural grafting in the rat model of Parkinson’s disease.
Nature (Lond) 1982;298:652–654.
2 Cicchetti F, Fodor W, et al: Immune parameters relevant to neuroxenograft survival in the primate
brain. Xenotransplantation 2003;10:41–49.
3 Deacon P, Schumacher J, et al: Histological evidence of fetal pig neural cell survival after trans-
plantation into a patient with Parkinson’s disease. Nat Med 1997;3:350–352.
4 Deacon T, Fodor W, et al: Xenotransplantation of transgenic fetal pig dopamine neurons to rats
and systemic prevention of host compliment-mediated cell lysis. Abstr Soc Neurosci 1998;
24:1056.
5 Duan W: Immunological and inflammatory responses against intrastriatal neural grafts in the rat.
Thesis, Lund University, 1997.
6 Fahn S, Freed C, Breeze W: Transplantation of embryonic dopamine neurons for severe Parkinson’s
disease. New Eng J Med 2001;334:710–719.
7 Fahn S, Green P, et al: Double blind controlled trial of human embryonic dopaminergic tissue
transplants in advanced Parkinson’s disease: Clinical outcomes. Neurology 1999;52:A405.
8 Faustman D, Coe C: Prevention of xenograft rejection by masking donor HLA class 1 antigens.
Science 1991;252:1700–1702.
9 Fink J, Schumacher J, Elias S, Isacson O: Porcine xenographs in Parkinson’s disease and
Huntington’s disease patients: Preliminary results. Cell Transplant 2000;9:273–278.
10 Freeman T: Porcine xenografts in patients with Parkinson’s disease. Abstract: American Association
of Neurological Surgeons 2003.
11 Freeman T, Wojak J, et al: Cross-species intracerebral grafting of embryonic swine dopaminergic
neuron. Prog Brain Res 1998;78:473–477.
12 Isacson O, Bjorklund L: Parkinson’s disease. Interpretations of transplantation study are erro-
neous. Nature Neurosci 2001;4.
13 Isacson O, Deacon P, Pakzaban P: Transplanted xenogenic neural cells in neurodegenerative dis-
ease models exhibit remarkable axonal targets specificity and distinct growth patterns of glial and
axonal fibres. Nat Med 1995;1:1189–1194.
14 Isacson O, Deacon T, Schumacher J: Immunobiology and neuroscience of xenotransplantation and
neurological disease. San Diego, Academic Press, 1998, pp 365–387.
15 Isacson O, Dunnett S, Bjorklund A: Graft-induced recovery in an animal model of Huntington’s
disease. Proc Natl Acad Sci USA 1986;83:2728–2732.
16 Kordower J, Freeman T, et al: Neuropathological evidence of graft survival and striatal reinnerva-
tion after the transplantation of embryonic mesencephalic tissue in a patient with Parkinson’s dis-
ease. New Eng J Med 1993;332:1118–1124.
17 Lindvall O, Widner H, et al: Transplant of fetal dopamine neurons in Parkinson’s disease. Ann
Neurol 1992;31:155–165.
18 Pakzaban P, Deacon T, Isacson O: A novel mode of immunoprotection of neuroxenotransplants:
Masking of donor major histocompatibility complex class 1 enhances transplant survival in the
CNS. Neuroscience 1995;65:983–986.
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19 Pakzaban P, Isacson O: Neuroxenotransplantation: Reconstruction of neuronal circuitry across
species barriers. Neuroscience 1994;62:989–1001.
20 Schumacher J, Ellias P, et al: Transplantation of embryonic porcine mesencephalic tissue in
patients with PD. Neurology 2000;54:1042–1050.
21 Shirai Y: Transplanting rat sarcoma in adult heterogenous animals. Jap Med World 1921;1:14–15.
22 Widner H, Lindhval O (eds): Basic and Clinical Aspects of Neuroscience, vol 5. Heidelberg,
Springer, 1993, pp 63–74.
James M. Schumacher, MD
Center for Movement Disorders
University of Miami School of Medicine, Miami, FL 33136 (USA)
Tel. ⫹1 305 243 4675, Fax ⫹1 305 243 3337, E-Mail jschumacher@med.miami.edu
medwedi.ru
Adeno-Associated Viral Vectors

for Clinical Gene Therapy
in the Brain
R. Jude Samulski, Jennifer Giles
Gene Therapy Center, Department of Pharmacology, University of
North Carolina at Chapel Hill, N.C., USA
Introduction
A number of recombinant viral vectors have been engineered for gene

transfer to the brain. The vectors in widest use for neuroscience applications
include herpes virus vectors, adenovirus vectors, and lentiviral vectors, in addi-
tion to the parvovirus vector, adeno-associated virus (AAV). Despite its rela-
tively small capacity of ⬍5 kb, AAV vectors have gained wide acceptance as a
preferred vector for gene transfer to the central nervous system (CNS), due to
its advantages of neurotrophism, nonpathogenicity, and stable in vivo gene
expression. AAV is one of the few viral vectors that have already proven itself as
a gene transfer vector for functional genomics as well as clinical applications in
gene transfer to the human brain.
AAV is a nonpathogenic virus that is not associated with any human viral
syndrome or disease. It depends on the presence of a helper virus, such as ade-
novirus or herpes virus, for replication. The wild-type AAV (wtAAV) has a
4.68-kb single-stranded DNA genome comprised of capsid (cap) and replica-
tion (rep) open reading frames flanked by inverted terminal repeats. Three
structural proteins, VP1, VP2, and VP3, are encoded by the single cap gene
using alternative splicing and alternative start codons. The AAV virion is com-
posed of VP1, VP2, and VP3 at a ratio of 1:1:10, respectively. The rep gene
codes for four overlapping proteins involved in AAV DNA replication and the
control of AAV gene expression. The two larger rep proteins, Rep78 and Rep68,
are controlled by the p5 promoter and are needed for viral DNA replication,
while the smaller Rep52 and Rep40 proteins are transcribed from the p19 pro-
moter and serve to facilitate the accumulation of single-stranded virus. The
inverted terminal repeats are the only cis-acting elements required for AAV
replication, packaging, integration, and rescue [1].
Production of Clinical-Grade AAV Vector
Recombinant AAV (rAAV) is an increasingly important gene therapy vec-

tor. Perhaps most beneficially, wtAAV is innocuous and has a known integra-
tion site at chromosome 19qter13.4 [2]. Long-term transgene expression is
facilitated by the ability of rAAV to persist in vivo episomally and possibly
also by integrating into the host genome. This long-term persistence is further
enhanced by the fact that AAV does not induce a cell-mediated immune
response in the host [3]. Adding to its appeal as a therapeutic vector, rAAV has
been shown to infect both dividing and nondividing cells in a broad range of
tissues, including muscle, liver, brain, and retina [4].
The rAAV plasmid is constructed by replacing the entire AAV coding
genome with a transgene expression cassette flanked by the viral inverted ter-
minal repeats. The rAAV plasmid is then used to transfect cells concurrently
with a helper virus infection and an AAV helper plasmid that contains the rep
and cap genes needed to supply the Rep and Cap proteins in trans. This cotrans-
fection procedure allows efficient rescue and encapsidation of the rAAV genome
from the recombinant vector plasmid [5] and production of rAAV vectors that
can then be used for gene therapy delivery. It is important to note that the same
protocol can be used for production of any of the AAV serotypes or modified
vectors. An Ad-free AAV production system, using cotransfection of plasmid
encoding the Ad helper genes, is a recent development that allows quick, easy
generation of rAAV vectors for typical lab-scale use [6]. In order to generate the
high quantities of virus that will be needed for clinical applications, cell lines
engineered to produce AAV vectors or alternative methods of transfection must
be developed. Inducible cell lines for AAV production are a current focus of
virology research. These cell lines, that contain integrated copies of some or all
of the AAV genes needed for packaging, utilize a variety of approaches to pro-
vide the helper genes and a vector in the host genome [7, 8].
While there are many advances being made in the development of gene
delivery systems targeting the CNS, production of clinical grade viral vectors
continues to be a bottleneck in the progression of these therapies to the clinic.
Production of viral vectors under conditions that satisfy FDA Good Laboratory
Practices and Good Manufacturing Practice guidelines introduces a wide range
of testing and facility modifications not needed for research-grade vectors. All
Adeno-Associated Viral Vectors for Gene Therapy 155
medwedi.ru
steps in the process must be thoroughly documented, from the plasmid and
mammalian cells used to produce the virus to the final viral vector itself.
Quality control and release assays for vectors to be used in the clinic are exten-
sive, and include, to name but a few, ELISA assays to determine the physical
titer of the virus, quantitative PCR to determine the genomic titer, determina-
tion of the infectious titer, assays for contamination by the mammalian cell line
used to generate the virus, silver stain/Coomassie blue gel staining to assay for
protein contamination, and Western blot to assess the ratio of capsid proteins
VP1, VP2, and VP3. The unprocessed ‘bulk harvest,’ which includes the mam-
malian cells, media, and unpurified virus, undergoes a series of quality control
assays as well. Those include tests for sterility, mycoplasma, and replication-
competent AAV and adenovirus. After a series of purification steps, the final
vector preparation is assayed for sterility, bacteria, fungi, endotoxin, and resid-
ual DNA.
One of the unfortunate consequences of the need for adherence to the
above regulations is a shortage in facilities that are able to produce clinical-
grade reagents for gene therapy trials. The National Institutes of Health (NIH)
have designated two U.S. national vector labs for the production of viral vectors
for the clinic, one at Indiana University that specializes in retroviral vectors and
one at Baylor University specializing in adenoviral vectors. These facilities
have succeeded in establishing the technical expertise required to generate viral
vectors and the resources to pay for extensive testing of the final product.
Unfortunately, a number of novel vectors that do not fall under the mainstream
production procedure require expertise typically located in the labs that derive
these new systems.
In the case of AAV, a Human Applications Lab (HAL) at the University of
North Carolina at Chapel Hill (UNC) is an academic facility which has suc-
ceeded in producing AAV for a clinical trial. The successment implementation
of the recent Canavan’s disease clinical trial highlights the need for such a facil-
ity to produce reagents for Phase I clinical trials. With fewer than 1,000 chil-
dren in the USA affected by the disease, it is not an attractive target for private
industry, while most academic institutions do not have Good Laboratory
Practices facilities. Facilities like the HAL at UNC fills a critical gap between
clinicians interested in gene therapy applications for rare genetic disorders and
the patients who have so much to gain through these pioneering clinical trials.
The amount of vector necessary to treat 21 human patients, on the order of mil-
liliters of the final product, has been scaled up from the quantities that are more
typical of experiments in animal models, on the order of microliters, and it is
expected that further scale-up for other clinical trials will be possible in the
future, especially as technical advances in large-scale AAV production at our
center are achieved.
Samulski/Giles 156
AAV Serotypes for Gene Transfer
AAV exists naturally as a variety of serotypes that have immunologically

unique properties. To date, a total of eight mammalian serotypes have been dis-
covered and tested as viral vectors [9–15]. AAV serotype 1 or AAV-1 was the
first to be isolated and characterized. Although isolated from the rhesus monkey,
epidemiological data indicates that it frequently infects humans as well; how-
ever, it has yet to be recovered from a human sample. AAV serotypes 2, 3, and
5 are human parvoviruses, and have a high level of infection in the general pop-
ulation, as shown in epidemiological studies. The most unique AAV serotype on
the nucleic acid level, AAV serotype 4, was isolated from the African green mon-
key and is rarely found in humans. It has, however, demonstrated the ability to
infect human cells in vitro [16]. AAV serotype 6 is not serologically unique, and
is more than 99% homologous to AAV-1 in its capsid proteins at the amino acid
level. An analysis of AAV-1 and AAV-2 nucleic acid sequences suggests that
AAV-6 is the product of a recombination event between these two serotypes [10].
Most recently, AAV serotypes 7 and 8 were isolated from nonhuman primates.
As would be suggested by the serological uniqueness of the AAV
serotypes, comparison of their capsids shows that they are indeed heteroge-
neous. Initial studies to evaluate the different AAV serotypes as gene delivery
vectors indicated that serotypes have unique tropisms and differing transduc-
tion efficiencies depending on the cell type transduced, when compared to
AAV-2 or against each other [17]. Until recently, the majority of the research
conducted using AAV-based vectors implored AAV-2. This serotype historically
has been used to study critical steps in AAV DNA replication, site-specific
recombination, and AAV viral gene expression [18]. For this reason, it was only
natural to extend upon this base of knowledge in the early development of AAV
vectors. Only after extensive use of AAV-2 vectors in vivo and the identifica-
tion of limitations in efficient transduction did attention turn to the other
serotypes. Each can be distinguished by the efficiency of transduction for spe-
cific target tissue when compared to traditional AAV-2 vectors. For example,
AAV serotype 5, but not AAV-2, binds to the apical surface of airway epithelia
and facilities gene transfer [12, 13]. AAV-1 appears more robust in muscle cells
while AAV-4 has been suggested to infect primarily ependymal cells when
introduced into the mouse brain [12, 13]. AAV serotypes 7 and 8 have been
shown to have muscle (AAV-7) and liver-specific tropism (AAV-8).
Surprisingly, only ninety amino acid differences exist between these two
serotypes, strongly suggesting that the capsid domain responsible for tissue tro-
pism can be narrowed down and eventually identified. Understanding these dif-
ferences and the capsid regions required for tropism for cells of the CNS will
be critical for developing effective therapies for neurometabolic disorders.
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Fig. 1. Ribbon structure of AAV-2 VP3.
The above evidence clearly points to the benefits of exploiting the natural
and unique tropisms of AAV serotypes other than AAV-2 to increase AAV-
mediated gene transfer efficiency in different cell types. In order to achieve the
most efficient gene delivery to the CNS, an approach that takes advantage of
different serotypes will be needed.
Crystal Structure of AAV and the Future of

Specific Targeting with AAV Vectors
The structure of parvovirus capsid proteins is now known. The structure of

six autonomous parvoviruses and one dependo-virus have been solved: B19 [19],
canine parvovirus (CPV) [20], feline panleukopenia virus (FPV) [21],
Galleria mellonella densovirus (an insect parvovirus) [22], Aleutian Mink
Disease parvovirus [23], minute virus of mice [24], and AAV-2 [25]. Sequence
alignment of the capsid genes of B19 and CPV/FPV shows only 23% amino acid
identity [26], yet these capsid proteins share extensive basic structural motifs.
The virions of these viruses are made up of sixty subunits, with the smallest
capsid protein making up the majority of the virion. They all share the eight
B-barrel motif with looped out regions between barrels (fig. 1) [20, 21, 26].
It is possible to change parvovirus tropism by swapping key capsid amino
acids. CPV and FPV share 98% sequence similarity within the capsid-coding
region [27]. However, these viruses have different host range infectivity [28].
Using recombinants of CPV and FPV capsid sequences, Parrish et al. [28] were
Samulski/Giles 158
BC1/C0 Loop
GH12/13 Loop
HI Loop GH10/11 Loop GH2/3 Loop
I Loop
Fig. 2. Ribbon diagram of AAV-2 VP3. Regions in bold from AAV-1, -2, -3, -7, and -8
alignment are highlighted in blue, many are surface-displayed and may reflect muscle versus
liver tropism differences. They are labeled according to Xie et al. [25].
able to map specific functions to epitopes on the capsid including determinants

of host range infectivity. Refinement of this work defined the determinants of
host range to amino acids 93 and 323 of VP2. Coding sequence of these amino
acids were introduced into FPV, which could then replicate in canine cells [29].
These data support the ability to interchange epitopes and tropism between
parvoviruses.
A comparison of parvovirus capsid structures indicates that they are quite
similar. AAV-2 VP3 has eight B-barrel motifs that are separated by looped out
regions (fig. 2) [26]. The known differences between AAV-2 and the other
serotypes may provide the information essential for understanding receptor
binding and entry step of AAV vectors. Similarly to the differences found
between CPV and FPV, amino acid sequences in loops 3 and 4 may explain the
differences in cellular receptors used by AAV2 and the other serotypes. The
functional domains can be identified by regions of viral capsid homology. All
serotypes of AAV have unique tropisms based on epitopes present on the virion
shell. However, the epitopes responsible for those tropisms are not well under-
stood. In the future it will be beneficial to understand which domains on the
virion are responsible for each serotype’s unique tropism, to improve targeting
of specific cell types.
The virions of all serotypes of AAV are assembled from a homologous set
of precursor capsid proteins. The alignment of amino acid sequence of the cap-
sid proteins illustrates the degree of homology between each serotype [12], and
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the domains within the capsid sequence with high or low levels of homology
have been identified [30]. Areas of low homology are of special interest since
they may hold the key to the determinants of the serotypes. Alignment of AAV
serotypes elucidates those domains that share the lowest degree of amino acid
homology. Prior to the availability of the crystal structure of AAV-2, we
exchanged domains between serotypes 1, 3, 4, and 5 from amino acid 440–603
(AAV-2 numbering) and AAV-2 (fig. 2). This domain occupies almost all of the
GH looped out domain, the largest and most variable domain between all
serotypes, including the recently discovered AAV-7 and AAV-8 as well as the
most autonomous parvoviruses. Using pair-wise comparisons this domain has
the highest level of homology between AAV-2 and AAV-3 (70%) and the least
homology between AAV-4 and AAV-5 (10%). Additionally, comparisons of the
autonomous CPV and the FPV shows that amino acid substitutions that resulted
in species-jumping from feline to canine are located in the homologous GH
loop. This leads us to make the assumption that those domains that are surface-
localized and have low homology between the AAV serotypes may be respon-
sible for tropism differences between AAV serotypes. This information will also
direct future chimeric vector design in order to incorporate phenotypes specific
to vector application.
Determining which amino acids are surface-displayed is essential for
understanding tropism. To approach this problem in a rational way, the newly
resolved AAV-2 crystal structure will be essential. The usefulness of the crystal
structure has been demonstrated with the positioning of targeting insertions into
the adenovirus knob HI loop. For the autonomous parvoviruses a wealth of
information has been revealed through comparisons of the amino acid
sequences and the crystal structure with respect to surface-display and tropism.
The availability of the crystal structure now provides a specific road map to
rational structural/functional analysis.
Delivery of Gene Therapy to the Brain
In spite of the tremendous growth in the field of gene therapy and the
numerous clinical trials currently underway, relatively few applications target
disorders of the CNS. This is due in large part to the complexity of the brain
and its circuitry, which is intolerant to even mild inflammation or toxicity.
Limited access to the brain itself makes direct injection difficult. Additionally,
the protection afforded to the CNS by the blood-brain barrier hinders global
delivery of viral vectors through venous injection or cerebrospinal fluid.
One of the first experiments in rodents to demonstrate the utility of rAAV
vectors in vivo was aimed at transduction of brain tissue into rats [31]. Many of
Samulski/Giles 160
Step 1 Receptor binding
FGFR
HSPG
␣v␤5
␣v␤5
H+
H+
Step 2 Nuclear entry
Step 3 DNA template
Fig. 3. Diagram of potential rate-limiting steps in efficient AAV transduction.
the recent advances in the understanding of rAAV vectors have come about
through the need to better understand in vitro and in vivo transduction.
Although several recent studies have shown great promise in terms of duration
of transgene expression in vivo, there has been a shortfall in transduction effi-
ciency, which was unexpected, based on previous results in vitro [32]. High
transduction efficiency is of particular importance in the treatment of global
neurometabolic disorders which require gene delivery to every affected cell in
order to be therapeutically useful.
Regardless of the serotype, all of the AAV vectors follow three basic steps
for productive infection (fig. 3). First, receptor binding to the cell membrane is
required; second, internalization and nuclear entry; and third, DNA template
formation. After receptor binding, internalization, and nuclear entry, AAV viri-
ons uncoat and release a single-stranded DNA template, which must convert to
a duplex intermediate before transcription can ensue. The efficiency of forming
the complementary strand can significantly impact vector transduction [33, 34].
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a wtAAV (ssDNA) b Duplex AAV (scDNA)
Fig. 4. Self-complementary AAV packages both strands.
There are two possible mechanisms by which single-stranded AAV

genomes can be converted to duplex templates. The first mechanism relies on
the reannealing of two single-stranded genomes of different polarity (⫹ and –).
Since AAV packages both strands with equal efficiency, this polarity may offer
a viable mechanism for solving the duplex template requirement. The second
(and usual) mechanism by which single-stranded AAV genomes can be con-
verted to duplex templates involves DNA replication. AAV productive infection
relies on the 145-bp hairpin terminal repeat and a self-priming mechanism for
viral DNA replication [35]. The terminal repeat exists as double-stranded DNA
duplex ‘T’ shaped structure and serves as an origin of replication for the single-
stranded viral template [35]. The single-stranded viral template and the termi-
nal repeat hairpin structures are required to form a duplex intermediate [36].
Second-strand synthesis is a rate-limiting step for rAAV transduction.
Evidence supporting this conclusion was found in experiments correlating the
induction of transgene expression with the conversion of the single-stranded
virion DNA to the duplex. Generation of a duplex DNA template is required
before transcription can ensue. Careful analysis of this process has now deter-
mined unique proviral intermediates (monomer, dimer, concatemeric struc-
tures, and circular molecules), all of which are derived from input
single-stranded viral DNA [3, 37–48]. The dimer length of vector molecules
originally characterized comprise duplex monomers, which are covalently
linked at one end and are identical to substrates characterized for wtAAV
[34, 36]. The characteristic lag of vector gene expression after infection in non-
dividing cells correlates with the formation of these duplex DNA intermediates
[34, 36, 40, 41, 47, 49]. Double-stranded vectors, (fig. 4) rather than the natu-
rally packaged singled-stranded molecule, could bypass the rate limiting step of
second-strand synthesis.
Recently, we have generated a novel double-stranded AAV (dsAAV) vector
and demonstrated that steps which influence traditional single-stranded AAV
Samulski/Giles 162
transduction (i.e., availability of host DNA pol) were not required for dsAAV
vectors, supporting the importance of the duplex intermediate as a rate-limiting
step in AAV transduction [50]. The use of dsAAV may be particularly useful in
the treatment of global CNS disorders that necessitate very high transduction
rates. At present, AAV vectors in vivo appear to have very little toxicity or
immune consequences after vector transduction [51]. Increased understanding
of the biology of AAV has led to the generation of preclinical data for the treat-
ment of neurological disorders and in the case of Canavan’s disease and
Parkinson’s disease, Phase I clinical trials.
Treatment of Focal Brain Disorders with rAAV Vectors
Huntington’s Disease
Like many other neurological disorders, Huntington’s disease (HD) is
caused by the degeneration of specific cell groups within the CNS. By directly
targeting these cell groups with gene transfer to express neurotrophic factors,
this degeneration may be reversed or avoided altogether. HD is manifested as
an array of motor, cognitive, and psychiatric disturbances caused by the degen-
eration of medium-sized spiny neurons in the striatum and cerebral cortex.
Glial cell-line-derived neurotrophic factor has been shown to prevent the loss
of striatal neurons in animal models of HD [52–56]. In an animal model of HD,
bilateral injections of a rAAV viral vector containing the glial cell-line-derived
neurotrophic factor transgene into the striatum showed marked protection of
striatal neurons and prevention of behavioral disturbances [57]. Because HD is
passed on through an autosomally dominant gene, persons who have not yet
sustained any neurological damage could be identified as having the disease
and receive gene therapy, making it possible to avoid the debilitating effects of
the disease altogether.
Seizure Disorders
Gene therapy with AAV also has been tested for the treatment of focal
seizure disorders. A study by Haberman et al. [58] shows that the delivery of
N-methyl D-aspartic acid receptor antisense using an AAV-derived vector can
modulate seizure disorder in vivo. However, it was also shown that using dif-
ferent promoters to drive the transgene expression resulted in completely oppo-
site physiological effects, increasing seizure sensitivity as opposed to reducing
it. In order to work around this effect, the gene for the inhibitory neuroactive
peptide, galanin, was delivered to cells using an AAV-derived vector, and con-
stitutively secreted through the use of the fibronectin signal sequence.
Importantly, the choice of promoter to drive the transgene did not impact the
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decrease in seizure sensitivity [59]. This approach provides a new option for the
long-term control of focal seizure disorders.
Parkinson’s Disease
Another devastating neurological disorder, Parkinson’s disease, is charac-
terized by the degeneration of the substantia nigra pars compacta and conse-
quently, reduced dopamine in the striatum. The resulting lack of inhibition of
the subthalamic nucleus (STN) contributes to the motor abnormalities typical
of the disease. Deep brain electrical stimulation of the STN has been shown to
effectively reduce symptoms of Parkinson’s disease. One alternative approach
for the treatment of Parkinson’s using gene therapy seeks to achieve the same
effect biochemically. GABA, the brain’s major inhibitory transmitter, can be
generated by two isoforms of glutamic acid decarboxylase. Stereotactic injec-
tion of rAAV carrying the glutamic acid decarboxylase transgenes into the
STN of adult parkinsonian rats resulted in neuroprotection of the STN and a
reduction in the excitatory phenotype of the disease. Robust expression of
the transgene was seen up to 5 months after injection, with no significant
immune response [60]. One of the two clinical trials currently underway that
utilize rAAV vectors to treat neurological disorders uses this vector in human
patients.
Global Gene Delivery with rAAV Vectors
Canavan’s Disease
The original clinical trial in which rAAV was first used as a vector for
gene delivery in humans is for the treatment of Canavan’s disease. This study,
led by Dr. Paola Leone at UMDNJ-Robert Wood Johnson Medical School, was
the first gene transfer clinical trial to use viral vectors to treat a neurodegen-
erative disorder. Canavan’s disease is an inherited disease, with autosomal
recessive inheritance, that shortens life expectancy to a few years. Symptoms
are generally first seen within the first 6 months of life and include megalo-
cephaly and developmental delays. As the disease progresses, mental retarda-
tion, spasticity and cortical blindness develop, culminating in seizures and
childhood death.
A lack of the enzyme aspartoacylase (ASPA) that hydrolyzes N-acetyl-
aspartic acid (NAA) into L-aspartate and acetate causes the damage associated
with Canavan’s [61, 62]. It is thought that the accumulation of metabolic pre-
cursors such as NAA, the function of which remains undetermined, is toxic and
leads to neurological damage [63]. It also appears that high levels of NAA
affect the phenotype of developing myelinating cells through a complex set of
Samulski/Giles 164
gene expression effects which are currently being worked out [Leone, pers.
commun.]. This clinical trial is intended to retard the damage of elevated NAA
levels by injecting rAAV carrying the aspartoacylase gene into the brain.
Preliminary data suggest beneficial effects of treatment on the biochemical and
clinical level, with an absence of adverse events.
dsAAV and Improved Global CNS Delivery?

Because global neurometabolic disorders require long-term transgene
expression, and in cases of intrinsically expressed gene products, transduction
of nearly every affected cell, the development of less intrusive delivery methods
is a key step in getting these treatments into the clinic. A recent study by Fu
et al. demonstrated the utility of the dsAAV vector for global CNS distribution
in a mouse model. An IV injection of 4 ⫻ 1011 particles of dsAAV2-expressing
green fluorescent protein preceded by an injection of 12.5% mannitol showed a
global distribution of the transgene in the brain and spinal cord 4–8 weeks after
injection. No green fluorescent protein expression was seen using dsAAV2 in
12.5% mannitol or with the viral vector without mannitol. Additionally, no
green fluorescent protein expression was seen when ssAAV was injected
intravenously preceded by the injection of mannitol. Through the use of
mannitol to transiently open the blood-brain barrier in conjunction with dsAAV
to increase transduction efficiency, minimally invasive intravenous injections
may provide another effective route to global gene delivery to the CNS, in
addition to intraparenchymal injection protocols.
Conclusion
Safe and efficient delivery of corrective gene therapy to the CNS using
rAAV vectors has great promise for the treatment of neurological disorders. An
array of recent developments including advances in production, the elucidation
of the rAAV crystal structure, and newly discovered serotypes will facilitate
further clinical applications. While the CNS presents unique challenges to the
development of effective therapies, the devastating nature of the diseases being
targeted should serve as an impetus to overcome the inherent hurdles.
Acknowledgements
Thanks to the members of the University of North Carolina, Chapel Hill (UNC) Gene
Therapy Core.
medwedi.ru
References
1 Berns KI: Parvorviridae and their replication; in Fields BN, Knipe DM (eds): Virology. New York,
Raven, 1990, pp 1743–1763.
2 Samulski RJ: Adeno-associated virus: Integration at a specific chromosomal locus. Curr Opin
Genet Dev 1993;3:74–80.
3 Xiao X, Li J, Samulski RJ: Efficient long-term gene transfer into muscle tissue of immunocom-
petent mice by adeno-associated virus vector. J Virol 1996;70:8098–8108.
4 Monahan PE, Samulski RJ: AAV vectors: Is clinical success on the horizon? Gene Ther
2000;7:24–30.
5 Samulski RJ, Chang LS, Shenk T: Helper-free stocks of recombinant adeno-associated viruses:
Normal integration does not require viral gene expression. J Virol 1989;63:3822–3828.
6 Xiao X, Li J, Samulski RJ: Production of high-titer recombinant adeno-associated virus vectors in
7 Qiao C, et al: Feasibility of generating adeno-associated virus packaging cell lines containing
inducible adenovirus helper genes. J Virol 2002;76:1904–1913.
8 Qiao C, et al: A novel gene expression control system and its use in stable, high-titer 293 cell-
based adeno-associated virus packaging cell lines. J Virol 2002;76:13015–13027.
9 Walters RW, et al: Incorporation of adeno-associated virus in a calcium phosphate coprecipitate
improves gene transfer to airway epithelia in vitro and in vivo. J Virol 2000;74:535–540.
10 Xiao W, et al: Gene therapy vectors based on adeno-associated virus type 1. J Virol 1999;73:
3994–4003.
11 Muramatsu S, et al: Nucleotide sequencing and generation of an infectious clone of adeno-
accociated virus 3. Virology 1996;221:208–217.
12 Chiorini JA, et al: Cloning of adeno-associated virus type 4 (AAV4) and generation of recombi-
nant AAV4 particles. J Virol 1997;71:6823–6833.
13 Chiorini JA, et al: Cloning and characterization of adeno-associated virus type 5. J Virol
1999;73:1309–1319.
14 Rutledge EA, Halbert CL, Russell DW: Infectious clones and vectors derived from adeno-associated
virus (AAV) serotypes other than AAV type 2. J Virol 1998;72:309–319.
15 Gao GP, et al: Novel adeno-associated viruses from rhesus monkeys as vectors for human gene
therapy. Proc Natl Acad Sci USA 2002;99:11854–11859.
16 Muzyczka N: Use of adeno-associated virus as a general transduction vector for mammalian cells.
Curr Top Microbiol Immunol 1992;158:97–129.
17 Rabinowitz JE, et al: Cross-packaging of a single adeno-associated virus (AAV) type 2 vector
genome into multiple AAV serotypes enables transduction with broad specificity. J Virol 2002;76:
791–801.
18 Berns KI, Hauswirth WW: Review: Adeno-associated viruses. Adv Virus Res 1979;25:407–449.
19 Agbandje-McKenna M, et al: The structure of human parvovirus B19 at 8 Å resolution. Virology
1994;203:106–115.
20 Tsao J, et al: The three-dimensional structure of canine parvovirus and its functional implications.
Science 1991;25:1456–1464.
21 Agbandje M, et al: Structure determination of feline panleukopenia virus empty particles. Proteins
1993;16:155–171.
22 Simpson AA, et al: The structure of an insect parvovirus (Galleria mellonella densovirus) at 3.7 Å
resolution. Structure 1998;6:1355–1367.
23 McKenna R, et al: Three-dimensional structure of Aleutian mink disease parvovirus: Implications
for disease pathogenicity. J Virol 1999;73:6882–6891.
24 Agbandje-McKenna M, et al: Functional implications of the structure of the murine parvovirus,
minute virus of mice. Structure 1998;6:1369–1381.
25 Xie Q, et al: The atomic structure of adeno-associated virus (AAV-2), a vector for human gene
therapy. Proc Natl Acad Sci USA 2002;99:10405–10410.
26 Chapman M, Rossmann M: Structure, sequence, and function correlations among parvoviruses.
Virology 1993;194:419–508.
Samulski/Giles 166
27 Martyn J, Davidson B, Studdert M: Nucleotide sequence of feline panleukopenia virus: Comparison
with canine parvovirus identifies host-specific differences. J Gen Virol 1990; 71: 2747–2753.
28 Parrish C, Aquadro C, Carmichael L: Canine host range and a specific epitope map along with
variant sequences in the capsid protein gene of canine parvovirus and related feline, mink, and rac-
coon parvoviruses. Virology 1988;166:293–307.
29 Chang S, Sgro J, Parrish C: Multiple amino acids in the capsid stucture of canine parvovirus coor-
dinately determine the canine host range and specific antigenic and hemagglutination properties.
J Virol 1992;66:6858–6867.
30 Rabinowitz JE, Samulski J, Adeno-associated virus expression systems for gene transfer. Curr
Opin Biotechnol 1998;9:470–475.
31 Kaplitt MG, et al: Preproenkephalin promoter yields region-specific and long-term expression in
adult brain after direct in vivo gene transfer via a defective herpes simplex viral vector. Proc Natl
Acad Sci USA 1994;91:8979–8983.
32 Flotte TR: Prospects for virus-based gene therapy for cystic fibrosis. J Bioenerg Biomembr 1993;
25:37–42.
33 Fisher KJ, et al: Transduction with recombinant adeno-associated virus for gene therapy is limited
by leading-strand synthesis. J Virol 1996;70:520–532.
34 Ferrari FK, et al: Second-strand synthesis is a rate-limiting step for efficient transduction by
recombinant adeno-associated virus vectors. J Virol 1996;70:3227–3234.
35 Berns K: Parvoviridae: The viruses and their replication; in Fields BN, Knipe PM, Howley EA
(eds): Virology. Philadelphia, Lippincott-Raven Publishers, 1996, pp 2173–2198.
36 Straus SE, Sebring ED, Rose JA: Concatemers of alternating plus and minus strands are interme-
diates in adenovirus-associated virus DNA synthesis. Proc Natl Acad Sci USA, 1976;73:742–746.
37 Duan D, et al: Lef1 transcription factor expression defines airway progenitor cell targets for in
utero gene therapy of submucosal gland in cystic fibrosis. Am J Respir Cell Mol Biol 1998; 18:
750–758.
38 Duan D, et al: Formation of adeno-associated virus circular genomes is differentially regulated by
adenovirus E4 ORF6 and E2a gene expression. J Virol 1999;73:161–169.
39 Duan D, et al: Structural analysis of adeno-associated virus transduction circular intermediates.
Virology 1999;261:8–14.
40 Snyder RO, Im DS, Muzyczka N: Evidence for covalent attachment of the adeno-associated virus
Rep protein to the ends of the AAV genome. J Virol 1990;64:6204–6213.
41 Snyder RO, Samulski RJ, Muzyczka N: In vitro resolution of covalently joined AAV chromosome
ends. Cell 1990;60:105–133.
42 Vincent-Lacaze N, et al: Structure of adeno-associated virus vector DNA following transduction
of the skeletal muscle. J Virol 1999;73:1949–1955.
43 McLaughlin SK, et al: Adeno-associated virus general transduction vectors: Analysis of proviral
structures. J Virol 1988;62:1963–1973.
44 Hong G, Ward P, Berns KI: Intermediates of adeno-associated virus DNA replication in vitro.
J Virol 1994;68:2011–2015.
45 Hoggan MD, et al: Continuous carriage of adenovirus associated virus genome in cell culture in
the absence of helper adenovirus. Proceedings of the Fourth Lepetite Colloquium, Mexico,
Cocoyac, 1972.
46 Flotte TR, Afione SA, Zeitlin PL: Adeno-associated virus vector gene expression occurs in non-
dividing cells in the absence of vector DNA integration. Am J Respir Cell Mol Biol 1994; 11:
517–521.
47 Clark KR, Sferra TJ, Johnson PR: Recombinant adeno-associated viral vectors mediate long-term
transgene expression in muscle. Hum Gene Ther 1997;8:659–669.
48 Berns KI, Linden RM: The cryptic life style of adeno-associated virus. Bioessays 1995;17:237–245.
49 Miao CH, et al: The kinetics of rAAV integration in the liver [letter]. Nat Genet 1998;19:
13–15.
50 McCarty DM, Monahan PE, Samulski RJ: Self-complementary recombinant adeno-associated
virus (scAAV) vectors promote efficient transduction independently of DNA synthesis. Gene Ther
2001;8:1248–1254.
medwedi.ru
51 Samulski RJ, Sally M, Muzyczka N (eds): Adeno-associated viral vectors; in Friedman T (ed): The
development of human gene therapy. Cold Spring Harbor, Cold Spring Harbor Laboratory Press,
1999, pp 131–172.
52 Perez-Navarro E, et al: Glial cell line-derived neurotrophic factor protects striatal calbindin-
immunoreactive neurons from excitotoxic damage. Neuroscience 1996;75:345–352.
53 Perez-Navarro E, et al: Intrastriatal grafting of a GDNF-producing cell line protects striatonigral
neurons from quinolinic acid excitotoxicity in vivo. Eur J Neurosci 1999;11:241–249.
54 Gratacos E, et al: Neuroprotection of striatal neurons against kainate excitotoxicity by neu-
rotrophins and GDNF family members. J Neurochem 2001;78:1287–1296.
55 Araujo DM, Hilt DC: Glial cell line-derived neurotrophic factor attenuates the excitotoxin-
induced behavioral and neurochemical deficits in a rodent model of Huntington’s disease.
Neuroscience 1997;81:1099–1110.
56 Araujo DM, Hilt DC: Glial cell line-derived neurotrophic factor attenuates the locomotor hypo-
function and striatonigral neurochemical deficits induced by chronic systemic administration of
the mitochondrial toxin 3-nitropropionic acid. Neuroscience 1998;82:117–127.
57 McBride JL, et al: Structural and functional neuroprotection in a rat model of Huntington’s dis-
ease by viral gene transfer of GDNF. Exp Neurol 2003;181:213–223.
58 Haberman R, et al: Therapeutic liabilities of in vivo viral vector tropism: Adeno-associated virus
vectors, NMDAR1 antisense, and focal seizure sensitivity. Mol Ther 2002;6:495–500.
59 Haberman RP, Samulski RJ, McCown TJ: Attenuation of seizures and neuronal death by adeno-
associated virus vector galanin expression and secretion. Nat Med 2003;9:1076–1080.
60 Luo J, et al: Subthalamic GAD gene therapy in a Parkinson’s disease rat model. Science 2002;
298:425–429.
61 Matalon R, et al: Aspartoacylase deficiency and N-acetylaspartic aciduria in patients with Canavan
disease. Am J Med Genet 1988;29:463–471.
62 Divry P, et al: N-acetylaspartic aciduria: Report of three new cases in children with a neurologi-
cal syndrome associating macrocephaly and leukodystrophy. J Inherit Metab Dis 1988;11:
307–308.
63 Hsich G, Sena-Esteves M, Breakefield XO: Critical issues in gene therapy for neurologic disease.
Hum Gene Ther 2002;13:579–604.
R. Jude Samulski, PhD

Gene Therapy Center, Department of Pharmacology
CB# 7352, 7119 Thurston Bowles, University of North Carolina at Chapel Hill
Chapel Hill, NC 27599–7352 (USA)
Tel. ⫹1 919 962 3285, Fax ⫹1 919 966 0907, E-Mail rjs@med.unc.edu
Samulski/Giles 168
Molecular Mechanisms of Epilepsy

and Gene Therapy
Albert Telfeiana, Juanita Celixb, Marc Dichterc
a
Division of Neurosurgery, Texas Tech University Medical Center, Lubbock, Tex.,
b
Department of Neurosurgery, University of Washington, Seattle, Wash.,
c
Department of Neurology, University of Pennsylvania, Philadelphia, Pa., USA
Introduction
‘Extreme remedies are appropriate for extreme diseases…’

Hippocrates (460–370 B.C.)
Epilepsy is a neurological disorder that afflicts 1–2% of the general

population and encompasses a variety of disorders with seizures [1]. To best
understand where we must go in the treatment of epilepsy, it is necessary to
understand first where we have failed. The prognosis for seizure control in
epilepsy with medication is good in ⬃60% of patients, and up to 40% of
individuals suffer from intractable pharmacoresistant epilepsy. There are over
twenty different anti-epileptic drugs available to the neurologist or neurosur-
geon to treat seizures, but patients not controlled on monotherapy with the first
anti-epileptic agent have only a 10% chance of being controlled with addi-
tional anti-epileptic agents, even when using medications that work by diverse
mechanisms [2].
Newer is not necessarily better in terms of drug regimens. Only a small
minority (⬍5%) of patients refractory to traditional drug therapy has been
reported to become seizure free with a new generation anti-epileptic drug [2].
The more we understand about the genetic basis of this disease, the more naïve
it appears that a single drug tailored to a specific channel or neurotransmitter
receptor will effectively cure epilepsy. In fact, it appears that the better drugs
have a wider basis in their mechanisms of action. Most clinically efficacious
anti-epileptic drugs possess a combination of various properties. While there
have been advances in the drugs available to control the seizures associated
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with epilepsy, to date there is no effective therapy for the prevention of epilepsy.
Generally, one third of all cases of epilepsy have a potential cause, the most
obvious being trauma, tumors, and stroke, but treatment seems to have done
little to prevent the process of increased intrinsic excitability, synchronization,
or synaptic connectivity that may be responsible for the development of
seizures. Here, we concentrate on how a better understanding of the molecular
mechanisms of epilepsy may guide future therapies, such as gene therapy, for
this disease.
Review of Pharmacotherapy in Epilepsy
Anti-epileptic drug therapy is the first-line treatment for epilepsy. Until the
1990s, there were only a handful of drugs available to treat the various seizure
disorders. The earliest drugs used to control seizures (e.g., bromides, pheno-
barbital, valproic acid) were identified inadvertently. Later, a more scientific
approach utilized animal models of epilepsy against which potential anti-
epileptic drugs were tested. The maximal electroshock (MES) model is used to
evaluate agents for the ability to decrease seizure severity and to identify those
drugs with efficacy in treating generalized tonic-clonic or partial seizures. The
pentylenetetrazole (PTZ) model is utilized to test for agents that increase
the seizure threshold and exert possible anti-absence seizure properties. While
the early anti-epileptic drugs identified by these models were understood to
function via action at the neural membrane or synapse, the animal models
could not elucidate the important mechanisms of action. It was not until the
introduction of more modern electrophysiological and pharmacological
research techniques that the effects of anti-epileptic drugs at the neural mem-
brane and synapse were determined.
The diligent study of anti-epileptic therapies lead to the discovery of the
principle mechanisms of action of the clinically efficacious drugs used to treat
seizure disorders. In general, anti-epileptic agents control the initiation,
maintenance, or propagation of epileptiform discharges through augmented
inhibition, suppressed excitation, or modulation of action potential ion current,
with effects at the level of localized neurons to entire neural networks.
Typically, anti-epileptic drugs act on one of four classes of neuron ion
channels: ␥-aminobutryic acid (GABAA) ligand-gated chloride channels,
glutamate ligand-gated sodium and calcium channels (NMDA, N-methyl-
D-aspartate; AMPA, ␣-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic
acid; and kainate), voltage-gated sodium channels, and voltage-gated T-type
(low threshold) calcium channels. Most of the agents with activity in the MES
model function through use-dependent inactivation of voltage-gated sodium
Telfeian/Celix/Dichter 170
channels. Drugs with efficacy in the PTZ model function via blockade of
T-type calcium channels or augmentation of GABAA receptor-mediated
chloride channels. Several of the recently developed anti-epileptic drugs that
have been introduced during the past decade have new or additional mecha-
nisms of action, targeting pre- and postsynaptic membrane-bound receptors
and enzymes that function in the metabolism of neurotransmitters. Still, there
are a few anti-epileptic agents whose mechanisms of action remain largely
unknown (e.g., topiramate, felbamate). As we continue to make advances in
cellular and molecular biology and gain a greater understanding of the
excitability and synchronization of neurons in circuit, a fuller appreciation for
the mechanisms of action of the efficacious anti-epileptic drugs will undoubt-
edly become more apparent.
Traditional Pharmacotherapy
Phenytoin, carbamazepine, and valproic acid are traditional anti-epileptic

drugs with primary actions at the voltage-gated sodium channel. Phenytoin acts
at the sodium channel in a voltage- and frequency-dependent manner. It prefer-
entially binds to and stabilizes the channel in the open inactive state. Selective
blockade of the inactive sodium channel prevents release of excitatory amino
acid neurotransmitters, particularly glutamate and aspartate, delays channel
recovery time, and slows propagation of electrical discharges. There is evidence
that sustained, high-frequency repetitive firing plays a major role in neuron
excitability, and that phenytoin functions to limit this repetitive firing [3]. As
sodium channels may be more susceptible to blockade when they are in the
open inactive state, preferential binding of phenytoin to the inactivated voltage-
gated sodium channel of the depolarized neuron during seizure activity is the
likely mechanism through which phenytoin acts to limit sustained repetitive
firing and terminate seizure activity. At high drug doses, phenytoin has addi-
tional clinically significant actions, including voltage-gated calcium channel
blockade and decreased presynaptic glutamate release.
The anti-epileptic properties of carbamazepine, initially developed as a
tricyclic antidepressant, and valproic acid were fortuitously discovered. Similar
to phenytoin, carbamazepine and valproic acid both function in a voltage- and
frequency-dependent manner to bind the inactive sodium channel and delay
recovery. Valproic acid also indirectly facilitates the inhibitory activity of
GABA via increased synaptic neurotransmitter levels, while carbamazepine’s
additional mechanisms of action include inhibition of norepinephrine reuptake,
decrease in intracellular cAMP levels through interaction with adenosine recep-
tors, and possible potentiation of GABA inhibition through interaction with the
Epilepsy and Gene Therapy 171
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GABAB receptor. The clinical importance of these additional mechanisms of
action remains to be determined.
The voltage-gated calcium channels consist of at least four types, L-type,
T-type, N-type, and P-type, with differing levels of voltage activation and
inactivation. Ethosuximide is an anti-epileptic drug with activity at the T-type
calcium channel, a low threshold voltage-gated channel. Ethosuximide
functions primarily at the T-type channels of thalamic neurons, reducing the
calcium current and interrupting the 3 Hz spike/wave thalamocortical action
potentials typical of absence seizures.
Activity at GABA ligand-gated chloride channels is the primary mecha-
nism of action of the most well known anti-epileptic drugs, barbiturates and
benzodiazepines. The GABAA receptor complex contains multiple modulatory
sites for site-specific interaction with various agents. The GABA receptor
subtypes have differential developmental and regional expression and varying
sensitivities to the diverse ligands. Phenobarbital is one of the oldest anti-
epileptic drugs. At the GABAA receptor chloride channel, it potentiates the
activity of GABA through enhanced duration of channel opening. At high drug
doses, the barbiturates also interact with the N-type voltage-gated calcium
channels to block the calcium influx and prevent release of excitatory amino
acid neurotransmitters. Benzodiazepines also function at the GABAA receptor,
where they increase the frequency of chloride channel opening without affect-
ing the duration of opening.
Advances in Pharmacotherapy
The recent introduction of new generation anti-epileptic drugs illustrates

our increased understanding of the multiple interrelated mechanisms by which
synchronous excitatory electrical activity can be initiated, maintained or prop-
agated, and enables a more rational approach to pharmacotherapy in epilepsy.
While some of the newer agents were discovered using the MES and PTZ
animal models of epilepsy, others were rationally designed to produce a spe-
cific molecular effect. Many newer agents have multiple mechanisms of action
and predominantly function via metabotropic or ionotropic channels, similar to
the traditional anti-epileptic drugs. Lamotrigine is a new generation drug that
functions at the presynaptic open inactive voltage-gated sodium channel to
delay channel recovery and decrease excitatory amino acid release. It may also
interact with the GABA receptor, and it has efficacy in treating certain seizure
types that cannot be explained by its primary mechanism of action.
Unexplained therapeutic effects are common with many of the new gener-
ation agents. Gabapentin, which was developed based on the theoretical role of
GABA in epileptogenesis, is a novel drug formed by the linkage of a cyclohexyl
group to GABA. This modification allows it to easily penetrate the blood-brain
barrier (BBB). As a structural analog of the endogenous inhibitory neurotrans-
mitter, gabapentin was designed to function via GABA-mimetic activity, but the
drug does not interact with GABA receptors in the CNS. Neither does it inter-
act with voltage-gated sodium or calcium channels, nor with NMDA ligand-
gated ion channels. It is unclear through which mechanism or combination of
mechanisms gabapentin exerts its anti-epileptic effect, but it does indeed have
anti-epileptic properties.
A novel class of second generation anti-epileptic drugs is active in modu-
lating local levels of GABA. Altered intracellular metabolism of endogenous
neurotransmitter and inhibition of reuptake from the synaptic cleft are the
mechanisms by which these new agents augment levels of inhibitory GABA
within the brain and ultimately alter the balance of excitation and inhibition.
Vigabatrin is an irreversible inhibitor of intracellular GABA transaminase, the
protein responsible for the metabolic degradation of GABA. Inhibition of
GABA degradation allows an increase in the local concentration of GABA and
potentiation of its physiological role in limiting excitatory neural activity in the
brain. Tiagabine increases synaptic GABA through blockade of neuronal and
glial GABA reuptake from the synaptic cleft.
Two of the new generation drugs were designed based on the hypothe-
sized role of glutamate neurotransmission in seizure generation. These agents
employ a novel mechanism of action with activity at the NMDA ligand-gated
calcium channel. The NMDA receptor is believed to play a fundamental role
in the initiation and propagation of epileptiform activity. NMDA receptor-
mediated blockade of the calcium channel results in inhibition of neuronal
hyperexcitability, with the potential to not only control seizure activity, but to
modulate the underlying epileptogenic defect. In practice, the NMDA recep-
tor antagonists have not proven efficacious in controlling seizure activity, and
the incidence of adverse effects with this class of drugs limits their clinical
usefulness.
The elucidation of the basic mechanisms of action of the anti-epileptic
drugs and the development of novel agents has afforded us a greater apprecia-
tion for the complex molecular mechanisms underlying neural excitability and
synchronization in epileptogenesis. While these drug discoveries allow clini-
cians to move from the empirical treatment of epilepsy to a more rational
approach to seizure control, they may not have a significant impact on the
effective treatment of intractable seizures. An approach to epilepsy treatment
based on innovative understandings of epileptogenic activity and novel avenues
of investigation holds the greatest promise for the future of epilepsy research
and the ultimate development of a cure.
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The Future of Pharmacotherapy in Epilepsy
The future of pharmacotherapy in epilepsy will be based on an appreciation

of the molecular mechanisms that result in abnormal synchronized excitatory
electrical activity in the brain. Understanding the basic principles of initiation,
maintenance, and propagation of seizure activity and their relationship to the
factors that influence susceptibility to spontaneous recurrent seizures offers
the potential for fundamental shifts in the paradigms of therapy in epilepsy.
Integration of our knowledge of pharmacogenetics, drug resistance mechanisms,
drug delivery systems, and cellular and gene therapy will allow us to develop a
more powerful approach to drug therapy in epilepsy. The most promising
strategies in pharmacotherapy should aim to propel us beyond treatment of the
symptoms of epilepsy, namely suppression of seizures, to prevention or cure of
this devastating neurological disease.
Pharmacogenetics: A New Approach to Drug Therapy
An increased understanding of both the molecular mechanisms of epilepsy

and the mechanisms of action of the anti-epileptic drugs has made it increasingly
possible to move away from the empirical treatment of epilepsy and employ a
more rational approach to pharmacotherapy. Yet our considerable knowledge of
the etiology of epilepsy, including the underlying neuropathological processes,
electrophysiology and biochemistry of a seizure, and specific therapeutic and
adverse effects of each anti-epileptic drug has not enabled clinicians to antici-
pate an individual patient’s response to a particular anti-epileptic agent. The
individual response to a specific drug is still empirical. As a result, patients with
refractory epilepsy undergo multiple trials of single or combination therapy with
significant risk of CNS or systemic toxicity and severe idiosyncratic reactions.
A novel approach to enhanced drug therapy in epilepsy is based upon our under-
standing of genetic polymorphism in drug metabolism. Genetic variations in
drug pharmacokinetics are a likely factor in refractory seizures due to either lack
of anti-epileptic drug efficacy or intolerable side effects.
Genetic differences may influence both the pharmacokinetics and pharmaco-
dynamic effects of a particular anti-epileptic drug. Abnormal drug metabolism can
be due to genetic polymorphisms that result in altered metabolic enzymes. Rapid
metabolizers may have low plasma concentrations of a drug despite appropriate
dosing and good adherence to therapy, and essentially appear to be refractory to
medical management. Slow metabolizers can experience high plasma concentra-
tions at normal drug dosages with resultant CNS and systemic toxicity that limits
the use of an anti-epileptic drug.
The effects of genetic polymorphisms of cytochrome P450 isoenzymes on
phenytoin metabolism have been studied extensively. In adult patients with
epilepsy, impaired CYP2C9 or CYP2C19 isoenzymes were associated with
altered phenytoin hydroxylation, which resulted in dramatically increased plasma
drug concentrations even at normal or low phenytoin doses [4]. Molecular studies
revealed two G to A point mutations in the CYP2C19 gene as the cause of
the defect in the functional CYP2C19 protein (de Morias et al., 1994a,b), and
multiple amino acid variants in the CYP2C9 protein have been described
(Kaminsky et al., 1992). Identification of the genetic mutations in CYP2C19
allows for widespread genotyping by simple polymerase chain reaction restriction
fragment length polymorphism for the defective isoenzyme. Identification of
genetic polymorphisms in anti-epileptic drug metabolism has broad implications
for the effective drug treatment of epilepsy. Knowledge of an individual’s
genotype has the potential to allow for tailored drug therapy and effective control
of seizures in someone who was once considered medically refractory due to
aberrant drug metabolism.
Idiosyncratic hypersensitivity reactions may also be influenced by alter-
ations in metabolic enzymes. It has been proposed that CYP450 anti-epileptic
drug bioactivation results in reactive metabolites that mediate the chemical
modification of detoxifying enzymes. The aberrantly modified enzyme leads to
deficient detoxification of anti-epileptic drug and a subsequent increase in the
availability of bioactivated drug. A host-dependent immune response is
believed to have a role in the complex series of events that lead to a hypersen-
sitivity reaction. Further characterization of the pathways involved may allow
for genotyping studies that will identify patients at risk for adverse effects
with anti-epileptic drug therapy, and permit clinicians to take a truly rational
approach to pharmacotherapy in epilepsy.
Pharmacoresistance in Epilepsy
Of patients who are refractory to first-line conventional anti-epileptic

drugs, less than 5% will attain good seizure control with use of the newer
agents, and a patient whose seizures cannot be adequately controlled with one
anti-epileptic drug has only a 5–10% chance of controlling their seizures with
multiple anti-epileptic agents [2]. In this population, multiple trials of drugs
with differing mechanisms of action do not improve the chance of a positive
response.
Given the character of global resistance to different anti-epileptic drugs
with varying mechanisms of action, it is unlikely that acquired alterations in the
multiple receptors upon which anti-epileptic drugs act can adequately explain
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pharmacoresistance. It is instead hypothesized that pharmacoresistant epilepsy
may be due to altered permeability of anti-epileptic drugs across the BBB and
that the mechanisms whereby drug access to the brain is limited are most likely
nonspecific [5]. It is further proposed that nonspecific drug resistance mecha-
nisms may represent adaptive changes in the epileptic brain [5]. As we continue
in search of an effective drug treatment for pharmacoresistant epilepsy, it is
crucial that we appreciate the full nature of drug resistance mechanisms and the
specific character of those mechanisms in the epileptogenic brain.
Our understanding of epilepsy continues to evolve and the pursuit of an
effective drug treatment for epilepsy continues to advance. But if we are to truly
make breakthroughs in the pharmacotherapy of epilepsy we must move beyond
the traditional line of investigation. An appreciation of the mechanisms of
pharmacoresistance in epilepsy is essential to the development of better
epilepsy therapy. The discovery of cellular membrane proteins that function in
chemotherapy-resistant neoplasms has advanced our understanding of drug
resistance in epilepsy. Extensive biochemical study of the molecular character
of these proteins has elucidated the mechanisms by which drugs are denied
access to or extruded from the intracellular space, and overexpression of
multidrug transporters in neoplastic cells has been shown to correlate with
resistance to chemotherapeutic drugs.
Multidrug transporters in the BBB have been described and alterations in
these drug transporters are one proposed mechanism of drug resistance in
epilepsy. P-glycoprotein and members of the multidrug resistance-associated
protein (MRP) family are the primary drug transporters identified to have a
role in drug-resistant epilepsy. P-glycoprotein is a transporter expressed in
endothelial cells of the BBB that functions in the active transport of lipophilic
molecules from the intracellular space into the vascular space. Studies of
epileptogenic tissue surgically removed from patients with intractable seizures
show an overexpression of P-glycoprotein at the epileptic focus [6] and exper-
iments in animals provide evidence for the role of P-glycoprotein in regulat-
ing the brain concentrations of multiple anti-epileptic drugs [7, 8]. The family
of proteins known as MRP also functions in the transport of lipophilic mole-
cules at the BBB. These proteins are normally expressed in various tissue
types throughout the body. In the brain, MRP expression is found normally in
capillary endothelial cells. Studies in human brain tissue have shown the
abnormal expression of MRP in neurons and glia. In surgically resected
lesional tissue from patients with refractory epilepsy due to hippocampal scle-
rosis, focal cortical dysplasia, and dysembryoplastic neuroepithelial tumors,
abnormal MRP expression was demonstrated in reactive astrocytes, dysplas-
tic neurons, and capillary endothelium [9], suggesting a physiological basis
for drug resistance.
While the overexpression of multidrug transporters in the brain has impli-
cations for the effective control of seizure activity with an anti-epileptic drug, the
inability to adequately control seizure activity may have etiological implications
for drug-resistant epilepsy. A recent study of seizure-induced expression of
P-glycoprotein in rodents demonstrates that both acute and chronic epileptic
activity significantly increases the level of P-glycoprotein mRNA [10]. These
findings suggest that uncontrolled seizures may contribute to pharmacoresistant
epilepsy. For the subpopulation of patients with a genetic predisposition to drug-
resistant epilepsy, the lack of effective pharmacotherapy to adequately control
seizure activity may be especially detrimental.
The overexpression of drug transporters in an epileptic focus appears to
promote pharmacoresistant epilepsy by limiting local access of anti-epileptic
drugs. In the future, novel pharmacotherapeutic approaches to epilepsy may
include the development of anti-epileptic drugs that are not substrates for
membrane permeability proteins and the adjunctive use of multidrug transport
inhibitors with traditional anti-epileptic drugs. The lipophilic nature of most
anti-epileptic drugs enhances their ability to penetrate the brain, but may inad-
vertently promote drug resistance in those patients that overexpress multidrug
transporters. The development of new drug delivery systems that effectively
deliver anti-epileptic agents to the brain, but avoid the potential to promote
multidrug transporter-mediated pharmacoresistant epilepsy, is an area that
deserves attention.
Epilepsy: An Autoimmune Disorder?
As the scientific community continues to search for the mechanisms that

underlie intractable epilepsy, our understanding of the complexity of factors
that influence the development of seizures continues to expand. In the late
1970s, based on empirical evidence from children with epilepsy who received
immunoglobulin for recurrent upper respiratory tract infections, the theory of
an immunological component of epilepsy was revisited. A decrease in the fre-
quency and severity of seizures was observed in this population. This prompted
a flourish of research in the area of immunological mechanisms in the central
nervous system.
Research has revealed new evidence that certain refractory seizure disor-
ders may be autoimmune mediated. The presence of specific antibodies has
been identified in Rasmussen’s encephalitis, noninflammatory focal epilepsy,
and pediatric ‘catastrophic’ epilepsy and research has shown these antibodies to
be directed towards the GluR3 subtype of the AMPA glutamate receptor [11].
Characterization of the antibodies indicates that some interact with the GluR3
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receptor at a site distant from the glutamate binding site and function in channel
opening and neuronal activation [12]. This provided the first evidence of
autoimmune-mediated activation of a metabotropic ion channel receptor in the
central nervous system. Further studies demonstrated the influence of IgG
immunoreactivity and complement activation on neuronal death [13]. Cortical
dysfunction via immune-mediated mechanisms may be due to the combination
of excitotoxic overactivation of the neuronal glutamate receptor and activation
of complement leading to neuron injury.
It is hypothesized that Rasmussen’s encephalitis is an autoimmune disease
that results in neuronal damage and subsequent development of seizures.
Animal studies revealed that rabbits immunized with a portion of the GluR3
protein demonstrated production of anti-GluR3 antibodies and development of
a Rasmussen’s type disorder characterized by inflammatory cerebral lesions
and recurrent seizures. In a study of pediatric patients with Rasmussen’s
encephalitis, serum GluR3 autoantibodies were identified and titers were
correlated with seizure frequency [14]. In both studies, a response to plasma
exchange to remove circulating anti-GluR3 antibodies was demonstrated. In
subsequent studies, a positive response to the use of intravenous immunoglob-
ulin in patients with the adult-onset variant of Rasmussen’s encephalitis was
observed [15, 16]. In separate animal studies, though, mice immunized with the
GluR3 peptide displayed significant pathological brain abnormalities, but no
seizure activity [17, 18], lending support to the hypothesis that production of
anti-GluR3 autoantibodies may be necessary for the development of excitotoxic
and complement-mediated neuronal damage in Rasmussen’s encephalitis, but
is not sufficient to play a primary role in development of epileptic seizures.
A causative autoimmune mechanism has also been hypothesized for the
intractable epilepsy associated with West’s syndrome, Lennox-Gastaut syndrome,
and Landau-Kleffner syndrome. While most of the support comes from noncon-
trolled studies, small case series, or single case reports, there is evidence that
immunotherapy with intravenous immunoglobulin can completely control
seizures in a portion of these patients.
There are multiple factors that may contribute to the development of
epilepsy as a result of autoimmune-mediated neuronal activity. The presence of
autoantibodies could have a role in modulating epileptic activity independent
of glutamate receptor activation. Immunoreactivity-associated epilepsy is seen
in Hashimoto’s and viral encephalitis where it is mediated by autoantibodies
against voltage-gated potassium channels (Lang and Vincent, 1996). Animal
studies have shown that mice immunized with GluR3B peptide demonstrate
production of anti-ssDNA antibodies at levels similar to those seen in the mouse
model of systemic lupus erythematosus [17, 18]. It has been documented that
systemic lupus erythematosus patients exhibiting high levels of anti-ssDNA
antibodies can experience seizures, but it is not known through what mechanism
anti-DNA reactivity influences epileptogenesis. As would be anticipated in a
complex disease process that results in epileptic activity, genetic factors are also
likely to influence susceptibility to autoimmune seizure development. Many
autoimmune disorders, such as systemic lupus erythematosus and juvenile onset
diabetes mellitus, are associated with genetic variations in MHC gene expres-
sion. There is evidence of an increased incidence of Rasmussen’s encephalitis in
patients with specific human leukocyte antigen haplotypes.
While the precise mechanisms whereby autoantibodies alter neural circuits
and influence seizure activity are being elucidated, there is evidence that
treating appropriately selected patients with immunotherapy is beneficial in
reducing seizure frequency and improving function. In select patients with
Rasmussen’s encephalitis, treatment with intravenous human immunoglobulin
or protein A immunoadsorption resulted in decreased seizure frequency,
improved cognitive function, and improvement on SPECT imaging [15].
Plasma exchange and intravenous IgG are beneficial in patients with antibody-
associated Hashimoto’s or viral encephalitis and epilepsy. Despite the lack of
definitive data as to the precise role of autoantibodies in seizure development,
empirical evidence does support a possible role for immunotherapy in select
types of intractable epilepsy. The influence of immunological mechanisms in
certain epilepsy syndromes provides support for the multifactorial nature of
seizure etiology and encourages researchers to consider novel approaches to
pharmacotherapy in epilepsy.
Neuroprotection: Preventing Epilepsy?
Traditionally, pharmacological neuroprotection is a concept primarily

associated with acute neurodegeneration due to cerebral ischemia or trau-
matic brain injury. The goal of drug therapy in these settings is to restore the
normal biochemical environment and protect neurons from the cytotoxic
effects of inflammation and hyperexcitability. Anti-thrombotic, thrombolytic,
and anti-inflammatory agents are used in stroke and head injury to prevent
the sequelae of such insults to the brain. Recent studies of anticoagulation
with unfractionated heparin following ischemia and brain trauma in animal
models show a decrease in lesion size and improvement in motor and cognitive
deficits [19]. Unfractionated heparin is believed to have not only anti-coagulant,
but anti-oxidant, anti-inflammatory, anti-excitatory, and neurotrophic effects that
may act synergistically to provide neuroprotection in acute brain insult. After
traumatic brain injury, neuroprotective therapy generally focuses on preven-
tion of secondary brain injury. Many pharmacotherapeutic agents are being
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investigated and a variety of promising therapeutic options have emerged,
including glutamate receptor antagonists, calcium channel antagonists, and
free radical scavengers.
A common theme in neuroprotection is prevention of seizures in patients who
are at increased risk for developing epilepsy. Potential causes of epilepsy include
cerebrovascular disease, perinatal hypoxia or ischemia, infection, febrile seizures,
tumors, congenital malformations, trauma, and status epilepticus. Underlying
genetic factors may also have a role in determining seizure susceptibility after an
insult. The ability of the anti-epileptic drugs to prevent epileptogenesis in at-risk
patients is largely unknown.
Clinical studies of phenytoin, phenobarbital, carbamazepine, and valproate
have failed to show a protective effect in the development of epilepsy after head
injury. Studies in animal models, though, have shown that valproate may be
effective in preventing epilepsy. These findings emphasize the complex multi-
factorial nature of seizure development and emphasize the need for multifaceted
treatment strategies in epilepsy. Identifying the fundamental mechanisms of
epileptogenesis may allow us to develop therapies that target the underlying
disease process and effectively alter the development or progression of epilepsy.
There is undoubtedly a cascade of disparate events that occurs in the
development of epilepsy. A primary insult in the setting of genetic suscepti-
bility may lead to fundamental structural or biochemical changes that result in
spontaneous seizures and epilepsy. Pharmacotherapeutic strategies that can alter
the sequelae of brain injury, influence the process of epileptogenesis, prevent
or terminate seizure activity, modify underlying pathology, or interfere with
multidrug resistance mechanisms do have an essential role in the successful
treatment of patients with epilepsy.
Defective Ion Channels: From Pathogenesis to Therapy
Further development of effective therapeutic strategies will depend upon

elucidation of genetic factors that influence epilepsy and novel approaches to
modulating genetic defects. Altered gene expression and mutated gene products
are known to play an etiological role in epilepsy. Genetic mutations have been
identified in some of the rare familial epilepsies as well as some types of
idiopathic epilepsy. Many of the identified genes encode for ligand- or voltage-
gated channels. A variety of paroxysmal disorders are due to defective ion
channels, or ‘channelopathies,’ including some of the myotonias and other
neuromuscular disorders, and long QT-syndrome. Defects in sodium, potassium,
calcium, chloride, or glycine-mediated channels have been identified in these
disorders. Epilepsy models employing sporadic mutant animal strains, genetically
engineered animals, and knockout animals, coupled with human studies have
helped elucidate the role of ion channel defects in epilepsy.
Ion channels have a recognized role in generating electrical currents and a
channel defect could easily alter the balance of excitation and inhibition in
neural networks, resulting in neuronal hyperexcitability and progression of
epileptogenesis. All of the genes identified in the human familial epilepsies
encode ion channels or auxiliary subunits. Genetic defects in ion channels not
only contribute to idiopathic epilepsy, but may also participate in the develop-
ment of post-traumatic epilepsy. Neuronal injury induces the expression of ion
channels, and overexpression of abnormal ion channels may lead to epilepsy
after a cerebral insult [20].
Recognition of some of the multiple genetic defects associated with
epilepsy illustrates the incredible heterogeneity of genes that function in pro-
ducing the epileptic phenotype. Just as the scientific community has adjusted
its view of cancer etiology to emphasize a multifactorial basis, the approach to
epilepsy research must consider the varied genetic and environmental factors
that underlie the mechanisms of epileptogenesis. It is unlikely that a single
mechanism is responsible for the development of seizures and progression to
epilepsy. A singular approach to epilepsy therapy based on the premise that a
single agent operating on a single mechanism can potentiate effective treatment
is fundamentally limited. An appreciation for the diversity of genetic influences
in seizure disorders will guide future therapeutic strategies.
Advances in Drug Delivery
The disappointing failure of the new anti-epileptic drugs to significantly

improve outcomes in the pharmacotherapeutic treatment of epilepsy has
prompted researchers to re-evaluate the current approach to seizure control
and pursue innovative avenues of investigation. Traditional approaches to
anti-epileptic drug development have focused on formulating new agents or
improving existing agents. A novel avenue of drug development focuses on
advanced delivery systems. From engineered drug reservoirs to implantable
drug pumps and synthetic polymers, these new methods of drug delivery may
prove successful in improving the efficacy while decreasing the systemic
effects of anti-epileptic drugs. Moreover, new drug delivery systems may have
applications beyond simple delivery of anti-epileptic drugs to delivery of cell
and gene therapy agents.
Special oral formulations of traditional anti-epileptic drugs have been
developed and are in widespread clinical use. Some of these new forms of
medication were designed for use in certain seizure settings or specific patient
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populations, while others were developed to achieve the elusive goal of
pharmacotherapy with enhanced efficacy and decreased systemic toxicity. For
emergency situations that require quick drug delivery and acute seizure exacer-
bations, such as occur in febrile seizures, traditional oral therapy is not an
effective mode of delivery. Transmucosal administration of anti-epileptic drugs
theoretically allows for faster drug delivery. A rectal gel formulation of
diazepam is available and commonly used in terminating acute seizure activity.
Nasal or buccal mucosa administration of anti-epileptic medication may also
achieve rapid delivery. While there are no formulations currently approved for
administration through these routes, the off-label use of liquid benzodiazepine
formulations via nasal and buccal routes is common practice.
Drug delivery through the respiratory mucosa is an attractive alternative
mode of therapy. The administration of inhaled drugs is a daily practice in the
induction of anesthesia and the treatment of acute asthma exacerbations.
Recently, inhaled chemotherapeutic agents have been used to treat lung cancer.
The engineering of micro- and nanoparticulate systems with an adhesive
coating may allow for better delivery to the alveoli and greater uptake across
the blood-air barrier, and holds promise for the development of an improved
anti-epileptic drug delivery system.
Drug administration in the pediatric population has been made easier with
the introduction of syrups, sprinkles, and chewable formulations of certain
anti-epileptic drugs. Depot forms of anti-epileptic drugs, such as transdermal
patches or subcutaneous implants, may also prove effective in improving phar-
macotherapy in the pediatric and other populations, but these drug formulations
are still only theoretical. While new anti-epileptic drug formulations increase
the treatment options available to patients with epilepsy and may improve
adherence to therapy, they have not had a significant impact on the proportion
of patients that achieve seizure control. Some researchers argue that the focal
delivery of anti-epileptic agents holds the most promise for significantly
improving seizure control in patients with epilepsy. This is an area of drug
development that has gained much attention in recent years.
The administration of a prodrug that is systemically inert and becomes
activated at the seizure focus holds the potential to inhibit seizure activity
with minimal systemic toxicity. The development of the prodrug DP-VPA is
already underway [21]. This engineered drug is composed of valproic acid
coupled to a phospholipid moiety that serves to inactivate the valproic acid.
At the seizure focus, abnormal neuronal activity results in elevated activity of
phospholipases, which function to cleave the phospholipid moiety and release
the activated drug.
Direct administration of anti-epileptic drugs into the CSF space provides
for the possibility of better prevention of seizure activity and may have a role
in termination of seizures. The use of an intraventricular or intrathecal catheter
to deposit drug directly into the CSF bypasses one of the major problems
in CNS drug delivery, penetration of the BBB, with the additional benefit of
decreased systemic toxicity. Subcutaneous infusion pump implants are already
in clinical use for the long-term intrathecal administration of analgesics for
chronic pain control and anti-spasticity medications following brain injury.
While the application of an intrathecal infusion pump system in the delivery of
anti-epileptic drugs is still speculative, a collaborative effort to investigate the
infusion of an NMDA antagonist via the intrathecal route is underway. Direct
infusion of drugs into the CNS has proven successful in certain settings, but
there are limitations to this therapy.
The delivery of anti-epileptic drugs directly into a seizure focus is an innov-
ative approach with immense potential to improve pharmacotherapy in intractable
epilepsy. Microinjection and microinfusion systems have been used to investigate
the effects of focal application of anti-seizure agents on seizure activity in
animals. Local perfusion of an anti-epileptic drug directly into the seizure focus
in an animal model of epilepsy was effective in attenuating ictal and interictal
events. Pioneering work in the development of a computer-controlled drug deliv-
ery system coupled with a seizure detection device is being conducted (Stein
et al., 2000). The automated system employs a seizure-prediction algorithm that
activates a programmable infusion pump to deliver a predetermined amount of
anti-epileptic drug directly into the seizure focus. Animal studies show the ability
of such a system to shorten seizure duration and prevent subsequent seizures. The
ability to prevent progression of partial to generalized seizures would prove
invaluable to many patients who suffer from intractable epilepsy. More powerful
detection algorithms hold the potential to deliver anti-epileptic agents into the
seizure focus before a seizure is clinically evident.
Engineered drug reservoirs such as liposomes and nanoparticles have broad
applications as delivery systems and are currently under investigation for use in
delivering anti-epileptic drugs to the CNS and directly to the seizure focus.
These inert carrier vehicles direct agents to a target tissue via ligand-receptor
binding, theoretically increasing potency at the target site while decreasing
toxicity in other tissues. The specific delivery and penetration parameters of
unmodified carriers vary depending upon the lipid composition of the vehicle.
In contrast, modified carriers are tagged with a ligand or receptor that functions
to increase vehicle affinity for a specific cell type, thus enhancing drug delivery
to the desired site of action. These tags include antibodies, hormones, cytokines,
toxins and engineered ligands.
A major obstacle in the development of a system of drug delivery to the
CNS is penetration of the BBB. The cerebral capillary endothelium and astro-
cyte foot processes that comprise this protective barrier play an important role
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in maintaining a constant chemical environment and shielding neurons from
toxic agents. Hydrophilic and large molecules are normally excluded from the
CNS by this impermeable barrier. Liposomes are generally unable to penetrate
normal endothelial pores (2 nm). The tight junctions and lack of pores that
characterize the cerebral endothelium present a significant impediment to the
vehicle-mediated delivery of drugs to the CNS.
Deliberate chemical disruption of the BBB is already used to gain direct
access to the CNS for the delivery of chemotherapy agents, and researchers are
investigating the use of electrical disruption as a similar means of gaining direct
CNS access [Orr, 2000]. It is known that seizures disrupt the BBB, which holds
interesting implications for the focal delivery of an anti-epileptic drug immedi-
ately preceding or at the onset of a seizure. Moreover, the methods of direct
CNS delivery of anti-epileptic drugs may also apply to the delivery of cell and
gene therapy agents.
An alternative approach to disruptive penetration of the CNS proposes the
use of active targeting of transport vectors to circumvent the protective nature
of the BBB. Endogenous peptides, modified proteins, and monoclonal
antibodies can be used to transport large, water-soluble molecules across the
BBB and deliver therapeutic agents directly to targets in the CNS. A recent
study demonstrated the CNS delivery of systemically administered vasoactive
intestinal peptide coupled to a monoclonal antibody [Bikel et al., 2001]. The
use of this approach to gain access across the BBB will depend upon the
identification of appropriate targets within the CNS. Its value as an effective
method of drug delivery in epilepsy will require the characterization of the
seizure focus and the discovery of common targets. As researchers continue to
elucidate the cellular and molecular nature of epileptogenic brain the prospects
for development of effective drug delivery strategies to treat epilepsy will
continue to improve.
The use of polymers to deliver anti-epileptic drugs directly to the seizure
focus is another method that has potential for seizure control in intractable
epilepsy. A polymer is a complex of drug in a dissolvable matrix. As the matrix
dissolves, drug is released into the immediate area. Polymers can be engineered
to vary dissolution rates in response to changes in the chemical environment.
The use of polymers as a strategy in drug delivery is being employed in the
treatment of recurrent glioblastoma multiforme. A polymer composed of an
alkylating agent is implanted in the tumor bed following resection and slowly
releases the chemotherapy agent directly into the malignant tissue. The use of
polymers in anti-epileptic drug delivery to the brain has been investigated in
animal models and was shown to decrease or attenuate seizure activity [22].
While this promising delivery strategy is still in development, the potential
requirement of multiple craniotomies may make its use impractical.
As the cellular and molecular mechanisms underlying epilepsy are eluci-
dated, it has become apparent that traditional strategies of pharmacotherapy
will continue to fail until we fully appreciate the complex interplay of multiple
factors that influence the development of treatment-resistant epilepsy.
Innovative methods of enhanced drug delivery do indeed hold promise for
effective treatment of intractable epilepsy, but even direct delivery of anti-
epileptic agents into a seizure focus does not attempt to modulate the funda-
mental physiological and structural changes that characterize epileptogenic
brain. While the development of novel drug delivery systems is primarily
directed towards improving the pharmacological treatment of epilepsy, these
strategies have been extended to the delivery of cell and gene therapy agents in
the hope of ultimately curing this multifaceted disease.
Current Alternatives to Pharmacotherapy
When pharmacotherapy fails to adequately control seizures there are a vari-

ety of adjunctive or alternative therapies available. While some may be considered
extreme, in the patient with intractable epilepsy these nonpharmacological thera-
pies offer the only possibility of a life free from seizures. Noninvasive adjunctive
therapy is limited to the ketogenic diet. The options for invasive treatment of
epilepsy are varied and include conventional surgical techniques, multiple subpial
transection, implantation of a vagus nerve stimulator, gamma knife radiosurgery,
and implantation of depth electrodes for deep brain stimulation.
Dietary Treatment of Epilepsy

The ketogenic diet has been utilized in the adjunctive treatment of refractory
epilepsy for over 75 years. Its pattern of use in treating intractable seizures has
varied and we are currently experiencing resurgence in the use of the ketogenic
diet to control seizures that are resistant to pharmacotherapeutic strategies. The
high-fat, low-protein, and low-carbohydrate diet produces a ketotic state similar
to that seen in starvation. The diet has been evaluated using the MES and PET
infusion models in a manner similar to anti-epileptic drugs. It has been shown that
ketosis functions to increase the seizure threshold, but there is no evidence that it
lessens the severity of seizures. The exact mechanisms whereby a ketotic state
produces an anti-epileptic effect are unclear.
Ketosis causes changes in overall brain energy metabolism, cell membrane
lipid composition, localized cerebral pH, and brain water content, though
the extent to which any one or a combination of these alterations influences
seizures is not certain. The results of recent work in the biochemical basis of the
ketotic effect suggest that alterations in brain excitatory amino acid metabolism
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may be primarily responsible for the anti-seizure property of the ketogenic diet.
Altered metabolism has the overall effect of shifting neuronal amino acid equi-
librium to favor glutamate production at the expense of aspartate production,
enhance synthesis of GABA from the increased pool of glutamate, increase
removal of glutamate from the synaptic cleft, and enhance release of glutamate
in the form of glutamine into the vascular space. The ultimate result of these
biochemical alterations is the ability to attenuate and possibly terminate the
development of seizures.
Some reports indicate the ketogenic diet to be at least as effective as the
anti-epileptic drugs in controlling certain seizure types, and even exceeds
pharmacotherapeutic efficacy in some cases. Studies of efficacy vary and
indicate that 14–46% of children on the diet experience a greater than 50%
reduction in seizure frequency and 7–54% of children achieve complete seizure
control [23]. While this can represent a significant improvement in seizure
activity, in practice it seldom offers freedom from the burden of daily debilitat-
ing seizures. What the ketogenic diet does offer is a new paradigm to think about
the molecular mechanisms of epilepsy. A change in the primary metabolic
substrate in the brain from glucose to ketones appears to alter the seizure thresh-
old in one area of brain without affecting the normal global function throughout.
The ketogenic diet has utility in treating seizures of multiple types and various
etiologies. This lends to the theory that there are biochemical pathways common
to the different seizure types and etiologies that make diverse seizure disorders
responsive to a single therapy. Elucidation of the fundamental changes in cere-
bral energy metabolism that underlie the development of seizures holds major
implications for the development of successful therapy for epilepsy.
Surgery: A Cure for Epilepsy?

For over a century, the surgical resection of epileptogenic lesions has
proven curative in certain types of epilepsy. At present, it remains the only
true cure for certain subtypes of this devastating disorder, but remains signif-
icantly underutilized. The remarkable advances in diagnostic tools available
to evaluate the epileptogenic brain and localize seizure foci have enabled sur-
gical intervention to play an increasingly important role in epilepsy therapy.
The use of magnetic resonance imaging, PET and SPECT functional imaging,
electrocorticography, and implanted depth electrodes allow better localization
of seizure foci. Coupled with refined surgical techniques, localization-related
refractory epilepsy may be particularly amenable to surgical therapy.
While the standard of medical management of epilepsy defines
intractable epilepsy as seizures that continue after 2 years of therapy with at
least two first-line anti-epileptic drugs, it is not uncommon for the patient
with refractory epilepsy to have been tried on numerous traditional and new
generation anti-epileptic drugs in various combinations for more than 2 years. In
the pediatric population there is an even greater reluctance to consider early
surgical intervention, as the natural history of seizures in this population may be
self-limited. The natural history of epilepsy is dependent upon several factors,
including the clinical presentation, epilepsy syndrome, and etiology of epilepsy.
The importance of recognizing self-limited disorders from those that are truly
refractory becomes evident when one considers the cognitive and psychosocial
consequences of intractable epilepsy. The ability to reduce the high morbidity
that accompanies both temporal- and extra-temporal localization-related
epilepsy provides a strong argument for early surgical intervention in children
with intractable seizures of these etiologies. There are also nonlocalized epilepsy
syndromes with severe seizures in which surgical intervention can have a
significant impact on the progression of the devastating neurodevelopmental
consequences.
The capacity to actually affect the course of progression of a seizure
syndrome is of considerable importance in the surgical therapy of intractable
epilepsy. Anti-epileptic drugs may be of great benefit in controlling the seizures
associated with a particular disorder, but they cannot offer any advantage
in altering the natural history of the epilepsy. A 30–35% recurrence rate of
seizures after the discontinuation of an anti-epileptic drug is not uncommon,
while surgical therapy for certain seizure disorders can reduce or completely
eliminate seizures in 70–90% of treated patients [24].
Surgical treatment of epilepsy is often considered radical and carries
known risks. A rigorous presurgical evaluation is essential to identify those
patients that will benefit from surgical intervention. Anatomical or functional
hemispherectomy is generally reserved for patients with progressive devastat-
ing nonlocalizing epilepsies who are not candidates for a localized resection. If
accomplished early enough, neuronal plasticity will allow for some recovery of
function in selected areas. As previously indicated, this intervention can have a
significant affect on the progression of neurodevelopmental consequences.
Corpus callosotomy has benefit in treating frequent intractable drop attacks
from both tonic and atonic seizures. Surgical intervention in this setting cannot
confer freedom from seizures and is instead aimed at improving quality of life.
Focal temporal cortical resection is effectively curative in appropriately
selected patients with intractable complex partial seizures originating in the
temporal lobe. Multiple surgical techniques have been employed, from
amygdalohippocampectomy to en bloc resection. Focal extratemporal cortical
resection is the most commonly employed surgical intervention in children.
Presurgical evaluation is complicated in this population as the seizure focus is
generally more difficult to localize in extratemporal as compared to temporal
lobe epilepsy, and the focus may be located in a silent region of the brain and
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clinically apparent only after spread to adjacent areas. In patients with a
seizure focus either completely or partially localized to eloquent cortex
multiple subpial transection allows for the surgical manipulation of epilepto-
genic cortex without significant neurological deficit. The procedure is designed
to disrupt horizontal neuronal connections without interrupting efferent cortical
fibers.
The successful surgical treatment of epilepsy depends on the identification
of the appropriate surgical candidate. In all cases, early identification of
suitable syndromes and timely surgical intervention offers the best possibility
for a normal life. In the future, perhaps, surgical intervention in epilepsy will
not be limited to removal of epileptogenic brain or mechanical disruption of
neural connections, but will instead be utilized in the cellular and genetic ther-
apy of epilepsy to fundamentally alter the pathological processes underlying
this debilitating disorder.
Vagus Nerve Stimulation

In 1997, a new treatment modality for intractable epilepsy was intro-
duced. Vagus nerve stimulation is FDA-approved for the adjunctive therapy
of refractory partial seizures. Patients who are not candidates for epilepsy
surgery or elect not to undergo intracranial surgery may benefit from the sub-
pectoral implantation of a programmable pulse generator and left mid-cervical
vagus nerve electrodes for continuous cyclical stimulation. Studies evaluating
the mechanism of action of vagus nerve stimulation indicate it functions via
immediate and long-term effects. Short-term changes in the nucleus of the
solitary tract and its connections cause synchronization and desynchronization
of electrical activity in the brain [Magnes et al., 1961; Peñaloza, 1964; Chase
et al., 1967]. The solitary tract nucleus has projections to the parabranchial
nucleus, hypothalamus, amygdala, infralimbic cortex, ventroposterior,
intralaminar, and midline thalamic nuclei, insular cortex, dorsal raphe, and
nucleus ambiguous. Long-term changes in cerebral blood flow and neuro-
transmitter concentrations have been documented [25, 26]. Increased nor-
adrenergic and serotoninergic activity, which functions to increase the seizure
threshold, has also been hypothesized to play a role [27]. Blinded randomized
controlled trials of efficacy of vagus nerve stimulation are methodologically
difficult, but longitudinal studies demonstrate a mean reduction in seizure
frequency of 22–48%, which varies with intensity of stimulation, with some
patients experiencing >75% reduction in seizures [28]. While this may repre-
sent a significant improvement in seizure status for an individual, vagus nerve
stimulation does not confer complete seizure control. A novel approach to
vagus nerve stimulation employs a transcutaneous stimulator for noninvasive
therapy of refractory partial seizures [29].
Gamma Knife Radiosurgery
Advances in our understanding of the molecular mechanisms of epilepsy
coupled with an increasing appreciation for the role of surgical treatment of
intractable seizures has lead to the application of a variety of novel surgical
techniques for control of seizures. Gamma knife radiosurgery allows for the
precise irradiation of a specific target with minimal radiation effects on
surrounding tissue. It is commonly used in the ablation of arteriovenous
malformations and neoplastic lesions. In the early 1980s animal experiments
established the role of ionizing radiation in restricting the spread of discharges
in the epileptic brain. A role for radiosurgery in epilepsy therapy was first
noted in a series of patients who underwent gamma knife surgery for the
treatment of cerebral arteriovenous malformations and showed a concomitant
improvement of seizures. Complete seizure control can be achieved after
radiosurgery treatment of a lesion with seizures at presentation, and a signifi-
cant decrease in seizure frequency can be seen in adults with low-grade
astrocytoma and intractable epilepsy following conformal radiotherapy.
Brachytherapy and conventional radiotherapy for low grade tumors with
refractory seizures can also have a significant impact on seizure frequency.
The success of gamma knife treatment of seizures associated with mass
lesions essentially introduced the possibility of radiosurgery as effective ther-
apy for focal epilepsy.
Less than a decade ago the first patient with intractable mesial temporal
lobe epilepsy (MTLE) was treated with gamma knife entorhinoamygdalohip-
pocampectomy. Since then, studies evaluating radiosurgery instead of micro-
surgery for MTLE indicate it is an efficacious and safe treatment option that
can reduce the morbidity associated with invasive surgical intervention [30].
The mechanism of action of gamma knife surgery is largely unknown.
Computed tomography and MRI imaging show radiation-induced structural
changes in the mesial temporal lobe, but the significance of these findings is
unclear. Clinical studies of gamma knife surgery suggest improvement of
seizures may represent an actual anti-epileptic effect independent of structural
alterations. Using animal models it has been demonstrated that non-necrotizing
doses of irradiation can improve seizures [31] and the anti-epileptic effect
increases with increasing doses [32]. Biochemically, it is theorized that radia-
tion inhibits protein synthesis, thus preventing maintenance of spontaneous
bursting in neurons [33], and has a differential effect on the inhibitory GABA
system and the excitatory amino acid system [31]. Developments in the tech-
niques for noninvasive surgery provide novel treatment options for intractable
epilepsy. Gamma knife radiosurgery offers the possibility of effective treatment
for certain types of refractory seizures with minimal impact on normal brain
function.
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Deep Brain Stimulation
Brain stimulation is a novel therapeutic strategy for intractable epilepsy
with the potential to effectively control seizures associated with certain
epilepsy types in patients who are not candidates for surgical resection.
Neuromodulation of brain structures by electrical stimulation has been used to
treat neurological disorders of movement and chronic pain. In the treatment
of Parkinson’s disease, high frequency stimulation of the thalamus, pallidum,
and subthalamic nucleus produces the same effect as ablative neurosurgery.
Previous attempts to influence seizure activity by electrical stimulation of deep
brain structures have targeted the caudate nucleus, anterior thalamus, centro-
median thalamic nucleus, posterior hypothalamus, and hippocampus. Results
have been varied and limited by study design. Recent attention to deep brain
stimulation in epilepsy therapy is focusing on the subthalamic nucleus as a
target for stimulation. While the inhibitory effect of high-frequency stimulation
of the subthalamic nucleus on the substantia nigra was initially based on the
theory that electrical stimulation inhibits function, several studies provide
evidence for the molecular mechanisms that underlie inhibition via electrical
stimulation. High-frequency stimulation of subthalamic neurons has been
shown to produce a long-lasting blockade of depolarization of voltage-gated
channels [34]. Both spontaneous and induced epileptiform activity has been
reduced or terminated by high-frequency cortical, subthalamic, and hippocam-
pal stimulation, and this inhibition of activity occurs when neurons are depo-
larized [35]. There is also evidence that high-frequency stimulation may activate
inhibitory GABAergic circuits in the basal ganglia and inhibit postsynaptic
activity in the subthalamic nucleus [36, 37].
Direct inhibition of deep brain structures may not be the only effect of
electrical stimulation. An excitatory effect of high-frequency stimulation has
also been supported. Functional imaging provides evidence for activation of
stimulated structures. Neurophysiological studies indicate the findings from
microelectrode recordings of stimulated structures are inconsistent with the
hypothesis that high-frequency stimulation inhibits the target structure [38, 39].
Stimulation of deep brain structures may also affect cortical activity through
anti-dromic connections. Using animal models, stimulation of the subthalamic
nucleus has demonstrated retrograde activation of the corticosubthalamic
pathway, a major afferent projection to the subthalamic nucleus, evidenced by
measurable cortical potentials [40, 41]. Preliminary studies in patients with
epilepsy demonstrated evoked cortical potentials after subthalamic nucleus
stimulation [38]. It is unclear precisely how retrograde cortical activation could
suppress seizure activity. Anti-dromic activation of cortical interneurons may
be the mechanism whereby cortical excitability is inhibited. Computer models
of high-frequency stimulation suggest simultaneous neuronal excitation and
inhibition may mediate the therapeutic effects of subthalamic nucleus stimula-
tion in epilepsy [42].
Stimulation of the subthalamic nucleus in the treatment of certain
intractable epilepsy types in humans has been successful in reducing seizures.
Modulation of glutamatergic subthalamic output may influence cortical
excitability by inactivation of the nigral control system as well as activation of
cortical GABAergic inhibition, resulting in suppression of seizure activity.
A greater understanding of the basic principles by which stimulation of deep
brain structures can influence the endogenous control systems in the brain and
modulate cortical excitability will promote the application of deep brain stim-
ulation to certain intractable epilepsies.
From Drug Delivery to Gene Delivery
Novel drug delivery strategies are being applied to the cellular and genetic
treatment of epilepsy. Cellular transplantation and delivery of genetic material
hold the potential to not only effectively treat intractable seizures, but also offer
the possibility of altering the fundamental defects that result in epilepsy.
Cellular Therapy for Epilepsy

Cell transplantation or replacement theoretically offers the possibility of
providing a continuous endogenous supply of a deficient neuromodulator to a
localized area of brain, essentially enabling the restoration of functional
neuronal connections. The feasibility of this innovative approach to treating
pharmacoresistant epilepsy has already been demonstrated. The intraventricu-
lar grafting of an adenosine-releasing synthetic polymer in an animal model
of epilepsy was shown to significantly decrease seizure activity [43]. As an
endogenous inhibitory neuromodulator, adenosine has the potential to influence
the development of synaptic connections and alter the balance of excitation and
inhibition in the epileptogenic brain. This pioneering work has important impli-
cations for cellular therapy of epilepsy. The grafting of stem cells or free cells
from another species has the potential to establish operative neural circuits in
areas of the brain that are intrinsically defective.
Several studies have investigated cell transplantation in animal models of
epilepsy. Neuronal grafts from fetal rats have been transplanted into rats mod-
eling temporal lobe epilepsy and amygdala-kindled rats and demonstrated the
ability to restore GABAergic interneuron connections and reduce epileptiform
after-discharges and clinical seizures, respectively [44–46]. More recently,
fibroblasts engineered to release adenosine and encapsulated into polymers
were grafted into the ventricles of kindled rats. The grafted rats demonstrated a
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significant reduction in kindled seizure activity and significant suppression of
after-discharges [47].
The transplantation of neuronal grafts as a therapeutic strategy in epilepsy
holds tremendous promise for effectively treating this disorder. Currently, one of
the major limitations of such a therapy is immunological rejection. As the brain
is an immunologically privileged site, a host-versus-graft immunoreaction
to transplanted cells can occur. The ability to utilize immature cells that are
immunologically neutral is one possibility in circumventing this problem.
Continued research in the areas of transplantation and neural stem cells is crucial
and will undoubtedly lead to advances in the application of this treatment option.
Strategies in Gene Therapy for Epilepsy

Many concepts in drug delivery and cell therapy can be applied to gene
therapy for epilepsy. What gene therapy offers as an epilepsy treatment strategy
is the possibility of correcting the defect that underlies epileptogenic brain and
essentially curing epilepsy. Just as we have adjusted our view of the pathogen-
esis of epilepsy to encompass a complex multifactorial etiology with genetic
influence, we must also revise our approach to the treatment of epilepsy to
include novel concepts in genetic therapy.
The use of animal models in the study of the molecular mechanisms that
underlie hyperexcitability and epileptogenesis has contributed significantly to our
understanding of the genetic basis of epilepsy. Spontaneous epileptic mutants
involving both mono- and polygenic inheritance allow researchers to progress
from phenotype to genotype and identify many of the genes involved in the devel-
opment of cortical hyperexcitability. The use of engineered transgenic mouse
models permits a genotype to phenotype approach that can enable the elucidation
of the critical steps in epileptogenesis and function in the systematic testing of
pharmacological therapies in epilepsy. Through the use of genetic animal models
we have gained tremendous insight into the genetic abnormalities that can influ-
ence the intrinsic excitability of epileptogenic brain, the mechanisms of altered
synaptic transmission, and disruptions in neural networks.
An estimated 40–50% of epilepsy and epilepsy syndromes are considered
idiopathic and presumed to have a genetic basis [1]. Most of the epilepsy syn-
dromes are not likely to be the result of a single genetic defect, but the outcome
of multiple factors, including a genetic abnormality. Researchers have identified
a number of genes with known causative roles in the pathogenesis of certain
epilepsy syndromes. Many of these syndromes are associated with metabolic
derangements or neurodegenerative disorders.
The genetic defects in several of the progressive myoclonus epilepsy
syndromes have already been elucidated. Unverricht-Lundoborg disease, also
known as progressive myoclonic epilepsy type I, is due to truncation or unstable
insertion in the gene encoding the protease cystatin B and MERRF, or
myoclonic epilepsy associated with ragged-red fibers, results from defects in
mitochondrial DNA. The genetic mechanisms underlying some of the familial
epilepsies have also been identified, including autosomal dominant nocturnal
frontal lobe epilepsy, benign familial neonatal convulsions, generalized
epilepsy with febrile seizures, and episodic ataxia with partial epilepsy. In all
of the familial epilepsies, the identified genes encode for entire cation channels
or their subunits, further evidence that channelopathies may have an essential
role in the development of seizure activity and progression to epilepsy.
Continued research in the molecular basis of the epilepsies will undoubtedly
elucidate additional mechanisms whereby genetic defects lend to the develop-
ment of epilepsy.
Elucidating the genetic mechanism underlying an epileptic disorder
provides for the possibility of effective treatment by replacing either the defec-
tive DNA or abnormal protein product. Autosomal dominant nocturnal frontal
lobe epilepsy is due to a genetic defect that results in a dysfunctional neuronal
nicotinic acetylcholine receptor ␣4 subunit. Knowledge of this defect offers
the possibility of a genetic therapy that could restore the CHRNA4 gene or
replace the abnormal receptor subunit. In the most prevalent epilepsies where
a single genetic defect is not the known etiology, gene therapy theoretically
remains a reasonable treatment option. Similar to cellular therapy, genetic
therapy in the setting of an unknown gene defect may prove therapeutic if
utilized as a modality of drug treatment. The introduction of genetic material
into the brain in this setting cannot correct underlying pathology, but it can
provide a continuous source of neurotransmitter to normalize the balance
between excitation and inhibition.
Strategies in gene therapy have been applied to the treatment of cerebral
neoplasia, neurodegenerative disorders, lysosomal storage disease, Parkinson’s
disease, and stroke with varying success. As a therapeutic approach in epilepsy,
gene therapy is an area of investigation still in its infancy. The successful
delivery of genetic material into the brain is based on strategies borrowed from
novel approaches used in drug delivery and cellular therapy. Penetration of the
BBB remains a formidable challenge, but many of the strategies utilized in drug
delivery, such as the coupling of modified proteins or monoclonal antibodies
to transport vectors, as previously discussed, can be applied to the delivery
of DNA.
There are several methods whereby DNA can be delivered to cells in the
brain. Both ex vivo and in vivo approaches to gene therapy are utilized in the
delivery of genetic material to the CNS. Ex vivo methods are characterized
by the introduction of transgenes into cells that are then grafted into the
brain, while in vivo methods employ vectors to introduce transgenes directly
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into cells in the brain. The use of viral vectors and the development of
engineered vectors, such as plasmids and peptide vehicles, represent the
variety of techniques whereby genetic material is delivered to the CNS.
Herpes simplex virus (HSV), adenovirus, adeno-associated virus (AAV),
retrovirus, and lentivirus are the commonly used viral vectors in gene
therapy. The neurotrophic nature of HSV makes it a good candidate for deliv-
ery of genetic material to the brain, but its long latency period and resultant
transient expression of gene products presents the major obstacle to its effec-
tive use as a genetic vector. The therapeutic use of HSV as a genetic vector
has been demonstrated in rodent models of neuroprotection in focal cerebral
ischemia. The use of an engineered gene that encodes a herpes simplex
enzyme designed to activate a prodrug, such as herpes simplex thymidine
kinase, is another strategy in gene therapy utilizing the HSV. Adenovirus
vectors are also in use in experimental models of gene therapy. Adenovirus is
known to infect both dividing and nondividing cells, which permits its use in
both rapidly dividing malignant brain tumors and neurological disorders of
the postmitotic CNS, such as Parkinson’s disease and epileptic disorders that
generally do not exhibit cell division. AAV shows great promise for effective
use in the delivery of genetic material to the brain. In vivo studies utilizing
recombinant AAV vectors have demonstrated long-term gene expression
without evidence of infection or immune response, and in a mouse model of
traumatic brain injury recombinant AAV was successfully delivered to the
hippocampus. The feasibility of vector-mediated gene transfer into the
epileptogenic brain has been demonstrated in both rats and humans. For
example, the tremor rat is a genetic mutant that exhibits absence-like seizures
and is used as a model of inherited epilepsy. This rat is now known to be an
animal model for Canavan disease. A deletion of the aspartoacylase
gene has been discovered in these animals and the resultant high levels of
N-acetyl-aspartate are understood to be responsible for the epileptic seizures
in tremor rats. Recent studies utilizing the intraventricular administration of
a recombinant adenovirus carrying the rat aspartoacylase gene demonstrated
significant inhibition of the generation of absence-like seizures in experi-
mental animals [48]. In human studies, the effective use of viral vectors to
mediate the transfer of genes into human epileptogenic brain slices has been
shown [49].
Nonviral methods of delivery of genetic material to the brain offer an
advantage over viral vectors, which can be limited by inadequate brain
penetration and ineffective cell transfection. The use of liposome-packaged
plasmids conjugated to a monoclonal antibody has been shown to successfully
cross the BBB and access the microvasculature and parenchyma of the brain.
Viral genetic material can also be packaged into liposomes, termed virosomes,
for delivery to the brain. Virosomes containing AAV plasmids have been
delivered into the ventricles in both primates and humans. Cationic vectors,
such as lipospermines and polyethylenimine, can also be used to facilitate the
transport of genetic material into the brain.
The current body of research in genetic therapy demonstrates the potential
for effective treatment of certain epilepsy disorders using a gene therapy
approach. The transfer of genetic material into neurons in a seizure focus and
the expression of inhibitory neurotransmitters or neuropeptides, membrane
transporters, postsynaptic receptor subunits, or antisense sequences provide for
the possibility of altering the path of signal transduction and inhibiting the
initiation, propagation, or maintenance of seizure activity. Gene therapy has the
potential to influence epileptogenesis due to a variety of causes, including
neurological injury, defective ion channels, or altered levels of neurotransmit-
ter or receptors. Further development of viral vectors that can be used to
transfer therapeutic genes offers the hope of a cure for certain epilepsy disor-
ders. Other avenues of investigation must focus on delivery of transgenes to the
target tissue.
The stereotaxic procedures of molecular neurosurgery provide a powerful
method of delivery of genetic material to cerebral tissue. Currently, stereotaxic
techniques are applied to neuroablative, neuroaugmentative, and neuroendo-
scopic procedures, as well as radiation dosing, anatomical-physiological cor-
relation in neuroimaging, tumor biopsy and resective therapy, and restorative
surgical therapy. Both point-in-space and volumetric techniques are utilized in
stereotaxic procedures, but volumetric stereotaxis provides many advantages
in molecular neurosurgery including localization of a target structure, concep-
tualization of the three-dimensional shape of a target structure, preoperative
planning of surgical approach and trajectory, and positional differentiation of
target and adjacent tissue. A stereotaxic neurosurgical approach to genetic
therapy in epilepsy can be utilized to deliver a transgene-containing viral
vector or genetically engineered cells to a seizure focus. When a focal seizure
origin cannot be identified the global delivery of genetic material via the
endovascular system can be accomplished using stereotaxic intraventricular or
intraparenchymal injection, interstitial infusion, or catheter-mediated delivery
of transgenic vectors.
As the molecular mechanisms of the epilepsies are elucidated, it is becom-
ing apparent that an aggressive approach to gene therapy in epilepsy must be
pursued. The delivery of genetic material to cerebral tissue offers a therapeutic
strategy that can alter the pathological basis underlying epileptogenesis in the
human brain. The possibility of a cure for certain epilepsy disorders is on the
horizon and we must continue to pursue innovative and novel approaches to
gene therapy for this devastating neurological disorder.
medwedi.ru
RNA Expression Profiling: Pharmacogenomics and
Disease ‘Fingerprinting’
The analysis of mRNA expression in individual cells provides a strategy to

compare the transcriptional profile of individual phenotypically characterized
neurons. This approach has been implemented in human and experimental
epilepsy models and in live as well as fixed cell types. The use of an oligo-dT
primer and T7 RNA polymerase permits amplification of a broad population of
expressed genes across many gene families. The size range and complexity of
the amplified mRNA provides a comprehensive view of differential gene
expression in single cells. Individual cell differences in gene expression could
be used to develop new targets for epilepsy pharmacotherapy, ‘personalize’ treat-
ment with existing drugs, or ‘fingerprint’ individuals for disease diagnostics.
The concept of ‘personalized medicine’ is now within reach due to the land-
mark innovation of the biochip and the wealth of information created by the
Human Genome Project. The massive amount of genomic information generated
by sequencing efforts could only be exploited by using complex bioinformatics
tools to comprehensively analyze systems at the DNA, RNA, and protein level.
These bioinformatics tools together with the data available from RNA expression
profiling using DNA chips has led to the comprehensive analyses of individual
clinical samples in an attempt to describe disease and disease risk at the molecu-
lar level and integrate data to facilitate clinical decision making.
Pharmacogenomics aims to optimize patient management by customizing
and synthesizing drugs based on genetic variations in drug response. Its thrust
is based on genome-based rational therapeutics that addresses interindividual
variations or polymorphisms affecting metabolism, receptors, and absorption
that can influence drug sensitivity, toxicity, and dosing. Potential benefits of
pharmacogenomics include increasing efficacy and preventing adverse drug
reactions, thus improving patient care and decreasing costs. All of these tech-
nologies are not yet in current clinical use and it is also too early to decide
whether molecular ‘fingerprints’ or genomic profiles will have the diagnostic
and prognostic power currently predicted.
One approach that we have taken to investigate new targets for epilepsy
pharmacotherapy has been to profile dentate gyrus granule cells from human
epilepsy tissue. Mesial temporal lobe sclerosis is a common pathological find-
ing in patients with medically intractable temporal lobe epilepsy. This disease
is characterized by extensive cell loss in the hilus and the hippocampal CA1 and
CA3 cell fields in addition to synaptic reorganization throughout the dentate
gyrus. The dentate granule cells from hippocampal slices of patients diagnosed
with medial temporal lobe sclerosis exhibit reduced synaptic inhibition with
concomitant hyperexcitability. These physiological changes have been studied
Control human dentate gyrus
a
Epileptic human dentate gyrus
b
GABA␣1 GABA␣2 GABA␣3 GABA␣4 GABA␣5 GABA␣6
GABA␤1 GABA␤2 GABA␤3 GABA␥2 GABA␥3 NOS
NMDA1a NMDA2a NMDA2b NMDA2c NMDA2d VGAT
GAD65 GAD67 GFAP pBS ␤-Actin GABA-T
GluR1 GluR2 GluR4 GluR5 GluR6 GluR7
GLT-1 Netrin-1 Netrin-2 Nestin BF1 BF2
HES INX CREB OTX-1 ARC 4B2
S49 EAAC1 c-fos c-jun AChE NCAM
c
Fig. 1. Radiolabeled amplified aRNAs are used as probes of small scale cDNA arrays
containing candidate genes of interest. For each 32P-labelled aRNA from a cell, duplicated slot
blots were used for each hybridization reaction. An mRNA expression profile could then be
obtained. a Expression profile for autopsy control dentate gyrus granule cell. b Expression
profile for medial temporal lobe epilepsy dentate gyrus granule cell. c Candidate genes and
controls used.
relative to the hippocampi of patients with temporal lobe tumors in which the
cell loss and synaptic reorganization are not seen. The synaptic reorganization
of both excitatory and inhibitory systems in the dentate gyrus of the hip-
pocampus may be an important mechanism that contributes to chronic limbic
seizures. Of interest is the role of neurotransmitter receptors and their uptake
sites in the generation of seizures in MTLE.
Differences in gene expression in temporal lobe epilepsy have been
reported from investigations on surgically removed hippocampi implicating an
up-regulation in the expression of excitatory neurotransmitter receptor genes
in a role in epileptogenesis. Increases in mGluR1 (Mathern 97, 98; Lynd 96;
Blumcke 00), mGluR2 (Blumcke 96), NMDAR2a (Mathern 99), NMDAR2b
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Fig. 2. Harvesting single cells. Human hippocampi fixed in paraformaldehyde were
stained with TA51, an antibody for neurofilament. First round synthesis of cDNA was done on
the histologically sectioned tissue. Single cells were identified and removed from the dentate
gyrus granule cell layer. A glass microelectrode is shown moving in to harvest a single granule
cell. Conversion to double-stranded cDNA, then amplification of this cDNA as radiolabeled
antisense mRNA was then performed.
(Mathern 99, 98) mRNA expression have all been reported in the dentate gyrus
from hippocampal surgical specimens. These findings support the hypothesis
that changes in hippocampal circuitry alter the postsynaptic gene expression in
a way that contributes to chronic seizure.
Our strategy has been to remove individual dentate gyrus granule cells
from fixed specimens (fig. 1) of surgically removed hippocampi from
patients with MTLE and autopsy hippocampi, stained with TA51, an antibody
for neurofilament. Radiolabeled aRNA from these cells was used to probe
cDNA arrays containing the GABAA ␣1–6, and ␤1–3 receptor subunits,
mGluR1–6, NMDAR 1A-B, NMDAR2A-D receptor subunits, GAD65,
GAD67, and VGAT. The relative intensity of each mRNA-cDNA hybrid is
then quantified (fig. 2). Selective differences can be found at the level of gene
expression in hippocampal dentate gyrus granule cells from MTLE patients
compared to nonseizure autopsy controls. Reduced transcription of select
receptors and increased expression of other subunits in MTLE may contribute
to epileptogenesis.
Although select differences in mRNA expression can be found in human
epilepsy tissue, it is the level of functional receptor protein, and any associated
regulatory component, which will determine the functional significance of
these findings. Routine biochemical analysis (e.g., Western blots) cannot be
performed at the single cell level in order to determine how protein expression
correlates with mRNA expression. Immunohistochemistry is a more qualitative
technique although it can be resolved at the single cell level. However, it cannot
be used for more than two simultaneous antigens and is still not very specific
for receptor subunits. It is likely, thus, that physiological or pharmacological
analyses will be required to determine the functional significance of the expres-
sion differences. Despite these difficulties, ultimately, in order to understand
epilepsy and develop highly targeted therapies, molecular characterization of
individual neuronal cell types in critical areas of the involved CNS is likely to
be necessary.
References
1 Hauser WA, Annegers JF, Kurland LT: Incidence of epilepsy and unprovoked seizures in Rochester,
Minnesota: 1935–1984. Epilepsia 1993;34:453–468.
2 Regesta G, Tanganelli P: Clinical aspects and biological bases of drug-resistant epilepsies. Epilepsy
Res 1999;34:109–122.
3 Macdonald RL, McLean MJ: Anticonvulsant drugs: Mechanisms of action. Adv Neurol 1986;44:
713–736.
4 Mamiya K, Ieiri I, Shimamoto J, et al: The effects of genetic polymorphisms of CYP2C9 and
CYP2C19 on phenytoin metabolism in Japanese adult patients with epilepsy: Studies in stereo-
selective hydroxylation and population pharmacokinetics. Epilepsia 1998;39:1317–1323.
5 Loscher W, Potschka H: Role of multidrug transporters in pharmacoresistance to antiepileptic drugs.
J Pharmacol Exp Ther 2002;301:7–14.
6 Abbott NJ, Khan EU, Rollinson CM, et al: Drug resistance in epilepsy: The role of the blood-brain
barrier. Novartis Found Symp 2002;243:38–47.
7 Potschka H, Fedrowitz M, Loscher W: P-Glycoprotein-mediated efflux of phenobarbital, lamotrigine,
and felbamate at the blood-brain barrier: Evidence from microdialysis experiments in rats. Neurosci
Lett 2002;327:173–176.
8 Sills GJ, Brodie MJ: Update on the mechanisms of action of antiepileptic drugs. Epileptic Disord
2001;3:165–172.
9 Sisodiya SM, Lin WR, Harding BN, Squier MV, Thom M: Drug resistance in epilepsy: Expression
of drug resistance proteins in common causes of refractory epilepsy. Brain 2002;125:22–31.
10 Rizzi M, Caccia S, Guiso G, et al: Limbic seizures induce P-glycoprotein in rodent brain:
Functional implications for pharmacoresistance. J Neurosci 2002;22:5833–5839.
11 Mantegazza R, Bernasconi P, Baggi F, et al: Antibodies against GluR3 peptides are not specific for
Rasmussen’s encephalitis but are also present in epilepsy patients with severe, early onset disease
and intractable seizures. J Neuroimmunol 2002;131:179–185.
12 Twyman RE, Gahring LC, Spiess J, Rogers SW: Glutamate receptor antibodies activate a subset
of receptors and reveal an agonist binding site. Neuron 1995;14:755–762.
13 He XP, Patel M, Whitney KD, Janumpalli S, Tenner A, McNamara JO: Glutamate receptor GluR3
antibodies and death of cortical cells. Neuron 1998;20:153–163.
14 Andrews PI, McNamara JO: Rasmussen’s encephalitis: An autoimmune disorder? Curr Opin
Neurobiol 1996;6:673–678.
15 Leach JP, Chadwick DW, Miles JB, Hart IK: Improvement in adult-onset Rasmussen’s encephalitis
with long-term immunomodulatory therapy. Neurology 1999;52:738–742.
16 Villani F, Spreafico R, Farina L, et al: Positive response to immunomodulatory therapy in an adult
patient with Rasmussen’s encephalitis. Neurology 2001;56:248–250.
17 Levite M, Hermelin A: Autoimmunity to the glutamate receptor in mice – A model for Rasmussen’s
encephalitis? J Autoimmun 1999;13:73–82.
medwedi.ru
18 Levite M, Fleidervish IA, Schwarz A, Pelled D, Futerman AH: Autoantibodies to the glutamate
receptor kill neurons via activation of the receptor ion channel. J Autoimmun 1999;13:61–72.
19 Stutzmann JM, Mary V, Wahl F, Grosjean-Piot O, Uzan A, Pratt J: Neuroprotective profile of
enoxaparin, a low molecular weight heparin, in in vivo models of cerebral ischemia or traumatic
brain injury in rats: A review. CNS Drug Rev 2002;8:1–30.
20 Westenbroek RE, Bausch SB, Lin RC, Franck JE, Noebels JL, Catterall WA: Upregulation of L-type
Ca2⫹ channels in reactive astrocytes after brain injury, hypomyelination, and ischemia. J Neurosci
1998;18:2321–2334.
21 Bialer M, Johannessen SI, Kupferberg HJ, Levy RH, Loiseau P, Perucca E: Progress report on new
anti-epileptic drugs: A summary of the Fifth Eilat Conference (EILAT V). Epilepsy Res 2001;43:
11–58.
22 Tamargo RJ, Rossell LA, Kossoff EH, Tyler BM, Ewend MG, Aryanpur JJ: The intracerebral
administration of phenytoin using controlled-release polymers reduces experimental seizures in
rats. Epilepsy Res 2002;48:145–155.
23 DiMario FJ Jr, Holland J: The ketogenic diet: A review of the experience at Connecticut Children’s
Medical Center. Pediatr Neurol 2002;26:288–292.
24 Snead OC 3rd: Surgical treatment of medically refractory epilepsy in childhood. Brain Dev
2001;23:199–207.
25 Henry TR, Votaw JR, Pennell PB, et al: Acute blood flow changes and efficacy of vagus nerve
stimulation in partial epilepsy. Neurology 1999;52:1166–1173.
26 Ben-Menachem E, Hamberger A, Hedner T, et al: Effects of vagus nerve stimulation on amino
acids and other metabolites in the CSF of patients with partial seizures. Epilepsy Res 1995;20:
221–227.
27 Rafael H, Moromizato P: Vagus stimulator for seizures. J Neurosurg 1993;79:636–637.
28 Valencia I, Holder DL, Helmers SL, Madsen JR, Riviello JJ Jr: Vagus nerve stimulation in pediatric
epilepsy: A review. Pediatr Neurol 2001;25:368–376.
29 Ventureyra EC: Transcutaneous vagus nerve stimulation for partial onset seizure therapy. A new
concept. Childs Nerv Syst 2000;16:101–102.
30 Regis J, Bartolomei F, de Toffol B, et al: Gamma knife surgery for epilepsy related to hypothalamic
hamartomas. Neurosurgery 2000;47:1343–1351; discussion 1351–1352.
31 Regis J, Kerkerian-Legoff L, Rey M, et al: First biochemical evidence of differential functional
effects following Gamma Knife surgery. Stereotact Funct Neurosurg 1996;66(suppl 1):29–38.
32 Mori Y, Kondziolka D, Balzer J, et al: Effects of stereotactic radiosurgery on an animal model of
hippocampal epilepsy. Neurosurgery 2000;46:157–165; discussion 165–168.
33 Chalifoux R, Elisevich K: Effect of ionizing radiation on partial seizures attributable to malignant
cerebral tumors. Stereotact Funct Neurosurg 1996;67:169–182.
34 Beurrier C, Bioulac B, Audin J, Hammond C: High-frequency stimulation produces a transient
blockade of voltage-gated currents in subthalamic neurons. J Neurophysiol 2001;85:1351–1356.
35 Bikson M, Lian J, Hahn PJ, Stacey WC, Sciortino C, Durand DM: Suppression of epileptiform
activity by high frequency sinusoidal fields in rat hippocampal slices. J Physiol 2001;531:
181–191.
36 Iribe Y, Moore K, Pang KC, Tepper JM: Subthalamic stimulation-induced synaptic responses in
substantia nigra pars compacta dopaminergic neurons in vitro. J Neurophysiol 1999;82:925–933.
37 Kayyali H, Durand D: Effects of applied currents on epileptiform bursts in vitro. Exp Neurol
1991;113:249–254.
38 Montgomery EB Jr, Baker KB: Mechanisms of deep brain stimulation and future technical devel-
opments. Neurol Res 2000;22:259–266.
39 Dostrovsky JO: Immediate and long-term plasticity in human somatosensory thalamus and its
involvement in phantom limbs. Pain 1999;suppl 6:S37–S43.
40 Maurice N, Deniau JM, Glowinski J, Thierry AM: Relationships between the prefrontal cortex and
the basal ganglia in the rat: Physiology of the corticosubthalamic circuits. J Neurosci 1998;18:
9539–9546.
41 Maurice N, Deniau JM, Menetrey A, Glowinski J, Thierry AM: Prefrontal cortex-basal ganglia
circuits in the rat: Involvement of ventral pallidum and subthalamic nucleus. Synapse 1998;29:
363–370.
42 McIntyre CC, Grill WM: Selective microstimulation of central nervous system neurons. Ann
Biomed Eng 2000;28:219–233.
43 Boison D, Scheurer L, Tseng JL, Aebischer P, Mohler H: Seizure suppression in kindled rats by
intraventricular grafting of an adenosine releasing synthetic polymer. Exp Neurol 1999;160:
164–174.
44 Loscher W, Ebert U, Lehmann H, Rosenthal C, Nikkhah G: Seizure suppression in kindling
epilepsy by grafts of fetal GABAergic neurons in rat substantia nigra. J Neurosci Res 1998;51:
196–209.
45 Shetty AK, Turner DA: Fetal hippocampal grafts containing CA3 cells restore host hippocampal
glutamate decarboxylase-positive interneuron numbers in a rat model of temporal lobe epilepsy.
J Neurosci 2000;20:8788–8801.
46 Shetty AK, Zaman V, Turner DA: Pattern of long-distance projections from fetal hippocampal
field CA3 and CA1 cell grafts in lesioned CA3 of adult hippocampus follows intrinsic character
of respective donor cells. Neuroscience 2000;99:243–255.
47 Huber A, Padrun V, Deglon N, Aebischer P, Mohler H, Boison D: Grafts of adenosine-releasing
cells suppress seizures in kindling epilepsy. Proc Natl Acad Sci USA 2001;98:7611–7616.
48 Seki T, Matsubayashi H, Amano T, et al: Adenoviral gene transfer of aspartoacylase into the tremor
rat, a genetic model of epilepsy, as a trial of gene therapy for inherited epileptic disorder. Neurosci
Lett 2002;328:249–252.
49 O’Connor WM, Davidson BL, Kaplitt MG, et al: Adenovirus vector-mediated gene transfer into
human epileptogenic brain slices: Prospects for gene therapy in epilepsy. Exp Neurol 1997;148:
167–178.
Albert Telfeian, MD, PhD

Neurosurgical Associates, LLP
3601 21st Street, Lubbock, TX 79410 (USA)
Tel. ⫹1 806 797 2222, E-Mail albert.telfeian@nsallp.com
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Emerging Treatment of
Neurometabolic Disorders
Roscoe O. Brady, Roscoe O. Brady, Jr.
Developmental and Metabolic Neurology Branch, National Institute of Neurological
Disorders and Stroke, National Institutes of Health, Bethesda, Md., USA
Introduction
Metabolic disorders are caused by mutations in genes that result in harm-

ful reductions of catalytic activity of any of the enzymes required for the nor-
mal ‘housekeeping’ functions of cells. Many of these conditions are
characterized by the accumulation of deleterious amounts of nondegraded
materials within lysosomes, and are commonly known as ‘lysosomal storage
disorders.’ Lysosomes are subcellular organelles which contain a plethora of
enzymes that are necessary for the biodegradation of subcellular materials.
These enzymes are preferentially active under acidic conditions that are char-
acteristic of the intraluminal milieu of lysosomes. Substances that undergo
lysosomal biodegradation include glycogen, mucopolysaccharides, and the
major class of lipids called sphingolipids. Because approaches to enzyme
replacement therapy (ERT) and gene therapy are particularly advanced in the
sphingolipid storage disorders, we shall limit this chapter primarily to consid-
erations of these conditions, although the basic techniques of gene and ERT
have wide applicability to many metabolic disorders of the brain. We shall indi-
cate briefly what has been accomplished to date, and then describe approaches
that we believe will be required for the effective treatment of patients in which
neurological involvement is a prominent cause of morbidity and mortality.
Background
Sphingolipids have a portion of their common structure comprised of the long

chain amino alcohol sphingosine (fig. 1a). In these lipids, a long-chain fatty acid
a Sphingosine
CH3-(CH2)12-CH⫽CH-CH-CH-CH2OH
OH NH2
D-erythro-trans-2-amino-4-octadecene-1,3-diol
b Ceramide
Sphingosine
CH3-(CH2)12-CH⫽CH-CH-CH-CH2OH
OH NH
CH3-(CH2)22-C⫽O
Fatty acid
c Glucocerebroside
Sphingosine-Glucose
Fatty acid
d Sphingomyelin
Sphingosine-Phosphocholine
Fatty acid
e Globotriaosylceramide
[Ceramidetrihexoside (GB3)]
Sphingosine-Glucose-Galactose-Galactose
Fatty acid
f Ganglioside GM2
Sphingosine-Glucose-Galactose-N-acetylgalactosamine
Fatty acid N-acetylneuraminic acid
g Ganglioside GM1
Sphingosine-Glucose-Galactose-N-acetylgalactosamine-Galactose
Fatty acid N-acetylneuraminic acid
Fig. 1. Structures of pertinent sphingolipids. a Sphingosine; b Ceramide;

c Glucocerebroside; d Sphingomyelin; e Globotriaosylceramide; f Ganglioside GM2;
g Ganglioside GM1.
Neurogenetic Diseases 203
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is linked to the nitrogen atom covalently bound to carbon atom two of sphingosine,
forming a moiety known as ceramide (fig. 1b). In all of the sphingolipid storage
disorders (except Farber’s disease) in which ceramide itself is the principal accu-
mulating substance, additional components are linked to the oxygen on carbon one
of the sphingosine moiety of ceramide. The most prevalent metabolic storage dis-
order of humans is Gaucher’s disease. Here, the principal accumulating substance
is glucocerebroside, comprised of ceramide to which a single molecule of glucose
is linked by a ␤-glycosidic bond (fig. 1c). Another common sphingolipid storage
disorder is Niemann-Pick’s disease. Here, the accumulating material is sphin-
gomyelin (fig. 1d). Still another prominent condition is Fabry’s disease in which
globotriaosylceramide (ceramidetrihexoside)(Gb3) accumulates in many organs
(fig. 1e). Of particular significance to neuroscientists are Tay-Sach’s disease in
which the ganglioside GM2 accumulates (fig. 1f ) and generalized gangliosidosis
in which ganglioside GM1 is the major accumulating metabolite (fig. 1g).
The nature of the metabolic abnormalities in the sphingolipid storage disor-
ders was established 38 years ago by Brady et al. [1, 2] with the demonstration
that the enzymatic defect in Gaucher’s disease was the insufficient activity of
glucocerebrosidase, the enzyme that catalyzes the hydrolytic cleavage of glucose
from glucocerebroside. There are three principal clinical phenotypes of
Gaucher’s disease. The first is Type 1 (nonneuronopathic) Gaucher’s disease in
which the CNS is not involved. The second is Type 2 (acute neuronopathic)
Gaucher’s disease that is characterized by early and extensive CNS damage. The
term neuronophagia is used in the context of Type 2 Gaucher, because of the
widespread destruction of neurons by monocytes that are attracted into the brain
from the circulation by cytokines that are elaborated by damaged neurons. The
third is Type 3 (chronic neuronopathic) Gaucher’s disease in which signs of CNS
involvement occur later than in Type 2 Gaucher patients. Neurological manifes-
tations in Type 3 patients may be confined to horizontal, or less frequently, ver-
tical gaze paresis. Some Type 3 patients also have progressive myoclonic epilepsy
that is notoriously difficult to control. The identification of the enzymatic defects
in Niemann-Pick’s disease [3], Fabry’s disease [4], generalized gangliosidosis
[5], Tay-Sach’s disease [6], and Krabbe’s disease [7] followed soon after the
elucidation of the enzymatic defect in Gaucher’s disease. This information was
used to develop widely used enzymatic assays for the diagnosis [8], carrier detec-
tion [9] and prenatal identification of fetuses at risk for these conditions [10–12].
Development of ERT
A long period of time elapsed before effective treatment for any of these
debilitating conditions became available. Once again, investigations into the
Brady/Brady 204
nature of Gaucher’s disease were of paramount importance. Since glucocere-
brosidase activity was less than normal in patients with this disorder, it
appeared to be a fairly rudimentary task to purify this enzyme and determine
whether its administration to Gaucher patients would be beneficial [13]. In
order to minimize the possibility of sensitizing recipients to the exogenous
protein, human placental tissue was initially used as the source of glucocere-
brosidase. This treatment strategy, whereby exogenous enzyme is administered
to a patient in whom a deficiency caused disease, was later termed ‘enzyme
replacement therapy.’ These initial studies, using protein extracted from human
tissue, were performed before the advent of recombinant DNA techniques made
it possible to clone a gene and express the resultant protein without the need for
biological source material.
The first major impediment in establishing the effectiveness of this thera-
peutic approach was the difficulty in obtaining sufficient quantities of purified
glucocerebrosidase to undertake clinical trials. Eventually, a limited amount of
the enzyme was isolated in a sufficiently pure form that was believed not to be
harmful if injected into patients with Gaucher’s disease. Small quantities of pla-
cental glucocerebrosidase were injected intravenously into 2 splenectomized
patients with Gaucher’s disease. Percutaneous liver biopsy was performed
before administering the enzyme and another the day after the injection. In both
patients, the quantity of glucocerebroside in the postinfusion biopsy specimens
was 26% less than that in the preinfusion biopsy samples [26]. Moreover, there
was a long-lasting reduction of glucocerebroside in the blood [14]. An addi-
tional year was required to isolate enough enzyme to examine its effect in a
third Gaucher patient. In this patient, only an 8% reduction of glucocerebroside
occurred in the liver following the enzyme delivery, and there was no change in
the blood level. Based on the amount of glucocerebroside in the biopsy samples
from the third recipient, it was deduced that insufficient glucocerebrosidase
had been administered to obtain a significant clearance of the accumulated
glucocerebroside.
Because of the difficulty in obtaining large quantities of the enzyme from
the placenta with the original methods of purification, a new isolation procedure
was developed by Furbish et al. [15] in the mid-1970s. It was found that gluco-
cerebrosidase obtained by this procedure was inconsistently delivered to lipid-
storing macrophages such as Kupffer cells in the liver. Macrophages have a
lectin on their surface that has a particularly high affinity for mannose-terminal
glycoconjugates [16]. In order to target glucocerebrosidase to these cells, the
oligosaccharide side chains of glucocerebrosidase were trimmed with three exo-
glycosidases to produce mannose as the terminal sugar [17]. In experimental
animals, this modification of glucocerebrosidase resulted in a 50-fold increase
in the uptake of enzyme by Kupffer cells. A clinical trial was conducted in which
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190 IU of mannose-terminal glucocerebrosidase were injected weekly over a
period of 6 months into 7 adults and one child with Type 1 Gaucher’s disease.
Only the child showed any beneficial clinical response [18]. A dose-response
study was carried out by performing percutaneous liver biopsies before and after
injecting the enzyme over a wide dosing range. The quantity of enzyme that con-
sistently produced a reduction of hepatic glucocerebroside was 60 IU/kg of body
weight. When this amount of mannose-terminal placental enzyme was given to
12 adults with Type 1 Gaucher’s disease every 2 weeks for a period of 6 months,
striking beneficial effects occurred in all of the recipients [19]. Based on
these findings, mannose-terminal glucocerebrosidase was approved for the treat-
ment of patients with Type 1 Gaucher’s disease by the U.S. Food and Drug
Administration on April 5, 1991. Recombinant glucocerebrosidase was sub-
sequently produced in Chinese hamster ovary cells. This product is biologically
equivalent to placental glucocerebrosidase [20] and was approved for the treat-
ment of Gaucher patients in the USA in 1994. ERT for Gaucher patients was
later approved in 55 countries. At this time, more than 4,000 patients with
Gaucher’s disease throughout the world are being treated by ERT, based on work
originating back in the 1960s.
Extension of this treatment to patients with neuronopathic forms of
Gaucher’s disease is of great importance. Reduction of hepatosplenomegaly
and skeletal improvement was universal in clinical trials in patients with Type 3
Gaucher’s disease, but improvement of the supranuclear gaze palsy manifested
by these patients has been inconsistent [21]. ERT also has been examined in
patients with Type 2 Gaucher’s disease. Again, systemic improvement occurred
in infants, but no amelioration of the CNS impairment was evident [22]. This
finding is not surprising since it has been known for many years that intra-
venously injected enzymes do not reach the brain because of the blood-brain
barrier [23]. Alternative delivery strategies have, therefore, been explored for
the treatment of patients with neuronopathic Gaucher’s disease.
Substrate Depletion
Inhibition of the formation of glucocerebroside (substrate depletion) was

proposed a number of years ago as a therapeutic strategy for the treatment of
metabolic storage disorders [24]. The effect of blocking glucocerebroside syn-
thesis with N-butyl-deoxynojirimycin (NB-DNJ) has recently been examined in
patients with Type 1 Gaucher’s disease, and some apparently salutary effects
have been reported [25]. N-butyl-deoxynojirimycin is a small-molecular-weight
compound that has been shown to reach the brain of experimental animals
when given orally in large doses. A trial of N-butyl-deoxynojirimycin has been
Brady/Brady 206
undertaken in patients with Type 3 Gaucher’s disease in whom the systemic
manifestations are controlled by intravenous administration of mannose-terminal
glucocerebrosidase. Abnormal saccadic eye movements of the recipients will be
monitored as a critical clinical endpoint. This general approach to substrate
inhibition is also relevant to other important lysosomal storage diseases such as
Tay Sach’s and Sandhoff’s diseases, and successful studies in animal models
have led to studies in human subjects. Advances in neurosurgical delivery
may help to increase the effectiveness of the approach and offset the current
limitations (e.g., toxicity, nontargeted delivery, insufficient penetration of target
tissue in the CNS) of systemic dosing of substrate inhibitors.
Intracerebral Injection of Mannose-Terminal

Glucocerebrosidase
It is likely that much, if not all, of the glucocerebroside that accumulates

in neurons in the brain of patients with Type 2 Gaucher’s disease originates from
the catabolism of larger sphingoglycolipids such as gangliosides (fig. 1f, g).
Ganglioside turnover is most active during the neonatal period of life.
Thereafter, it decreases to a constant level that is approximately 5% of the max-
imum velocity. Glucocerebrosidase activity in patients with Type 2 Gaucher’s
disease is very low, usually in the range of 1–2% of normal [2]. It is conceiv-
able that these patients might improve if glucocerebrosidase could be supplied
to the brain during the neonatal period to catabolize glucocerebroside in neu-
rons during this critical period of development. Investigators in the Surgical
Neurology Branch of the National Institute of Neurological Disorders and
Stroke (NINDS) have developed a technique called convection-enhanced deliv-
ery to deliver proteins in solution directly to the brain [27]. It was desirable to
determine whether glucocerebrosidase could be delivered to the brain by this
technique. An investigation was carried out to examine the safety of the proce-
dure and the distribution of intracerebrally injected glucocerebrosidase in nor-
mal rats [28]. The procedure was found feasible and innocuous to the recipient
animals. Glucocerebrosidase was carried by convective flow along white matter
fiber tracts from the site of administration in the striatum to the cerebral cortex
(fig. 2). The half-life of injected glucocerebrosidase in the brain was ⬃9 h,
which is comparable to that in other major organs such as the liver following
intravenous administration. The enzyme was specifically taken up by neurons,
precisely the cells that appear to require it to prevent neuronophagia that is a
hallmark of this condition (fig. 3). The reason for the selective delivery to
neurons is believed to be due to a mannose lectin on their surface [29, 30] that
is present in a lesser amount but is qualitatively similar to that on macrophages.
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Glucocerebrosidase diffusion in rat brain
Enzymatic activity mU
12 Injection site
11
10
9
8
aft 7 fore
fore aft
6
5
4
Striatum 3 Striatum
Left hemisphere 2 Right hemisphere
1
Control side Infused side
Fig. 2. Distribution of human glucocerebrosidase in the brain of normal rats following

intracerebral injection of the enzyme. [Reproduced from 28 with permission of Wiley-Liss,
a subsidiary of John Wiley & Sons].
Fig. 3. Immunohistochemical staining of human glucocerebrosidase in neurons of nor-

mal rats following intracerebral injection of the enzyme. [Reproduced from 28 by permis-
sion of Wiley-Liss, a subsidiary of John Wiley & Sons].
The safety and intracerebral distribution of glucocerebrosidase will be exam-

ined in nonhuman primates. If the results of these investigations are favorable,
it may be worthwhile exploring this approach for the treatment of patients with
Type 2 Gaucher’s disease during the neonatal period. It is likely that a combi-
nation of intracerebral and intravenous administration of the enzyme will be
necessary. Moreover, it may be useful to include a substrate-depleting agent
in the treatment regimen to reduce glucocerebroside formation. If intracere-
bral administration of glucocerebrosidase proves beneficial in patients with
Brady/Brady 208
neuronopathic Gaucher’s disease, it is likely that this approach will be extended
to a number of other human metabolic disorders in which the CNS is involved.
Again, intracerebral delivery will require neurosurgical expertise for clinical
trials and will require the development of additional instrumentation and tech-
nologies to uniformly disperse the enzyme in the brain. Some areas for future
development from a neurosurgical perspective might involve improved col-
loidal formulations of the enzyme solution as well as pumps and dispersion
(injection) devices in the brain.
Gene Therapy
Gene therapy has been examined in patients with Type 1 Gaucher’s disease
using retroviral transduction of bone marrow stem and progenitor cells. In the
first recipient, no expression of the transgene was detected. In the second sub-
ject, expression of the transgene was detected over a period of several months
[31]. Several steps are necessary before gene therapy for patients with Type 1
Gaucher’s disease becomes realistic. Among the initial goals that must be
reached are: (1) more effective transduction of stem and progenitor cells with
the gene of interest; (2) selective enrichment of transduced cells before their
reintroduction into patients; (3) development of procedure(s) for the delivery
and implantation of the transduced cells into the patient’s bone marrow, and
(4) elimination of harmful effects of retroviruses including various forms of
leukemia [32, 33] which have been attributed to gene therapy in the context of
X-linked severe combined immunodeficiency.
The use of self-inactivating lentiviral vectors has recently come under
investigation. The principal advantages of lentivirus vectors are: (1) efficient
integration into the genomes of target cells; (2) sustained long-term gene
expression; (3) no apparent immune response, and (4) ability to infect nondi-
viding cells such as neurons. Delivery of genes into nondividing cells with
pseudotyped, high-titer, replication-defective HIV-1 vector has already been
achieved [34]. This strategy was improved by the construction of a herpes sim-
plex virus VP22 fusion protein that greatly increased the intercellular delivery
of the test protein [35]. A logical extension of this investigation was the use of
such a construct to deliver genes and their protein products to nondividing cells
in the CNS [36]. Use of herpes simplex virus VP22 greatly enhanced the deliv-
ery of proteins between cells. Incorporation of the neuron-specific enolase
promoter resulted primarily in the transduction of neurons within the CNS.
These encouraging findings have led to the development of a lentivirus gene
construct containing the human glucocerebrosidase gene and VP22. It is
expected that the fusion product will assist in the intercellular transport of the
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therapeutic protein to a major portion of the brain. If this hypothesis is sub-
stantiated, it is expected to provide a significant impetus for serious considera-
tion of gene therapy for patients with neuronopathic Gaucher’s disease.
Whether the results of such investigations can be translated to other metabolic
storage disorders remains to be established. The development and exploration
of gene therapy in authentic animal analogs of human enzyme deficiency con-
ditions should significantly accelerate our sense of the potential of this
approach and hopefully reveal any unanticipated difficulties prior to their appli-
cation to patients. In addition to lentivirus, a number of other viral vectors and
nonviral gene delivery systems must be considered. As time goes by, the limi-
tations of effective gene therapy are more related to technical obstacles that are
gradually being overcome, rather than fundamental problems with the gene
therapy approach. One can envision a future time in which the promise of ERT
and gene transfer are fully realized through advances in neurosurgical delivery
and improvements in vector design.
References
1 Brady RO, Kanfer JN, Shapiro D: Metabolism of glucocerebrosides. II. Evidence of an enzymatic
deficiency in Gaucher’s disease. Biochem Biophys Res Commun 1965;18:221–225.
2 Brady RO, Kanfer JN, Bradley RM, Shapiro D: Demonstration of a deficiency of glucocerebroside-
cleaving enzyme in Gaucher’s disease. J Clin Invest 1966;45:1112–1115.
3 Brady RO, Kanfer JN, Mock MB, Fredrickson DS: The metabolism of sphingomyelin. II.
Evidence of an enzymatic deficiency in Niemann-Pick disease. Proc Natl Acad Sci USA 1966;55:
366–369.
4 Brady RO, Gal AE, Bradley RM, Martensson E, Warshaw AL, Laster L: Enzymatic defect in
Fabry’s disease. Ceramidetrihexosidase deficency. N Engl J Med 1967;276:1163–1167.
5 Okada S, O’Brien JS: Generalized gangliosidosis: Beta-galactosidase deficiency. Science 1968;
160:1002–1004.
6 Kolodny EH, Brady RO, Volk BW: Demonstration of an alteration of ganglioside metabolism in
Tay-Sach’s disease. Biochem Biophys Res Commun 1969;37:526–531.
7 Suzuki K, Suzuki Y: Globoid cell leucodystrophy (Krabbe’s disease): Deficiency of galactocere-
broside beta-galactosidase. Proc Natl Acad Sci USA 1970;66:302–309.
8 Kampine JP, Brady RO, Kanfer JN, Feld M, Shapiro D: The diagnosis of Gaucher’s disease and
Niemann-Pick disease using small samples of venous blood. Science 1967;155:86–88.
9 Brady RO, Johnson WG, Uhlendorf BW: Identification of heterozygous carriers of lipid storage
diseases. Am J Med 1971;51:423–431.
10 Brady RO, Uhlendorf BW, Jacobson CB: Fabry’s disease: Antenatal detection. Science 1971;172:
174–175.
11 Epstein CJ, Brady RO, Schneider EL, Bradley RM, Shapiro D: In utero diagnosis of Niemann-
Pick disease. Am J Hum Genet 1971;23:533–535.
12 Schneider EL, Ellis WG, Brady RO, McCulloch JR, Epstein CJ: Infantile (Type II) Gaucher’s
disease: In utero diagnosis and fetal pathology. J Pediatr 1972;81:1134–1139.
13 Brady RO: Sphingolipidoses. N Engl J Med 1966;275:312–318.
14 Brady RO, Pentchev PG, Gal AE, Hibbert SR, Dekaban AS: Replacement therapy for inherited
enzyme deficiency: Use of purified glucocerebrosidase in Gaucher’s disease. N Engl J Med
1974;291:989–993.
Brady/Brady 210
15 Pentchev PG, Brady RO, Gal AE, Hibbert SR: Replacement therapy for inherited enzyme
deficiency: Sustained clearance of accumulated glucocerebroside in Gaucher’s disease following
infusion of purified glucocerebrosidase. J Mol Med 1975;1:73–78.
16 Furbish FS, Blair HE, Shiloach J, Pentchev PG, Brady RO: Enzyme replacement therapy in
Gaucher’s disease: Large-scale purification of glucocerebrosidase suitable for human administra-
tion. Proc Natl Acad Sci USA, 1977;74:3560–3563.
17 Stahl PD, Rodman JS, Miller MJ, Schlesinger PH: Evidence for receptor-mediated binding of
glycoproteins, glycoconjugates, and lysosomal glycosidases by alveolar macrophages. Proc Natl
Acad Sci USA 1978;75:1399–1403.
18 Brady RO, Furbish FS: Enzyme replacement therapy: Specific targeting of exogenous enzymes to
storage cells; in Martonosi AT (ed): Membranes and Transport. New York, Plenum, 1982, vol 2,
pp 587–592.
19 Barton NW, Furbish FS, Murray GJ, Garfield M, Brady RO: Therapeutic response to intravenous
infusions of glucocerebrosidase in a patient with Gaucher disease. Proc Natl Acad Sci USA
1990;87:1913–1916.
20 Barton NW, Brady RO, Dambrosia JM, DiBisceglie AM, Doppelt SH, Hill SC, Mankin HJ,
Murray GJ, Parker RI, Argoff CE, Grewal RP, Yu K-T: Replacement therapy for inherited enzyme
deficiency – Macrophage-targeted glucocerebrosidase for Gaucher’s disease. N Engl J Med
1991;324:1464–1470.
21 Grabowski GA, Barton NW, Pastores G, Dambrosia JM, Banerjee TK, McKee MA, Parker C,
Schiffmann R, Hill SC, Brady RO: Enzyme therapy in Gaucher disease Type 1: Comparative effi-
cacy of mannose-terminated glucocerebrosidase from natural and recombinant sources. Ann
Intern Med 1995;122:33–39.
22 Altarescu G, Hill S, Wiggs E, Jeffries N, Kreps C, Parker CC, Brady RO, Barton NW, Schiffmann R:
The efficacy of enzyme replacement therapy in patients with chronic neuronopathic Gaucher’s
disease. J Pediatr 2001;138:539–547.
23 Prows CA, Sanchez N, Daugherty C, Grabowski GA: Gaucher disease: Enzyme therapy in the
acute neuronopathic variant. Am J Med Genet 1997;71:16–21.
24 Johnson WG, Desnick RJ, Long DM, Sharp HL, Krivit W, Brady B, Brady RO: Intravenous injec-
tion of purified hexosaminidase A into a patient with Tay-Sach’s disease. Birth Defects Orig Artic
Ser 1973;IX:120–124.
25 Radin NS: Inhibitors and stimulators of glucocerebroside metabolism. Prog Clin Biol Res 1982;
95:357–370.
26 Cox T, Lachmann R, Hollak C, Aerts J, van Weekly S, Hrebicek M, Platt F, Butters T, Dwek R,
Moyses C, Gow I, Elstein D, Zimran A: Novel oral treatment of Gaucher’s disease N-butyl-
deoxynojirimycin (OGT 918) to decrease substrate biosynthesis. Lancet 2000;355: 1481–
1485.
27 Bobo RH, Laske DW, Akbasak A, Morrison PF, Dedrick RL, Oldfield EH: Convection-enhanced
delivery of macromolecules in the brain. Proc Natl Acad Sci USA 1994;91:2076–2080.
28 Zirzow GC, Sanchez OA, Murray GJ, Brady RO, Oldfield EH: Delivery, distribution and neuronal
uptake of exogenous mannose-terminal glucocerebrosidase in the intact rat brain. Neurochem Res
1999;24:301–305.
29 Burudi EM, Regnier-Vigouroux A: Regional and cellular expression of the mannose receptor in
the post-natal developing mouse brain. Cell Tissue Res 2001;303:334–339.
30 Schueler U, Kaneski C, Murray G, Sandhoff K, Brady RO: Uptake of mannose-terminal gluco-
cerebrosidase in cultured human cholinergic and dopaminergic neuron cell lines. Neurochem Res
2002;27:325–330.
31 Dunbar CE, Kohn DB, Schiffmann R, Barton NW, Nolta J, Esplin J, Pensiero M, Long Z, Lockey C,
Emmons RVB, Cski S, Leitman S, Kreps CB, Carter C, Brady RO, Karlsson S: Retroviral transfer
of the glucocerebrosidase gene into CD34⫹ cells from patients with Gaucher disease: In vivo detec-
tion of transduced cells without myeloablation. Hum Gen Ther 1998;9:2629–2640.
32 Hacein-Bey-Abina S, von Kalle C, Schmidt M, Le Deist F, Wulffraat N, McIntyre E, Radford I,
Villeval JL, Fraser CC, Cavazzana-Calvo M, Fischer A: A serious adverse event after success-
ful gene therapy for X-linked severe combined immunodeficiency. N Engl J Med 2003;348:
255–256.
medwedi.ru
33 Verma IM: A voluntary moratorium? Mol Ther 2003;7:141.
34 Reiser J, Harmison G, Kluepfel-Stahl S, Brady RO, Karlsson S, Schubert M: Transduction of
non-dividing cells using pseudotyped defective high-titer human immunodeficiency virus type 1
particles. Proc Natl Acad Sci USA 1996;93:15266–15271.
35 Lai Z, Han I, Zirzow GC, Brady RO, Reiser J: Intercellular delivery of a herpes simplex virus
VP22 fusion protein from cells infected with lentiviral vectors. Proc Natl Acad Sci USA 2000;97:
11297–11302.
36 Lai Z, Brady RO: Gene transfer into the central nervous system in vivo using a recombinant
lentivirus vector. J Neurosci Res 2002;67:363–371.
Roscoe O. Brady, MD
Building 10 Room 3D04, National Institutes of Health
9000 Rockville Pike, Bethesda, MD 20892–1260 (USA)
Tel. ⫹1 301 496 3285, Fax ⫹1 301 496 9480, E-Mail bradyr@ninds.nih.gov
Brady/Brady 212
Gene Therapy for Parkinson’s Disease

Piotr Hadaczek, Marcel Daadi, Krystof Bankiewicz
Molecular Therapy Laboratory, Department of Neurological Surgery,
University of California, San Francisco, Calif., USA
Why Gene Therapy for Parkinson’s Disease?
The main existing pharmacological therapy for Parkinson’s disease (PD)

centers on replacement of dopamine (DA) by administration of the DA precursor
L-dopa. In many cases, agents that prolong the action of DA by preventing its
breakdown are also used to potentiate L-dopa effects [1]. Current problems asso-
ciated with L-dopa treatment include motor fluctuations and choreic or dystonic
involuntary movements (dyskinesias), which are superimposed on underlying
breakthrough symptoms of bradykinesia, rigidity, and postural instability [2].
With the inevitable progression of the disease, L-dopa loses its initial effects of
symptom relief. The major limitations of L-dopa treatment are 3-fold: inability to
achieve site-specific delivery, which results in unwanted side effects and limits
the amount of drug which can be given [3]; nonsustained drug levels within the
central nervous system (CNS), thought to contribute to unpredictable ‘on-off’
effects [4, 5]; and progressive degeneration of DA-secreting nerve cells during
treatment [6]. Development of new therapeutic approaches to PD must address
these inadequacies of L-dopa. Most importantly, L-dopa addresses the biochem-
ical sequelae of PD but does not address the underlying causes. Therefore, a
primary goal for therapy of PD is the development of neuroprotective therapy
which will slow down and prevent the death of neurons in substantia nigra (SN).
Gene therapy encompasses any technique whereby an absent or faulty
gene is replaced by a working one, so that a cell can make the correct enzyme
or protein and consequently eliminate the cause of the disease. As a result, gene
transfer may serve as a compensation for missing or defective protein expres-
sion. Several features of PD make it particularly suited for a gene therapy-based
approach to treatment: (1) The pathology of the disease has been well charac-
terized (loss of dopaminergic neurons and degeneration of the nigrostriatal
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circuitry); (2) the initial pathology is confined to a discrete location within the
brain where stereotactic targeting is possible (i.e., global gene transfer is not
required in early stages); (3) disease processes such as apoptosis occurring
within the SN may be prevented with a gene transfer approach, and (4) established
animal models are available for testing clinical efficacy, safety and prognostic
assessments.
Gene therapy models for PD have focused on two treatment strategies. One
is the replacement of biosynthetic enzymes for DA synthesis. It has been hypoth-
esized that the transfer of genes involved in DA production would help to ame-
liorate the direct motor symptoms of the disease by the sustained local delivery
of this neurotransmitter. The biochemistry of the DA synthesis involves several
enzymes and cofactors. The rate-limiting enzyme in DA production is tyrosine
hydroxylase (TH), which converts the amino acid tyrosine to L-dopa. L-dopa is
then converted to DA by the aromatic amino acid decarboxylase (AADC) [7].
Another cofactor that is essential for DA metabolism is 6-tetra-hydrobiopterin,
the level of which is limited by the availability of the enzyme GTP-cyclohydro-
lase 1 (GTPCH-1) [8, 9]. In theory, these enzymes could be genetically manipu-
lated to produce increased DA levels. Some studies have reported a benefit from
such enzyme replacement therapy, but others have challenged the relevance of
providing biosynthetic enzymes to a milieu in which the cells are dying and
incapable of properly using DA in any case.
Another treatment strategy is providing neurotrophic factors for protection
and restoration of dopaminergic neurons, thereby preventing them from further
degeneration. Within each of these separate strategies, both in vivo (direct transfer
of the gene into brain) and ex vivo (transplantation of genetically engineered cells)
approaches have been considered.
In vivo Approach
Neurodegenerative diseases like PD are chronic; therefore, treatment needs
to be longlasting. This situation makes PD particularly suited to treatment with
viral vectors, where a single application of a vector can result in prolonged, stable
transgene expression of relevant enzymes or growth factors. In vivo gene ther-
apy involves direct gene transfer into the host somatic cell via viral vectors or
a liposome vehicle [10]. There are several advantages of direct gene transfer
over cellular transplantation: (1) vector delivery may be less invasive for the
synaptic circuitry and brain parenchyma; (2) there is limited risk of unregulated
cellular proliferation; (3) tissue-specific delivery systems for regulatable trans-
gene expression can be designed, and (4) multiple genes can be administered at
the same time.
If gene therapy is to become a truly practical mode of treatment of PD, the
therapeutic gene will need to be expressed for a sustained length of time and it
Hadaczek/Daadi/Bankiewicz 214
will require stable transduction and maintenance of gene expression in the
targeted region of the brain and limited immune response to the vector and the
target product. Various vector systems represent different features (table 1) that
have to be carefully considered before clinical application.
Both viral and nonviral vectors have become an important and powerful
tool for gene delivery to the human nervous system. Neurodegenerative diseases
like PD are suitable candidates for ‘molecular neurosurgery’ approaches because
they are localized and specific regions of the brain are responsible for their
development. Researchers have developed many different virus-based systems
to manipulate subtle neuronal cell biochemistry and physiology. Crucial issues
that need to be taken into consideration include transgene transduction effi-
ciency, adverse tissue responses, targeting specificity, and regulation of trans-
gene expression. Issues related to vector toxicity, long-term expression, gene
regulation, vector production, CNS administration, and axonal transport will
need to be addressed to develop an optimal gene delivery system for PD.
Herpes Simplex Virus (HSV) – Based Vectors

Herpes Simplex Virus 1 (HSV-1) has some features, which make it attrac-
tive as a vehicle for the delivery of therapeutic genes to the nervous system.
HSV-1 is neurotropic and viral genomes persist as extrachromosomal elements.
A neuronal-specific HSV promoter is capable of remaining active during viral
latency, making HSV-based vector systems less susceptible to promoter silencing.
Additional modifications to the viral genome (e.g., removal of genes responsi-
ble for the lytic cycle) reduce the cytotoxicity of the vector [11]. One technical
advantage of the HSV genome for vector construction is that viral genes are
almost entirely found as contiguous transcribable units, which makes their genetic
manipulation relatively straightforward. The large-size viral genome of 152 kb
permits insertion of a large size transgene and the ability to deliver multiple
therapeutic genes via a single vector source.
In general, HSV-based vector systems can be assigned to one of two major
categories, either recombinant viral vectors or defective viral vectors. The
recombinant HSV vectors carry a foreign gene in the native viral genome. They
lack essential viral genes crucial for replication, but retain their ability to enter
into the latency state within neuronal cells. The other type of HSV vector, the
plasmid-based amplicon (‘defective’ HSV vector) contains approximately 1%
of the HSV-1 genome and its backbone includes an eukaryotic plasmid modified
by the addition of an HSV origin of replication (ori) and packaging sequence.
This system requires a helper virus such as the wild-type HSV for high-level
transgene expression and packaging [12]. Titers of amplicon stocks are typi-
cally lower than those of recombinant vectors (⬃106–107 units). Since defective
HSV vector stock preparations may contain helper virus, the use of such vectors
Gene Therapy for Parkinson’s Disease 215
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Table 1. Review of viral vectors used in gene therapy systems
Hadaczek/Daadi/Bankiewicz
Vector HSV HSV Ad Minimal Lentivirus AAV

amplicon Ad ‘gutless’ (HIV)
Diameter 200 nm 200 nm 70–100 nm 70–100 nm 80 nm 20 nm

Size of viral 152 kb minimal (only HSV 30–40 kb 500 bp 7 kb 4.7 kb
genome replication and
packaging origins)
Insert capacity ⬍30 kb ⬎50 kb ⬍8 kb 36 kb 7 kb 4.8 kb
Occurrence in cell episomal episomal episomal episomal integrated episomal/
in genome integrated
in genome
Vector type recombinant defective recombinant defective defective defective
Viral contamination yes yes yes minimal no minimal
during production
Immunogenicity high high high low low low
Titers (TU/ml) 1011 107 1012 107 107 1012
Major advantage large capacity of large capacity of high titers low immunogenicity/ ability to low
transgene; high transgene; low toxicity; large transduce immunogeni-
titers immunogenicity/ capacity of transgene dividing and city/toxicity
toxicity nondividing cells
Major limitation transient low titers triggers immune tedious production possible small size of
expression/triggers response/transient conversion to insert
immune response expression HIV-1; random
genomic
integration
216
in vivo may result in the expression of cytotoxic gene products from the helper
virus, leading to neuropathological effects. Progress in reducing cytotoxicity
includes improvements in the packaging system such as increasing the ratio
between defective viral vector and helper; usage of helper virus with a larger
deletion in IE3 (immediate early gene); using helper-free packaging systems;
and improving purification of amplicon from helper virus.
Studies using HSV vectors for gene therapy in PD have had mixed results.
In a rat 6-hydroxydopamine (6-OHDA) PD model, HSV-based vectors contain-
ing the TH gene were delivered to the rat striatum and the animals appeared to
demonstrate behavioral and biochemical recoveries for one year [13, 14]. Using
the same animal model, neuroprotective effects on dopaminergic neurons have
been demonstrated using glial derived neurotrophic factor (GDNF) and the apop-
tosis inhibitor Bcl2 with HSV-derived vectors. It was also shown that cotransfec-
tion of HSV-GDNF and HSV-Bcl2 had additive neuroprotective properties [15].
Both vector systems have shown reduction of amphetamine-induced rotations
in the 6-OHDA rat model of PD.
As mentioned, the main limitations of HSV systems include CNS cyto-
toxic effects and poor long-term gene expression, with limited number of cells
expressing the transgene [13]. For these reasons, clinical use of HSV-1 appears
to be impractical unless changes in vector design are implemented.
Adenovirus Vectors
Adenoviruses (Ad) have been a popular vehicle for gene transfer. Their
attractive features include the capacity to accommodate large transgene inserts
up to 36 kb and the ability to infect a wide variety of cell types and species
(including postmitotic cells). The four main cell types in the brain which can be
transduced by Ad vectors are neurons, astrocytes, oligodendrocytes, and epen-
dymal cells [16, 17]. Recombinant Ad vectors have focused on deletions of E1,
E2, E3, and E4 genes to reduce immunogenicity [11]. In humans, perhaps the
most important quality is that Ad is not associated with any neoplastic disease
and causes relatively mild, self-limiting illness in immunocompetent individuals
(respiratory infection, keratoconjunctivitis, gastroenteritis). The development
of Ad vectors of first, second and third generations are all based on deletions of
one or more of these genes.
Replication-deficient Ad vectors are propagated on special cell lines that
provide functions of the early transcription units. The first-generation vectors
(lacking E1) can still induce substantial inflammation, despite being replicant-
deficient. Both viral proteins and therapeutic proteins were found to be targets
for immune attack. Despite the lack of E1, viral proteins are expressed on first-
generation vectors at levels sufficient to elicit a T-cell response [18]. Unfortunately,
in addition, even the therapeutic gene will often be recognized as foreign by the
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host. To bypass such an immune response, use of tissue-specific promoters has
been proposed [19]. To improve the utility of Ad vectors for gene therapy, inves-
tigators have further modified the virus by mutating or deleting regions E2-E4
(second- and third-generation Ad vectors) [20–23]. Ads have not gained wide-
spread use because of their inconsistent performance, probably due to the insta-
bility of the deleted vector genome [24]. Deletion of all Ad protein-coding
sequences is possible with fully deleted Ad vectors (minimal or ‘gutless’ Ad
vectors). The only Ad sequences that need be retained are ⬃500 bp of cis-acting
DNA elements, including the viral inverted terminal repeats located at each end
of the genome and the viral packaging signal. Current methods for producing
gutless Ad involve its coreplication in the presence of a second helper virus that
provides replicative functions. The advantages of this system are increased cloning
capacity (⬃37 kb), increased safety, and potentially reduced immune responses
due to elimination of viral sequences.
The episomal nature of Ad often means that ultimately the transgene will be
expelled from the cytosol during cell division. However, the genome may persist
as an episome in nondividing cells (neurons) with sustained transgenic expres-
sion for longer than one year [25]. Nevertheless, repeat vector administration is
probably required in order to boost transgene expression levels to initial levels.
Unfortunately, in most cases administration to immunocompetent individuals
results in the formation of anti-Ad neutralizing antibodies (directed at the vector
capsid) which presents a significant barrier to vector readministration [26–28].
In vivo use of Ad vectors in PD animal models has focused on delivery of
either Ad-TH or Ad-GDNF. When Ad vector encoding the TH gene was intro-
duced into the striatum of 6-OHDA-lesioned rats, a reduced frequency of
apomorphine-induced rotational behavior was observed [29]. TH expression,
being confined predominantly to astrocytes, was demonstrated only for
1–2 weeks following gene transfer and an inflammatory response with gliosis was
detected. More recent experiments with Ad vector encoding TH under the con-
trol of the repressible tetracycline regulatory system (‘tet off’) also showed that
this vector mediates synthesis of TH in striatal cells. Transgene expression was
observed in a large proportion of cells for at least 17 weeks, resulting in a sig-
nificant overall reduction of apomorphine-induced rotation for at least 30 days.
However, after 6 weeks, the pre- and postinjection outcomes were comparable
[30]. In studies with multisite partitioned delivery of Ad-TH, Leone et al. [31]
showed a correlation between the numbers of TH-immunoreactive cells and the
loss of apomorphine-induced rotation, with a near-linear relationship between
TH expression and phenotypical recovery. Those data suggested that only a frac-
tion of striatal cells need to be transduced in order to exert phenotypical effects.
Neuroprotective effects on dopaminergic neurons have been demonstrated when
Ad-GDNF vectors were delivered [32, 33], with increased survival of SN
dopaminergic neurons and preservation of dopaminergic innervation to the
striatum [33]. Studies by Connor et al. [34] also demonstrated that GDNF
expressing Ad injected into the striatum and SN of aged rats, one week prior to
6-OHDA lesioning, allowed the production of GDNF at DA nerve terminals.
However, only striatal injections of Ad-GDNF protected against the develop-
ment of behavioral deficits characteristic of unilateral DA depletion. These
results show that increased levels of striatal, but not solely nigral, GDNF
biosynthesis prevents DA neuronal loss and protects DA terminals from oxida-
tive damage from 6-OHDA lesioning.
The development of gutless Ad from first-generation vectors was inevitable
because of the shortcomings of the latter. For now, gutless Ad appears to be a
promising vector platform for genetic diseases where long-term gene expression
is required. With the advancement of vectorology, Ad-based delivery systems
may be amenable to clinical applications in the future, but many problems remain
such as immunological sensitization.
Lentivirus-Based Vectors
Lentiviral (LV) vectors are derived from a group of pathogenic retroviruses,
which include human immunodeficiency virus (HIV). The retroviral machinery
requires the conversion of the RNA genome to double-stranded DNA, mediated
by the reverse transcriptase enzyme that is present in the infectious virion. The
last step of the replication cycle leads to the integration of the provirus into the
host genome. Once integrated, the provirus is ready to be expressed. The first
retroviral vectors used for gene transfer were murine leukemia virus. Their use
in the CNS was largely limited to ex vivo gene therapy as they were not able to
transduce nondividing neuronal cells [11]. Lentiviral-based vectors share the
properties of commonly used retroviruses with additional advantages: they can
infect both dividing and terminally differentiated cells such as neurons; they
have a large cloning capacity (⬎9 kb); they can be stably integrated into the
genome of the target cells; and they do not encode viral proteins that can trigger
an immune response. In current versions of HIV-1-based LV vectors, up to 60%
of the viral genome has been eliminated and only three or four of the nine genes
of HIV-1 are retained [35, 36]. Viral particles are generated by transient trans-
fection of 293T cells with a three-plasmid system consisting of packaging,
envelope, and transfer vectors [37, 38]. Splitting of the viral genome limits the
formation of replication-component particles [35]. Through integration, retro-
viral vectors offer the opportunity of long-lasting expression, a major advantage
in the treatment of genetic diseases. The level of expression in the brain can be
further increased by the introduction of postregulatory elements that stabilize
nascent RNA transcripts [39, 40]. In most versions of LV vectors, the particles
are pseudotyped with the G envelope protein of vesicular stomatitis virus,
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which gives the vector the capacity to infect a broad range of tissues including
nervous tissues, and is probably responsible for their high affinity for neurons
[37, 38, 41–43].
Because retroviral vectors integrate into the host genomic DNA, there are
some major biosafety issues. The first is that nonspecific integration represents
a potential source of genetic mutation. Host genomic integration with lentivirus
appears to be a random process and important host cellular genes may be dis-
rupted or activated [44–46]; however, in ex vivo human gene therapy for severe
combined immunodeficiency X-1, a retrovirus was found to have preferentially
inserted into an oncogene sequence in 2 separate patients. Other risks may
result from the vector preparation itself (i.e., toxicity of viral proteins or com-
pounds derived from the production system). Generating replication-competent
retrovirus also is a major concern; primate studies had highlighted this poten-
tial risk of [47]. To minimize the risk of such recombinants, a self-inactivating
version of LV has been developed [40, 43]. The self-inactivating design results
in the removal of the major part of the viral transcriptional elements prior to
integration, which also minimizes the chance that genes adjacent to the vector
integration site will become activated.
As with other gene transfer vectors, immunogenicity of retroviral vectors
needs to be studied further before widespread clinical applications are possible.
LV gene transfer into the monkey nigrostriatal system has been shown to induce
minor perivascular cuffing, but without an apparent inflammatory response
[48]. In Fisher rats, after intraportal infusion into the liver of more than 8 ⫻ 108
transducing units of an LV, a mortality rate of 74% was observed [49], clearly
unacceptable for clinical implementation.
Despite these caveats, significant advances in defective LV systems have
provided a new perspective on gene delivery to the brain. Use of LVs in PD ani-
mal models has permitted delivery of GDNF to the striatum or SN. For exam-
ple, Deglon et al. [39] were the first to examine lenti-GDNF delivery in a rodent
model of PD. Their study indicated a significant sparing of nigral neurons after
unilateral injection of lenti-GDNF over the SN. Georgievska et al. [50] simi-
larly demonstrated structural and functional protection in 6-OHDA-lesioned
rats with a LV. Similar results were observed in a mouse model of PD [51] by a
different group. In a MPTP monkey model of PD, Kordower et al. [52] tested
LV for intracerebral GDNF delivery. In treated animals, severe motor deficits were
partially corrected, loss of dopaminergic neurons in SN was partially spared,
and striatal dopamine innervation was preserved up to 70–80%. Consistent with
these results, striatal 18F-fluorodopa uptake [Positron Emission Tomography
(PET) prior to euthanasia] was increased by 300% in the lenti-GDNF-treated
striatum. This work certainly supports the eventual use of lentivirus-GDNF
treatment in the gene therapy of PD, though issues of long-term efficacy and
toxicity must first be addressed. In another in vivo approach by Azzouz et al.
[53], a self-inactivating minimal LV expressing TH, AADC, and GTPCH-1 in a
single transcriptional unit was designed. After stereotactic delivery into the
DA-denervated striatum of the 6-OHDA-lesioned rat, sustained expression of each
enzyme and production of catecholamines was detected, resulting in significant
reduction of apomorphine-induced motor asymmetry during testing. Expression
of each enzyme in the striatum was observed for up to 5 months after injection.
These data indicate that production of three catecholaminenergic enzymes by a
single LV can achieve functional improvement in 6-OHDA-lesioned rats. These
results are somewhat tempered by work from other groups suggesting that multiple
enzymatic delivery in 6-OHDA PD rats with gene transfer did not appear to cre-
ate any additive effect beyond TH gene transfer alone [Janson, pers. commun.].
HIV-2 derived LVs have been used extensively for gene delivery to human
neuronal cells. HIV-2 appears to be slightly less pathogenic than HIV-1 and
because of limited sequence homology; cross-packaging of HIV-2 vectors into
HIV-1 cores will minimize recombination between sequences in the transfer and
packaging vectors. Gene transfer of AADC gene using the above-mentioned
system was first examined in vitro by D’Costa et al. [54]. SVG cells (human
neuronal cells immortalized by SV40 transformation) were transduced by both
HIV-1 and HIV-2 based vectors carrying a cassette containing the AADC gene.
Subsequently, gene transfer was evaluated by determining the ability of the
transduced cells to convert L-dopa into DA. This conversion was measured in
the intracellular compartment as well as in the secreted form in the supernatant.
The results showed that both HIV-1 and HIV-2 AADC vectors successfully
imparted the ability on transduced cells to efficiently convert L-dopa into DA.
It was noted that the observed higher transduction for HIV-1 cross-packaged
vectors was partly due to the higher titer of the latter vector. This approach pro-
vides the ability to combine gene transfer and standard drug treatment. These
outcomes suggest that efficient HIV-2 vectors with a therapeutic transgene self-
packaged in HIV-2 cores, or cross-packed in HIV-1 cores, can be generated for
the future treatment of PD.
Adeno-Associated Virus-Based Vectors

Adeno-associated viral (AAV) vectors are favorable candidates as gene
delivery vehicles. They have many advantageous properties for gene therapy
applications. For example, the parental virus does not cause disease; no viral
genes are included in AAV recombinants and therefore, host immune response
is minimized, the vectors transduce dividing or nondividing cells and a wide
range of cells and tissues; expression can persist, mediating impressive long-
term gene expression. One main limitation of AAV vectors is their small trans-
gene capacity of ⬍5 kb per particle.
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Wild-type AAV is the smallest (⬃20 nm) and simplest of the DNA replication
defective viruses (Parvoviruses). The nonenveloped wild-type AAV particle
contains a linear single-stranded DNA genome of 4.7 kb encapsidated with a
simple three-protein capsid. The conversion of the single-stranded DNA genome
to a double-stranded molecule is an important event required for the efficient
function of AAV as a delivery vehicle. Its rate largely depends on the physio-
logical state of the cell and may be a limiting factor for the transduction effi-
ciency. Wild-type AAV has been shown to stably integrate into a single specific
site within chromosome 19q13.3-ter [55]. Latent persistence occurs when AAV
infects cells in the absence of helper virus (Ad or HSV). When cells containing
an AAV provirus are superinfected with a helper virus possessing trans-acting
elements (necessary for replication and packaging), the integrated AAV genome
is ‘rescued’ and replicated to yield progeny AAV particles.
There is divergence in tropism among various AAV serotypes (types 1–5)
[56]. For example, recombinant AAV-5 and AAV-2 preferentially transduce neu-
rons. Viral receptors strictly define the specific tissue tropism of a particular
viral serotype. The general principles of AAV vector construction are based on
the substitution of the AAV coding sequence with foreign DNA (transgene) to
generate a vector plasmid. Only the AAV inverted terminal repeats flanking the
transgene cassette must be retained intact. Current methods to produce stocks of
defective AAV often use a human cell line (typically 293 cells) that is cotrans-
fected with an AAV vector and a helper plasmid containing the AAV coding
sequences (rep and cap genes flanked by Ad). The transfected cells are subse-
quently superinfected with Ad plasmid, which serves as a helper virus. The result
of this system is a mixture of AAV vector particles and Ad particles. The Ad can
be inactivated by temperature (56oC for one hour) and separated by CsCl-density
centrifugation. Other, more recent, methods for obtaining high titers of AAV
with no contamination by helper Ad have been developed [57, 58]; these typi-
cally use ‘triple transfection’ with a rAAV vector and two helper plasmids that
serve the replicative roles of Ad and the packaging role of the AAV wild-type
sequence. Purification of AAV is critical for clinical trials. A variety of chro-
matographic methods (e.g., ion exchange, antibody and heparin affinity resins)
have been used in both conventional chromatography and HPLC systems.
Vectors derived from recombinant AAV appear to exist as episomes and
have not been shown to integrate to a significant degree. After AAV particles
enter the nucleus, the vectors become circularized and ligated into larger con-
catameric molecules. Most of such molecules appear to persist for prolonged
periods, perhaps even for the lifetime of the cell in the case of nondividing cells
such as neurons. This phenomenon may help to explain long-term expression of
transgenes delivered to the cell by recombinant AAV. Recombinant AAV vectors
are considered one of the safest viral delivery systems, with minimal induction
of innate immune response. Nevertheless, there are reports describing generation
of humoral neutralizing antibody responses to AAV capsid proteins following
systemic delivery. This response may reduce the efficiency of the transduction, a
consideration for systemic readministration [59, 60].
AAV vectors were first introduced into clinical trials for the treatment of
cystic fibrosis [61, 62], using inhaled delivery for the treatment of the lungs. In
the brain, two clinical protocols have been initiated thus far using AAV, for
Canavan disease [Janson C, pers. commun.] and PD [During M, pers. commun.].
In preclinical work, long-term expression of AAV transgenes has been demon-
strated in the CNS, including in the SN, globus pallidus, and striatum [63–71].
Mandel and colleagues [72, 73] examined the neuroprotective effects of intran-
igral AAV-GDNF injected 3 weeks before or immediately after intrastriatal
6-OHDA lesions. Significant neuroprotective effects were observed on the his-
tological level in both versions of that experiment. However, no functional
recovery was detected. More recently, Kirik et al. [67] examined the regional
effects of AAV-GDNF delivery. A 6-month period of sustained expression was
reported. Interestingly, GDNF expression and its protective effect were observed
at both injection sites (nigra and striatum), but preservation of striatal dopamin-
ergic fibers occurred only with striatal injection of the vector. Functional recov-
ery also occurred only when AAV-GDNF was transduced into the striatum. It
appears, therefore, that protection of dopaminergic terminals in the striatum is
a critical feature in promoting functional recovery.
Another approach using AAV vectors, replacement of DA biosynthetic
enzymes, has been examined in various animal models of PD. Injection of an
AAV vector containing the TH gene resulted in expression of TH enzyme in
neurons as early as 24 h postinjection and persisted up to 7 months [66]. That
study was among the first attempts to use enzyme replacement strategy with AAV
as the delivery system. More recent studies have confirmed the performance of
AAV both in terms of efficiency and the absence of cytotoxicity [74]. AAV-TH
alone, however, was reported by one group to produce neither significantly ele-
vated L-dopa levels nor significant behavioral improvements [63]. In addition
to TH, that group found that gene transfer for other enzymes (AADC, GTPCH-1)
was necessary for efficient DA production. Replacement of two or even three
crucial enzymes in PD can be therapeutic. Indeed, behavioral recovery and
effective dopamine production was achieved in combination with therapy with
AAV-TH and AAV-AADC [75]. In turn, triple transduction with AAV-TH, AAV-
AADC, and AAV-GTPCH 1 showed improved rotational behavior lasting at
least 12 months, and elevated DA production in rat striatum, compared with dou-
ble transduction with AAV-TH and AAV-AADC [69]. This strategy extended
the preclinical exploration to a primate model of PD and also showed some
behavioral improvement with restoration of DA synthesis [76].
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AAV/Lac-Z AAV/AADC
AADC-IR AADC-IR
Fig. 1. Convection-enhanced delivery (CED) of AAV2-AADC. Efficient technique for

vector delivery is required for successful gene transfer. Convection-enhanced delivery can
distribute AAV-2 vector in a nontraumatic and uniform fashion within monkey striatum [71].
Immunostaining of the monkey brain section for AADC shows the extent of transduction
with AAV2-AADC (right). The section from the control monkey transduced with AAV2-
LacZ (left). Residual immunoreactivity (IR) for AADC is seen only in nucleus accumbens
which is spared in PD.
An alternative approach of combined drug and gene transfer proposed by

Bankiewicz and colleagues [70, 71] is based on the premise that a reduction in
AADC might contribute to the loss of L-dopa therapeutic efficacy. Therefore,
gene transfer to restore the decarboxylating capacity of L-dopa may result in a
therapeutic gain with continued L-dopa dosage. In a MPTP monkey model
[71], AAV2-AADC injected alone in the striatum was found to confer long-term
(3.5 years) expression of the AADC gene (fig. 1, 2) with robust conversion of
peripheral L-dopa to DA and some behavioral improvement. Modulating intras-
triatal DA levels, by combination of AADC gene delivery and oral adjustments
of L-dopa dosage, may provide a treatment strategy that could prolong L-dopa
efficacy and reduce side effects seen from chronic high-dose oral drug therapy.
Because DA levels are difficult to regulate after single or multiple gene trans-
duction, the AADC and L-dopa approach is inherently more safe, though long-
term efficacy is still unproven.
Fig. 2. Sustained AADC gene expression following AAV2-AADC gene delivery in PD
monkey. AAV2-mediated gene expression can be long lasting (over 3.5 years) [147]. Picture
was taken at 9 months post-transduction. Due to the neurotropic nature of AAV2, mostly
medium spiny neurons are targeted.
An interesting approach to correct the physiological circuit affected by PD

with AAV was recently proposed by Luo et al. [77]. The basis of their study
design was the idea that marked improvement of the motor symptoms of PD
occurs following subthalamic nucleus (STN) ablation or high-frequency stimula-
tion. The projection axons from the STN end in excitatory synapses on target
neurons in the SN pars reticulata, a major output pathway to the thalamus. Luo
et al. generated AAV vectors containing two isoforms of glutamic acid decar-
boxylase (GAD65, GAD67), an enzyme which is responsible for conversion of
glutamate to gamma amino butryic acid. Adult male rats were stereotactically
injected into the STN with AAV-GAD vectors. Expression of transgenes was
observed up to 5 months posttransduction. Transduced neurons, when driven by
electrical stimulation, produced mixed inhibitory responses associated with the
gamma amino butryic acid release. Three weeks after surgery, the ipsilateral
medial forebrain bundle was lesioned with 6-OHDA, while control animals
received AAV-GFP (green fluorescent protein) or PBS. These lesions led to
impaired general locomotor activity and apomorphine-induced rotations con-
tralateral to the denervated side in control animals. In the GAD65-treated rats,
however, abnormal apomorphine-induced rotation was decreased by 65%.
Immunohistochemical data revealed that 80% of dopaminergic neurons survived
in the ventral tegmental area and 35% in the SN pars compacta. These results sug-
gest that neurons generally considered excitatory and glutamatergic can express
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GAD transcripts. Hence, AAV-GAD gene transfer into excitatory neurons may
have clinical potential for the treatment of PD or other conditions associated with
excessive excitation. One major concern regarding this study has been that AAV
can spread from the site of administration via axonal transport, and thus expres-
sion could adversely influence neurons beyond the targeted site. Moreover, data
in a primate model has not been made public and it will be important to confirm
results from the rat model in a large-animal model that more accurately reflects
the human physiology. In a Phase I study that was initiated at Long Island Jewish
Hospital (USA), ablative surgery in the STN is proposed in case of any adverse
effects involving uncontrolled expression of the GAD transcript.
Hybrid Vectors
As all vector systems have certain advantages and disadvantages, researchers
have tried to combine elements from different viruses to create hybrid vectors
with the most advantageous features for gene delivery into the CNS. Problems
with current viral vectors include toxicity of viral proteins, difficulty in regu-
lating transgene expression, and poor efficiency of transgene delivery and sta-
bility in host cells. New generations of chimeric viral vectors will be focused
on targeting of specific tissues and cell types; achieving stable and regulated
transgene expression through integration into the host genome or maintenance as
episomal elements; accommodating large transgenes; retaining high-transduction
efficiency; and minimizing adverse cytotoxic and/or immune responses. Different
versions of chimeric delivery systems have already been proposed: Ad/EBV
hybrid vectors, HSV/EBV/RV hybrid amplicon vectors, Ad/RV, Ad/AAV, HSV/
AAV, and others. Costantini et al. [78] used a HSV/AAV hybrid system and
showed high-transduction efficiency and stability in culture.
Nonviral Approaches for Gene Therapy of PD
All the limitations of viral gene delivery systems (mentioned above)

emphasize the need for alternative therapies with high effectiveness, specificity,
and minimal side effects. Therefore, nonviral vectors have become an attractive
option for gene delivery. Their low immunogenicity and easy large-scale pro-
duction capability are among their most important characteristics.
Naked DNA
It was demonstrated by Wolff and coworkers [79] that simple administra-
tion of ‘naked’ or free DNA by intramuscular injection resulted in a fairly high
level of expression in muscle. Later studies confirmed naked DNA gene trans-
fer in other tissues (e.g., lung, heart, liver, kidney). The most likely mechanism
for cellular entry of such foreign DNA is based on receptor-mediated endocytosis
[80]. While syncytial muscle fibers can readily uptake naked DNA, other tis-
sues such as brain cells are not nearly as permissive, limiting this approach.
Nevertheless, a number of routes and methods have been proposed for delivery
of naked DNA into peripheral tissues, which may also apply to peripheral
nerves. Examples include topical or intradermal; direct injections into deeper
tissues, including intratumoral injections; and intravenous or intra-arterial.
Most studies with naked DNA have focused on intratumoral injections as a pos-
sible anti-tumor strategy. For example, Coll et al. [81] showed that injection of
naked DNA carrying Bax or p53 genes into a xenograft model of human lung
non-small cell carcinoma could inhibit tumor growth. Naked DNA can be used
as a DNA tumor vaccine; one such study showed anti-tumor immunity when
naked DNA encoding the tumor antigen carcinoembryonic antigen or CEA was
delivered by intrasplenic administration [82]. The transfer of naked DNA is
gaining growing acceptance as a form of nonviral gene therapy; however, this
technique is not sufficiently efficient in the brain.
Lipid Vectors
Liposomes have been used as drug carriers for many years. Several differ-
ent liposomal formulations have been used in clinical trials. Cationic lipid is the
most commonly used for such a purpose. To further stabilize liposomal struc-
ture, various polymers (commonly polyethyleneglycol or PEG) have been used,
which may result in improved pharmacokinetics and biodistribution [83]. Cationic
liposome-DNA complexes (plasmid DNA encapsulated in liposomal vesicle)
are the most studied nonviral gene delivery systems in humans. After reaching
the target cell, the DNA is carried across the plasma membrane, either by fusion
or by endocytosis. Subsequently, DNA must be released from the endosome
into the cytoplasm to avoid degradation in the lysosomes. Finally, the DNA
must relocate from the cytoplasm into the nucleus to direct the expression of
the gene products. All of these three steps (entry into the cell, escape into the
cytoplasm, entry into the nucleus) are the main areas for chemists to design
optimal formulations of lipids for gene delivery into cells. In the 6-OHDA rat
model, Zhang et al. [84] demonstrated that it is possible to normalize brain TH
enzyme activity by liposomal gene transfer via intravenous administration. The
TH gene was encapsulated in 85-nm polyethylene glycol immunoliposomes
and targeted to the brain with a monoclonal antibody to the rat transferrin
receptor. In this manner, the gene was successfully delivered across the blood-
brain barrier and the plasma membrane. Three days after intravenous adminis-
tration, striatal TH activity was normalized in association with a 70% reduction
in apomorphine-induced rotation behavior, an approach that was repeated by
others [10, 85].
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Polymer-DNA Complexes (Polyplexes)
Similar to cationic lipidic vectors, polycationic polymers can interact with
the negative phosphate groups of DNA. These polyplexes protect DNA from
degradation and enhance DNA uptake into the cell, resulting in efficient gene
transfer. Cells take up condensed particles through a number of natural processes
such as endocytosis, pinocytosis, or phagocytosis. Similar to lipid vectors, a
polyplex has to pass the plasma and nuclear membranes. Different strategies
have been proposed to improve transfection efficiency, improve specific target-
ing (e.g., conjugation with different ligands), prolong gene expression (e.g.,
insertion of regulatory sequences), and minimize toxicity. The most common
polymers for DNA delivery include poly-L-lysine, protamine, polyethylenimine,
and dendrimer. Several groups have already used polyplexes in animal models
for cancer, an important application of gene therapy. Polyethylenimine/DNA
complexes were also found to be efficient for in vivo gene transfer into neurons
after stereotactic injection into the brain [86, 87]. A study by Wang et al. [88]
demonstrated that polyethylenimine/DNA complexes migrate by retrograde
axonal transport to neuronal cell bodies after being internalized by nerve termi-
nals in the muscle, and confirmed the feasibility of nonviral gene delivery to the
CNS via peripheral injection sites. This approach may have a number of clinical
applications including PD, but specificity remains a problem.
Regulation of Gene Expression
Many proteins of therapeutic value posses a narrow window for optimal

mode of action and have side effects and toxicities when overproduced. Therefore,
gene therapy systems that introduce expression of an endogenous protein ide-
ally should be regulated in vivo to achieve sustained transgene expression. For
example, in the case of PD, too much DA production as a result of excessive
DA biosynthetic enzyme expression can result in unmanageable dyskinesias
and other serious side effects [89–91].
Early gene delivery systems generally relied on viral promoters to drive
constitutive expression [11]. Disadvantages include loss of transgene expres-
sion over time and lack of well-regulated expression. Using promoters that are
specific for particular cell types and tissues is one method of gene regulation,
as their presence in a physiologically specific environment prevents gene silencing
or shutdown of expression. The neuron-specific enolase, enkephalin, Purkinje
cell-specific L7 protein, and myelin-basic protein promoters have been used as
transcriptional activators in viral vectors to express transgenes in neurons, cere-
bellar Purkinje cells, and oligodendrocytes [92–95]. Xu et al. [96] compared a
range of different mammalian CNS expression cassettes in AAV vectors using
different promoter sequences. The highest expression of reporter genes occurred
when endogenous, nonviral promoters such as neuron-specific enolase and
beta-actin were used in AAV-based vectors delivered into rat brain. The com-
monly used basal CMV promoter was found to be the weakest of those tested
in vitro and in vivo. The choice of the proper promoter, therefore, is an impor-
tant component of successful transgene expression.
In a retroviral-mediated gene transfer system, Cortez et al. [97] used a glial
fibrillary acidic protein promoter, whose activity is up-regulated in areas of
gliosis often characteristic for PD. When astrocytes were transduced with the
TH gene and implanted into the striatum of rats lesioned with 6-OHDA, a
significant reduction in the turning behavior occurred for at least 4 weeks after
grafting. The glial fibrillary acid protein promoter is of interest for gene ther-
apy for neurodegenerative disorders, as it is active in the CNS throughout adult
life and may serve as a disease-specific activator, since expression increases
following many types of brain insults. It is important to investigate additional
promoters to express transgenes in subpopulations of neurons most affected in
neurodegenerative diseases such as dopaminergic neurons in PD.
Gene expression can be manipulated by introducing a hybrid gene formed
by linking a regulatory element upstream of the gene to be transcribed. One
such strategy is to use a small-molecule drug that can cross the blood-brain barrier
to act on drug-dependent promoters which directly activate or repress target
gene transcription. Current drug-dependent gene-regulation systems use three
general types of transcription factors: (1) drug-responsive elements (e.g., tetracy-
cline, rapamycin); (2) nuclear hormone receptors (e.g., glucocorticoid-regulated
systems), and (3) heterodimeric proteins (i.e., chemical-induced dimerization).
At this time, the most commonly exploited transgene regulation systems use
tetracycline as the activator or suppressor. The tetracycline-repressible system
(‘tet-off’) works via negative control: the expression of the target gene is on in
the absence of tetracycline and off in its presence [98]. The repressible system
requires two gene sequences, the tetracycline transactivator (tTA) and the target
gene that contains a promoter with tetracycline-binding sites (tetracycline oper-
ator, TetO). In the absence of an antibiotic, tTA has affinity for the TetO sites
and stimulates transcription of the transgene (mode ON). When tetracycline is
present, tTA protein changes its conformation and reduces its affinity for TetO
sites so that the transcription is shut down [99]. The magnitude of the transgene
repression in vivo can be as high as ⬃100-fold. The tetracycline-inducible system
(‘tet-on’) uses positive control and works in the opposite manner [99]. The rtTA
(reverse tTA) gene encodes a protein that has a very low affinity to TetO sites;
however, when an antibiotic is added, the rtTA protein is converted to an active
form which gains the ability to bind TetO sites and activates transgene expres-
sion. A similar principle is applied in anti-progestin or other hormone-inducible
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systems. In the presence of an inducer, transgene expression is ‘on,’ while in
the absence of a hormone, the promoter is not activated and the expression is
effectively blocked.
An advantage of drug-controlled gene transfer systems is that genes can be
delivered in a relatively dose-dependent manner with more consistent and pre-
dictable expression. As these are only the first steps in controlling expression
of transgenes, it is important to understand the limitations of regulated gene
therapy systems before applying them in clinical trials. More studies in animal
models should address issues of safety. The ideal solution would be to develop
a system that would place a transgene under the control of both a tissue-specific
promoter and a disease-specific promoter. The first reports of such advanced
systems have already been published [100] and may soon be used in human
studies.
Targeting of Viral Vectors
Viral surface proteins that bind to the specific cell receptors work as the
primary means of initiating cellular attachment. The expression of specific
surface molecules produces the tissue and cellular tropism for particular viral
vectors. Most viral vectors transduce a relatively broad spectrum of host cells.
The main goal of targeted gene therapy is to specifically infect a single cell type
or group of cells and the choice of the right vector is crucial for specific and
targeted gene delivery. For example, there are eight natural AAV serotypes
which have been studied for gene transfer (AAV-1–8). In the majority of stud-
ies for PD, the neurotropic AAV-2 vector was used. AAV-5 has much broader
tropism and also drives efficient gene expression in astrocytes and epithelial
cells [56]. Other serotypes demonstrate preference for skeletal muscle (AAV-1),
neurons (AAV-3), or ependymal cells (AAV-4).
It is possible to modify viral surface structure by attaching or conjugating
receptor ligands or antibodies. Restricting the vector’s ability to infect unwanted
tissues decreases nonspecific infectivity, which is, of course, an unwelcome
result of every in vivo gene therapy strategy. Incorporation of the vesicular
stomatitis viral glycoprotein has been shown to increase infectivity of retrovirus
[101–103], but decreases with HSV virions [104]. It is possible to generate an
Ad vector expressing a chimeric fiber protein which alters the recognition pro-
file of the virus. In the CNS, one could design a strategy with a fiber protein
conjugated to a neurotrophic factor. This would preferentially target the vector
to neurons expressing the receptor for the conjugated neurotrophic factor. The
enhancement of the affinity of the virus for a particular cell type by modification
of the viral coat could result in lowering the number of viral vector particles to
be used in vivo, an added advantage especially because it may have an important
effect on the immune response against immunogenic vectors like Ad.
Nonviral vectors (e.g., DNA-polyplexes, liposomes) have practically no
selectivity at the level of their incorporation into cells; therefore, introducing
specific ligands has been the major solution in designing targeted gene deliv-
ery. Attachment or incorporation of antibodies is a commonly used strategy.
Receptor targeting increases transduction efficiency of disease-affected cells,
while decreasing gene delivery to nontarget cells. This is perhaps of importance
in PD where a very specific and isolated subset of neurons in the nigra and
striatum is primarily affected. Of course, the use of selected promoters that are
active only within subsets of cells or the use of cell-type-specific drug-inducible
promoters, are solutions to the problem of nonspecificity in this context.
The ex vivo Approach
Ex vivo gene transfer offers the potential for persistent and regulated local
and widespread delivery of therapeutic agents into the CNS. Several strategies
utilizing genetically engineered cells for treating PD are currently under inves-
tigation. These strategies consist of introducing therapeutic genes to cells and
grafting the modified cells into the diseased brain region. In PD models, genet-
ically modified cells may be aimed at DA replacement in the denervated stria-
tum, whereby the therapeutic cells are transduced with multiple genes that
encode for the enzymes and cofactors involved in the biosynthesis of DA; or
protecting the remaining midbrain dopaminergic cells that are still functional
from degeneration. The therapeutic effect also may be aimed at rescuing DA
cells that have begun the process of degeneration through the production of
local trophic factors. Among growth factors that have been described to support
the survival and/or regeneration of the midbrain DA neurons are brain-derived
neurotrophic factor, basic fibroblastic growth factor (bFGF), insulin-derived
growth factor, and glial cell line-derived neurotrophic factors (i.e., GDNF, neur-
turin, persephin, artemin). GDNF has the most prominent and selective effect
in rescuing midbrain DA neurons, increasing DA activity and improving behav-
ioral deficits of both rodent and primate models of PD.
Source of Cells for ex vivo Gene Therapy
There are multiple potential sources of cells for ex vivo gene therapy. The
cells used as delivery vehicles must meet at least three basic criteria: (1) they
should not form tumors in vivo; (2) they should graft at the site of the diseased
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brain area, and (3) they should not elicit a strong host immune response.
Autologous cells isolated from patient biopsies meet these criteria and neural
cells derived from the brain in particular represent an attractive source. However,
this source is unpractical because of the difficulties in isolating and maintain-
ing neuronal cells in culture and obtaining adequate numbers for clinical appli-
cations. Myoblasts, skin fibroblasts, and bone marrow stem cells all have been
considered for ex vivo gene therapy for PD. While myoblasts demonstrated
some limited success, bone marrow stem cells transduced with either L-dopa or
TH demonstrated limited neuronal differentiation and functional integration
within host tissue. Skin fibroblasts have been a popular source for autologous
cells; they demonstrated good survival in a primate model of PD, with expres-
sion of transgene that lasted for several months [74]. However, long-term gene
expression by grafted fibroblasts has not been shown to be successful in rat
models of PD. The reason for this failure may be due to inflammatory cytokine
reaction to traumatic changes in the host tissue. It is important to note that these
cells do not process the cellular machinery to store and release DA; to be com-
petent for such a function within the host striatum, fibroblasts would need to be
transduced with DA transporters and other genes involved in DA storage and
release mechanisms [74, 105, 106]. Indeed, Lee et al. [106] cotransduced rat
fibroblasts with both vesicular monoamine transporter-2 and AADC genes and
demonstrated that these cells were then capable of converting L-dopa to DA and
of storing DA. Transplantation of these engineered fibroblasts into a rat model
of PD resulted in efficient DA production and storage.
Other cell lines have been explored as a vehicle for gene transfer in PD
preclinical studies, which include immortalized fibroblasts, immortalized fetal
astrocytes, schwanoma and glioma cell lines, and endothelial cells [107–111].
The cells were engineered to produce enzymes or trophic factors and then grafted
into the striatum of PD model. While these cell lines survived and expressed the
transgene after implantation, most of them also gave rise to tumors, initiated
immunological rejection, or did not integrate and died.
Neural precursor cells are capable of giving rise to neurons, astrocytes and
oligodendrocytes, and migrating and integrating into the local circuitry. These
cells are preferred for grafting applications, as they approximate the normal
physiological activity of neural cells. One approach is to isolate purified midbrain
dopaminergic neurons by using a cell type-specific live monitoring technique.
To achieve this selective isolation, Sawamoto et al. [112] generated transgenic
mice and rats expressing GFP under the control of a 9-kb rat TH promoter. The
authors demonstrated that expression of GFP protein was specific to DA
neurons in the mesencephalon. The rat fetal midbrain was dissected out and dis-
sociated cells were sorted using the fluorescent activated cell sorting. This
purification step yielded an enriched population of TH-GFP positive neurons
(⬎60%). This sorted and enriched population of DA neurons improved the
behavioral deficits of 6-OHDA lesioned rats. A similar approach was applied
by the same group to isolate midbrain precursor cells [113]. The GFP reporter
gene was under control of the neural-specific second intronic enhancer of the
nestin gene. Nestin GFP mice showed strong fluorescence in the ventricular
zone where precursors are known to reside. Neurospheres generated from the
fluorescent activated cell-sorted nestin-GFP precursors were implanted into the
6-OHDA rat model of PD and 5 weeks after transplantation the rats demon-
strated a significant behavioral improvement.
Other studies have used nonmidbrain-derived stem cells transduced with
the transcription factor Nurr1 (which promotes a dopaminergic neuronal
phenotype) in order to induce stable midbrain DA lineage. For instance, to
induce a dopaminergic cell lineage, Wagner et al. [114] transduced C17.2 cells
[115] with Nurr1 and incubated the cultured cells with soluble factors derived
from ventral mesencephalic type 1 astrocytes. This treatment resulted in the
induction of dopaminergic fate in 80% of the total cells [114]. The genetic mod-
ification of stem cells with Nurr1 was also required to efficiently convert
embryonic stem (ES) cells to DA neurons. Kim et al. [116] demonstrated that
overexpression of Nurr1 alone promoted a 10-fold increase in the number of
TH-expressing neurons. The administration of Shh (Sonic hedgehog) and FGF8
resulted in an additional 5.6-fold increase in the proportion of TH-positive neu-
rons. The ability of these newly induced TH-positive neurons to synthesize and
release DA was demonstrated using HPLC. After implantation into the
6-OHDA lesioned rats, TH-positive neurons survived and extended processes
within the host parenchyma. These cells were postmitotic as demonstrated by
the absence of the cell proliferation marker expression Ki-67. In other studies,
Nurr1 ES cells grafted in parkinsonian rats improved their rotational behavioral
and motor skills as tested in the adjusting step, cylinder, and paw-reaching tests.
Fetal Mesencepahlic DA Neurons

In both rodent and monkey models of PD, midbrain-derived fetal tissue
implants are able to survive, extend neurites, make functional synaptic contact
with host neurons, and secrete DA leading to a dramatic improvement in behav-
ioral deficits [117–120]. Based on these studies, clinical trials of primary fetal
nigral cell transplantation for medically intractable PD were initiated in the
1980s. Open-label clinical trials with mesencephalic DA neurons obtained from
6- to 9-week-old aborted human fetuses demonstrated graft survival, DA stor-
age and release (assayed with PET), and significant and persistent improvement
as measured with Unified Parkinson Disease Rating Scales (UPDRS) relative
to the baseline. Histopathological demonstration of striatal DA reinnervation
was obtained for a period extending up to 10 years [121–125]. These encouraging
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findings have been recently called into question, however, by the first double-blind
placebo-controlled clinical trial [89].
This large, placebo-controlled study involving sham surgeries enrolled 40
patients, 20 transplant subjects and 20 controls who underwent sham surgery. The
graft survival rate in transplanted patient was 85%, without the use of immuno-
suppression. Young patients (⬍60 years) demonstrated 28% and 34% improve-
ment in UPDRS total and motor ‘off’ components, respectively. However, despite
cell survival and DA production demonstrated with PET, clinical improvement
was not as dramatic in patients in the typical age range for PD (⬎60 years). Of
greatest concern were 5 patients who developed severe dyskinesias and/or
dystonia in the absence of levodopa. This graft-induced dyskinesia was termed
‘runaway’ dyskinesia because it was an uncontrolled, off-medication side effect.
The authors’ interpretation was that the side effects were due to graft overgrowth
and an excess of DA production. Subsequently, it has been argued that this
conclusion was somewhat simplistic and the negative outcome of this study is
partially attributable to extended culture of donor tissue, unconventional neuro-
surgical procedures, and an absence of immunosuppression [126, 127].
Nevertheless, together with a study by Fahn and coworkers [89] using stan-
dardized cell transplantation procedures and assessment protocols, Hagell et al.
[128] observed a pronounced ‘runaway’ dyskinesias due to fetal mesencephalic
DA cell grafts. As with the trial by Fahn, these dyskinesias persisted after with-
drawal of L-dopa and DA agonists [128]. In the latter study, the authors argued
that the ‘runaway’ dyskinesias are not caused by overgrowth of grafted cells,
but could be due to micrograft DA spillover which overstimulated supersensi-
tive receptors outside the graft-innervated area. These authors also speculated
that the development of ‘runaway’ dyskinesias could be due to the extended
storage or culture of donor tissue before grafting or to transplantation-evoked
changes in host striatum or nondopaminergic components of the grafts. More
recently, a third report on the second double-blind, placebo-controlled trial of
fetal nigral transplants [91] demonstrated a failure to induce significant motor
improvement relative to placebo. Moreover, patients who received grafts also
developed severe ‘runaway’ dyskinesias that tended to appear 6–12 months
after transplantation. Patients with a higher dose (4 donor grafts) showed
improvement at 6 and 9 months and deteriorated thereafter, coincident with the
termination of cyclosporine intake, suggesting a possible immune reaction
against the graft. Indeed, activated microglia immunostaining with CD45 anti-
body demonstrated an increased immune reaction particularly surrounding the
graft deposits compared to the placebo. Significant improvement was noted in
patients with milder disease at baseline, UPDRS ⱕ 49. The development of
runaway dyskinesias did not depend on differences in fluorodopa uptake, nor
on the dose of cells implanted.
A study by Ma et al. [129] had suggested that higher fluorodopa uptake
coincides with dyskinesia side effects. Contrary to Hagell’s [128] interpreta-
tion, the use of fresh donor tissue (less than 48 h storage) did not prevent the
development of debilitating side effects [91]. In view of the new risks of trans-
plantation that were exposed, Fahn et al. recommended against using fetal
nigral cells as a cell-based therapy for PD at this time, yet suggest that patients
with milder disease may benefit from such a strategy if they receive grafts with
a higher number of surviving cells and a more prolonged immunosuppressant.
It appears likely that overproduction and focal pulsatile delivery of DA within
the dennervated striatum can lead to the development of uncontrolled dyskine-
sias, and future use of DA tissue grafts will need to address this issue of DA
regulation and immune regulation.
Stem Cells: ES and Brain-Derived

The use of stem cells for ‘cell therapy’ of PD requires directing the cells
toward dopaminergic lineages before transplantation or at early stages of grafting.
This preconditioning of stem cells has been tested in vitro using growth factors,
cytokines, and conditioned media for forebrain-derived neural precursors [130,
131] and for midbrain cells as well [132–135]. Transplantation of nonimmor-
talized stem cells into parkinsonian animal models has led to survival, inte-
gration, and expression of TH, the rate-limiting enzyme of DA synthesis, and
to behavioral improvement of the lesioned animals [133,135–138]. Clonally
derived neural stem cells [115] were shown to spontaneously differentiate into
TH-expressing cells in a rat model of PD, a characteristic that is dependant on
the culture confluency of the clone and the host’s microenvironment [139].
DA neurons can be derived form ES cells using a multistep protocol. In a
study by Kawasaki et al. [140], ES cells were maintained in an undifferentiated
state in media supplemented with serum and leukemia inhibitory factor. To
direct differentiation toward the DA lineage, ES cells were cocultured with PA6
stromal cells for 8 days and then ascorbate was added to the media for 6–12
days. With this treatment, 16% of the total cell population was converted into
DA neurons expressing TH- and DA lineage-specific transcription factors Nurr1
and Ptx3. After implantation into the 6-OHDA rat model of PD, the ES-derived
neurons maintained the DA phenotype and did not form tumors. However,
behavioral analysis of these animals was not reported. In another study by Lee
et al. [141], ES cells were maintained in serum and media containing leukemia
inhibitory factor. Removing leukemia inhibitory factor for 4 days and then grow-
ing the cells on adhesive substrate for 24 h induced the formation of embryoid
bodies. Stem cells were subsequently expanded in serum-free media for 6–10
days before inducing DA neurons with bFGF, sonic hedgehog (Shh-N), and
FGF8 for an additional 6 days. Maturation of DA neurons was established by
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culturing the cells in media supplemented with cAMP and ascorbate. Under
these conditions 23% of the total cells were dopaminergic. These DA neurons
expressed transcription factors characteristic of midbrain DA neurons; however,
they were not assessed for their functional ability to reverse behavioral deficits
in PD animal model.
Naïve ES cells also have been implanted into 6-OHDA denervated rat
striatum [142]. In this study, ES cells gave rise to TH-immunoreactive neurons
that also expressed DA transporter and AADC. Using PET and functional MRI,
the grafted ES cells demonstrated the appropriate dopaminergic neuronal prop-
erties which paralleled behavioral recovery demonstrated with the apomorhine
rotational test [142]. This study also reported, however, that ES cell transplan-
tation led to the generation of lethal teratomas in 20% of implanted animals.
While these findings are encouraging, further in vitro manipulation of ES cells
and long-term posttransplant survival studies are required to provide assurance
that tumor formation does not occur, an unacceptable outcome in a disease with
existing drug and surgical therapies. One possible technology to control both
growth rate and lineage of the cells before transplantation is genetic modifica-
tion, such as providing a repressible regulatory unit or suicide gene that could
be induced as required.
Detection of Gene Expression
PET
Neuroimaging techniques and behavioral analyses make it possible to
assess in vivo the state of the DA system in patient and animal models.
Functional studies provide valuable information about the structure and func-
tion of DA neurons and the effects of therapeutic approaches. These techniques
permit quantitative measurement of changes in DA terminals, receptors, and
release of DA in vivo. PET and the use of specific radiolabeled ligands can non-
invasively quantify pre- and postsynaptic markers of the DA system. Many of
these tracers bind selectively to specific transporters, such as DA transporter or
the vesicular monoamine transporter 2. The type of ligand utilized will deter-
mine the information we can obtain about a particular system [143]. Such non-
invasive techniques are ideal for longitudinal studies in experimental models of
PD. Conceivably, they could be used to monitor the progress in effectiveness of
gene therapy approaches by monitoring the transgene expression.
Studies from our lab demonstrated that PET was successfully applied to
monitor AADC expression introduced by AAV-based vector [71]. Another
study from our laboratory in the MPTP monkey model of PD demonstrated the
ability to distinguish between dopaminergic changes in the putamen and the SN
compacta using PET scanning [144]. The previously cited study by Kordower
et al. [48] with AAV-GDNF used PET to monitor therapeutic effects of gene
transfer in a MPTP model. The choice of imaging ligands is especially impor-
tant because the use of growth factors and other therapeutic interventions often
specifically target either the terminals or cell bodies. In vivo detection of gene
expression, as seen in studies after AADC gene transfer in MPTP-treated mon-
keys, is important because it provides quantitative assessment of gene transfer.
This approach applies for both in vivo and in vitro gene transfer where duration,
levels, and location of AADC gene expression can be detected.
Microdialysis
In vivo intracerebral microdialysis has been used in rats, nonhuman pri-
mates, and humans to monitor the extracellular level of neurotransmitters. This
method can be very useful to evaluate alterations in brain metabolism. Micro-
dialysis probes, connected to microinjection pumps, are stereotactically inserted
into targeted points in the brain. Artificial cerebrospinal fluid is administered
at a slow rate and dialysates are collected to microtubes for chemical analysis.
The amount of neurotransmitters in each fraction is determined by HPLC.
Alternatively, by directly sampling cerebrospinal fluid from the lateral ventri-
cles, the levels of amino acids can be measured. Microdialysis was used during
stereotaxic thalamic surgery for PD tremor for neurochemical characterization
of the target area [145]. Studies by Fedele et al. [146] confirmed that this method
might be used in PD to measure amino acid release in human basal ganglia.
Using this approach one can assess the level of DA restoration via gene therapy.
The very same procedure was successfully used by Pernaute et al. [70] to eval-
uate the functional effect of AAV-mediated gene transfer of aromatic L-amino
acid decarboxylase into the striatum of 6 OHDA-lesioned rats.
Conclusions
PD is characterized primarily by the degeneration of a specific population

of neurons in SN and a decline in local neurotransmitter synthesis. Replacement
therapy with the DA precursor L-dopa has been a mainstay of therapy for PD.
However, L-dopa addresses only the biochemical consequences of the disease
and leads to long-term side effects such as dyskinesias. Prevention of further loss
of dopaminergic neurons by neuroprotection, perhaps using gene transfer, is one
alternative approach to treatment. Advances in cellular and genetic engineering
also will permit stem cell transplants to replace neurons once they have been
lost. Gene therapy for PD based on in vivo or ex vivo strategies is realistic, but
will depend on the progress that is made over the years to come.
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References
1 Fahn S: Is levodopa toxic? Neurology 1996;47:S184–S195.

2 Hurtig HI: Problems with current pharmacologic treatment of Parkinson’s disease. Exp Neurol
1997;144:10–16.
3 Harder S, Baas H, Rietbrock S: Concentration-effect relationship of levodopa in patients with
Parkinson’s disease. Clin Pharmacokinet 1995;29:243–256.
4 Chase TN, Juncos J, Serrati C, Fabbrini G, Bruno G: Fluctuation in response to chronic levodopa
therapy: Pathogenetic and therapeutic considerations. Adv Neurol 1987;45:477–480.
5 Obeso JA, Grandas F, Herrero MT, Horowski R: The role of pulsatile versus continuous dopamine
receptor stimulation for functional recovery in Parkinson’s disease. Eur J Neurosci 1994;6:889–897.
6 Melamed E, Hefti F: Mechanism of action of short- and long-term L-DOPA treatment in parkin-
sonism: Role of the surviving nigrostriatal dopaminergic neurons. Adv Neurol 1984;40:149–157.
7 Nagatsu T, Levitt M, Udenfriend S: Conversion of L-tyrosine to 3,4-dihydroxyphenylalanine by
cell-free preparations of brain and sympathetically innervated tissues. Biochem Biophys Res
Commun 1964;14:543–549.
8 Nagatsu T, Yamaguchi T, Rahman MK, Trocewicz J, Oka K, Hirata Y, Nagatsu I, Narabayashi H,
Kondo T, Iizuka R: Catecholamine-related enzymes and the biopterin cofactor in Parkinson’s dis-
ease and related extrapyramidal diseases. Adv Neurol 1984;40:467–473.
9 Nagatsu T, Ichinose H: Molecular biology of catecholamine-related enzymes in relation to
Parkinson’s disease. Cell Mol Neurobiol 1999;19:57–66.
10 Imaoka T, Date I, Ohmoto T, Nagatsu T: Significant behavioral recovery in Parkinson’s disease
model by direct intracerebral gene transfer using continuous injection of a plasmid DNA-liposome
complex. Hum Gene Ther 1998;9:1093–1102.
11 Hermens WT, Verhaagen J: Viral vectors, tools for gene transfer in the nervous system. Prog
Neurobiol 1998;55:399–432.
12 Spaete RR, Frenkel N: The herpes simplex virus amplicon: A new eucaryotic defective-virus
cloning-amplifying vector. Cell 1982;30:295–304.
13 During MJ, Naegele JR, O’Malley KL, Geller AI: Long-term behavioral recovery in parkinsonian
rats by an HSV vector expressing tyrosine hydroxylase. Science 1994;266:1399–1403.
14 Geller AI, Yu L, Wang Y, Fraefel C: Helper virus-free herpes simplex virus-1 plasmid vectors for
gene therapy of Parkinson’s disease and other neurological disorders. Exp Neurol 1997;144:
98–102.
15 Natsume A, Mata M, Goss J, Huang S, Wolfe D, Oligino T, Glorioso J, Fink DJ: Bcl-2 and GDNF
delivered by HSV-mediated gene transfer act additively to protect dopaminergic neurons from
6-OHDA-induced degeneration. Exp Neurol 2001;169:231–238.
16 Akli S, Caillaud C, Vigne E, Stratford-Perricaudet LD, Poenaru L, Perricaudet M, Kahn A,
Peschanski MR: Transfer of a foreign gene into the brain using adenovirus vectors. Nat Genet
1993;3:224–228.
17 Davidson BL, Allen ED, Kozarsky KF, Wilson JM, Roessler BJ: A model system for in vivo gene
transfer into the central nervous system using an adenoviral vector. Nat Genet 1993;3:219–223.
18 Yang CC, Xiao X, Zhu X, Ansardi DC, Epstein ND, Frey MR, Matera AG, Samulski RJ: Cellular
recombination pathways and viral terminal repeat hairpin structures are sufficient for adeno-
associated virus integration in vivo and in vitro. J Virol 1997;71:9231–9247.
19 Pastore L, Morral N, Zhou H, Garcia R, Parks RJ, Kochanek S, Graham FL, Lee B, Beaudet AL:
Use of a liver-specific promoter reduces immune response to the transgene in adenoviral vectors.
Hum Gene Ther 1999;10:1773–1781.
20 Zhou H, O’Neal W, Morral N, Beaudet AL: Development of a complementing cell line and a sys-
tem for construction of adenovirus vectors with E1 and E2a deleted. J Virol 1996;70:7030–7038.
21 Gorziglia MI, Kadan MJ, Yei S, Lim J, Lee GM, Luthra R, Trapnell BC: Elimination of both E1
and E2 from adenovirus vectors further improves prospects for in vivo human gene therapy. J Virol
1996;70:4173–4178.
22 Krougliak V, Graham FL: Development of cell lines capable of complementing E1, E4, and
protein IX defective adenovirus type 5 mutants. Hum Gene Ther 1995;6:1575–1586.
23 Yeh P, Dedieu JF, Orsini C, Vigne E, Denefle P, Perricaudet M: Efficient dual transcomplementation
of adenovirus E1 and E4 regions from a 293-derived cell line expressing a minimal E4 functional
unit. J Virol 1996;70:559–565.
24 Lieber A, He CY, Kirillova I, Kay MA: Recombinant adenoviruses with large deletions generated
by Cre-mediated excision exhibit different biological properties compared with first-generation
vectors in vitro and in vivo. J Virol 1996;70:8944–8960.
25 Zermansky AJ, Bolognani F, Stone D, Cowsill CM, Morrissey G, Castro MG, Lowenstein PR:
Towards global and long-term neurological gene therapy: Unexpected transgene dependent, high-
level, and widespread distribution of HSV-1 thymidine kinase throughout the CNS. Mol Ther
2001;4:490–498.
26 Dong JY, Wang D, Van Ginkel FW, Pascual DW, Frizzell RA: Systematic analysis of repeated
gene delivery into animal lungs with a recombinant adenovirus vector. Hum Gene Ther 1996;7:
319–331.
27 Kaplan JM, St George JA, Pennington SE, Keyes LD, Johnson RP, Wadsworth SC, Smith AE:
Humoral and cellular immune responses of nonhuman primates to long-term repeated lung expo-
sure to Ad2/CFTR-2. Gene Ther 1996;3:117–127.
28 St George JA, Pennington SE, Kaplan JM, Peterson PA, Kleine LJ, Smith AE, Wadsworth SC:
Biological response of nonhuman primates to long-term repeated lung exposure to Ad2/CFTR-2.
Gene Ther 1996;3:103–116.
29 Horellou P, Vigne E, Castel MN, Barneoud P, Colin P, Perricaudet M, Delaere P, Mallet J: Direct
intracerebral gene transfer of an adenoviral vector expressing tyrosine hydroxylase in a rat model
of Parkinson’s disease. Neuroreport 1994;6:49–53.
30 Corti O, Sanchez-Capelo A, Colin P, Hanoun N, Hamon M, Mallet J: Long-term doxycycline-
controlled expression of human tyrosine hydroxylase after direct adenovirus-mediated gene transfer
to a rat model of Parkinson’s disease. Proc Natl Acad Sci USA 1999;96:12120–12125.
31 Leone P, McPhee SW, Janson CG, Davidson BL, Freese A, During MJ: Multi-site partitioned
delivery of human tyrosine hydroxylase gene with phenotypic recovery in Parkinsonian rats.
Neuroreport 2000;11:1145–1151.
32 Choi-Lundberg DL, Lin Q, Chang YN, Chiang YL, Hay CM, Mohajeri H, Davidson BL, Bohn MC:
Dopaminergic neurons protected from degeneration by GDNF gene therapy. Science 1997;275:
838–841.
33 Bilang-Bleuel A, Revah F, Colin P, Locquet I, Robert JJ, Mallet J, Horellou P: Intrastriatal injection
of an adenoviral vector expressing glial-cell-line-derived neurotrophic factor prevents dopaminer-
gic neuron degeneration and behavioral impairment in a rat model of Parkinson disease. Proc Natl
Acad Sci USA 1997;94:8818–8823.
34 Connor B, Kozlowski DA, Schallert T, Tillerson JL, Davidson BL, Bohn MC: Differential effects
of glial cell line-derived neurotrophic factor (GDNF) in the striatum and substantia nigra of the
aged Parkinsonian rat. Gene Ther 1999;6:1936–1951.
35 Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, Naldini L: A third-generation
lentivirus vector with a conditional packaging system. J Virol 1998;72:8463–8471.
36 Zufferey R, Nagy D, Mandel RJ, Naldini L, Trono D: Multiply attenuated lentiviral vector achieves
efficient gene delivery in vivo. Nat Biotechnol 1997;15:871–875.
37 Naldini L, Blomer U, Gage FH, Trono D, Verma IM: Efficient transfer, integration, and sustained
long-term expression of the transgene in adult rat brains injected with a lentiviral vector. Proc Natl
Acad Sci USA 1996;93:11382–11388.
38 Naldini L, Blomer U, Gallay P, Ory D, Mulligan R, Gage FH, Verma IM, Trono D: In vivo gene deliv-
ery and stable transduction of nondividing cells by a lentiviral vector. Science 1996;272:263–267.
39 Deglon N, Tseng JL, Bensadoun JC, Zurn AD, Arsenijevic Y, Pereira de Almeida L, Zufferey R,
Trono D, Aebischer P: Self-inactivating lentiviral vectors with enhanced transgene expression as
potential gene transfer system in Parkinson’s disease. Hum Gene Ther 2000;11:179–190.
40 Zufferey R, Dull T, Mandel RJ, Bukovsky A, Quiroz D, Naldini L, Trono D: Self-inactivating
lentivirus vector for safe and efficient in vivo gene delivery. J Virol 1998;72:9873–9880.
41 Blomer U, Naldini L, Kafri T, Trono D, Verma IM, Gage FH: Highly efficient and sustained gene
transfer in adult neurons with a lentivirus vector. J Virol 1997;71:6641–6649.
medwedi.ru
42 Cisterni C, Henderson CE, Aebischer P, Pettmann B, Deglon N: Efficient gene transfer and
expression of biologically active glial cell line-derived neurotrophic factor in rat motoneurons
transduced with lentiviral vectors. J Neurochem 2000;74:1820–1828.
43 Miyoshi H, Blomer U, Takahashi M, Gage FH, Verma IM: Development of a self-inactivating
lentivirus vector. J Virol 1998;72:8150–8157.
44 Li Z, Dullmann J, Schiedlmeier B, Schmidt M, von Kalle C, Meyer J, Forster M, Stocking C,
Wahlers A, Frank O, Ostertag W, Kuhlcke K, Eckert HG, Fehse B, Baum C: Murine leukemia
induced by retroviral gene marking. Science 2002;296:497.
45 Marshall E: Clinical research. Gene therapy a suspect in leukemia-like disease. Science 2002;
298:34–35.
46 Murre C: Intertwining proteins in thymocyte development and cancer. Nat Immunol 2000;1:
97–98.
47 Donahue RE, Kessler SW, Bodine D, McDonagh K, Dunbar C, Goodman S, Agricola B, Byrne E,
Raffeld M, Moen R: Helper virus induced T cell lymphoma in nonhuman primates after retroviral
mediated gene transfer. J Exp Med 1992;176:1125–1135.
48 Kordower JH, Bloch J, Ma SY, Chu Y, Palfi S, Roitberg BZ, Emborg M, Hantraye P, Deglon N,
Aebischer P: Lentiviral gene transfer to the nonhuman primate brain. Exp Neurol 1999;160:1–16.
49 Park F, Ohashi K, Chiu W, Naldini L, Kay MA: Efficient lentiviral transduction of liver requires
cell cycling in vivo. Nat Genet 2000;24:49–52.
50 Georgievska B, Kirik D, Rosenblad C, Lundberg C, Bjorklund A: Neuroprotection in the rat
Parkinson model by intrastriatal GDNF gene transfer using a lentiviral vector. Neuroreport 2002;
13:75–82.
51 Bensadoun JC, Deglon N, Tseng JL, Ridet JL, Zurn AD, Aebischer P: Lentiviral vectors as a gene
delivery system in the mouse midbrain: Cellular and behavioral improvements in a 6-OHDA
model of Parkinson’s disease using GDNF. Exp Neurol 2000;164:15–24.
52 Kordower JH, Emborg ME, Bloch J, Ma SY, Chu Y, Leventhal L, McBride J, Chen EY, Palfi S,
Roitberg BZ, Brown WD, Holden JE, Pyzalski R, Taylor MD, Carvey P, Ling Z, Trono D, Hantraye P,
Deglon N, Aebischer P: Neurodegeneration prevented by lentiviral vector delivery of GDNF in
primate models of Parkinson’s disease. Science 2000;290:767–773.
53 Azzouz M, Martin-Rendon E, Barber RD, Mitrophanous KA, Carter EE, Rohll JB, Kingsman SM,
Kingsman AJ, Mazarakis ND: Multicistronic lentiviral vector-mediated striatal gene transfer of
aromatic L-amino acid decarboxylase, tyrosine hydroxylase, and GTP cyclohydrolase I induces
sustained transgene expression, dopamine production, and functional improvement in a rat model
of Parkinson’s disease. J Neurosci 2002;22:10302–10312.
54 D’Costa J, Harvey-White J, Qasba P, Limaye A, Kaneski CR, Davis-Warren A, Brady RO,
Bankiewicz KS, Major EO, Arya SK: HIV-2 derived lentiviral vectors: Gene transfer in Parkinson’s
and Fabry disease models in vitro. J Med Virol 2003;71:173–182.
55 Kotin RM, Menninger JC, Ward DC, Berns KI: Mapping and direct visualization of a region-specific
viral DNA integration site on chromosome 19q13-qter. Genomics 1991;10:831–834.
56 Okada T, Nomoto T, Shimazaki K, Lijun W, Lu Y, Matsushita T, Mizukami H, Urabe M, Hanazono Y,
Kume A, Muramatsu S, Nakano I, Ozawa K: Adeno-associated virus vectors for gene transfer to
the brain. Methods 2002;28:237–247.
57 Xiao X, Li J, Samulski RJ: Production of high-titer recombinant adeno-associated virus vectors in
58 Urabe M, Ding C, Kotin RM: Insect cells as a factory to produce adeno-associated virus type 2
vectors. Hum Gene Ther 2002;13:1935–1943.
59 Chirmule N, Xiao W, Truneh A, Schnell MA, Hughes JV, Zoltick P, Wilson JM: Humoral immunity
to adeno-associated virus type 2 vectors following administration to murine and nonhuman pri-
mate muscle. J Virol 2000;74:2420–2425.
60 Halbert CL, Standaert TA, Wilson CB, Miller AD: Successful readministration of adeno-associated
virus vectors to the mouse lung requires transient immunosuppression during the initial exposure.
J Virol 1998;72:9795–9805.
61 Flotte T, Carter B, Conrad C, Guggino W, Reynolds T, Rosenstein B, Taylor G, Walden S, Wetzel R:
A phase I study of an adeno-associated virus-CFTR gene vector in adult CF patients with mild
lung disease. Hum Gene Ther 1996;7:1145–1159.
62 Wagner JA, Messner AH, Moran ML, Daifuku R, Kouyama K, Desch JK, Manley S, Norbash AM,
Conrad CK, Friborg S, Reynolds T, Guggino WB, Moss RB, Carter BJ, Wine JJ, Flotte TR,
Gardner P: Safety and biological efficacy of an adeno-associated virus vector-cystic fibrosis trans-
membrane regulator (AAV-CFTR) in the cystic fibrosis maxillary sinus. Laryngoscope 1999;
109:266–274.
63 Mandel RJ, Rendahl KG, Spratt SK, Snyder RO, Cohen LK, Leff SE: Characterization of intras-
triatal recombinant adeno-associated virus-mediated gene transfer of human tyrosine hydroxylase and
human GTP-cyclohydrolase I in a rat model of Parkinson’s disease. J Neurosci 1998;18:4271–4284.
64 McCown TJ, Xiao X, Li J, Breese GR, Samulski RJ: Differential and persistent expression pat-
terns of CNS gene transfer by an adeno-associated virus (AAV) vector. Brain Res 1996;713:99–107.
65 Leff SE, Spratt SK, Snyder RO, Mandel RJ: Long-term restoration of striatal L-aromatic amino
acid decarboxylase activity using recombinant adeno-associated viral vector gene transfer in a
rodent model of Parkinson’s disease. Neuroscience 1999;92:185–196.
66 Kaplitt MG, Leone P, Samulski RJ, Xiao X, Pfaff DW, O’Malley KL, During MJ: Long-term gene
expression and phenotypic correction using adeno-associated virus vectors in the mammalian
brain. Nat Genet 1994;8:148–154.
67 Kirik D, Rosenblad C, Bjorklund A, Mandel RJ: Long-term rAAV-mediated gene transfer of GDNF
in the rat Parkinson’s model: Intrastriatal but not intranigral transduction promotes functional
regeneration in the lesioned nigrostriatal system. J Neurosci 2000;20:4686–4700.
68 Lo WD, Qu G, Sferra TJ, Clark R, Chen R, Johnson PR: Adeno-associated virus-mediated gene
transfer to the brain: Duration and modulation of expression. Hum Gene Ther 1999;10:201–213.
69 Shen Y, Muramatsu SI, Ikeguchi K, Fujimoto KI, Fan DS, Ogawa M, Mizukami H, Urabe M,
Kume A, Nagatsu I, Urano F, Suzuki T, Ichinose H, Nagatsu T, Monahan J, Nakano I, Ozawa K:
Triple transduction with adeno-associated virus vectors expressing tyrosine hydroxylase,
aromatic-L-amino-acid decarboxylase, and GTP cyclohydrolase I for gene therapy of Parkinson’s
disease. Hum Gene Ther 2000;11:1509–1519.
70 Sanchez-Pernaute R, Harvey-White J, Cunningham J, Bankiewicz KS: Functional effect of adeno-
associated virus mediated gene transfer of aromatic L-amino acid decarboxylase into the striatum
of 6-OHDA-lesioned rats. Mol Ther 2001;4:324–330.
71 Bankiewicz KS, Eberling JL, Kohutnicka M, Jagust W, Pivirotto P, Bringas J, Cunningham J,
Budinger TF, Harvey-White J: Convection-enhanced delivery of AAV vector in parkinsonian
monkeys; in vivo detection of gene expression and restoration of dopaminergic function using
pro-drug approach. Exp Neurol 2000;164:2–14.
72 Mandel RJ, Spratt SK, Snyder RO, Leff SE: Midbrain injection of recombinant adeno-associated
virus encoding rat glial cell line-derived neurotrophic factor protects nigral neurons in a progressive
6-hydroxydopamine-induced degeneration model of Parkinson’s disease in rats. Proc Natl Acad
Sci USA 1997;94:14083–14088.
73 Mandel RJ, Snyder RO, Leff SE: Recombinant adeno-associated viral vector-mediated glial cell
line-derived neurotrophic factor gene transfer protects nigral dopamine neurons after onset of
progressive degeneration in a rat model of Parkinson’s disease. Exp Neurol 1999;160:205–214.
74 Bankiewicz KS, Leff SE, Nagy D, Jungles S, Rokovich J, Spratt K, Cohen L, Libonati M, Snyder RO,
Mandel RJ: Practical aspects of the development of ex vivo and in vivo gene therapy for Parkinson’s
disease. Exp Neurol 1997;144:147–156.
75 Fan DS, Ogawa M, Fujimoto KI, Ikeguchi K, Ogasawara Y, Urabe M, Nishizawa M, Nakano I,
Yoshida M, Nagatsu I, Ichinose H, Nagatsu T, Kurtzman GJ, Ozawa K: Behavioral recovery in
6-hydroxydopamine-lesioned rats by cotransduction of striatum with tyrosine hydroxylase and
aromatic L-amino acid decarboxylase genes using two separate adeno-associated virus vectors.
Hum Gene Ther 1998;9:2527–2535.
76 Muramatsu S, Fujimoto K, Ikeguchi K, Shizuma N, Kawasaki K, Ono F, Shen Y, Wang L,
Mizukami H, Kume A, Matsumura M, Nagatsu I, Urano F, Ichinose H, Nagatsu T, Terao K,
Nakano I, Ozawa K: Behavioral recovery in a primate model of Parkinson’s disease by triple trans-
duction of striatal cells with adeno-associated viral vectors expressing dopamine-synthesizing
enzymes. Hum Gene Ther 2002;13:345–354.
77 Luo J, Kaplitt MG, Fitzsimons HL, Zuzga DS, Liu Y, Oshinsky ML, During MJ: Subthalamic
GAD gene therapy in a Parkinson’s disease rat model. Science 2002;298:425–429.
medwedi.ru
78 Costantini LC, Jacoby DR, Wang S, Fraefel C, Breakefield XO, Isacson O: Gene transfer to the
nigrostriatal system by hybrid herpes simplex virus/adeno-associated virus amplicon vectors.
Hum Gene Ther 1999;10:2481–2494.
79 Danko I, Williams P, Herweijer H, Zhang G, Latendresse JS, Bock I, Wolff JA: High expression
of naked plasmid DNA in muscles of young rodents. Hum Mol Genet 1997;6:1435–1443.
80 Budker V, Budker T, Zhang G, Subbotin V, Loomis A, Wolff JA: Hypothesis: Naked plasmid DNA
is taken up by cells in vivo by a receptor-mediated process. J Gene Med 2000;2:76–88.
81 Coll JL, Negoescu A, Louis N, Sachs L, Tenaud C, Girardot V, Demeinex B, Brambilla E,
Brambilla C, Favrot M: Antitumor activity of bax and p53 naked gene transfer in lung cancer:
In vitro and in vivo analysis. Hum Gene Ther 1998;9:2063–2074.
82 White SA, LoBuglio AF, Arani RB, Pike MJ, Moore SE, Barlow DL, Conry RM: Induction of
anti-tumor immunity by intrasplenic administration of a carcinoembryonic antigen DNA vaccine.
J Gene Med 2000;2:135–140.
83 Papahadjopoulos D, Allen TM, Gabizon A, Mayhew E, Matthay K, Huang SK, Lee KD, Woodle MC,
Lasic DD, Redemann C, Martin FJ: Sterically stabilized liposomes: Improvements in pharmaco-
kinetics and antitumor therapeutic efficacy. Proc Natl Acad Sci USA 1991;88:11460–11464.
84 Zhang Y, Calon F, Zhu C, Boado RJ, Pardridge WM: Intravenous nonviral gene therapy causes
normalization of striatal tyrosine hydroxylase and reversal of motor impairment in experimental
parkinsonism. Hum Gene Ther 2003;14:1–12.
85 Segovia J, Vergara P, Brenner M: Astrocyte-specific expression of tyrosine hydroxylase after
intracerebral gene transfer induces behavioral recovery in experimental parkinsonism. Gene Ther
1998;5:1650–1655.
86 Boussif O, Lezoualc’h F, Zanta MA, Mergny MD, Scherman D, Demeneix B, Behr JP: A versa-
tile vector for gene and oligonucleotide transfer into cells in culture and in vivo: Polyethylenimine.
87 Abdallah B, Hassan A, Benoist C, Goula D, Behr JP, Demeneix BA: A powerful nonviral vector
for in vivo gene transfer into the adult mammalian brain: Polyethylenimine. Hum Gene Ther
1996;7:1947–1954.
88 Wang S, Ma N, Gao SJ, Yu H, Leong KW: Transgene expression in the brain stem effected by
intramuscular injection of polyethylenimine/DNA complexes. Mol Ther 2001;3:658–664.
89 Freed CR, Greene PE, Breeze RE, Tsai WY, DuMouchel W, Kao R, Dillon S, Winfield H, Culver S,
Trojanowski JQ, Eidelberg D, Fahn S: Transplantation of embryonic dopamine neurons for severe
Parkinson’s disease. N Engl J Med 2001;344:710–719.
90 Koh JY, Gwag BJ, Lobner D, Choi DW: Potentiated necrosis of cultured cortical neurons by
neurotrophins. Science 1995;268:573–575.
91 Olanow CW, Goetz CG, Kordower JH, Stoessl AJ, Sossi V, Brin MF, Shannon KM, Nauert GM,
Perl DP, Godbold J, Freeman TB: A double-blind controlled trial of bilateral fetal nigral trans-
plantation in Parkinson’s disease. Ann Neurol 2003;54:403–414.
92 Andersen JK, Frim DM, Isacson O, Breakefield XO: Herpesvirus-mediated gene delivery into the
rat brain: Specificity and efficiency of the neuron-specific enolase promoter. Cell Mol Neurobiol
1993;13:503–515.
93 Kaplitt MG, Kwong AD, Kleopoulos SP, Mobbs CV, Rabkin SD, Pfaff DW: Preproenkephalin pro-
moter yields region-specific and long-term expression in adult brain after direct in vivo gene transfer
via a defective herpes simplex viral vector. Proc Natl Acad Sci USA 1994;91:8979–8983.
94 Hashimoto M, Aruga J, Hosoya Y, Kanegae Y, Saito I, Mikoshiba K: A neural cell-type-specific
expression system using recombinant adenovirus vectors. Hum Gene Ther 1996;7:149–158.
95 Peel AL, Zolotukhin S, Schrimsher GW, Muzyczka N, Reier PJ: Efficient transduction of green
fluorescent protein in spinal cord neurons using adeno-associated virus vectors containing cell
type-specific promoters. Gene Ther 1997;4:16–24.
96 Xu R, Janson CG, Mastakov M, Lawlor P, Young D, Mouravlev A, Fitzsimons H, Choi KL, Ma H,
Dragunow M, Leone P, Chen Q, Dicker B, During MJ: Quantitative comparison of expression with
adeno-associated virus (AAV-2) brain-specific gene cassettes. Gene Ther 2001;8:1323–1332.
97 Cortez N, Trejo F, Vergara P, Segovia J: Primary astrocytes retrovirally transduced with a tyrosine
hydroxylase transgene driven by a glial-specific promoter elicit behavioral recovery in experi-
mental parkinsonism. J Neurosci Res 2000;59:39–46.
98 Gossen M, Bujard H: Tight control of gene expression in mammalian cells by tetracycline-responsive
promoters. Proc Natl Acad Sci USA 1992;89:5547–5551.
99 Hinrichs W, Kisker C, Duvel M, Muller A, Tovar K, Hillen W, Saenger W: Structure of the Tet
repressor-tetracycline complex and regulation of antibiotic resistance. Science 1994;264:418–420.
100 Chyung YH, Peng PD, Kay MA: System for simultaneous tissue-specific and disease-specific
regulation of therapeutic gene expression. Hum Gene Ther 2003;14:1255–1264.
101 Aiken C: Pseudotyping human immunodeficiency virus type 1 (HIV-1) by the glycoprotein of
vesicular stomatitis virus targets HIV-1 entry to an endocytic pathway and suppresses both the
requirement for Nef and the sensitivity to cyclosporin A. J Virol 1997;71:5871–5877.
102 Bartz SR, Vodicka MA: Production of high-titer human immunodeficiency virus type 1 pseudotyped
with vesicular stomatitis virus glycoprotein. Methods 1997;12:337–342.
103 Arai T, Takada M, Ui M, Iba H: Dose-dependent transduction of vesicular stomatitis virus G protein-
pseudotyped retrovirus vector into human solid tumor cell lines and murine fibroblasts. Virology
1999;260:109–115.
104 Anderson DB, Laquerre S, Ghosh K, Ghosh HP, Goins WF, Cohen JB, Glorioso JC: Pseudotyping
of glycoprotein D-deficient herpes simplex virus type 1 with vesicular stomatitis virus glycopro-
tein G enables mutant virus attachment and entry. J Virol 2000;74:2481–2487.
105 Kang UJ, Fisher LJ, Joh TH, O’Malley KL, Gage FH: Regulation of dopamine production by
genetically modified primary fibroblasts. J Neurosci 1993;13:5203–5211.
106 Lee WY, Chang JW, Nemeth NL, Kang UJ: Vesicular monoamine transporter-2 and aromatic
L-amino acid decarboxylase enhance dopamine delivery after L-3, 4-dihydroxyphenylalanine
administration in Parkinsonian rats. J Neurosci 1999;19:3266–3274.
107 Shimohama S, Rosenberg MB, Fagan AM, Wolff JA, Short MP, Breakefield XO, Friedmann T,
Gage FH: Grafting genetically modified cells into the rat brain: Characteristics of E. coli beta-
galactosidase as a reporter gene. Brain Res Mol Brain Res 1989;5:271–278.
108 Wolff JA, Fisher LJ, Xu L, Jinnah HA, Langlais PJ, Iuvone PM, O’Malley KL, Rosenberg MB,
Shimohama S, Friedmann T, Gage FH: Grafting fibroblasts genetically modified to produce
L-dopa in a rat model of Parkinson disease. Proc Natl Acad Sci USA 1989;86:9011–9014.
109 Tornatore C, Baker-Cairns B, Yadid G, Hamilton R, Meyers K, Atwood W, Cummins A, Tanner V,
Major E: Expression of tyrosine hydroxylase in an immortalized human fetal astrocyte cell
line; In vitro characterization and engraftment into the rodent striatum. Cell Transplant 1996;5:
145–163.
110 Keir SD, Miller J, Yu G, Hamilton R, Samulski RJ, Xiao X, Tornatore C: Efficient gene transfer
into primary and immortalized human fetal glial cells using adeno-associated virus vectors:
Establishment of a glial cell line with a functional CD4 receptor. J Neurovirol 1997;3:322–330.
111 Horellou P, Brundin P, Kalen P, Mallet J, Bjorklund A: In vivo release of dopa and dopamine from
genetically engineered cells grafted to the denervated rat striatum. Neuron 1990;5:393–402.
112 Sawamoto K, Nakao N, Kakishita K, Ogawa Y, Toyama Y, Yamamoto A, Yamaguchi M, Mori K,
Goldman SA, Itakura T, Okano H: Generation of dopaminergic neurons in the adult brain from mes-
encephalic precursor cells labeled with a nestin-GFP transgene. J Neurosci 2001;21: 3895–3903.
113 Sawamoto K, Nakao N, Kobayashi K, Matsushita N, Takahashi H, Kakishita K, Yamamoto A,
Yoshizaki T, Terashima T, Murakami F, Itakura T, Okano H: Visualization, direct isolation, and
transplantation of midbrain dopaminergic neurons. Proc Natl Acad Sci USA 2001;98:6423–6428.
114 Wagner J, Akerud P, Castro DS, Holm PC, Canals JM, Snyder EY, Perlmann T, Arenas E: Induction
of a midbrain dopaminergic phenotype in Nurr1-overexpressing neural stem cells by type 1 astro-
cytes. Nat Biotechnol 1999;17:653–659.
115 Snyder EY, Deitcher DL, Walsh C, Arnold-Aldea S, Hartwieg EA, Cepko CL: Multipotent neural
cell lines can engraft and participate in development of mouse cerebellum. Cell 1992;68:33–51.
116 Kim JH, Auerbach JM, Rodriguez-Gomez JA, Velasco I, Gavin D, Lumelsky N, Lee SH, Nguyen J,
Sanchez-Pernaute R, Bankiewicz K, McKay R: Dopamine neurons derived from embryonic stem
cells function in an animal model of Parkinson’s disease. Nature 2002;418:50–56.
117 Bjorklund A, Stenevi U: Reconstruction of the nigrostriatal dopamine pathway by intracerebral
118 Perlow MJ, Freed WJ, Hoffer BJ, Seiger A, Olson L, Wyatt RJ: Brain grafts reduce motor abnor-
malities produced by destruction of nigrostriatal dopamine system. Science 1979;204:643–647.
medwedi.ru
119 Redmond DE, Sladek JR Jr, Roth RH, Collier TJ, Elsworth JD, Deutch AY, Haber S: Fetal neuronal
grafts in monkeys given methylphenyltetrahydropyridine. Lancet 1986;1:1125–1127.
120 Bankiewicz KS, Plunkett RJ, Jacobowitz DM, Porrino L, di Porzio U, London WT, Kopin IJ,
Oldfield EH: The effect of fetal mesencephalon implants on primate MPTP-induced parkinson-
ism. Histochemical and behavioral studies. J Neurosurg 1990;72:231–244.
121 Freeman TB, Olanow CW, Hauser RA, Nauert GM, Smith DA, Borlongan CV, Sanberg PR, Holt DA,
Kordower JH, Vingerhoets FJ: Bilateral fetal nigral transplantation into the postcommissural
putamen in Parkinson’s disease. Ann Neurol 1995;38:379–388.
122 Hauser RA, Freeman TB, Snow BJ, Nauert M, Gauger L, Kordower JH, Olanow CW: Long-term
evaluation of bilateral fetal nigral transplantation in Parkinson disease. Arch Neurol 1999;56:
179–187.
123 Kordower JH, Freeman TB, Snow BJ, Vingerhoets FJ, Mufson EJ, Sanberg PR, Hauser RA,
Smith DA, Nauert GM, Perl DP, Olanow CW: Neuropathological evidence of graft survival and
striatal reinnervation after the transplantation of fetal mesencephalic tissue in a patient with
Parkinson’s disease. N Engl J Med 1995;332:1118–1124.
124 Kordower JH, Freeman TB, Chen EY, Mufson EJ, Sanberg PR, Hauser RA, Snow B, Olanow CW:
Fetal nigral grafts survive and mediate clinical benefit in a patient with Parkinson’s disease. Mov
Disord 1998;13:383–393.
125 Piccini P, Brooks DJ, Bjorklund A, Gunn RN, Grasby PM, Rimoldi O, Brundin P, Hagell P,
Rehncrona S, Widner H, Lindvall O: Dopamine release from nigral transplants visualized in vivo
in a Parkinson’s patient. Nat Neurosci 1999;2:1137–1140.
126 Brundin P, Dunnett S, Bjorklund A, Nikkhah G: Transplanted dopaminergic neurons: More or
less? Nat Med 2001;7:512–513.
127 Nikkhah G: Neural transplantation therapy for Parkinson’s disease: Potential and pitfalls. Brain
Res Bull 2001;56:509.
128 Hagell P, Piccini P, Bjorklund A, Brundin P, Rehncrona S, Widner H, Crabb L, Pavese N, Oertel WH,
Quinn N, Brooks DJ, Lindvall O: Dyskinesias following neural transplantation in Parkinson’s dis-
ease. Nat Neurosci 2002;5:627–628.
129 Ma Y, Feigin A, Dhawan V, Fukuda M, Shi Q, Greene P, Breeze R, Fahn S, Freed C, Eidelberg D:
Dyskinesia after fetal cell transplantation for parkinsonism: A PET study. Ann Neurol 2002;52:
628–634.
130 Carpenter MK, Cui X, Hu ZY, Jackson J, Sherman S, Seiger A, Wahlberg LU: In vitro expansion
of a multipotent population of human neural progenitor cells. Exp Neurol 1999;158:265–278.
131 Daadi MM, Weiss S: Generation of tyrosine hydroxylase-producing neurons from precursors of
the embryonic and adult forebrain. J Neurosci 1999;19:4484–4497.
132 Ling ZD, Potter ED, Lipton JW, Carvey PM: Differentiation of mesencephalic progenitor cells into
dopaminergic neurons by cytokines. Exp Neurol 1998;149:411–423.
133 Studer L, Tabar V, McKay RD: Transplantation of expanded mesencephalic precursors leads to
recovery in parkinsonian rats. Nat Neurosci 1998;1:290–295.
134 Yan H, Bunge MB, Wood PM, Plant GW: Mitogenic response of adult rat olfactory ensheathing
glia to four growth factors. Glia 2001;33:334–342.
135 Sanchez-Pernaute R, Studer L, Bankiewicz KS, Major EO, McKay RD: In vitro generation
and transplantation of precursor-derived human dopamine neurons. J Neurosci Res 2001;65:
284–288.
136 Arenas E: Stem cells in the treatment of Parkinson’s disease. Brain Res Bull 2002;57:795–808.
137 Svendsen CN, Caldwell MA, Shen J, ter Borg MG, Rosser AE, Tyers P, Karmiol S, Dunnett SB:
Long-term survival of human central nervous system progenitor cells transplanted into a rat model
of Parkinson’s disease. Exp Neurol 1997;148:135–146.
138 Carvey PM, Ling ZD, Sortwell CE, Pitzer MR, McGuire SO, Storch A, Collier TJ: A clonal line
of mesencephalic progenitor cells converted to dopamine neurons by hematopoietic cytokines:
A source of cells for transplantation in Parkinson’s disease. Exp Neurol 2001;171:98–108.
139 Yang M, Stull ND, Berk MA, Snyder EY, Iacovitti L: Neural stem cells spontaneously express
dopaminergic traits after transplantation into the intact or 6-hydroxydopamine-lesioned rat. Exp
Neurol 2002;177:50–60.
140 Kawasaki H, Mizuseki K, Nishikawa S, Kaneko S, Kuwana Y, Nakanishi S, Nishikawa SI, Sasai Y:
Induction of midbrain dopaminergic neurons from ES cells by stromal cell-derived inducing activity.
Neuron 2000;28:31–40.
141 Lee SH, Lumelsky N, Studer L, Auerbach JM, McKay RD: Efficient generation of midbrain and
hindbrain neurons from mouse embryonic stem cells. Nat Biotechnol 2000;18:675–679.
142 Bjorklund LM, Sanchez-Pernaute R, Chung S, Andersson T, Chen IY, McNaught KS, Brownell AL,
Jenkins BG, Wahlestedt C, Kim KS, Isacson O: Embryonic stem cells develop into functional
dopaminergic neurons after transplantation in a Parkinson rat model. Proc Natl Acad Sci USA
2002;99:2344–2349.
143 Sanchez-Pernaute R, Brownell AL, Isacson O: Functional imaging of the dopamine system:
In vivo evaluation of dopamine deficiency and restoration. Neurotoxicology 2002;23:469–478.
144 Eberling JL, Bankiewicz KS, Jordan S, VanBrocklin HF, Jagust WJ: PET studies of functional
compensation in a primate model of Parkinson’s disease. Neuroreport 1997;8:2727–2733.
145 Meyerson BA, Linderoth B, Karlsson H, Ungerstedt U: Microdialysis in the human brain: Extra-
cellular measurements in the thalamus of parkinsonian patients. Life Sci 1990;46:301–308.
146 Fedele E, Mazzone P, Stefani A, Bassi A, Ansaldo MA, Raiteri M, Altibrandi MG, Pierantozzi M,
Giacomini P, Bernardi G, Stanzione P: Microdialysis in Parkinsonian patient basal ganglia: Acute
apomorphine-induced clinical and electrophysiological effects not paralleled by changes in the
release of neuroactive amino acids. Exp Neurol 2001;167:356–365.
147 Bankiewicz KS, Daadi MM, Pivirotto P, Bringas J, Sanchez-Pernaute R, Herscovitch P, Carson R,
Eckelman W, Cunningham J, Reutter B, VanBrocklin HF, Eberling JL. Long term evaluation of
AAV/AADC gene transfer in parkinsonian monkeys. 33rd Annual Meeting of Society for Neuro-
science, 2003, Washington DC. Neuroscience 2003, p. Program No. 299.214.
Dr. Krystof Bankiewicz

Department of Neurosurgery, University of California San Francisco
MCB, 1855 Folsom Street, Room 225, San Francisco, CA 94103 (USA)
Tel. ⫹1 415 502 3132, Fax ⫹1 415 514 2777, E-Mail kbank@itsa.ucsf.edu
medwedi.ru
Simplifying Complex
Neurodegenerative Diseases
by Gene Chip Analysis
Clemens R. Scherzera, Steven R. Gullansa, Roderick V. Jensenb
a
Laboratory for Functional Genomics, Center for Neurologic Diseases,
Harvard Medical School, Brigham and Women’s Hospital, Cambridge, Mass., and
b
Department of Physics, Wesleyan University, Middletown, Conn., USA
Genes for many autosomal dominant or recessive neurodegenerative dis-

eases have been already identified. However, little is known about the complex
genetics behind the vast majority of sporadic or ‘idiopathic’ neurodegenerative
diseases. These diseases are likely to be caused by the combinatorial effect of
several susceptibility genes acting in concert with environmental risk factors.
Identifying the relevant genes, elucidating their molecular function, and defin-
ing targets for neuroprotective drugs pose great challenges and will require
novel scientific methodologies. These genetic strategies will help to bring the
benefits of the recent genomic revolution to the clinic and the operating room,
by developing treatment strategies for neurodegenerative diseases.
Traditional scientific approaches have always focused on serial studies of
one gene at a time. For complex diseases that are caused by a multiplicity of
susceptibility genes, high-throughput analysis of many genes in parallel is a
more efficient and informative approach, though cost considerations have been
a major problem in the past. Gene chips or ‘microarrays’ attach probes for tran-
scripts of tens of thousands of genes onto a rigid support such as a glass slide
and permit a comprehensive genome-wide analysis of transcript changes.
This chapter will discuss how gene chip technology can be applied to the
investigation of neurodegenerative diseases. We will address how gene chips can
identify candidate disease-modifying genes and prioritize susceptibility genes
for genotyping in complex neurodegenerative diseases. We envision that in the
future, gene chip analysis will efficiently detect the molecular fingerprints asso-
ciated with distinct clinical states and will define unique gene activity profiles
or ‘mRNA barcodes’ for specific clinical traits. In clinical practice, these tools
may assist in the diagnosis and prognosis of neurodegenerative diseases, more
accurately predict individual treatment responses, and be used as markers of dis-
ease risk in presymptomatic subjects.
Current Best Practices of Microarray Technique:

Refining Modifier Candidates
Primary Screen
In our experience, many investigators would like to use a combination of
microarrays, bioinformatics, and simple validation experiments to define a
short list of one to ten high-priority candidate genes. A stepwise filtering
process is generally applied to the initial microarray datasets. We typically start
with error models tailored to the specific microarray platforms, to optimize
quantification of the gene expression levels. We also recommend a stringent
three-step statistical analysis to minimize false positives due to biological or
technical variation and to correct for multiple testing.
First, a selective intensity filter is applied to exclude genes with low
hybridization signal intensities, because false-positive results are particularly
high for low-intensity genes. With Affymetrix gene arrays, we generally require
that the gene ‘Average Difference’ or ‘Signal’ be greater that the ‘Target Intensity’
(defined as the trimmed-mean expression level on the array) for at least one
sample in the study. This will focus further analysis on the 30–40% most abun-
dant transcripts. Second, a ratio threshold (generally fold changes of >1.5–2.0)
is applied to eliminate small changes in expression that are of unclear technical
and biological significance. Although smaller fold-changes may be statistically
significant they are very difficult to verify by other means (e.g., quantitative
polymerase chain reaction with reverse transcription; RT–PCR). Finally, a
t-like test statistic is used to identify genes that are expressed differentially on
the basis of confidence values or P values [1]. Permutation tests (e.g.,
Significance Analysis of Microarrays [2]) are performed to estimate the signif-
icance of the test statistic and to correct for multiple testing. The number of
false positives expected by chance alone is determined by repeatedly permuting
the samples’ class labels and computing t statistics for all genes in the scram-
bled data.
Secondary Screen
To qualify each gene further after the primary microarray assessment, a
secondary screen may be required to independently confirm the observed
changes in gene expression. If the primary screen results in a relatively short
Simplifying Complex Neurodegenerative Diseases 247
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list (less than fifty genes), quantitative RT–PCR can be performed (on samples)
for technical validation. Investigators may further prioritize genes as candidate
targets on the basis of their organismal roles; for example, hormones may be
favored as potential therapeutic proteins, or receptors or enzymes that are
amenable to modulation by small-molecule drugs may be chosen (for further
study). For genes with unknown or unclear functions, prioritizing those of great-
est physiological relevance requires further analysis such as quantitative
RT–PCR or protein expression analysis. Western blot or immunohistochemistry
are preferred for protein analysis, but an antibody is not always readily avail-
able. The secondary screening process may obtain a more detailed dissection of
the biological process using time series, more diverse biological samples, and
anatomical specificity.
Shotgun Microarrays
Secondary screens become labor intensive, time consuming, and expensive
if a large list of genes need be confirmed. Therefore, we have begun to use mul-
tiple microarray platforms for efficient technical validation of large numbers of
differentially expressed genes. Different high-density oligonucleotide plat-
forms (e.g., Affymetrix, Amersham, Agilent) spot distinct probes for the genes
interrogated and have distinct technical advantages and weaknesses. Our results
suggest that for the more highly expressed transcripts, 70–80% of the >2-fold
gene expression changes are concordant when the same RNA sample is run on
Affymetrix and Amersham arrays. In our opinion, the current optimal sec-
ondary screen takes advantage of two independent high-density oligonucleotide
platforms in a cross-validation strategy that we term ‘shotgun’ or sequential
microarray analysis.
Error Minimization
When using microarrays to identify differentially expressed genes, it is
important to recognize the inherent error caused by technical and biological
variations. Reproducibility and sensitivity problems can generate both false-
negative and false-positive results. But these issues can be addressed readily
through robust experimental design, rigorous statistical analysis, the use of bio-
logical and technical replicates, and independent verification by quantitative
RT–PCR or other microarray platforms. Although microarrays represent a pow-
erful tool for forming initial hypotheses, it is essential to consider the limita-
tions of interpreting biological responses through measurements of mRNA
abundance alone. Measurements of mRNA do not directly reflect protein quan-
tities, enzyme activities, or extranuclear signal transduction. Microarray exper-
iments also may fail to resolve true ‘modifier genes’ from homeostatic responses
that attempt to restore the original state of the system. Generally, microarray
Scherzer/Gullans/Jensen 248
measurements fail to resolve cause from effect. Thus, successful use of microar-
ray technology requires that sources of error be controlled carefully in the
design and execution of experiments.
Biological Validation
The primary microarray screen will identify a shortlist of high-priority
modifier candidates. Each type of selective profiling identifies differentially
expressed genes characteristic for a particular RNA source. The choice of
(tissue) source and controls will modify the biases flowing into the results of
the screen. Invariably, validation experiments will be indicated to distinguish
microarray-derived candidates that are strong modifiers of the disease process
and to overcome the limitations of each RNA source.
Several approaches can be taken to validate and to prioritize candidate
modifiers once a shortlist has been identified. Among the most important are
gene knockout and knock-in strategies in cells and model organisms, because
these can replicate more closely the actions of potential modifiers and identify
phenotypic changes and mechanisms. For a high-throughput genetic validation
of microarray candidates, simple model organisms such as yeast, flies, and
worms are most frequently used.
An elegant application of this strategy resulted in the discovery of a new
modifier candidate for multiple sclerosis (MS). Microarray analysis of MS
lesions yielded new modifiers of MS that were validated in autoimmune
encephalomyelitis [3]. In a landmark study [3], Lawrence Steinman’s group at
Stanford defined microarray-derived modifiers of human MS. By combining
expression analysis and high-throughput sequencing of expressed sequence
tags in a rat model of MS and human MS plaque tissue, they found an increase
in osteopontin mRNA abundance in both human and rat tissues. The biological
role of osteopontin in the progression of MS was then further validated in
knockout mice: osteopontin-deficient mice were resistant to the progressive
MS subtype and had significantly more remissions compared to wild-type
mice. Using microarrays as a screening tool, osteopontin is now a promising
novel drug target for blocking progressive MS in humans.
Prioritizing Candidate Suppressors or Enhancers of

Neurodegeneration through Gene Chip Analysis
Selective Vulnerability Profiles

When using microarrays to discover modifier genes in neurodegenerative
diseases, genome-wide mRNA expression profile is determined in postmortem
brain tissue from patients. The investigator applies a series of noise filters and
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significance statistics to identify candidate modifier genes that are differen-
tially expressed in patient tissue out of the tens of thousands of genes interro-
gated by human genome arrays.
Neurodegenerative processes are highly selective for specific neuronal
populations and brain regions and are often associated with characteristic
histological lesions. Each neurodegenerative disease preferentially affects
distinct neuronal populations and distinct brain regions and is associated with
hallmark histopathological lesions. This vulnerable neuronal population is often
distributed in distinct brain regions. For example, in Parkinson’s disease (PD)
dopaminergic neurons localized to the substantia nigra pars compacta are
predominantly affected, while dopaminergic cells in other brain regions are less
vulnerable. Regional and cellular profiling techniques have been developed that
are tailored to investigate the selective regional and cellular vulnerability of
neurodegenerative diseases.
Expression analysis of vulnerable brain regions (regional profiling), vul-
nerable neuronal or glial populations (cellular profiling), or characteristic his-
tological lesions such as MS plaques [3] (lesion profiling) has lead to intriguing
results reflecting the strengths and weaknesses of each approach.
Regional Profiling
Nonspecific gene expression changes related to neuronal loss or reactive
glial proliferation must be considered in the interpretation of gene expression
in affected brain regions. Hauser et al. [in preparation] have used disease
controls with dopaminergic cell loss such as progressive supranuclear paralysis
to control cell loss not specific to PD pathogenesis. Alternatively, expression
changes of neuronal markers such as neurofilaments or of neuronal specific
subpopulations such as tyrosine hydroxylase and other dopamine biosynthesis
enzymes, and glial markers such as glial fibrillary acidic protein, may be used
to estimate the range of gene expression changes accounted for by unspecific
cell loss and gliosis alone. Validation of regional expression changes in vulner-
able neuronal populations by double-labeling immunohistochemistry or double-
labeling in situ hybridization can address this concern. Analysis of gene
expression in patients ‘at risk’ or at presymptomatic disease stages could reduce
some of these biases but tissue availability and diagnostic uncertainty limit this
approach.
Cellular Profiling
Laser-capture microdissection (LCM) of vulnerable neuronal populations
allows direct sampling of the neuronal population of interest under the micro-
scope [4–6]. LCM controls for some biases associated with regional profiling
such as reactive gliosis or nonspecific neuron loss. Distinct considerations
guide the interpretation of LCM expression profiles. During interpretation of
results, one must take into account whether gene expression changes observed
are specific to the disease in question or whether they may be generally found
in dying neurons irrespective of the specific disease process. Comparison with
cellular profiles in disease controls could help to estimate this bias. In addi-
tion, a selection bias might be introduced by LCM; cellular profiling might
select for neurons less affected in the disease process. This is particularly
a concern if advanced disease stages are profiled. For example, in PD, an
estimated 70% of nigral neurons have died prior to the onset of clinical symp-
toms [7]. Dopaminergic neurons that survive the disease process and thus
are found in postmortem tissue might reflect a particularly resistant sub-
population rather than reflecting the transcription profile of vulnerable
dopaminergic cells. The cellular gene expression profile thus might identify
transcripts of genes conferring enhanced resistance within the vulnerable cell
population.
Extraneuronal Profiling
A novel approach to avoid some of these limitations has made use of
altered gene expression in peripheral tissues of patients with neurodegenerative
diseases. In this paradigm, neurodegenerative diseases are approached as a
systemic disease with systemic changes in the expression of disease-modifying
and susceptibility genes that act in a combinatorial fashion with localizing
factors unique to vulnerable neuronal populations and lead to selective
neurodegeneration. Biochemical and transcriptional alterations in peripheral
tissues such as platelets [8], lymphocytes [9, 10], fibroblasts [11] and muscle of
neurodegenerative patients have been extensively documented in Alzheimer’s
disease (AD), PD, and other neurodegenerative diseases. Indeed, most genes
implicated in familial AD [8, 11–13] and familial PD [14, 15] are ubiquitously
expressed.
To gain insight into the molecular basis of these alterations, we [23]
screened differential gene expression in lymphoblasts of controls and two
independent groups of AD patients using cDNA microarrays. This genomic
screen identified six differentially expressed genes. One of the six genes (LR11)
is a novel neuronal ApoE receptor and thus an excellent candidate modifier.
Subsequent validation experiments in the brain indicated that LR11 was
enriched in vulnerable cortical and hippocampal pyramidal neurons in human
control brains, and that it was concentrated in neuronal endosomal-lysosomal
compartments. In striking contrast to normal tissue, LR11 was diminished in
AD brains with dramatic reductions in surviving neurons. In cultured cells,
LR11 overexpression markedly reduced extracellular A␤ levels, providing a
mechanistic link between LR11 and A␤ clearance [Levey, unpubl. observations].
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Thus, changes in LR11 expression in AD lymphoblasts and brain, and its
effects on extracellular A␤, suggested an important role for this apoE receptor
in AD pathogenesis.
Detection of Neuroprotective Targets:

Gene Chip Analysis of Early Disease Stages
Candidate Modifier Screen in Animal Models
Toxic and genetic animal models of neurodegenerative diseases faithfully
replicate key features of human neurodegenerative diseases. Microarray analysis
of tissue from animal models, which is generally more available than human tis-
sue samples, allows for dissection of the molecular machinery involved in pro-
gressive neurodegeneration. In extension of the ‘static’ gene expression snapshot
detectable in human postmortem tissue representative of the disease endpoint,
transgenic animal models allow for detection of the ‘dynamic’ range of gene
expression changes during the disease progression, at any selected timepoints
when the animals are sacrificed. This approach is particularly valuable in the
analysis of chronic progressive neurodegenerative diseases. Pathology may
begin several years prior to the onset of clinical symptoms and progresses from
early disease stages associated with low morbidity and good response to
medications to clinically debilitating end stages associated with the depletion
of select neuronal populations. Specimens from animal models can capture
these changes over the entire course of a disease, in statistically meaningful
numbers.
For example, in PD, tremor and bradykinesia develop only after an esti-
mated 70% of vulnerable dopaminergic neurons in the substantia nigra have
already died during the presymptomatic stage, spanning a period of years [7].
It is a fundamental goal for the neurologist to develop medications that stop or
slow disease progression at presymptomatic or early disease stages. Modeling
changes in presymptomatic or early symptomatic stages is especially crucial for
understanding molecular pathogenesis and, perhaps even more importantly, for
identifying therapeutic targets that might help to slow the disease process
before it reaches the threshold for clinical symptoms.
In one model of PD, Drosophila expressing human ␣-synuclein (␣S) car-
rying the disease-linked A30P mutation in a panneural pattern faithfully repli-
cate age-dependent onset and chronic progression of human PD. Transgenic ␣S
Drosophila develop adult-onset, progressive degeneration of dopaminergic
cells, with widespread Lewy body inclusions and impaired locomotor function
as monitored by progressive loss of climbing ability [16]. Loss of dopaminer-
gic neurons and inclusion formation are first detected at 10 days of age, while
at day 1 post-eclosion, the A30P-␣S Drosophila are still histologically and
behaviorally normal.
To identify gene expression changes at presymptomatic, early and advanced
disease stages, our group hybridized RNA extracted from fly heads to high-
density oligonucleotide arrays spotted with probes representing the entire
Drosophila genome. In presymptomatic ␣S transgenics, microarray analysis
was more sensitive than conventional neuropathological techniques in elucidat-
ing disease-associated changes [17]. It was interesting that despite a ‘normal’
phenotype at this stage, in the one-day-old ␣S transgenics, transcription of thirty
six genes was significantly and reproducibly dysregulated. These abnormalities
presaged neuronal loss, Lewy body-like inclusion formation, and locomotor
impairment at later stages. We found that the ␣S signature genes are dysregu-
lated independent of disease stage in both presymptomatic and symptomatic
animals (fig. 1). This suggests that parts of the molecular machinery dysregu-
lated during symptomatic disease stages is already altered in presymptomatic
transgenics prior to the onset of neurodegeneration (fig. 1). Thus, temporal
profiling of progressive gene expression changes in neurodegenerative disease
models provides unbiased starting points for defining disease mechanisms and
for identifying potential targets for neuroprotective drugs at preclinical stages.
Discovery of Susceptibility Genes by Converging

Arraying and Mapping
Genes controlling a certain clinical trait may cause variation in the trait
through differential transcription due to DNA polymorphisms [18] that regulate
transcription. Microarray analysis can assist traditional linkage analysis by
identifying polymorphic transcription and in prioritizing candidate susceptibil-
ity genes. The correlation structure between transcript abundance and classical
genetic linkage has been used to identify susceptibility loci for complex
diseases such as diabetes [19] and asthma [20]. Most recently, convergence of
gene expression and linkage analysis implicated a novel gene, glutathione
S-transferase omega in the control of age-at-onset of AD [24].
A genetic linkage screen for age-at-onset in AD and PD has identified
several chromosomal regions that may harbor novel age-at-onset genes [21].
The most interesting finding was a ⬃15 cM linkage region on chromosome
10q. This linkage peak was large, spanning over fifteen megabases and several
hundred genes. Gene expression analysis probing for 22,000 human genes on
RNA from 6 AD patient hippocampus and matched normal controls was
performed to identify genes with polymorphic transcription in AD versus
control brain. Fifty-two genes were identified that demonstrated significant
differences in gene expression levels between AD and controls. Four of these
fifty-two genes were physically located in the chromosome 10q linkage region.
Genotyping fourteen single nucleotide polymorphisms in 1773 AD and
635 PD patients spanning these 4 candidates, and one functionally related
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a b
f
e
isoform, identified allelic association (p ⫽ 0.001) for age-at-onset with glu-
tathione S-transferase omega, one of the 4 microarray-defined candidates.
Thus, integration of the independent genetic linkage, gene expression, and
allelic association evidence implicated a novel gene as a significant biological
factor in the control of age-at-onset in AD.
The Practice of Genomic Neurosurgery: Diagnosis,

Subtype-Classification, and Treatment Personalization
through Microarray-Derived Biomarkers
The prospect of neuroprotective therapy has highlighted the crucial need
for disease-specific biomarkers that identify patients at early stages and allow
monitoring of disease progression. In addition, biomarkers for disease subtypes
are needed to efficiently design clinical trials for neuroprotective drugs.
In our opinion, transcription levels of susceptibility, disease-modifying, and
treatment-modifying genes will result in defined gene expression ‘barcodes’
based on haplotypes or single-nucleotide polymorphisms. These gene expres-
sion barcodes will serve to diagnose patients with neurodegenerative diseases,
to classify disease subtypes, and to predict treatment responses. Finally, using
bioinformatics techniques such as the ‘gene ratios’ [22], a small number of genes
will be extracted from the gene expression patterns that best define a clinical
state. This small subset of genes will then be assayed by simple and widely avail-
able standard laboratory techniques such as quantitative real-time PCR.
Fig. 1. Gene expression changes presage neurodegeneration in a Drosophila model of

PD (from [17]). a 51 signature genes tightly associated with A30P-␣-synuclein expression
independent of disease stage are clustered by hierarchical average-linkage analysis and
visualized in a colorgram. The branches of the dendrogram comprising the cluster of four
independent samples of presymptomatic 1-day-old transgenics are highlighted in pink.
Expression levels higher than the mean are displayed in red, lower than the mean in blue.
b–d While histology and behavior are normal in presymptomatic 1-day-old ␣S-transgenics,
microarray profiles reveal a PD-specific expression signature. Graphs show the average fold
change of select genes in different functional classes at day 1, 10, and 30 for ␣S transgenics
(left panels) and tau transgenics (right panels). In R406W-tau transgenics, expression of the
␣S-signature genes is generally unchanged (changes not significant by SAM). Time points
representing symptomatic stages of PD pathology are shaded gray. Signature expression of
down-regulated lipid genes (b, and green font in a), up-regulated membrane transporters
(c, orange font), and defense response genes (d, blue font) is detectable at the presympto-
matic stage. e Using the ␣S-associated signature genes as classifiers, blinded hierarchical
average-linkage analysis correctly distinguishes the eight ␣S samples from tau transgenics
and, as expected, from normal controls. f Progressive up-regulation of a set of energy genes
also begins in presymptomatics. This increase may be a compensatory response different
from the energy genes uniquely down-regulated at day 1 (fig. 1a). [Reproduced with per-
mission from Hum Mol Genet 2003;12:2457–2466, Oxford University Press.]
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Acknowledgements
We thank Dr. Mel Feany at Brigham and Women’s Hospital, Harvard Medical School,
for her helpful comments and critical review of the manuscript. The authors are supported by
grants from the Harvard Center for Neurodegeneration and Repair, the Paul B. Beeson
Career Development Award in Aging Research, the George C. Cotzias Memorial Fellowship
from the American Parkinson Disease Association (to C.R.S.), and the Michael J. Fox
Foundation (C.R.S, S.R.G., R.V.J).
References
1 Slonim DK: From patterns to pathways: Gene expression data analysis comes of age. Nat Genet
2002;32(suppl):502–508.
2 Tusher VG, Tibshirani R, Chu G: Significance analysis of microarrays applied to the ionizing
radiation response. Proc Natl Acad Sci USA 2001;98:5116–5121.
3 Chabas D, Baranzini SE, Mitchell D, Bernard CC, Rittling SR, Denhardt DT, Sobel RA, Lock C,
Karpuj M, Pedotti R, et al: The influence of the proinflammatory cytokine, osteopontin on autoim-
mune demyelinating disease. Science 2001;294:1731–1735.
4 Bahn S, Augood SJ, Ryan M, Standaert DG, Starkey M, Emson PC: Gene expression profiling in
the post-mortem human brain – No cause for dismay. J Chem Neuroanat 2001;22:79–94.
5 Wittliff JL, Erlander MG: Laser capture microdissection and its applications in genomics and pro-
teomics. Methods Enzymol 2002;356:12–25.
6 Kamme F, Salunga R, Yu J, Tran DT, Zhu J, Luo L, Bittner A, Guo HQ, Miller N, Wan J, Erlander
M: Single-cell microarray analysis in hippocampus CA1:Demonstration and validation of cellular
heterogeneity. J Neurosci 2003;23:3607–3615.
7 Fearnley JM, Lees AJ: Ageing and Parkinson’s disease: Substantia nigra regional selectivity. Brain
1991;114:2283–2301.
8 Di Luca M, Colciaghi F, Pastorino L, Borroni B, Padovani A, Cattabeni F: Platelets as a periph-
eral district where to study pathogenetic mechanisms of Alzheimer disease: The case of amyloid
precursor protein. Eur J Pharmacol 2000;405:277–283.
9 Ibarreta D, Urcelay E, Parrilla R, Ayuso MS: Distinct pH homeostatic features in lymphoblasts
from Alzheimer’s disease patients. Ann Neurol 1998;44:216–222.
10 Caronti B, Tanda G, Colosimo C, Ruggieri S, Calderaro C, Palladini G, Pontieri FE, Di Chiara G:
Reduced dopamine in peripheral blood lymphocytes in Parkinson’s disease. Neuroreport 1999;
10:2907–2910.
11 Citron M, Vigo-Pelfrey C, Teplow DB, Miller C, Schenk D, Johnston J, Winblad B, Venizelos N,
Lannfelt L, Selkoe DJ: Excessive production of amyloid beta-protein by peripheral cells of symp-
tomatic and presymptomatic patients carrying the Swedish familial Alzheimer disease mutation.
12 Li QX, Fuller SJ, Beyreuther K, Masters CL: The amyloid precursor protein of Alzheimer disease
in human brain and blood. J Leukoc Biol 1999;66:567–574.
13 Schlossmacher MG, Ostaszewski BL, Hecker LI, Celi A, Haass C, Chin D, Lieberburg I, Furie
BC, Furie B, Selkoe DJ: Detection of distinct isoform patterns of the beta-amyloid precursor
protein in human platelets and lymphocytes. Neurobiol Aging 1992;13:421–434.
14 Shin EC, Cho SE, Lee DK, Hur MW, Paik SR, Park JH, Kim J: Expression patterns of alpha-
synuclein in human hematopoietic cells and in Drosophila at different developmental stages. Mol
Cells 2000;10:65–70.
15 Sunada Y, Saito F, Matsumura K, Shimizu T: Differential expression of the parkin gene in the
human brain and peripheral leukocytes. Neurosci Lett 1998;254:180–182.
16 Feany MB, Bender WW: A Drosophila model of Parkinson’s disease. Nature 2000;404:
394–398.
17 Scherzer CR, Jensen RV, Gullans SR, Feany MB: Gene expression changes presage neurodegen-
eration in a Drosophila model of Parkinson’s disease. Hum Mol Genet 2003;12:2457–2466.
18 Schadt EE, Monks SA, Drake TA, Lusis AJ, Che N, Colinayo V, Ruff TG, Milligan SB, Lamb JR,
Cavet G, et al: Genetics of gene expression surveyed in maize, mouse and man. Nature 2003;
422:297–302.
19 Eaves IA, Wicker LS, Ghandour G, Lyons PA, Peterson LB, Todd JA, Glynne RJ: Combining
mouse once genic strains and microarray gene expression analyses to study a complex trait: The
NOD model of type 1 diabetes. Genome Res 2002;12:232–43.
20 Karp CL, Grupe A, Schadt E, Ewart SL, Keane-Moore M, Cuomo PJ, Kohl J, Wahl L, Kuperman D,
Germer S, et al: Identification of complement factor 5 as a susceptibility locus for experimental
allergic asthma. Nat Immunol 2000;1:221–226.
21 Li YJ, Scott WK, Hedges DJ, Zhang F, Gaskell PC, Nance MA, Watts RL, Hubble JP, Koller WC,
Pahwa R, et al: Age at onset in two common neurodegenerative diseases is genetically controlled.
Am J Hum Genet 2002;70:985–993.
22 Gordon GJ, Jensen RV, Hsiao LL, Gullans SR, Blumenstock JE, Ramaswamy S, Richards WG,
Sugarbaker DJ, Bueno R: Translation of microarray data into clinically relevant cancer diagnostic
tests using gene expression ratios in lung cancer and mesothelioma. Cancer Res 2002;62:
4963–4967.
23 Scherzer CR, Offe K, Gearing M, Rees HD, Fang G, Heilman CJ, Schaller C, Bujo H, Levey AI,
Lah JJ: Loss of apolipoprotein E receptor LR11 in Alzheimer disease. Arch Neurol 2004;61:
1200–1205.
24 Li YJ, Oliveira SA, Xu P, Martin ER, Stenger JE, Scherzer CR, Hauser MA, Scott WK, Small GW,
Nance MA, Watts RL, Hubble JP, Koller WC, Pahwa R, Stern MB, Hiner BC, Jankovic J, Goetz
CG, Mastaglia F, Middleton LT, Roses AD, Saunders AM, Schmechel DE, Gullans SR, Haines JL,
Gilbert JR, Vance JM, Pericak-Vance MA: Glutathione S-transferase omega-1 modifies age-at-
onset of Alzheimer disease and Parkinson disease. Hum Mol Genet 2003;12:3259–3267.
Clemens R. Scherzer, MD
Laboratory for Functional Genomics, Center for Neurologic Diseases
Harvard Medical School, Brigham and Women’s Hospital
65 Landsdowne Street, Suite 327, Cambridge, MA 02319 (USA)
Tel. ⫹1 617 768 8697, Fax ⫹1 617 768 8595, E-Mail cscherzer@partners.org
medwedi.ru
Molecular Pathology of Dementia

Emerging Treatment Strategies
Gunnar K. Gouras
Department of Neurology and Neuroscience, Laboratory of Alzheimer’s Disease
Neurobiology, Weill Medical College of Cornell University, New York, N.Y., USA
Introduction
Dementia is defined as a progressive and abnormal decline in cognition,

typically over months to years. Delirium differs from dementia in that it is an
acute or subacute impairment in cognitive abilities, often caused by a reversible
toxic or metabolic insult. Neurodegenerative diseases of aging that cause demen-
tia are a growing public health concern as life expectancy increases. Alzheimer’s
disease (AD), the most common cause of dementia, currently afflicts about
4 million Americans. Annual costs associated with the care of patients with AD
to our society (USA) have been estimated to exceed USD 100 billion annually,
and will only increase unless new therapeutic approaches are devised.
Major categories in the differential diagnosis of dementia include diverse
neurodegenerative diseases, toxic-metabolic encephalopathies, vascular
dementia, structural lesions and dementia of depression (table 1). Currently,
causes of dementia warranting surgical interventions include brain masses, sub-
dural hematoma and hydrocephalus. At times, brain tumors, such as a frontal
glioma or meningioma, can present with mainly gradual cognitive impairment. An
important and neurosurgically treatable cause of dementia, presenting with grad-
ual memory impairment in the elderly, is normal pressure hydrocephalus, which is
characterized by the triad of gait impairment, dementia, and urinary incontinence.
Obstructive hydrocephalus presenting with dementia may be secondary to the
obstruction of CSF flow, as may be caused by a colloid cyst of the third ventricle
or aqueductal stenosis.
It is interesting that recent work aimed at decreasing the symptoms or pro-
gression of Alzheimer’s have looked at shunting CSF fluid as one possible
approach (e.g., Eunoe, COGNIShunt System), and clinical trials at Stanford
Table 1. Major categories of dementia
Neurodegenerative diseases
• Alzheimer’s disease
• Diffuse Lewy body disease and Parkinson’s disease
• Frontotemporal dementia (Pick’s disease; corticobasal ganglionic degeneration)
• Huntington’s disease
• Creutzfeldt-Jakob disease (prion diseases)
• Progressive supranuclear palsy
• Other neurodegenerative diseases (SCAs; lipid storage diseases; demyelinating diseases)
Vascular dementia
Toxic-metabolic encephalopathy (endocrine, infectious, nutritional, toxins)
Normal pressure hydrocephalus
Structural lesions
• Subdural hematoma
• Brain tumor
• Obstructive hydrocephalus
University propose a role of CSF purification or clearance as a potential neu-

rosurgical treatment option for selected patients, outside of those with normal
pressure hydrocephalus. Other neurosurgical approaches for dementia include
the delivery of in vivo or ex vivo gene therapy in the form of recombinant
enzymes or growth factors, as well as stem cell transplants to regenerate lost
neurons and axons. In this chapter, we will provide an overview of neuro-
degenerative diseases, the most common cause of dementia in the elderly, and
discuss some emerging biological treatment strategies including various mole-
cular neurosurgical approaches.
Central Role of Amyloid Beta Peptide (‘Abeta’) in AD
Over the past two decades there has been tremendous progress in better
understanding of the molecular biology, pathology, and genetics of AD [8, 23].
The discovery of the peptide sequence of -amyloid (Abeta), the principle com-
ponent of senile plaques [7, 16] initiated the modern era of molecular biology
research into AD. Despite these advances, current treatment for AD remains
strictly palliative. There is only one class of medication that is currently F.D.A.
approved for the treatment of AD, the cholinesterase inhibitors, and these drugs
do not appear to be as effective in mid- to late-stage AD. The use of these drugs
evolved from neurochemical studies conducted in the 1970–80s which demon-
strated reductions of cholinergic neurotransmission in AD brain tissue, espe-
cially in the basal forebrain.
Molecular Pathology of Dementia 259
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Of the two hallmarks of AD neuropathology, neurofibrillary tangles and
senile plaques, the neurofibrillary tangles have been viewed as less specific
to AD because they are found in a variety of other neurodegenerative dis-
eases. For example, familial mutations in tau, the principle component of neu-
rofibrillary tangles, are associated with genetic forms of fronto-temporal
dementia. Nonetheless, indirect evidence suggests that tau may be essential
to the loss of neurons which occurs in AD [28]. It is interesting that both
Abeta and tau may be elevated following traumatic brain injury, particularly
chronic brain injury, suggesting that they may constitute a response to injuri-
ous stimuli.
The importance of Abeta to the AD disease process was strengthened by
the discovery of mutations within the Abeta precursor protein (-APP) gene
that segregate with autosomal dominant forms of early onset familial AD
(FAD). Subsequently, transgenic mice harboring human APP with FAD muta-
tions were developed that reproduce AD-like brain amyloidosis [6, 11]. The
discovery of autosomal dominant FAD mutations in presenilin (PS) 1 (chromo-
some 14) and 2 (chromosome 1) also led to the findings that these forms of
FAD invariably lead to increased generation of the longer Abeta42 form of
Abeta [23]. Abeta peptides range in length up to 42 or 43 amino acids. Both the
Abeta N-terminus and C-terminus reveal heterogeneity, but tend to be mainly
referred to in the literature as Abeta 1–40 (Abeta40) and Abeta 1–42 (Abeta42)
peptides. The slightly extended Abeta42 aggregates more readily and is the
main constituent of senile plaques in AD [9].
The mechanism whereby Abeta is involved in AD pathogenesis remains
controversial. Numerous investigators have demonstrated that Abeta isoforms
are neurotoxic when added to cultured neurons in vitro and when injected into
the brain of experimental animals in vivo [29]. Accordingly, neuritic plaques
found in the extracellular space in AD brains are presumed to be toxic to
surrounding neurons and their processes. Recent evidence suggests that AD,
analogous to a growing number of diverse neurodegenerative diseases, is also
characterized by the intracellular accumulation of its disease-linked Abeta
peptide. Indeed, a recent immunoelectron microscopy study observed accumu-
lation of Abeta within neurons, especially within distal neuronal processes
of transgenic APP mice with aging prior to and with the onset of plaque
pathology [30].
Currently, increases in Abeta oligomers (mainly soluble Abeta40 but also
insoluble Abeta42) are viewed as important neurotoxic intermediates in the
development of neuronal dysfunction [13, 23]. Increased soluble Abeta levels
appear to be the best Abeta correlate of cognitive dysfunction from mild
cognitive function through more severe stages of AD [18; fig. 1 for schema of
AD pathogenesis]. A major focus of AD research continues to be to better
Gouras 260
FAD mutations High cholesterol
Apo E
(APP.PS1, PS2) head trauma
↑Soluble A
Aging ↑Insoluble A oligomers
Neuronal and synaptic dysfunction MCI
A plaques and NFTs
Loss of nerve cells and synapses Dementia
Fig. 1. Schema of AD pathogenesis.
understand the biochemical mechanisms by which the genetically disease-

linked APP/Abeta, PS, and apolipoprotein-E are involved in neurobiology and
the AD disease process. Overall, there is growing optimism that with major
advances in our understating of neurodegenerative diseases of aging, therapies
for these incurable disorders are not far away [8; table 2; fig. 2].
Inhibitors of ␥- and ␤-Secretase
The increasing evidence for the importance of Abeta accumulation in AD

has made reduction of Abeta a leading target for AD therapy. Strategies to
accomplish Abeta reduction include inhibition of the proteases that lead to the
cleavage of Abeta from its larger precursor, -APP (fig. 3). The -site APP
cleaving enzyme (BACE) was discovered to be responsible for the initial cleav-
age of APP at the N-terminus of Abeta that first produces an APP C-terminal
fragment or CTF [31]. PS is viewed as critical for the subsequent -secretase
cleavage of the CTFs that generates the 40 or 42 C-terminal ends of Abeta [23].
Given that BACE knockout mice appear normal while PS1 knockout mice are
embryonically lethal [25], and reports linking PS to cleavages of several other
important proteins, BACE inhibition currently is the leading therapeutic target
for Abeta inhibition [2]. In common with other therapies directed at the CNS,
efficient delivery of such secretase inhibitors could be critical and may depend
on placement of indwelling infusion devices into the CSF or brain parenchyma
to bypass the blood brain barrier.
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Amyloid precursor protein (APP) metabolism
-secretase -secretase
(BACE)
N C
1 11 17 40/42
-secretase
or
Amyloidogenic CTF
1 40/42
4 kD A
11 40/42 -amyloid
3.4 kD A
Nonamyloidogenic
sAPP CTF
17 40/42
3 kD p3 p3
Fig. 2. Amyloid precursor protein (APP) metabolism.
Table 2. Outline of therapeutic treatment strategies for AD
Cholinesterase inhibitors (i.e., Aricept, Exelon, Reminyl)

Anti-Abeta approaches
• BACE inhibition
• gamma-secretase inhibition (inhibition of PS, Aph1, PEN2 and/or Nicastrin)
• anti-Abeta vaccine
• cholesterol-lowering agents
• anti-aggregation compounds (e.g., beta-sheet breakers)
• promoters of Abeta degrading enzymes (neprilysin, IDE, neutral endopeptidase)
Anti-oxidants (e.g., Vitamin E)
Anti-inflammatory medications (e.g., NSAIDs)
Estrogen replacement therapy → NO LONGER RECOMMENDED!
Anti-tau strategies
Anti-apoptosis agents
Neurotrophic factors (e.g., NGF)
CSF shunting
Stem cells
Gene therapy
Gouras 262
Abeta Vaccine: Theory and Practice
Other Abeta-based therapeutic approaches that are under investigation

include anti-Abeta vaccines, in which Abeta is deliberately administered with
an adjuvant in order to stimulate the body’s natural immune response to clear
the substance from the blood and the brain. Initial excitement demonstrating
reductions of cerebral Abeta plaques in transgenic FAD APP mice in response
to intravenous infusion of Abeta or anti-Abeta antibodies was seriously damp-
ened by the discontinuation of a clinical trial of Abeta infusion into AD patients
secondary to the occurrence of encephalitis in ⬃5% of patients. Despite this
setback for an anti-Abeta vaccine, reports indicating cognitive stabilization in
some patients with high anti-Abeta antibody titers provide some encourage-
ment [10]. Overall, an immunological approach to AD has received great inter-
est in the field, but remains a therapeutic direction in AD that is less well
understood than others. Further research on the neuroimmunology of Abeta
may help elucidate the mechanism whereby anti-Abeta antibodies may benefit
patients with AD. Some investigators have postulated that anti-Abeta antibod-
ies in the systemic circulation may act as a ‘peripheral Abeta sink’ that could
draw Abeta out of the brain [5]. Theoretically, evidence for the sink hypothesis
could also be viewed as a mechanism whereby neurosurgical CSF shunting [27]
may have therapeutic potential for AD, since it would be expected to divert
Abeta out of the brain.
Other Strategies Based on Reduction of Abeta
Retrospective studies suggest that intake of cholesterol-lowering

3-hydroxy-3-methyl glutaryl coenzyme A reductase inhibitors, commonly
known as statins, are associated with protection against AD. In addition, treat-
ment of APP transgenic mice with a high fat cholesterol diet increases, while
treatment with statins reduces, Abeta plaque pathology [21, 22]. There is a
compelling biological mechanism by which cholesterol influences Abeta
generation, since the critical -secretase cleavage of Abeta occurs within the
membrane lipid bilayer and could be modulated by higher or lower cholesterol;
supportive evidence comes from drops in Abeta levels in cultured cells that par-
allels transgenic mice studies. Prospective clinical trials to assess the efficacy
of cholesterol-lowering strategies for the prevention and/or treatment of AD are
ongoing, and preliminary results suggest an effect of statin treatment.
In the past, retrospective studies suggested that estrogen replacement for
postmenopausal women might reduce the incidence of AD. Studies in tissue
culture and on APP transgenic mice support the efficacy of both estrogen and
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Therapeutic anti-A targets in Alzheimer’s disease
Cholesterol Immunotherapy
modulators
- and ? -sheet
-secretase breakers
inhibitors
Intra- A
Antioxidants cellular plaques
A
Gonadal ?
Metal
hormones Signal chelators
transduction Modulators of
modulators inflammation
Fig. 3. Sites for intervention vis-à-vis Abeta for Alzheimer’s disease: treatment strate-
gies aimed at reducing Abeta are either directed at APP/Abeta metabolism within nerve cells
or Abeta associated with extracellular plaques.
noninflammatory treatments. Unfortunately, prospective studies of patients

with mild-to-moderate AD have failed to demonstrate benefits for estrogen.
Indeed, patients treated with estrogen experienced more adverse side effects,
especially vascular complications such as deep vein thrombosis and heart
attack. In addition, estrogen intake increases the risk for the development of
breast and uterine cancer. Moreover, the Women’s Health Initiative Memory
Study (WHIMS) [32] showed that rather than helping as had been originally
proposed, estrogen-progesterone hormone replacement therapy was harmful
and actually increased the occurrence of Alzheimer’s dementia over the treat-
ment period.
The use of anti-inflammatory medications such as NSAIDs has also been
proposed as a possible treatment option for incipient AD, given that experi-
mental evidence supports the role of various inflammatory processes in AD,
including the role of activated microglia and brain cytokine release. However,
given a lack of clinical data to support this approach and bearing in mind the
results of estrogen clinical trials which appear to contradict earlier in vitro and
in vivo work, caution is warranted in recommending anti-inflammatory drugs
at this time. It appears that inflammatory processes do affect the amount of
Abeta produced in cell culture and mouse models of AD, yet, prospective,
randomized clinical trials are required to validate this approach.
Metal chelation has been proposed in the past as a treatment for AD, since
certain divalent or trivalent metals appear to increase aggregation of Abeta
in vitro and the antibiotic clioquinol was reported to reduce plaque pathology
in transgenic APP mice [4]. However, metal accumulation is very likely to be
Gouras 264
an epiphenomenon of other changes and there are no convincing data to sup-
port the role of metals in the pathology of AD or the use of chelation therapy.
Signal transduction modulators such as GSK3, CDK5, PI3K, or PKC mod-
ulators have been proposed as treatment strategies for AD, acting via mecha-
nisms that may affect both Abeta and tau.
Augmenting the various proteases involved in degrading Abeta in brain is
another possible treatment approach. Reduced clearance and degradation of
Abeta may be more important for most cases of AD than the increased genera-
tion of Abeta associated with the relatively rare early onset forms of AD. Some
important enzymes in the brain that have been demonstrated to degrade Abeta
in vitro or in vivo include neprilysin, insulin degrading enzyme, neutral
endopeptidase, and other metalloproteinase enzymes. Protease augmentation in
targeted brain areas would be particularly amenable to an in vivo or an ex vivo
gene transfer approach.
Excitotoxicity is a general phenomenon involved in neuronal cell damage,
in which glutamatergic, excitatory transmission leads to excessive cell stimula-
tion, pathological Ca influx, and associated processes such as apoptosis.
Despite failure of other glutamatergic antagonists in the past to prevent
neurodegeneration, the N-methyl-D-aspartate (NMDA) antagonist memantine
was reported in a prospective clinical study to improve function in patients with
moderate to severe AD [20], though the biological mechanism by which it may
be beneficial for AD is not known. NMDA-mediated excitotoxicity is one
potential mechanism for protective effects of NMDA antagonists, though the
role of NMDA toxicity has not been precisely defined in the pathophysiology
and treatment of many neurodegenerative diseases. A recent neurobiological
study suggested that NMDA antagonists may provide therapeutic benefit in AD
by other biological effects on Abeta and neuronal physiology [12].
Increasing evidence supports the hypothesis that oxidative stress is a
mechanism whereby aging is the major risk factor for age-related neurodegen-
erative diseases [1]. While the biochemical pathways involved in the physio-
logically relevant oxidative stress associated with age-related diseases of the
brain require more definition, effects on the progression of AD in a large
double-blind clinical study following treatment with the anti-oxidant vitamin E
suggests that this approach requires further study [24]. Because oxidative
species may accentuate or accelerate the negative effects of Abeta deposition, it
is possible that lowering oxidative stress may have synergistic effects on lower-
ing Abeta.
Gene Therapy for Alzheimer’s?

The understanding that a significant component of AD, especially early
onset forms of AD, are inherited or that almost half of the more typical late
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onset cases of AD are associated with a single genetic risk factor, the
apolipoprotein E4 allele, has led to a growing awareness that gene therapy may
be an important direction for future therapy. Other neurodegenerative diseases
are known to have, and are increasingly being found to have, complex genetic
etiologies. Since a common theme in many neurodegenerative diseases (e.g.,
AD, Huntington’s disease, Parkinson’s disease) is the abnormal intracellular
accumulation of insoluble peptides, the most direct method to reduce the
expression of a disease-linked protein is reduced transcription of the protein,
increased transcription of proteases involved in the degradation of a given
protein, or reduction in proteases involved in the generation of toxic proteolytic
fragments (e.g., generation of Abeta from APP).
In addition to standard gene transfer using viral and nonviral vectors,
siRNA has emerged as a powerful new method to reduce gene transcription
and is being employed in experimental strategies to reduce protein accumula-
tion associated with neurodegenerative diseases such as AD. Effective gene
therapy will require improvements in vectors to reliably transduce cells in the
CNS while minimizing local inflammation and/or tissue damage. Once techni-
cal obstacles for safe and effective gene transfer to the CNS can be accom-
plished, manipulation of genes may one day revolutionize the treatment of
neurodegenerative diseases. Stem cells are increasingly being explored as a
vector to deliver ex vivo gene transfer and may be useful for dementias to
replenish neurons that are destroyed as a result of a neurodegenerative disease.
Neurotrophic factors, important in the development of the nervous system, con-
tinue to be produced in adulthood and have been proposed as a therapeutic
option for neurodegenerative diseases such as AD, particularly in combination
with stem cell-based ex vivo gene therapy.
Too Much Focus on Abeta?

Despite this preponderance of anti-amyloid approaches for treating AD,
there is not unanimous agreement in the field whether the focus on Abeta is
justified. Arguments against the primary role of Abeta in AD include the
following: only a small group of AD patients have mutations in APP (1%);
Abeta plaques do not correlate with cognitive dysfunction as well as synaptic
loss; and significant amounts of A plaques can be found in postmortem brains
of people without clear clinical evidence of dementia.
It should also be pointed out that despite its apparently deleterious role in
AD, Abeta40 (i.e., the majority of soluble Abeta), Abeta42 (i.e., largely insoluble
Abeta fraction), and other proteolytic fragments of Alzheimer’s precursor protein
such as sAPPalpha or sAPPbeta may have important physiological functions.
APP knockout mice were originally reported to have normal brains, but subse-
quent work has demonstrated abnormalities in synaptic markers in APP knockout
Gouras 266
mouse brain and decreased neurite extension in cultured APP knockout mouse
neurons [19]. Indeed, recent work has indicated that neuronal activity increases
Abeta secretion, which in turn may provide a negative feedback to modulate fur-
ther neuronal activity [12]. It was reported that transection of the perforant path,
the major outflow tract from the entorhinal cortex to hippocampus, reduced
plaque pathology in the hippocampus of transgenic APP mice [15, 26]. Because
APP can be transported down axons by fast axonal transport [14], normal trans-
port of APP may be required to develop plaques in the terminal fields of axonal
projections. Therapeutic implications include a concern that treatments which
interfere with axonal transport may reduce plaques in transgenic mouse models
of beta-amyloidosis, and therefore appear to have therapeutic potential for AD
while in fact they may be detrimental to neurons and not beneficial in AD.
Aggregation of Insoluble Proteins in Neurodegenerative Diseases

There are a number of major similarities among diverse neurodegenerative
diseases of aging, which include age-related development of aberrant cellular
accumulation and aggregation of insoluble proteins in vulnerable brain regions;
a role for oxidative stress; and the occurrence of inflammation. While it is still
debated whether intra- or extracellular aggregates are directly toxic to the cell
or are an attempt by the cell to defend itself, the prevailing view is that such
aggregates within aging cells of the brain are likely to be detrimental.
Mutations associated with Parkinson’s disease on two different proteins, parkin,
a ubiquitin ligase, and ubiquitin C-terminal hydrolase L1, have pointed to a role
for the ubiquitin-proteasome degradation pathway for intracellular proteins in
neurodegenerative diseases [17]. Evidence also indicates that endosomal-
lysosomal system abnormalities occur early in the development of AD [3].
These multiple lines of evidence indicate that the reduced ability of selective
neuronal populations to efficiently degrade disease-linked protein aggregates are
a final common pathway in neurodegenerative diseases of aging.
Future neurosurgical interventions that may be critical for the treatment of
neurodegenerative diseases such as AD could include placement of infusion
devices for delivery of treatments as diverse as gene therapeutic agents (i.e.,
viral vectors), protease inhibitors, anti-Abeta antibodies to bypass the blood-
brain barrier, stem cells, and CSF shunting to reduce levels of soluble brain
Abeta in patients with AD.
Acknowledgments
The author thanks Dr. Michael T. Lin for his critical reading of the chapter and helpful
discussions.
medwedi.ru
References
1 Beal MF: Mitochondria, free radicals, and neurodegeneration. Curr Opin Neurobiol 1996;6:
661–666.
2 Cai H, Wang Y, McCarthy D, Wen H, Borchelt DR, Price DL, Wong PC: BACE1 is the major beta-
secretase for generation of Abeta peptides by neurons. Nat Neurosci 2001;4:233–234.
3 Cataldo AM, Peterhoff CM, Troncoso JC, Gomez-Isla T, Hyman BT, Nixon RA: Endocytic path-
way abnormalities precede amyloid beta deposition in sporadic Alzheimer’s disease and Down
syndrome: Differential effects of APOE genotype and presenilin mutations. Am J Pathol 2000;
157:277–286.
4 Cherny RA, Atwood CS, Xilinas ME, Gray DN, Jones WD, McLean CA, Barnham KJ, Volitakis I,
Fraser FW, Kim Y, Huang X, Goldstein LE, Moir RD, Lim JT, Beyreuther K, Zheng H, Tanzi RE,
Masters CL, Bush AI: Treatment with a copper-zinc chelator markedly and rapidly inhibits beta-
amyloid accumulation in Alzheimer’s disease transgenic mice. Neuron 2001;30:665–676.
5 DeMattos RB, Bales KR, Cummins DJ, Dodart JC, Paul SM, Holtzman DM: Peripheral anti-
A beta antibody alters CNS and plasma A beta clearance and decreases brain A beta burden in a
mouse model of Alzheimer’s disease. Proc Natl Acad Sci USA 2001;98:8850–8885.
6 Games D, Adams D, Alessandrini R, Barbour R, Berthelette P, Blackwell C, Carr T, Clemens J,
Donaldson T, Gillespie F, et al: Alzheimer-type neuropathology in transgenic mice overexpressing
V717F beta-amyloid precursor protein. Nature 1995;373:523–527.
7 Glenner GG, Wong CW: Alzheimer’s disease: Initial report of the purification and characterization
of a novel cerebrovascular amyloid protein. Biochem Biophys Res Commun 1984;120:885–890.
8 Golde TE: Alzheimer disease therapy: Can the amyloid cascade be halted? J Clin Invest 2003;
111:11–18.
9 Iwatsubo T, Odaka A, Suzuki N, Mizusawa H, Nukina N, Ihara Y: Visualization of A beta 42(43)
and A beta 40 in senile plaques with end-specific A beta monoclonals: Evidence that an initially
deposited species is A beta 42(43). Neuron 1994;13:45–53.
10 Hock C, Konietzko U, Streffer JR, Tracy J, Signorell A, Muller-Tillmanns B, Lemke U, Henke K,
Moritz E, Garcia E, Wollmer MA, Umbricht D, de Quervain DJ, Hofmann M, Maddalena A,
Papassotiropoulos A, Nitsch RM: Antibodies against beta-Amyloid slow cognitive decline in
Alzheimer’s disease. Neuron 2003;38:547–554.
11 Hsiao K, Chapman P, Nilsen S, Eckman C, Harigaya Y, Younkin S, Yang F, Cole G: Correlative mem-
ory deficits, Abeta elevation, and amyloid plaques in transgenic mice. Science 1996;274:99–102.
12 Kamenetz F, Tomita T, Hsieh H, Seabrook G, Borchelt D, Iwatsubo T, Sisodia S, Malinow R: APP
processing and synaptic function. Neuron 2003;37:925–937.
13 Klein WL, Krafft GA, Finch CE: Targeting small Abeta oligomers: The solution to an Alzheimer’s
disease conundrum? Trends Neurosci 2001;244:219–224.
14 Koo EH, Sisodia SS, Archer DR, Martin LJ, Weidemann A, Beyreuther K, Fischer P, Masters CL,
Price DL: Precursor of amyloid protein in Alzheimer disease undergoes fast anterograde axonal
transport. Proc Natl Acad Sci USA 1990;87:1561–1565.
15 Lazarov O, Lee M, Peterson DA, Sisodia SS: Evidence that synaptically released beta-amyloid
accumulates as extracellular deposits in the hippocampus of transgenic mice. J Neurosci 2002;22:
9785–9793.
16 Masters CL, Simms G, Weinman NA, Multhaup G, McDonald BL, Beyreuther K: Amyloid plaque
core protein in Alzheimer disease and Down syndrome. Proc Natl Acad Sci USA 1985;82:
4245–4249.
17 McNaught KS, Olanow CW: Proteolytic stress: A unifying concept for the etiopathogenesis of
Parkinson’s disease. Ann Neurol 2003;53(suppl 3):S73–S84.
18 Naslund J, Haroutunian V, Mohs R, Davis KL, Davies P, Greengard P, Buxbaum JD: Correlation
between elevated levels of amyloid beta-peptide in the brain and cognitive decline. JAMA
2000;283:1571–1577.
19 Perez RG, Zheng H, Van der Ploeg LH, Koo EH: The beta-amyloid precursor protein of
Alzheimer’s disease enhances neuron viability and modulates neuronal polarity. J Neurosci 1997;
17:9407–9414.
Gouras 268
20 Reisberg B, Doody R, Stoffler A, Schmitt F, Ferris S, Mobius HJ: Memantine Study Group.
Memantine in moderate-to-severe Alzheimer’s disease. N Engl J Med 2003;348:1333–1341.
21 Refolo LM, Pappolla MA, LaFrancois J, Malester B, Schmidt SD, Thomas-Bryant T, Tint GS,
Wang R, Mercken M, Petanceska SS, Duff KE: A cholesterol-lowering drug reduces beta-amyloid
pathology in a transgenic mouse model of Alzheimer’s disease. Neurobiol Dis 2001;8:890–899.
22 Refolo LM, Malester B, LaFrancois J, Bryant-Thomas T, Wang R, Tint GS, Sambamurti K,
Duff K, Pappolla MA: Hypercholesterolemia accelerates the Alzheimer’s amyloid pathology in a
transgenic mouse model. Neurobiol Dis 2000;7:321–331.
23 Selkoe DJ: Alzheimer’s disease: Genes, proteins, and therapy. Physiol Rev 81:741–766.
24 Sano M, Ernesto C, Thomas RG, Klauber MR, Schafer K, Grundman M, Woodbury P, Growdon J,
Cotman CW, Pfeiffer E, Schneider LS, Thal LJ: A controlled trial of selegiline, alpha-tocopherol, or
both as treatment for Alzheimer’s disease. The Alzheimer’s Disease Cooperative Study. N Engl J
Med 1997;336:1216–1222.
25 Shen J, Bronson RT, Chen DF, Xia W, Selkoe DJ, Tonegawa S: Skeletal and CNS defects in
Presenilin-1-deficient mice. Cell 1997;89:629–639.
26 Sheng JG, Price DL, Koliatsos VE: Disruption of corticocortical connections ameliorates amyloid
burden in terminal fields in a transgenic model of Abeta amyloidosis. J Neurosci 2002;22:
9794–9799.
27 Silverberg GD, Levinthal E, Sullivan EV, Bloch DA, Chang SD, Leverenz J, Flitman S, Winn R,
Marciano F, Saul T, Huhn S, Mayo M, McGuire D: Assessment of low-flow CSF drainage as a
treatment for AD: Results of a randomized pilot study. Neurology 2002;59:1139–1145.
28 Trojanowski JQ, Lee VM: The role of tau in Alzheimer’s disease. Med Clin North Am 2002;86:
615–627.
29 Yankner BA: Mechanisms of neuronal degeneration in Alzheimer’s disease. Neuron 1996;16:
921–932.
30 Takahashi RH, Milner TA, Li F, Nam EE, Edgar MA, Yamaguchi H, Beal MF, Xu H, Greengard P,
Gouras GK: Intraneuronal Alzheimer abeta42 accumulates in multivesicular bodies and is associ-
ated with synaptic pathology. Am J Pathol 2002;161:1869–1879.
31 Vassar R, Bennett BD, Babu-Khan S, Kahn S, Mendiaz EA, Denis P, Teplow DB, Ross S,
Amarante P, Loeloff R, Luo Y, Fisher S, Fuller J, Edenson S, Lile J, Jarosinski MA, Biere AL,
Curran E, Burgess T, Louis JC, Collins F, Treanor J, Rogers G, Citron M: Beta-secretase cleavage
of Alzheimer’s amyloid precursor protein by the transmembrane aspartic protease BACE. Science
1999;286:735–741.
32 Shumaker SA, Legault C, Rapp SR, Thal L, Wallace RB, Ockene JK, Hendrix SL, Jones BN,
Assaf AR, Jackson RD, Kotchen JM, Wassertheil-Smoller S, Wactawski-Wende J (for the WHIMS
Investigators): Estrogen plus progestin and the incidence of dementia and mild cognitive impair-
ment in postmenopausal women. JAMA 2003;289:2651–2662.
Gunnar K. Gouras, MD
Assistant Professor, Department of Neurology & Neuroscience
Director, Laboratory of Alzheimer’s Disease Neurobiology
Weill Medical College of Cornell University, 525 East 68th Street, New York, NY 10021 (USA)
Tel. 1 212 746 6598, Fax 1 212 746 8741, E-Mail gkgouras@med.cornell.edu
medwedi.ru
Expanding the Role of Deep

Brain Stimulation from Movement
Disorders to Other Neurological
Diseases
Massimo Leone, Angelo Franzini, Giovanni Broggi,
Gennaro Bussone
Istituto Nazionale Neurologico Carlo Besta, Milano, Italy
Introduction
Deep brain stimulation (DBS) is a relatively new treatment modality, first

developed in the 1980s by Benabid and colleagues [3] in France, that until
recently has been limited to the treatment of complex movement disorders such
as Parkinson’s disease through stimulation of the thalamus, globus pallidus, or
subthalamic nucleus. However, recent work suggests that techniques of intra-
cranial stimulation may have much wider applications in diseases that are not
well managed by any other treatments. Among the new targets for DBS are
intractable epilepsy, intractable dystonia, and intractable headache [1–3]. Because
the molecular effects of DBS are still largely unknown, it is possible that DBS
may also have applications in neurodegenerative and psychiatric diseases; as
the molecular basis of disorders such as Alzheimer’s disease or schizophrenia
are elucidated, and effects of DBS on gene transcription and cellular activity are
better understood, additional applications in different brain regions may become
apparent. It is quite plausible that DBS will offer primary or adjunct treatment
options for a variety of complex neurological disorders in the future, many of
which the neurosurgeons are not accustomed to treating, but which nevertheless
are prevalent and debilitating, perhaps offering new career opportunities for
neurosurgeons specializing in functional and restorative neurosurgery. In the
special case of hypothalamic DBS, the topic of this chapter, applications out-
side of intractable cluster headache (CH) may include complex neuroendocrine
Table 1. The cluster headache attack, diagnostic criteria
A. At least five seperate attacks fulfilling criteria below

B. Severe or very severe unilateral orbital, supraorbital, and/or temporal pain
lasting 15 min to 3 h if untreated
C. Headache is accompanied by at least one of the following:
1. Ipsilateral conjunctival injection and/or lacrimation
2. Ipsilateral nasal congestion and/or rhinorrhea
3. Ipsilateral eyelid edema
4. Ipsilateral forehead and facial sweating
5. Ipsilateral miosis and/or ptosis
6. Sense of restlessness or agitation
or pain disorders. The field of hypothalamic microinstrumentation is in its

infancy, and these studies are a first step toward defining the roles and limita-
tions of DBS outside of the basal ganglia.
CH is an interesting clinical syndrome, the excruciating severity of which is
not widely appreciated. It is a primary pain syndrome characterized by unilateral
and incapacitating headache attacks and also associated with ipsilateral auto-
nomic phenomena [4]. Its prevalence is less than one per 1000 and males are
more affected than females, the ratio being about 3:1 [5, 6]. There are two main
forms of the disease, episodic and the chronic. About 80% of CHs occur in the
episodic form; in this case, the attacks are grouped in so-called ‘cluster periods’
usually lasting 1–2 months at a time. In the chronic form, attacks occur for more
than one year without remission or with remissions lasting less than one month.
The duration of single attacks ranges from 15 min to 3 h at a time, from once
every other day to almost continually at eight times a day [1]. Often, attacks
appear at the same hours of the day or night and (for unknown reasons) cluster
periods often start in autumn and spring [7]. Circadian rhythmicity is a clinical
landmark of the syndrome and for this reason it also has been nicknamed as
‘clock headache.’ The excruciating pain is orbital, supraorbital, and/or temporal
in location. At least one of the following autonomic phenomena are present dur-
ing the attack, ipsilateral to the pain: severe conjunctival injection and/or lacrima-
tion, nasal congestion and/or rhinorrhea, eyelid edema, forehead and facial
sweating, miosis and/or ptosis, and a sense of restlessness or agitation (table 1).
Pathophysiology of CH and Links to the Hypothalamus
The pain of CH is probably the most severe known to humans, similar

in severity to subarachnoid hemorrhage. Accordingly, it has been termed the
Hypothalamic DBS: Therapeutic Approach for Intractable Cluster Headache 271
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‘suicide headache.’ Until a few years ago, CH was still regarded as a vascular
headache. In the last years much has been learned about its pathophysiology
and as a consequence the vascular theory of the disease has been discarded. The
observation of specific events during the headache attacks involving both cra-
nial nerves and the brain suggests that the neural phenomena are much more
relevant to the pathophysiology of CHs. Cluster pain is probably initiated by the
activation of the first (ophthalmic) division of the trigeminal nerve, also asso-
ciated with an increase of calcitonin-gene related peptide in the ipsilateral jugu-
lar blood during the attack. Autonomic symptoms are due to the activation of
the cranial parasympathetic outflow from the VIIth cranial nerve, associated
with an increase of vasoactive intestinal polypeptide in the ipsilateral jugular
blood during the attack [8].
The relapsing-remitting course of cluster attacks, mainly in autumn and
spring, and the clockwise regularity of single attacks initially suggested that the
hypothalamus, site of circadian phenomena or the ‘biological clock,’ might play
a crucial role in the pathophysiology of this form of headache [9]. A number of
neuroendocrinological abnormalities have been reported in CH [10–12], lending
further support to this hypothesis. The first direct evidence showing a pivotal
role of the hypothalamus in CH came from positron emission tomography (PET)
studies. An increased regional blood flow in the posterior inferior ipsilateral
hypothalamic gray matter during the acute stage of cluster attack has been shown
both in nitroglycerine-induced attacks [13] and in spontaneous cluster attacks
[14]. In a voxel-based magnetic resonance imaging (MRI) study, an increased
neuronal density was found in the same brain region that was known to be
activated in the PET studies, again ipsilateral to the pain [15]. These structural
changes were seen independent of the headache state, suggesting an inherent
dysfunction of the hypothalamus in CH rather than an epiphenomenon.
Although CH was previously described as a vascular headache, and vas-
cular phenomena also appears to be involved, the striking circadian rhythmicity
of this strictly unilateral pain syndrome cannot be explained by a simplistic
vascular hypothesis. The case report we present in this chapter illustrates the
relevance of the hypothalmus to the pathophysiology of CH. We report on a
patient suffering from chronic intractable CHs on the left side, who had initially
received complete surgical section of the left trigeminal sensory root to relieve
the pain [16, 17]. After the operation, he was completely anesthetic over the
entire left trigeminal distribution and the left corneal reflex was absent but
he continued to have cluster attacks. Blink reflexes of the left supraorbital nerve
produced neither ipsilateral nor contralateral blink reflex responses. With
the complete section of the left trigeminal sensory root, the brain cannot perceive
vasodilatation or a peripheral neural inflammatory process; as a consequence,
none of these peripheral structures, be they neural or vascular, are necessary for
Leone/Franzini/Broggi/Bussone 272
an attack to happen [18]. It may be concluded that CH is generated primarily
from within the brain.
Pharmacological Treatments
The pharmacological treatment of CH is aimed at aborting the ongoing

attacks (i.e., acute therapy) and preventing recurrent attacks during the cluster
period (i.e., prophylaxis) [19, 20]. The gold standard in the treatment of acute
attacks of CH is the subcutaneous administration of the 5HT1B/D agonist sum-
atriptan [21], also used in migraine attacks. Alternatively inhalation of 100%
oxygen can be used. Verapamil [22], lithium carbonate [23], methysergide [24]
and cortisone are the most effective drugs to prevent the incidence of CH.
Valproic acid, topiramate, gabapentine, naratriptan, melatonin and local appli-
cation of civamide or anesthesia of the greater occipital nerve may also be of
some help [25].
Surgical Treatment
Radiofrequency thermocoagulation of the trigeminal ganglion has been

reported to be effective in about 75% of chronic drug-resistant CH patients
[26–29]. Other procedures on the trigeminal nerve have been tempted with
inconsistent results [30, 31]. Complications include recurrence of headache, in
which case, a repeat procedure may be necessary [28]. Other surgical sequelae
are corneal analgesia with resulting potential corneal infection or opacification,
and anesthesia dolorosa.
DBS of the Hypothalamus
PET has shown the activation of the ipsilateral posterior inferior hypothal-
amic gray matter during CH attacks [13, 14], which is apparently specific for
the condition [32], while voxel-based morphometric MR has documented alter-
ations in the same area [15], strongly suggesting that the CH generator is
located there. We reasoned that the stereotactic stimulation of this area might
prevent the activation and hopefully relieve intractable forms of CH [16, 17].
The first hypothalamic implantation using DBS to relieve intractable chronic
CH was done in July 2000 [16]. Due to the brilliant results, both in term of pain-
free state and absence of relevant adverse events, 13 new patients have been
implanted so far. A summary of the first implanted patient follows.
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The First Patient Implanted with Hypothalamic DBS
At the time of treatment, this patient was a 39-year-old right-handed man

who had suffered daily CH attacks for 5 years [16]. The attacks lasted from
30 min up to 4 h at a time, occurred two to eight times a day, and were associ-
ated with striking oculo-facial phenomena. The majority of the attacks were on
the right side and sometimes on the left, but they were never bilateral. Cerebral
MR, MR angiography, and catheter angiography were unremarkable. He became
completely drug resistant. He was operated on four times on the right trigemi-
nal nerve; after the last thermal rhizotomy, the right side headache attacks
ceased, but from that moment, the left-side attacks worsened, again with striking
autonomic phenomena. This new onset of CH was completely drug refractory.
In addition, the patient was blind on the right as a result of vitreous humor
hemorrhage and left trigeminal surgery was highly contraindicated by the risk
of corneal sequelae, which could have left the patient totally blind.
Stereotactic electrode implantation targeting the posterior inferior ipsilateral
hypothalamic gray matter was then proposed. After informed consent the opera-
tion was performed under local anesthesia using a CRW frame on July 14, 2000.
The electrode (Medtronic 3089, Minneapolis, Minn., USA) was inserted at coor-
dinates 6 mm posterior to the anterior commissure-posterior commissure mid-
point, 2 mm left of the midline, and 8 mm below the commissural plane [15, 16].
Intraoperative electrical stimulation induced no side effects. The permanent gen-
erator (Soletra, Medtronic, Minneapolis, Minn., USA), embedded in a subclav-
icular pocket, was connected by subcutaneous tunnelization. Therapeutic
stimulation was in continuous unipolar mode. The position of the permanent elec-
trode was verified by postoperative MR. When stimulated at 180 Hz, 3 V, 60-␮s
pulse width, the attacks disappeared after 48 h [16]. Twice, unknown to the
patient, the stimulator was switched off and the left side attacks reappeared within
48 h later. When the stimulator was turned on again, the attacks disappeared 48 h
later [16]. More than 4 years after the operation, the patient remains pain free [17].
Patient Selection for Hypothalamic DBS
First of all, it should be kept in mind how debilitating CH can be, when it
has a chronic course and does not respond to any pharmacological treatments.
All patients who received hypothalamic implantation suffered daily attacks in
the years before the operation, notwithstanding all kinds of prophylactic drugs,
including high dosage steroids. They did not tolerate the painful condition
and true to the name ‘suicide headache,’ 2 of the patients had attempted suicide.
Another patient had a myocardial infarction while waiting to be operated,
probably induced by her very high intake of daily triptans (up to 78 mg injectable
sumatriptan/day); thus, she could not take triptans anymore and had to increase
daily steroid consumption to control in some way the pain episodes. Three weeks
after increased dosage of steroids, she died because of repeated gastrointestinal
bleeding. Another patient also had myocardial infarction probably related to
triptan overuse and had to increase steroid daily intake; in a few months he had
developed, among other side effects, a myopathy in the legs and became unable
to climb stairs or to stand from the sitting position. Steroids were tapered and
strength in the leg gradually improved, but the CHs worsened. He was asked to
restart steroids but the myopathy worsened (confirmed with EMG). Again,
steroids were stopped, symptoms of myopathy improved, but CHs worsened. In
light of these side effects of standard treatments, it is easy to understand how
much chronic CHs may interfere with patients’ life.
The hypothalamic DBS operation should be considered only in patients
with chronic CH for at least 2 years, with daily attacks [33]. From a clinical
point of view, 2 years is sufficient time to apply the full range of available CH
prophylactic medications [19, 33] and to exclude that a remission period does
not occur spontaneously. At present, DBS should be considered only in patients
with strictly unilateral CH (no side shift), since contralateral attacks are well
known to develop in CH patients after procedures on the trigeminal nerve [33].
Even though we have implanted 2 patients on both sides because they suffered
from intractable bilateral chronic CH, bilateral DBS cannot be recommend at
this time until more experience on unilateral DBS has been accumulated [33].
Before considering this operation, it is very important to closely monitor the
patient over a period of time to verify the diagnosis and assess the attacks first-
hand; hence, the patient has to be admitted in order to witness the attacks.
Candidates for hypothalamic DBS must be psychologically stable, with a normal
psychological profile. Neuropsychological profiling has to be tested before the
operation and periodically afterwards. It is also important to exclude any intracra-
nial abnormality that may contraindicate DBS or which could be a potential
underlying cause of CH [33] by performing cranial MRI with gadolinium, cranio-
cervical transition, MRI arterial and venous angiography, and CT of the skull
base. The patient must be informed that continuous hypothalamic stimulation
might have effects on his/her fertility and sexual behavior, although this aspect
has not been well studied. At the present stage, the effects of hypothalamic stim-
ulation on pregnancy are completely unknown and for this reason, we recommend
that pregnant patients should not receive DBS. After electrode implantation, the
stimulator is turned on only when typical spontaneous CH attack has occurred
[33]. A list of primary contraindications to hypothalamic DBS also are listed in
table 2. All surgical candidates are informed of the classic surgical procedures
that are available for the treatment of intractable chronic CH (open microvascular
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Table 2. Criteria for electrode implants in intractable chronic cluster headache
1. CCH diagnosed according to IHS criteria; in addition both of the following:

a) CCH for at least 24 months
b) Attacks should normally occur on a daily basis
2. Attacks must have always been strictly unilateral
3. Patients must be hospitalized to witness attacks and document their characteristics
4. All state of the art drugs for CH prophylaxis must have been tried in sufficient
dosages (unless contraindicated or have unacceptable side effects, etc.) alone and in
combination, where applicable. These comprise verapamil, lithium carbonate, methy-
sergide, valproate, topiramate, gabapentin, melatonin (where available), pizotifen,
indomethacin and steroids
5. Normal psychological profile
6. No medical/neurological conditions contraindicating DBS including:
a) Recent myocardial infarction
b) Cardiac arrhythmia
c) Cardiac malformation
d) Epilepsy
e) Stroke
f) DBS for other reasons
g) Degenerative disorder of central nervous system
h) Arterial hypertension or hypotension, not controlled by drugs
i) Autonomic nervous system disorder
j) Endocrinological illnesses
k) Major disturbance in electrolyte balance (e.g., due to renal insufficiency or
hyperaldosteronism)
7. Normal neurological examination except for symptoms characteristic of CH
(e.g., persistent Horner’s syndrome)
8. Normal CT scan (base of the skull window). Normal cerebral MRI including
cranio-cervical transition and MRI arterial and venous angiography
9. Neurosurgical team experienced at performing stereotactic implant of electrodes
10. Patient should not be pregnant
11. Ethics Committee/Institutional Review Board approval
12. Patient informed and gives written consent
decompression/lesion of cranial nerves in the cerebellopontine angle and percu-

taneous radiofrequency trigeminal rhizotomy). They can choose among the vari-
ous surgical options once detailed informations on the procedures are given.
The Surgical Protocol
Ipsilateral Posterior Hypothalamus Electrode Implantation

Affixing a stereotactic apparatus (Leksell frame, Elekta, Sweden) is per-
formed under local anesthesia. If sedation is required, low doses of midazolam
(0.05–0.1 mg/kg) or propofol (0.5–1 mg/kg) are used [34]. Antibiotic treatment
was given to all patients during the perioperative period. A preoperative MRI
(brain axial volumetric fast spin echo inversion recovery) is used to obtain high-
definition anatomical images that allow for the precise determination of the
AC-PC line. MR images are fused with 2 mm thick CT slices obtained under
stereotactic conditions by using an automated technique that is based on a
mutual-information algorithm (Frame-link 4.0, Sofamor Danek Stealthstation,
Medtronic, Memphis, Tenn., USA). The workstation also provides stereotactic
coordinates of the target: 5 mm behind the mid-commissural point, 8 mm below
this point and 2 mm lateral from the midline [34].
A rigid cannula is inserted through a precoronaric paramedian burr hole
and positioned up to 10 mm from the target. This cannula is used both as a guide
for microrecording (Lead Point Medtronic, Minneapolis, Minn., USA) and for
the placement of the definitive electrode (DBS-3389, Medtronic, Minneapolis,
Minn., USA). Macrostimulation (1–7 V, 60 ␮s, 180 Hz) is used to evaluate poten-
tial side effects. All patients subjected to stimulus intensities higher than 4 V have
shown ocular deviation that was followed by verbal reports of extreme propor-
tions (‘I feel near to death’; ‘I am at the edge of the end’, etc). When other side
effects could be ruled out at standard parameters of stimulation, the guiding
cannula was then removed and the electrode was secured to the skull with
microplates. The extension was then connected to the electrode, tunneled, and
brought out percutaneously for subsequent trial stimulations.
On the day following surgery, an additional MR study is done for the pur-
pose of checking the electrode position. After 3–15 days of trial stimulation, the
electrodes are then connected to a pulse generator (Itrel II, Medtronic) posi-
tioned subcutaneously into the subclavicular area. The following parameters of
chronic stimulation have been employed: frequency 180 Hz and a 60-␮s pulse
width, with gradually increasing amplitude values [16, 17].
Clinical Results of Hypothalamic DBS
The results of this study are presented in table 3. All the patients have
achieved near-complete or complete pain relief, as a result of the long-term
high-frequency hypothalamic stimulation that was continued in the follow-up
evaluations [35]. Eight out of the 13 implanted patients have remained pain
free/almost pain free without any medication (table 3), while 5 needed low doses
of methysergide or verapamil to be pain free/almost pain free. It should be
noted that these same drugs had been completely ineffective prior to the operative
procedure. We observed no noxious side effects from chronic high-frequency
hypothalamic stimulation nor did we observe any acute complications from the
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Table 3. Chronic intractable cluster headache sufferers who received hypothalamic implantation
Pt. no and Age Chronic Side Implant. Date of Amplitude Drugs

gender CH since date improvement (V)
Pt. 1 M a) 39 1997 Left July 14, 2000 Pain free 1 month later 3.0 None
Pt. 1 M b) 40 Right May 31, 2001 Immediately pain free 0.5
Pt. 2 M 50 1997 Left November 17, Pain free 2 months later. 1.1 Methysergide
2000 Occasional attacks in the 3–4.5 mg/day
last 9 months
Pt. 3 F 63 1994 Left May 22, 2001 Pain free 2 months later 3.0 Verapamil
80–360 mg/day
Pt. 4 M 52 1997 Right October 11, Pain free 4 months later. 3.1 Methysergide
2001 July 2002 electrode 4.5 mg/day
replacement
Pt. 5 M 30 2000 Left March 22, Pain free 2 months later 1.4 None
2002
Pt. 6 M 46 2000 Left May 31, 2002 Pain free 1 month later 2.8 Verapamil
360 mg/day
Pt. 7 F a) 27 2001 Left September 12, Pain free 1 month later 2.0 None
2002
Pt. 7 F b) 27 2002 Right January 9, Pain free 5 months later 1.3
2003
Pt. 8 M 25 2001 Right July 10, 2003 2 brief attacks in the last 2.1 None
30 days
Pt. 9 M 43 2000 Left July 29, 2003 2 attacks/day, very brief 2.5 None
duration and intensity:
no sumatriptan need!
Pt. 10 M 46 2001 Right July 30, 2003 Pain free 3 weeks after 1.5 Verapamil
360 mg/day
Pt. 11 M 50 2001 Right August 26, 3 attacks per week 2.0 None
2003
Pt. 12 M 36 2000 Left September 25, From 10 attacks/day to 2.7 None
2003 2/day (decreasing
frequency and pain
intensity)
Pt. 13 M 24 2001 Left October 15, From 10–12 attacks/day 2.1 None
2003 to 3/day (decreasing
frequency and pain
intensity)
implant procedure [35]. Patient 1 presented with some signs of mild hypersexual
and hyperphagic behavior prior to the operation, which seemed to be resolved
by stimulation [17]. In fact, this patient showed a 25-kg weight reduction at the
18 months follow-up.
Discussion
Less than 20% of CHs have a chronic course and about 20% of the chronic
forms become drug resistant. In such cases, a surgical option has to be consid-
ered. Until a few years ago, surgical treatment of CH was not done, due to limited
knowledge about the pathophysiology of the disease, and was essentially based
on the interruption of the autonomic pathways [36–40] (i.e., greater superficial
petrosal nerve, intermedius nerve section, sphenopalatine ganglion lesions)
and/or on a partial or total trigeminal lesion [29–31, 41–46] (i.e., thermal rhi-
zotomy, glycerolysis, direct nerve sectioning, peripheral avulsions). There
appears, however, to be a direct relationship between sensory deficit and sub-
sequent discomfort with facial numbness, keratitis, dysesthesias and sometimes
anesthesia dolorosa and success rate using classical surgical approaches. In
addition to these troubling side effects, the recurrence rate of CH remains high
[29–31, 41–46] and even a complete trigeminal deafferentiation can be followed
by the persistence of attacks of CH [18]. Microvascular decompression of the
trigeminal nerve could obtain pain relief without lesioning nervous structures
but unfortunately, the long-term results of these procedures is unsatisfying [26].
For many years CH was considered and treated as having a peripheral
vasogenic origin. However, the striking circadian and circannual rhythmicity of
the disease indicated that the hypothalamus was probably involved in the patho-
genesis of CH. The recent functional PET [13, 14] and morphological, voxel-
based morphometry MRI [15] studies shed light on a new pathophysiological
process to explain CH in which the posterior inferior hypothalamic gray matter
could be the cluster generator [13]. If a central dysfunction involving hypothal-
amic circuitry is linked to CH, it seems reasonable to question whether surgical
strategies may be used to rebalance the unbalanced or disturbed circuits.
According to the current models of basal ganglia circuitry, the akinetic and
rigid symptoms of Parkinson’s disease result from the hyperactivity of the
globus pallidus internus and substantia nigra pars reticulata, as a consequence
of an increased glutamatergic drive from a disinhibited subthalamic nucleus.
Although the precise mechanisms of high-frequency DBS remain unknown, the
therapeutic effect found after long-term high-frequency DBS in Parkinson’s
disease seems to be a result of the inhibitory effect of current delivery to subthal-
amic nucleus hyperactive neurons [47]. It is possible to suggest that a similar
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mechanism may explain DBS effectiveness in relieving chronic CH. In fact, the
observed increased blood flow at the hypothalamic level observed during
CH attacks may originate from an increased neuronal activity at that level.
Notwithstanding the hypothesis that hypothalamic DBS rebalances the hyper-
active hypothalamus (accounting for the therapeutic effect in our patients), a
more generic analgesic effect coming from an activation of pain-modulating
pathway, such as the one involving the release of endogenous opiates, cannot be
excluded at the present stage. Future work will look at the effects of DBS on
the mechanisms of pain.
Over 30 years ago, other authors targeted the hypothalamus to relieve painful
conditions [48–50]. Surgical procedures on the posteromedial hypothalamus
were published by Sano and colleagues [49, 50] in the 1970s to treat otherwise
untreatable facial pain and behavioral disorders such as violence and aggres-
sion, in the days when so-called psychosurgery was in vogue. Intraoperative
high-frequency stimulation of the posteromedial hypothalamus produced anal-
gesic effects, autonomic responses such as hypertension, tachycardia, respira-
tory suppression, hyperpnea, tachypnea and mydriasis as well as somatomotor
responses. No such effects were observed in our series of CH patients, probably
because of differences in both the targeting and the stimulation parameters that
have been used [16, 17, 34, 35]. Now that the physiological basis of disorders
such as CH and other pain syndromes are able to be precisely measured with
new techniques such as PET, fMRI, and intraoperative microdialysis monitor-
ing, it is hoped that functional and restorative neurosurgery will re-emerge from
the discredited shadows of early 1960s psychosurgeries, much in the same
manner, that basal ganglia surgery has enjoyed a renaissance, since a decades-
long hiatus through the 1960s and 1970s until the pioneering work of Laitinen
and colleagues in the 1980s [51].
Conclusions and Future Directions in Hypothalamic DBS
In this chapter, we report the first large series of successfully treated chronic
CH sufferers using long-term high-frequency hypothalamic stimulation. These
results provide clear evidence that the hypothalamic stimulation offers a safe
and effective treatment for CH without any of the troublesome side effects asso-
ciated with peripheral nerve lesioning procedures. The rationale underlying
hypothalamic DBS in CH is based on more advanced morpho-functional stud-
ies pointing to the hypothalamus as the ‘CH generator.’ It is hypothesized that
the prolonged hypothalamic stimulation rebalances the genetic and cellular
mechanisms that leading to hyperfunctioning hypothalamic neurons. It should
be underscored that this is the first direct therapeutic application of neuroimaging
functional data in a primary headache syndrome, and such that the surgical
approach used is reversible (by turning off or altering current) in the event of
serious complications. Other conditions in which hypothalamic DBS may provide
a useful experimental model in the future include complex neuro-endocrine and
pain disorders.
References
1 Vonck K, Boon P, Achten E, De Reuck J, Caemaert J: Long-term amygdalohippocampal stimulation

for refractory temporal lobe epilepsy. Ann Neurol 2002;52:556–565.
2 Hodaie M, Wennberg RA, Dostrovsky JO, Lozano AM: Chronic anterior thalamus stimulation for
intractable epilepsy. Epilepsia 2002;43:603–608.
3 Benabid AL, Koudsie A, Benazzouz A, Vercueil L, Fraix V, Chabardes S, Lebas JF, Pollak P: Deep
brain stimulation of the corpus luysi (subthalamic nucleus) and other targets in Parkinson’s disease.
Extension to new indications such as dystonia and epilepsy. J Neurol 2001;248(suppl 3):III37–47.
4 Headache Classification Committee of The International Headache Society. The International
Classification of Headache Disorders, ed 2. Cephalalgia 2004;24:1–195.
5 D’Alessandro R, Gamberini G, Benassi G, Morganti G, Cortelli P, Lugaresi EI: Cluster headache
in the Republic of San Marino. Cephalalgia 1986;6:159–162.
6 Manzoni GC, Terzano MG, Bono G, Micieli G, Martucci N, Nappi G: Cluster headache – Clinical
findings in 180 patients. Cephalalgia 1983;3:21–30.
7 Kudrow L: The cyclic relationship of natural illumination to cluster period frequency. Cephalalgia
1987;7:76–78.
8 Goadsby PJ, Edvinsson L: Human in vivo evidence for trigeminovascular activation in cluster
headache: Neuropeptide changes and effects of acute attacks therapies. Brain 1994;117:427–434.
9 Kudrow L: Cluster Headache, Mechanism and Management, ed 1. New York, Oxford University
Press, 1980.
10 Waldenlind E, Gustafsson SA, Ekbom K, Wetterberg L: Circadian secretion of cortisol and mela-
tonin in cluster headache during active cluster periods and remission. J Neurol Neurosurg
Psychiatry 1987;50:207–213.
11 Leone M, Bussone G: A review of hormonal findings in cluster headache. Evidence for hypo-
thalamic involvement. Cephalalgia 1993;13:309–317.
12 Leone M, D’Amico D, Moschiano F, Fraschini F, Bussone G: Melatonin vs placebo in the
prophylaxis of cluster headache: A double-blind pilot study with parallel group. Cephalalgia 1996;
16:494–496.
13 May A, Bahra A, Büchel C, Frackowiak RS, Goadsby PJ: Hypothalamic activation in cluster
headache attacks. Lancet 1998;352:275–278.
14 Sprenger T, Boeker H, Toelle TR, Bussone G, May A, Leone M: Specific hypothalamic activation
during a spontaneous cluster headache attack. Neurology 2003;in press.
15 May A, Ashburner J, Buchel C, McGonigle DJ, Friston KJ, Frackowiak RS, Goadsby PJ: Correlation
between structural and functional changes in brain in an idiopathic headache syndrome. Nat Med
1999;5:836–838.
16 Leone M, Franzini A, Bussone G: Stereotactic stimulation of posterior hypothalamic gray matter
in a patient with intractable cluster headache. N Engl J Med 2001;345:1428–1429.
17 Leone M, Franzini A, Broggi G, May A, Bussone G: Long-term follow up of bilateral hypothala-
mic stimulation for intractable cluster headache. Brain 2004;in press.
18 Matharu MS, Goadsby PJ: Persistence of attacks of cluster headache after trigeminal nerve root
section. Brain 2002;125:976–984.
19 Dodick D, Rozen T, Goadsby P, Silberstein S: Cluster headache. Cephalalgia 2000;20:787–803.
20 Dodick DW, Capobianco DJ: Treatment and management of cluster headache. Curr Pain Headache
Rep 2001;5:83–91.
medwedi.ru
21 Ekbom K, Monstad I, Prusinski A, Cole JA, Pilgrim AJ, Noronha D: Subcutaneous sumatriptan
in the acute treatment of cluster headache: A dose comparison study. The Sumatriptan Cluster
Headache Study Group. Acta Neurol Scand 1993;88:63–69.
22 Leone M, D’Amico D, Frediani F, Moschiano F, Grazzi L, Attanasio A, Bussone G: Verapamil in
the prophylaxis of episodic cluster headache: A double-blind study vs. placebo. Neurology 2000;
54:1382–1385.
23 Bussone G, Leone M, Peccarisi C, Micieli G, Granella F, Magri M, Manzoni GC, Nappi G:
Double blind comparison of lithium and verapamil in cluster headache prophylaxis. Headache
1990;30:411–417.
24 Curran DA, Hinterburger H, Lance JW: Methysergide. Res Clin Stud Headache 1967;1:74–122.
25 May A, Leone M: Up to date on cluster headache. Curr Opin Neurol 2003;16:333–340.
26 Lovely TJ, Kotsiakis X, Jannetta PJ: The surgical management of chronic cluster headache.
Headache 1998;38:590–594.
27 Mathew NT, Hurt W: Percutaneous radiofrequency trigeminal gangliorhizolysis in intractable
cluster headache. Headache 1988;28:328–331.
28 Jarrar RG, Black DF, Dodick DW, Davis DH: Outcome of trigeminal nerve section in the treatment
of chronic cluster headache. Neurology 2003;60:1360–1362.
29 Taha Jm, Tew JM Jr: Long-term results of radiofrequency rhizotomy in the treatment of cluster
headache. Headache 1995;35:193–196.
30 Pieper DR, Dickerson J, Hassenbusch SJ: Percutaneous retrogasserian glycerol rhizolysis for treat-
ment of chronic intractable cluster headaches: Long-term results. Neurosurgery 2000;46:363–368.
31 Watson CP, Morley TP, Richardson JC, Schutz H, Tasker RR: The surgical treatment of chronic
cluster headache. Headache 1983;23:289–295.
32 May A, Bahra A, Büchel C, Frackowiak RS, Goadsby PJ: PET and MRA findings in cluster
headache and MRA in experimental pain. Neurology 2000;55:1328–1335.
33 Leone M, May A, Franzini A, Broggi G, Dodick D, Rapoport A, Goadsby PJ, Schoenen J,
Bonavita V, Bussone G: Deep brain stimulation for intractable chronic cluster headache: Proposals
for patient selection. Cephalalgia 2004;in press.
34 Franzini A, Ferroli P, Leone M, Broggi G: Stimulation of the posterior hypothalamus for treatment
of chronic intractable cluster headaches: First reported series. Neurosurgery 2003;52:1095–1099.
35 Leone M, Franzini A, D’Amico D, Grazzi L, Rigamonti A, Mea E, Broggi G, Bussone G: Long-term
follow-up of hypothalamic stimulation to relieve intractable chronic cluster headache. Neurology
2004;62(suppl 5):355.
36 Gardner WJ, Stowell A, Dutlinger R: Resection of the greater superficial petrosal nerve in the
treatment of unilateral headache. J Neurosurg 1947;4:105–114.
37 Sachs E Jr: Further observations on surgery of the nervus intermedius. Headache 1969;9:159–161.
38 Sachs E Jr: The Role of nervus intermedius in facial neuralgia: Report of four cases with obser-
vations on the pathways for taste, lacrimation and pain in the face. J Neurosurg 1968;28:54–60.
39 Stowell A: Physiologic mechanisms and treatment of histaminic or petrosal neuralgia. Headache
1970;9:187–194.
40 Sweet WH: Surgical treatment of chronic cluster headache. Headache 1988;28:669–670.
41 White JC, Sweet WH: Periodic migrainous neuralgia; in Pain and the Neurosurgeon: A Forty-Year
Experience. Springfield, Charles C Thomas, 1969, pp 345–434.
42 Wilkins RH, Morgenlander JC: Results of surgical treatment of cluster headache: Initial relief
followed by recurrence. Neurosurgery 1991;31:91–106.
43 Maxwell RE: Surgical control of chronic migrainous neuralgia by trigeminal gangliorhizolysis.
J Neurosurg 1982;57:459–466.
44 Morgenlander JC, Wilkins RH: Surgical treatment of cluster headache. J Neurosurg 1990;72:
866–871.
45 North RB, Kidd DH, Piantadosi S, Carson BS: Percutaneous retrogasserian glycerol rhizotomy:
Predictors of success and failure in treatment of trigeminal neuralgia. J Neurosurg 1990;72:
851–856.
46 O’Brien MD, MacCabe JJ: Trigeminal nerve section for unremitting migrainous neuralgia; in
Rose FC, Zilkha KJ (eds): Progress in Migraine Research. London, Pitman, 1981, vol 1, pp 185–187.
47 Benazzouz A, Gross C, Feger J, Boraud T, Bioulac B: Reversal of rigidity and improvement in
motor performance by subthalamic high-frequency stimulation in MPTP-treated monkeys. Eur J
Neurosci 1993;5:382–389.
48 Fairman D: Hypothalamotomy as a new perspective for alleviation of intractable pain and regres-
sion of metastatic malignant tumors; in Fusek I, Kunk Z (eds): Present Limits of Neurosurgery.
Prague, Avicenum, 1972, pp 525–528.
49 Sano K: Sedative neurosurgery with special reference to posteromedial hypothalamus. Neurol
Med Chir (Tokyo) 1962;4:112–142.
50 Sano K, Mayanagi Y, Sekino H, Ogashiwa M, Ishijima B: Results of stimulation and destruction
of the posterior hypothalamus in man. J Neurosurg 1970;33:689–707.
51 Laitinen LV: Brain targets in surgery for Parkinson’s disease. Results of a survey of neurosurgeons.
J Neurosurg 1985;62:349–351.
Massimo Leone, MD
Istituto Nazionale Neurologico Carlo Besta
Via Celoria 11, IT–20133 Milano (Italy)
E-Mail leone@istituto-besta.it
medwedi.ru
Molecular Mediators of Pain

Priya Chaudhary, Kim Burchiel
Department of Neurological Surgery, L472,
Oregon Health & Science University, Portland, Oreg., USA
Introduction: What Is Pain?
Pain is an universal, subjective and unpleasant sensation. Acute and sub-

chronic pain has a protective role as it warns of tissue damage. However, chronic
and severe pain offers no survival advantage and causes suffering. Particularly
in chronic pain, sensory processing from an affected region becomes abnormal
and innocuous stimuli (e.g., thermal, touch/pressure) that would normally not
cause pain may do so (i.e., allodynia) or noxious stimuli may elicit exaggerated
perceptions of pain (i.e., hyperalgesia). In addition, sensations similar to electric
tingling or shocks (i.e., paresthesias) and/or sensations having unpleasant qual-
ities (i.e., dysesthesias) may be elicited by normal stimuli.
Pain is initiated by specialized sensory nociceptors in the peripheral tissues
in response to noxious stimuli [1, 2]. The dorsal root ganglion (DRG) neurons
provide a site of communication between the periphery and the spinal cord.
Peripheral nociceptors have the machinery for encoding noxious stimuli into
action potentials. Central terminals mediate synaptic transmission as well as
presynaptic modulation. At the spinal cord level, pain impulses undergo substan-
tial modulation by local mechanisms and by projections from the supraspinal
structures (inhibition and facilitation). The processed signal is transmitted to the
brainstem and thalamic sites and finally to the cerebral cortex, where it elicits the
sensation of pain [3, 4].
Nociceptors are sensory nerve endings which respond to stimuli, which
threaten or are capable of causing tissue damage. In addition to activating cen-
tripetal discharge, the nociceptive stimuli cause primary afferent (sensory)
fibers to release endogenous chemicals. Cutaneous primary afferent nociceptor
fibers can be classified into three types, C, A␤ and A␦ based on their soma
diameter, structure and conduction velocity of their axon. Multiple classes of
Pathological condition
Nerve injury
Chemical and inflammatory mediators
Transcription Splicing Translation

DNA Pre-mRNA mRNA Protein
Alternative Stabilization
splicing destabilization
Post-translational
modifications like
glycosylation
Functional protein phosphorylation
(pain mediators) dephosphorylation
Fig. 1. Schematic demonstration of gene expression steps subjected to possible regu-

lation during pain [redrawn from 177].
C and A␦ exist with differing sensory properties. There are two main categories
of A␦ and C nociceptors: A␦ mechanical nociceptors and C-polymodal noci-
ceptors. A␦ mechanical nociceptors are activated by mechanical stimuli that
damage the tissue. C-polymodal nociceptors are capable of responding to
mechanical, thermal and chemical stimuli. The other nociceptor types include
A␦ mechanoheat nociceptors, A␦ and C cold nociceptors and C mechanical
nociceptors.
Acute pain corresponds to the activation of nociceptors with little inter-
vention from higher modulatory mechanisms. However, injury by physical,
chemical or immunological means also causes long-term alterations in the
expression levels of excitatory mediators, neuropeptides, neurotransmitters,
inhibitory neuromodulators, neurotrophic factors, peripheral terminal receptor
functions, and signal transduction molecules (fig. 1). These substances exert a
variety of actions on local tissue, vasculature, and the afferent fibers.
Acute nociceptive, inflammatory and neuropathic pain to some degree, all
depends on the activation of primary afferent neurons in the DRG and trigem-
inal ganglion. A variety of mediators are involved in the central transmission.
These substances, which are capable of altering the properties of nociceptors,
are broadly termed as modulators (fig. 2). In this chapter, we will describe var-
ious mediators like amines (e.g., histamine, serotonin), kinins (e.g., bradykinin;
BK), prostanoids (e.g., prostaglandins; PGs), cytokines (e.g., interleukins, tumor
necrosis factor; TNF), neuropeptides [e.g., substance P (SP), and calcitonin
gene related peptide (CGRP)], energy sources [e.g., adenosine triphospate (ATP)],
Molecular Mediators of Pain 285
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Inhibitory influences:
Opioids (␮, ␦, ␬) GABA (GABA B)
␣2-adrenoceptor (␣2c) Orphanin (ORL1)
Adenosine (A1) Somatostatin
Cannabinoids (CB1, CB2)
Immune cells
Mast cell
Platelets
Sensory neuron
Tissue injury
inflammation
Dorsal root
ganglion
Blood
Sensory nerve vessel Sympathetic
Spinal cord ending nerves
Excitatory influences:
Prostanoids (EP, IP) Adrenoceptor (␣2A)
Bradykinin (B1, B2) Glutamate (NMDA, AMPA, KA)
Histamine (H1) Acetylcholine (N)
Serotonin (5-HT1, 5-HT2, 5-HT3, 5-HT4) Adenosine (A2a, A3)
ATP (P2X3) Tachykinins (NK1, NK2)
TRPV1 Nerve growth factor (TrkA)
Sodium channels
Fig. 2. Excitatory and inhibitory influences on peripheral nerve activity by mediators

released by tissue injury and inflammation and by a variety of agents acting on neurorecep-
tors [178].
diffusible gas molecules (e.g., nitric oxide; NO), ions (e.g., H⫹, Na⫹, K⫹), and
neurotrophins [NT; e.g., nerve growth factor (NGF)]. Modulators such as opi-
oids, opioid receptors, cannabinoid receptors, and somatostatin are also
described (fig. 3, table 1).
Effective treatments for pain can be developed by understanding the cellu-
lar mechanisms, molecular mechanisms, and mediators that produce pain. This
chapter highlights the importance of pain mediators and modulators in devel-
oping novel approaches for the treatment of pain. Since gene therapy promises
to provide new, effective and innovative solutions for the treatment of pain, we
describe some aspects of how gene therapy can be used in pain treatment. We
have organized mediators and modulators into categories such as neurorecep-
tors, ion channels, excitatory receptors, inhibitory receptors, immune media-
tors, peptides, inflammatory mediators, growth factors and signal transduction
molecules, while recognizing that they work in concert to create the sensation
of pain.
Chaudhary/Burchiel 286
PG ATP Histamine 5-HT
EP3 P2Y2 H1 5-HT4/7
CGRP½ CGRP
BK B2 (B1)
H2 Histamine
SP NK1
A2A Adenosine
PLC
AC EP2/4 PG
Dorsal
horn [Ca2⫹]i
gK⫹ ␣2 NAD
NK1 SP
gCa2⫹ gNa⫹
NMDA 5-HT2A
EAA
5-HT
AMPA/Kainate 5-HT3
P2X2/ ASIC Capsaicin VDCC

P2X3
ATP Protons Vanilloids
Fig. 3. Roles of diverse receptors and intracellular signals in mediating pain at the
polymodal C fiber terminal [4].
Table 1. Receptors localized on primary afferent fibers and their ligands

from neuronal and non-neuronal origins [181]
Receptors associated with nociceptors

ATP, neurokinin-1, GABA, neuropeptide Y, acetylcholine, somatostatin,
prostaglandin E, cholecystokinin, adrenergic, 5-HT, glutamate, bradyinin,
noradrenaline, capsaicin, opioid, angiotensin II, adenosine (A1 and A2),
cannabinoid and menthol receptors
Ligands with non-neuronal sources
Acetylcholine, ATP, prostaglandin E, opioids, adenosine, glutamate,
bradykinin, noradrenaline, serotonin
Ligands in nociceptors
Substance P, opioid, ATP, adenosine, neuropeptide Y, glutamate,
cholecystokinin, somatostatin
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Neuronal Pain Receptors
Vanilloid (Capsaicin) Receptors: VR1/TRPV1

TRPV1 belongs to the transient receptor potential (TRP) family of ion
channels [5–7]. It is a nonspecific cation channel expressed preferentially in
small sensory neurons. TRPV1-expressing neurons are divided into two groups:
peptidergic, expressing SP and CGRP; and nonpeptidergic, expressing P2X3
purinoceptor. TRPV1 detects painful stimuli activated by heat and tissue acido-
sis, or H⫹ ions [8]. Endogenous substances released from activated immune
cells during inflammation also activate TRPV1 and lead to CGRP release. BK,
PGE2, and NGF are compounds released during inflammation and are also
known to modulate the activity of TRPV1 [9, 10]. Certain cannabinoids, which
are products of lipoxygenase pathways of arachidonic acid metabolism (e.g.,
12- or 15-hydroxyperoxy-eicosatetraenoic acid) and N-arachidonyl dopamine
are endogenous ligands, which activate TRPV1. TRPV1 and its ligands have
an important role in mediating inflammatory pain. TRPV1 knockout mice
have demonstrated that TRPV1 is important for inflammation-induced hyperal-
gesia [11, 12].
The natural vanilloid capsaicin, an ingredient of hot pepper, activates
TRPV1 and has been most commonly used to study the properties of TRPV1
receptors [5]. Most mechanistic studies of capsaicin-induced activation of noci-
ceptive neurons have been made using cultured sensory neurons and isolated
nerves in vitro. Capsaicin causes depolarization, during which there is an
increase in membrane permeability to cations (Ca2⫹, Na⫹). Vanilloids have a
biphasic response consisting of an initial excitatory response and a refractory
phase or desensitization [13]. Capsazepine, a competitive antagonist, and ruthe-
nium red, a noncompetitive antagonist, both block TRPV1. ATP, protein
kinase C (PKC), and protein kinase A (PKA) can also modulate the properties
of the TRPV1 channels. ATP acts as an allosteric factor and enhances the effect
of capsaicin on rat TRPV1 channels [14]. PKC can lead to the activation of cap-
saicin receptor even in the absence of ligands such as H⫹ or heat [15]. PKA
sensitizes the receptor to vanilloids and anandamide [16].
Analyses of mutations in TRPV1 have been the key to understanding the
function of this receptor. Site-directed mutagenesis experiments identified a
glutamic acid residue (Glu600) near the putative pore which is thought to
serve as a key regulatory site, setting the sensitivity to noxious stimuli in
response to changes in extracellular proton concentration [17]. Jordt et al.
[17] also showed that protons, vanilloids, and heat promote channel opening
through separate pathways, since mutations at E648 selectively abolish proton-
evoked channel activation without diminishing responses to other noxious
stimuli.
The example of anandamide, a cannabinoid that plays a dual role in the
body, serves to indicate the complexity of the vanilloid system. It can act as a
pro- or anti-inflammatory ligand depending on whether it activates TRPV1 or
cannabinoid receptor B1 (CB1). Anandamide acts as a full agonist of human
TRPV1 [18] and inhibits capsaicin-induced CGRP release in skin sensory
afferents and from the dorsal horn, possibly through the activation of CB1 [19,
20]. Understanding the functioning of the receptor (open and closed states) and
signal transduction pathways by which TRPV1 is activated could lead to the
identification of novel pain-relieving targets. Alternatively, down-regulation of
TRPV1 expression or factors promoting the closed state or desensitization of
the channel represents a promising therapeutic strategy for novel analgesic
drugs (table 1).
Cannabinoid Receptors
Cannabinoid and vanilloid receptors are colocalized in the primary
sensory neurons and the dorsal horn of the spinal cord [7]. CB1, CB2 are
G-protein coupled receptors (GPCRs). The CB1 receptors are expressed in
areas involved in modulation of nociception such as periaqueductal grey, spinal
cord dorsal horn, and the DRG. CB2 receptors are expressed in nonneuronal
cells such as mast cells and other immune cells. Behavioral tests indicate that
cannabinoids have anti-nociceptive effects in animal models of acute pain
and in persistent pain following peripheral inflammation [21] or nerve injury
[22]. It is now undisputable that cannabinoid receptor modulation has thera-
peutic value in anti-nociception, although concomitant modulatory activity of
dopaminergic systems may have adverse psychotropic effects.
Anandamide (an endo-cannabinoid) is formed from the hydrolysis of a phos-
pholipid precursor catalyzed by a phospholipase D and is inactivated via reuptake
by anandamide membrane transporter (AMT) and enzymatic hydrolysis by fatty
acid amide hydrolase enzyme. Anandamide activates the CB1 receptor in the brain
and also acts as a full agonist of TRPV1 [18, 23]. Since anandamide and capsaicin
share the same TRPV1-binding site, compounds which influence the activity of
AMT may facilitate the action of anandamide at the TRPV1. Activation of AMT
thus enhances the activity of anandamide at the TRPV1, and AMT inhibitors block
the anandamide activity. Since the anandamide-binding site on CB1 is extracellu-
lar, AMT could play an important role in distributing anandamide between the
intra- and extracellular compartments and activating TRPV1 or CB1 [24].
Cold Receptors
Mammals detect temperature effects with specialized neurons in the periph-
eral nervous system (PNS). Cold and menthol-sensitive receptor (TRPM8)
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belongs to the TRP family of excitatory ion channels and functions as a trans-
ducer of cold stimuli [25]. About 10% of trigeminal ganglion neurons express this
cold receptor, and thus it is still possible that cold excites the sensory neurons by
activating the cold and menthol-sensitive receptor as well as by modulating other
excitatory and inhibitory channels present on these neurons. TRPM8 was the first
molecule to be identified that responds to cold temperatures and stimulation with
menthol. ANKTM1 has recently been characterized as a cold-activated channel.
This channel has a lower activation temperature compared to the cold and men-
thol receptor, TRPM8. ANKTM1 shares little amino acid similarity with TRPM8.
ANKTM1 is found in a subset of nociceptive sensory neurons where it is coex-
pressed with TRPV1 (the capsaicin/heat receptor) but not TRPM8 [26].
Understanding the role of TRPM8 and ANKTM1 may help to identify a target for
therapeutic applications using cold receptors. Cold treatment is already used as a
method of relief from pain, due in part to its effect on inflammation. In some
cases, hypersensitivity to cold can lead to cold allodynia in patients suffering
from neuropathic pain, and this also constitutes a therapeutic rationale.
Proteinase-Activated Receptors
Proteinases like thrombin and trypsin not only act as degradative enzymes,
but also act as signaling molecules that regulate proteinase-activated receptors
(PAR) [27, 28]. Proteinases are released during inflammatory processes. Prote-
olytic cleavage of the extracellular amino terminus of PAR exposes a tethered
ligand domain, which acts as a receptor-activating ligand. Synthetic peptides
corresponding to this proteolytically revealed new N-terminal domain (PAR-
activating peptides) constitute selective agonists for these receptors. The PAR
receptor family is known to have four members PAR1, PAR2, PAR3 and PAR4
which are all G-protein coupled [29]. PARs are expressed on endothelium,
platelets, inflammatory cells, fibroblasts and nociceptive primary afferents [30].
Several studies suggest that these receptors might be mediators of neuro-
genic inflammation and may cause nociception. PAR agonists produce thermal
and mechanical hyperalgesia, which is diminished in mice lacking the NK-1
receptor [27, 28]. In the DRG, more than half of the neurons expressing PAR2
also coexpress CGRP and SP, which play a role in vasodilatation and inflam-
matory responses. Thus, proteases and PARs may play a previously unknown
novel role in pain. This may have a potential for developing therapeutic targets
in inflammation and pain.
Adrenoceptors
Adrenoceptors (ARs) mediate some of the main actions of the natural
catecholamines, epinephrine and norepinephrine. ARs include ␣1, ␣2, ␤1, ␤2
and ␤3. ARs are members of the much larger family of GPCRs, which include
muscarinic cholinergic receptors, serotonin receptors, dopamine receptors, neu-
rokinin receptors as well as the photoreceptor rhodopsin. ARs are distributed at
sites, which are associated with the transfer of sensory, nociceptive informa-
tion. For example ␣2 AR subtypes have been found in the DRG, superficial lam-
inae of the spinal cord, and the thalamic nuclei. Different subtypes of ␣2 ARs
play a role either in anti-nociception or have been implicated in causing hyper-
algesia. Clonidine is an ␣2-adrenergic agonist that has been used as analgesic
agent to control severe, acute and chronic pain conditions following epidural or
spinal administration. Clonidine relieves hyperalgesia in patients with sympa-
thetically maintained pain but has no effect on sympathetically independent
pain. Clonidine not only produces significant analgesia on its own but also
potentiates the analgesia produced by opiates [31]. More research is needed to
understand the roles played by individual AR subtypes.
Cholinergic Receptors
Acetylcholine activates cholinergic receptors, both muscarinic acetyl-
choline receptors and nicotinic acetylcholine receptors (nAChR). At least five
primary mACh receptor subtypes are known (M1–M5). They are GPCRs. M1,
M3, and M5 mediate their effects through increases in intracellular calcium,
whereas M2 and M4 mediate their effects through decreases in cAMP produc-
tion. Nicotinic receptors on the other hand are ligand-gated channels and at
least 11 nAChR subunits ␣2–␣9 and ␤2–␤4 have been identified. The activation
of neuronal nAChR produces significant increases in intracellular Ca2⫹ and
may play a role in cellular signaling. These receptors are known to produce
spinal and supraspinal analgesia. The central anti-nociceptive effects of nico-
tine, a neuronal nAChR agonist, have been known for many years. Epibatidine
is the most potent known agonist at several nicotinic receptor subtypes and
mediates anti-nociceptive effects. However, it is too toxic for use in humans
[32]. Only recently a potent nAChR agonist, ABT-594 was shown to have anti-
nociceptive properties equal in efficacy to those of morphine [33].
Ion-Gated Channels
ATP-Gated Ion Channels

Micromolar concentrations of extracellular ATP (nucleotide) activate sensory
neurons via ATP-gated ion channels, cell surface receptors known as P2 receptors
[34]. P2 receptors are classified into two categories: the P2X family consisting of
ligand-gated cation channels and the P2Y family made up of the GPCRs [35].
Seven subtypes of P2X and eight subtypes of P2Y family have been identified
[36]. P2X receptors are found as homomultimeric or heteromultimeric channels.
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P2X3 is expressed selectively by nociceptors in the DRG, predominantly
on the nonpeptidergic neurons [37]. DRG neurons respond to ATP by an
increase in the free intracellular calcium or by depolarization. P2X3 immunore-
active terminals have also been detected in the lamina II of the dorsal horn. P2Y
receptors are expressed on large DRGs and produce action potentials by light
touch. These observations suggest that P2 receptors play a role in signal trans-
duction of pain from the periphery to the spinal cord.
P2X3 receptors are down-regulated following peripheral nerve injury (e.g.,
sciatic nerve cut) and their expression can be regulated by glial cell-derived
neurotrophic factor (GDNF) [37]. In contrast to the sciatic nerve cut example,
the P2X3 receptor is up-regulated in the trigeminal ganglion after nerve injury
[38]. In human embryonic kidney cells expressing the P2X2 homomer or
P2X2/P2X3 heteromer, acidification (pH ⬍ 6.3) increased the ATP-induced
current [39]. Since inflammation causes a decrease in the tissue pH, these ATP
receptors may play an important role in inflammatory pain. Suramin and
PPADS are nonspecific blockers of the P2X1, P2X2, P2X3, P2X5 receptors at
micromolar concentrations and other P2X receptors at higher concentrations
[40]. The more specific inhibitor Trinitrophenyl-ATP, selectively inhibits P2X1,
P2X3 receptors and heteromeric channels that contain one of these receptors
subunits [41]. Thus, the P2X antagonist at the sensory terminal may help in
reducing pain caused due to inflammation.
Adenosine Receptors
Adenosine and ATP influence pain transmission at peripheral and spinal
sites. Four adenosine receptor types have been cloned: the A1, A2a, A2b and A3
receptors. The A2a receptor is found in the large neuronal cells of the rat DRG.
At the peripheral nerve terminals adenosine A1 receptor activation causes anti-
nociception [42] and adenosine A2 receptor activation produces pronociception
[42]. Adenosine A3 receptor activation produces pain behaviors due to release
of histamine and 5-HT from mast cells and subsequent actions on the sensory
nerve terminal [43].
Acid-Sensing Ion Channels

Acid-sensing (proton-gated) ion channels (ASICs) use protonation for the
activation of ionic current suggesting the importance of pH regulation in the
normal functioning of the nervous system. Severe tissue acidosis that accom-
panies inflammation is painful and sensory neurons respond to acidic tissue pH
with increased firing. Proton-gated cation channels in sensory nerve endings
are thought to be responsible for the activation of nociceptive afferents by acid.
Members of the ASIC family include ASIC1a, splice variant ASIC1b (BNC2),
ASIC2a (MDEG1, BNC1), ASIC2b (MDEG2), ASIC3 (DRASIC- dorsal root
ASIC), and ASIC4 [44]. These ion channels can form homo- or heteromulti-
mers with other ASICs and are expressed widely in the nervous system. These
channels are sensitive to amiloride at high concentrations and are selective for
Na⫹. No specific blockers for these individual channels have been suggested.
Only recently, psalmotoxin 1 was used to inhibit currents through ASIC1a [45].
The discovery of blockers for these channels is important to evaluate the role
played by these channels in nociception.
Potassium Channels
K⫹ channels form the largest family of ion channels. The common feature
of all K⫹ channels is the presence of a conserved motif called the P domain.
The 2P domain, leak/background K⫹ channels are non-voltage-gated channels.
These background channels are widely distributed in the nervous system. The
2P K⫹ channels play an essential role in setting the neuronal membrane poten-
tial and in tuning the action potential duration. They are represented by TWIK-1,
TWIK-2 (weak inward rectifiers), TREK-1, TREK-2 (Twik-related K⫹ chan-
nel), TRAAK (TWIK-related arachidonic acid-stimulated K⫹; lipid-sensitive
mechano-gated K⫹ channels) and TASK-1, TASK-2, TASK-3 (TWIK-related
acid sensing K⫹ channel; acid-sensitive outward rectifiers), [46, 47].
The TREK-1, TREK-2 and TRAAK channel activity is elicited by increasing
mechanical pressure. These channels are also reversibly opened by polyunsatu-
rated fatty acids including arachidonic acid. TREK-1 is opened by intracellular
acidosis, membrane stretch, cell swelling, arachidonic acid and heat [48, 49].
PGE2 and cAMP can close the channel by a PKA-mediated phosphorylation of
Ser333. Since TREK-1 is present in sensory neurons as well in the hypothalamus,
it is a good candidate as a temperature sensor [50]. TASK-1, TASK-2 and TASK-3
are sensitive to variations of extracellular pH in the physiological range. TASK-3
operates in the pathophysiological range of pH, closes at pH 6.0 and cytosolic
arachidonic acid (10 ␮M), which suggests that it may play a role in inflammation
[51]. The recent demonstration that TASK-1, TREK-1 and TREK-2 channels are
activated by inhalational general anesthetics, and that TRAAK is activated by the
neuroprotective agent riluzole, indicates that this novel class of K⫹ channels are
interesting targets for new therapeutic developments [52].
Sodium Channels
Na⫹ channels are important in electrogenesis within primary sensory neu-
rons. These channels are involved in multiple functions like transduction, signal
amplification, and genesis of action potentials [53]. There is growing evidence
that modulation of these currents/channels is an endogenous mechanism used to
control neuronal excitability [54]. Voltage-gated Na⫹ channels, which produce the
inward membrane current necessary for regenerative action potential production
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have emerged as targets in the study of pathophysiology of pain and in the search
for new pain therapies. Nine voltage gated Na⫹ channel ␣ subunits (Nav 1.1–1.9)
and three subunits (␤1–3) have been cloned [55]. Based on the sensitivity to
tetrodotoxin (TTX), these currents are divided into two types, TTX-sensitive
and -resistant channels. The TTX-sensitive channel is present in all sensory
neurons. TTX-resistant SNS/PN3/Nav1.8 and NaN/SNS2/Nav1.9 channels have
been detected in small diameter, unmyelinated sensory afferent neurons [2].
The ratio of these two types of Na⫹ channels can have a profound effect on
excitability. The slowly inactivating, rapidly repriming SNS/PN3/Nav1.8 channel
is the most likely candidate for the repetitive firing of the injured peripheral
nerve [56]. Several studies have demonstrated that TTX-resistant Na⫹ channels
can be modulated by inflammatory molecules such as PGs and serotonin through
the cAMP-PKA cascade. Down-regulation of TTX-resistant Na⫹ channels
(Nav1.8 and Nav1.9) and up-regulation of TTX-sensitive Type III Na⫹ channels
(Nav1.3) has been detected after nerve injury [57–60].
Local anesthetics such as lidocaine and mexiletine or anticonvulsants such
as carbamazepine and phenytoin have been used in the treatment of neuropathic
pain, although clinical performance has been hindered by a number of side
effects. There is interest in delineating mechanisms underlying membrane
excitability, action potential generation and transmission in nociceptive neurons
[54, 58]. Targeting sodium channels (especially the Nav1.8 and Nav1.9) in the
periphery could be a novel opportunity for producing analgesia without having
major side effects in the central nervous system (CNS).
Excitatory Receptors
Glutamate Receptors
Glutamate receptors play a key role in pain perception. Glutamate acts
through ionotropic glutamate receptors (iGluRs, coupled to ion channels) and
metabotropic glutamate receptors (mGluRs, coupled to intracellular secondary
messengers). Animal studies indicate that glutamate in the periphery plays an
important role in response to inflammatory agents such as intraplantar forma-
lin [61] and causes pain-related behaviors [62]. The nociceptive-specific pri-
mary afferent fibers are a source of peripheral glutamate [63].
Ionotropic glutamate receptors include those activated by ␣-amino-3-
hydroxy-5-methyl-4-isoxzolepropionic acid, N-methyl-D-aspartate (NMDA) and
kainate. The NMDA receptor (NMDA-R) is distinctive and unique. It acts as both
ligand and voltage-gated, and is selectively permeable to Ca2⫹ ions. As a conse-
quence, NMDA-R mediated alterations in intracellular Ca2⫹ levels regulate a vari-
ety of signaling pathways, ranging from localized, acute effects on receptor and
channels activities to long-term effects on nuclear gene transcription. The involve-
ment of peripheral NMDA-R in inflammatory nociception offers an attractive tar-
get for antagonists that do not cross the blood brain barrier. Such agents might be
powerful anti-nociceptive agents without having the CNS side effects [64].
There are eight cloned mGluRs (mGluR1–8), divided into three groups
based on sequence similarity, pharmacology and intracellular effector systems.
Group I consists of mGluR1 and mGluR5, group II is made up of mGluR2 and
mGluR3, and group III has mGluR4, mGluR6, mGluR7, mGluR8 [65]. The
mGluRs are activated upon the release of glutamate in the dorsal horn subse-
quent to the activation of sensory neurons. mGluRs are also activated in the
peripheral primary afferent terminals of sensory neurons in response to inflam-
matory stimulus and in experimental neuropathic pain elicited by ligation of
L5/L6 spinal nerves [66, 67]. Group I mGluRs act through the activation of
phospholipase C, which leads to the release of calcium from intracellular stores
and activation of PKC. mGluRs also activate other kinases that can modulate
the function of vanilloid receptors, TTX-resistant channels that have been
implicated in the production of pain.
The mGluRs contribute to nociceptive processes such as hyperalgesia since
receptor antagonists attenuate pain. mGluR5 antagonists [SIB-1757, 2-methyl-
6-(phenylethynyl)-pyridine (MPEP)] and mGluR1 receptor antagonists
[7-(hydroxyimino)cyclo-propa[b]chromen-1a-carboxylate ethyl ester (CPCCOEt),
2-methyl-4-carboxyphenylglycine (LY367385)], cause reversal of pain symp-
toms [66–68]. The reduction of hyperalgesia by mGluR antagonists is important
in designing drugs that could target the painful neuropathies or inflammatory
pain conditions. Elucidation of the underlying molecular mechanisms by which
the glutamate receptors enhance pain sensitivity, may lead to designing
inhibitors of glutamate release, selective glutamate receptor antagonists or the
inhibitors of intracellular glutamate-activated pathways [69–71].
Inhibitory Receptors
g-Amino-Butyric Acid Receptors

␥-amino-butyric acid (GABA) is a major inhibitory neurotransmitter
and acts via three receptor subtypes, GABAA, GABAB, and GABAC [72].
Endogenous peripheral GABA arises from primary afferent fibers (glutamate is
converted to GABA by glutamate decarboxylase). GABAA receptors, present
on some unmyelinated afferent axons [73] are, therefore, involved in modulat-
ing pain signaling.
The GABAmimetic agents have a broad spectrum of pharmacological
actions, including analgesia. Both directly acting (GABAA and GABAB
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agonists) and indirectly acting GABAergic agents (GABA uptake inhibitors
and GABA-transaminase inhibitors) produce analgesia [74]. Gabapentin, a
synthetic structural analog of GABA, given systemically, is clinically effective
in chronic neuropathic pain conditions [75, 76]. Pharmacological actions of
gabapentin are unclear. It is known to interact with an auxiliary subunit of
voltage-sensitive Ca2⫹ channel and modulation of GABA and glutamate
synthesis [77].
Immune Mediators
Cytokines
The role of neuroinflammation and neuroimmune activation in pain
involves the infiltration of immune cells to the site of injury (CNS or PNS) and
activation of endothelial cells, microglia, and astrocytes. Activation of these
cells leads to the production of cytokines and chemokines [78]. Proinflam-
matory cytokines like interleukin (IL-1, IL-6) and TNF have been implicated in
the genesis and maintenance of pain [79–83]. Proinflammatory cytokines can
exaggerate pain responses by directly acting on the cytokine receptors found on
neurons or by indirectly stimulating the release of other substances that could
act on neurons. Cytokines can cause neuronal hyperexcitability, via alterations
in ion channels. DRG neurons are known to express TNF receptor type I (TNF-
RI) and interleukin receptor I [84, 85]. Thus, DRG neuronal response during
pain is affected by the surrounding inflammatory cytokines. In parallel, anti-
inflammatory cytokines such as IL-4, IL-10, IL-13 and IL-1ra are produced and
reduce hyperalgesic effects of the proinflammatory cytokines that are initially
produced. Inflammatory pain, therefore, is the result of interplay between
hyperalgesic and analgesic mediators. Drugs such as immunosuppressants
influencing this interplay may also impair endogenous hyperalgesic and anal-
gesic mechanisms.
TNF␣, IL-1␣ and IL-1␤ are the first cytokines involved in Wallerian
degeneration. Indirectly, these cytokines further regulate macrophage recruit-
ment, myelin removal, survival of PNS neurons, regeneration, and pain through
the regulation of NGF production [86, 87]. Increased levels of spinal inter-
leukins have been detected following spinal nerve transection, L5 nerve root
injury [88], peripheral nerve injury, acute peripheral inflammation (formalin or
zymosan subcutaneous injections in the hind paw) [89], experimental traumatic
spinal cord injury in rats [90] and TNF␣ injection in the sciatic nerve [82]. The
role of cytokines in neuropathic pain is further demonstrated by the ability of
corticosteroids (immunosuppressants), thalidomide, and anti-inflammatory
cytokine IL-10 to alleviate neuropathic pain [91, 92].
Peptide Mediators: Opioids
Opioid Peptides
Dynorphins, enkephalins and ␤-endorphins (␤-EP) are the main groups of
opioid peptides. Recently, a novel group of endogenous opioid peptides have been
discovered in the brain and named endomorphins, including endomorphin-1 and
endomorphin-2 [93]. These opioid peptide-containing neurons have been found
in the thalamus, periaqueductal grey, cortex and spinal cord, regions involved in
nociceptive responses.
Opioids act through opioid receptors. Opioid receptors are found on the
primary afferent terminals, DRG and on immune cells. Three members of the
opioid receptor family cloned in the early 1990s include ␦-opioid receptor,
␮-opioid receptor and ␬-opioid receptor. Endomorphins have been shown to
induce analgesia via ␦-opioid receptors [94]. These three receptors belong to a
family of seven transmembrane GPCRs and share considerable homologies. In
addition to the well-established opioid receptors, an orphan opioid-like recep-
tor 1 has been cloned. Nociceptin, a novel opioid-like heptadecapeptide, is
believed to be the endogenous ligand for opioid-like receptor 1. The activation
of these receptors causes reduction in excitability and decreased propagation of
action potentials in the sensory neurons [95].
The role of opioids in the anti-nociceptive processes has been well docu-
mented for many centuries and opioids are arguably the earliest and most use-
ful medicines known to man. However, using opioids for chronic and
neuropathic pain remains somewhat controversial. Clinical evidence suggest
that neuropathic pain is not opioid resistant but that only reduced sensitivity to
systemic opioids is observed, i.e., an increase in opioid dose is needed to obtain
significant analgesia. This reduced efficiency may be due to changes in spinal
opioid receptors or signal transduction pathways [96]. The important problem
in administering chronic opioids to control pain is the development of tolerance
and dependence. Problems of tolerance are not observed, however, with periph-
erally applied opioids [97].
Nonopioid Peptides
SP
SP is one of the most intensively studied sensory neuropeptides, an unde-
capeptide belonging to the tachykinin peptide family, which includes SP and
neurokinin A/B. These peptides act through neurokinin receptors, NK1, NK2,
and NK3. A subpopulation of DRG neurons synthesizes and transports SP to
the spinal cord where it is released upon noxious stimulation. Released SP
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interacts with neurons of lamina I, which express the SP receptor (SPR or
neurokinin receptor). This receptor is expressed on spinothalamic and spino-
brachial neurons located in the lamina I, suggesting that these neurons play a
role in nociception. Increases in NK1 levels in the superficial laminae of the
dorsal horn have been detected in the sciatic nerve cut and inflammatory animal
models [98, 99]. Upon binding, SP and SPR are internalized [100]. This SP
induced internalization of SPR has been exploited as a means of entry into
spinal cord neurons in experimental models of pain treatment. SP was conju-
gated to the ribosome-inactivating protein saporin. This SP-conjugated neuro-
toxin resulted in the death of NK1 positive neurons, which led to the inhibition
of hyperalgesia [101]. The data suggest that a small population of SPR-expressing
neurons are important in the maintenance of hyperalgesia; however, the role of
a variety of other non-SPR receptors present on these cells should not be under-
estimated [102]. The novel approach of receptor internalization and introduc-
tion of therapeutic compounds may be of future use in targeting of spinal
neurons involved in transmitting chronic pain.
CGRP
Probably the most abundant neuropeptide in small sensory neurons, acti-
vation of sensory unmyelinated neurons by noxious stimuli evokes the release
CGRP from peripheral nerve endings. CGRP exerts its effects through CGRP1
and CGRP2 receptors, both of which are coupled to adenylyl cyclase.
Administration of neutralizing antibody to CGRP in the spinal cord produces
analgesia [103].
Neurotensin
Neurotensin (NT) is a brain-gut tridecapeptide with dual functionality. It
acts as a neurotransmitter/neuromodulator in the nervous system, and as a
paracrine and circulating hormone in the periphery. NT acts through three
receptors, NTS1, NTS2, and NTS3. NTS1 and NTS2 belong to a family of
GPCRs with seven transmembrane domains, whereas NTS3 is a single trans-
membrane domain protein. Most of the known peripheral and central effects of
NT are mediated through NTS1. NT receptors have been demonstrated on small
DRG neurons. Sciatic nerve transection causes a marked decrease in the
number of NT receptor mRNA-positive small neurons in DRGs, NT mRNA-
positive neurons in the dorsal horn, and NT-immunocreactive cell bodies and
fibers in laminae I-II. Thus, axotomy causes down regulation of several NT sys-
tems at the spinal level, suggesting that the possible effects of NT on primary
sensory neurons is attenuated after peripheral axotomy [104, 105]. NT admin-
istration into the CSF produces dose-related anti-nociceptive responses [106],
which may represent a possible NT-mediated approach to pain relief.
Somatostatin
Somatostatin or somatotrophin-release inhibiting factor was first isolated
as a 14-amino acid peptide that reduced the release of growth hormone from the
pituitary. Later, it was found to act as a neuromodulator in the mammalian
CNS. It is now known to have an inhibitory effect on nociceptors. Five somato-
statin receptor genes have been cloned, SST1–5 [107]. These receptors are
G-protein coupled with seven transmembrane domains, which interact with a
wide range of downstream signaling targets [108]. Experimental and clinical
data suggest that the SST2 receptor might be involved in nociceptive transmis-
sion at the central and peripheral sites. Thus, SST2-selective drugs may prove
to be important analgesics [109].
Neuropeptide Y
Neuropeptide Y (NPY) is a 36-amino acid peptide having diverse biological
activity. NPY was originally isolated from the mammalian brain tissue and its
three receptors (Y1, Y2, Y3) are relatively abundant in the brain and spinal cord
[110]. The anti-nociceptive property of NYP is due to the inhibition of SP release
from the primary afferent fibers [111]. Elevated levels of NPY are detected in the
spinal gray matter and the DRG after sciatic nerve transection.
Galanin
Galanin is a 29-amino acid peptide expressed in the DRG and spinal dorsal
horn interneurons and regulated by nerve injury and peripheral inflammation.
Three G-protein coupled galanin receptor subtypes have been identified:
GAL1–3. The role of galanin in pain processing at the spinal level appears to
be quite complex. It has been known to produce both facilitatory [112, 113] and
inhibitory [114] effects on nociceptive behaviors. This peptide is overexpressed
in sensory neurons following peripheral nerve damage. The precise role of the
peptide was unclear until the generation of a galanin-knockout mouse. Galanin
is now known to act as a neuromodulator and is also important in regeneration
[115]. Galanin influences pain processing at the dorsal horn level, particularly
via GAL1 (inhibitory) receptors on dorsal horn neurons in response to pain
arising from nerve injury (neuropathic pain). GAL1 receptor agonists could
perhaps be used to treat neuropathic pain [116]. A better understanding of the
role of galanin and its receptors may lead to potential therapeutic treatment
options.
Cholecystokinin
This peptide originally was isolated from mammalian gastrointestinal tract
and was later detected in brain. It is also present in primary sensory neurons.
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Cholecystokinin A and B (CCKA and CCKB) receptors are found in the
CNS and PNS. CCKB receptor is predominant in the brain, spinal cord and
DRG. The peptide and CCKB receptor levels increase after peripheral axotomy
[117, 118].
Chemical and Inflammatory Mediators
BK
Biologically active kinins, including bradykinin (BK) and kallidin, are
short-lived peptide mediators predominantly generated by the enzymatic
action of kallikreins on kininogen precursors [119]. Kinins are involved in
neurogenic inflammation through the activation of A␦ and C fibers. A diverse
spectrum of physiological and pathological actions attributed to local kinin
production is a consequence of the activation of GPCRs. Kinins act through
B1 and B2 receptors. B1 receptors are not normally expressed but are
expressed in pathological conditions and by the proinflammatory agents
such as lipopolysaccharides and cytokines. B2 receptors are expressed
constitutively in the PNS and CNS. Kinins also act partly through nonreceptor-
mediated release of histamine and 5-hydroxytryptamine (5-HT) from
mast cells and sensitize primary afferents through interactions with inflam-
matory mediators such as cytokines and PGs leading tohyperalgesia and
allodynia [120].
Various transduction pathways have been suggested for the effects of BK.
BK-mediated increase in membrane excitation (depolarization) reflects an
increase in cation membrane conductance due to Na⫹. This action involves the
production of diacyl glycerol, increase in intracellular Ca⫹⫹ and activation of
PKC. The increase in calcium levels could cause neuropeptide release, PG
synthesis and stimulation of NO synthase [121]. A new pathway for BK
effects is through the activation of capsaicin receptors via the production of
12-lipoxygenase metabolites [122].
Experiments have found B2 to have proalgesic actions on sensory neurons.
B2 receptor antagonists are effective in their anti-nociceptive properties. However,
given that B2 receptors play an important role in the control of physiological
processes such as the cardiovascular system, the blockade of B2 receptors has
many undesirable side effects. B1 receptors are activated under inflammatory
conditions and B1 receptor antagonists are thus being developed to control
inflammatory pain. Considering these facts and the widespread distribution
of kinin receptors in many tissues, it is no surprise that the therapeutic potential
of kinins and kinin receptor antagonists remains the focus of numerous
investigations.
PGs
Prostanoids include PGD2, PGE2, PGF2␣, PGI2 and thromboxane A2. The
enzyme PGH synthase (PGHS), also known as cyclooxygenase (COX) cat-
alyzes the synthesis of PG from arachidonic acid. Arachidonic acid is kept
esterified by enzymes until mobilized by phospholipases (PLA2). COX1 is the
constitutively active isoform generating PG required for cellular function.
COX2 generates huge amounts of PG under pathological conditions.
High concentrations of PGs contribute to the excitation of neurons through
the suppression of a K⫹ conductance and the increase in Na⫹ (and Ca2⫹)
conductance, which leads to an increase in neuropeptide release from C fiber
terminals. Prostanoids exert a variety of actions on various tissues and cells.
PGs act at peripheral sensory neurons and at central sites within the spinal cord
and brain to evoke hyperalgesia [123]. Of the many species of PG known, PGE2
and PGI2 are the major contributors to hyperalgesia.
There are at least 8 types and subtypes of the prostanoid receptors in
mouse and man: DP, EP1, EP2, EP3, EP4, FP, IP, TP [124]. The PG receptors
belong to a GPCR superfamily of seven-transmembrane spanning proteins.
Nonsteroidal anti-inflammatory drugs (NSAIDs; e.g., aspirin, indomethacin,
ibuprofen), block PGH synthase-derived PG synthesis and are commonly used
analgesics and anti-inflammatory agents [125]. Aspirin blocks substrate access
and orientation at the COX active site by covalently acetylating a serine
residue. The coxibs, e.g., celecoxib (Celebrex) and rofecoxib (Vioxx), are
newer selective COX-2 inhibitors that have been used clinically for managing
pain [126]. COX-1-derived ‘homeostatic’ PGs are not inhibited by the
coxibs. Second-generation coxibs, e.g., valdecoxib and etoricoxib, are under
development.
Serotonin (5-HT)
Serotonin or 5-HT is a neurotransmitter involved in various physiological
processes. There exist at least 14 subtypes of 5-HT receptors known to be
encoded by distinct genes. Splice variants of many of the subtypes have also
been identified resulting in the discovery of at least thirty distinct protein
products that recognize 5-HT as their physiological ligand [127]. The 5-HT
receptors have been divided into seven subfamilies by convention. The 5-HT1,
5-HT2, 5-HT4, 5-HT5, 5-HT6, and 5-HT7 receptors couple to G-proteins,
whereas the 5-HT3 receptors are 5-HT-gated ion channels [128].
Primary afferent fibers (C and A␦) are excited by 5-HT, which appears to
involve the activation of 5-HT3 receptors directly gating ion channels perme-
able to Na⫹ (and K⫹). Like the B2 receptors, 5-HT3 receptors are coupled to
PLC and initiate changes in the afferent fibers involving diacylglycerol-induced
activation of PKC and IP3-induced increases in intracellular Ca2⫹. 5-HT3
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receptor antagonists have anti-nociceptive properties in several inflammatory
pain models. The understanding of the serotonergic analgesic system will help
in the development of new nonopioid, nonaddictive analgesics.
Histamine
Histamine, a constituent of mast cells, can activate polymodal nociceptors
and release pain-related neuropeptides. Histamine generally elicits an itchy
sensation rather than pain; however, higher concentrations may induce pain.
Pharmacological studies have suggested that a subgroup of primary sensory
neurons is responsive to histamine via the H1 receptor, which is coupled to
PLC [129].
Growth Factors
NTs
NTs are molecules promoting the survival, growth and maintenance of
neurons. NGF, brain-derived neurotrophic factor, NT-3, NT4/5 are important
NTs (i.e., growth regulators) essential for the development and maintenance of
sensory neurons. NT receptor p75 (i.e., low affinity receptor capable of bind-
ing to all NTs) and a family of tyrosine kinases TrkA (i.e., binds NGF), TrkB
(i.e., binds brain-derived neurotrophic factor and NT4/5) and TrkC (i.e., binds
NT3) are located on adult sensory neurons. All the three Trk receptors show
discrete but partly overlapping distributions to subpopulations of primary sen-
sory neurons.
Peripheral nerve injury results in apoptosis of DRG neurons and down-
regulation of TrkA in DRG and spinal cord [130]. Administration of exogenous
NGF counteracts the degenerative changes in the NGF-responsive axotomized
neurons [130, 131]. Recent evidence suggests that NGF is a peripherally
produced mediator of some persistent inflammatory pain states. It has also been
demonstrated that administration of NGF produces thermal and mechanical
hyperalgesia [132].
GDNF
GDNF, a member of the transforming growth factor-␤ → (TGF-␤→)
superfamily, is a trophic factor with important effects on the primary sensory
neurons [133, 134]. GDNF mediates its actions through a multicomponent
receptor system composed of a glycosyl-phosphatidylinositol-linked protein
(designated GDNFR-␣ or GFR␣-1), a ligand-binding domain, and the trans-
membrane protein tyrosine kinase Ret, which acts as the signal-transducing
domain. About a third of the primary sensory neurons express Ret mRNA [135].
GDNF can both prevent and reverse signs of neuropathic pain. It reduces
ectopic discharges in damaged sensory neurons by normalization of expression
of sodium channel [57].
Vasoactive Intestinal Peptide (VIP) and Pituitary Adenylyl Cyclase

Activating Polypeptide (PACAP)
PACAP and VIP are members of the vasoactive intestinal peptide/ secretin/
glucagon family of peptides with neurotransmitter, neuroprotective, and neu-
rotrophic functions. PACAP is widely expressed in many central and peripheral
neurons [136], in trigeminal ganglion [137], in gastrointestinal tract, and
adrenal glands. It is expressed in two alternatively processed forms PACAP-27
and PACAP-38 and exerts its effects through three different receptors: PAC1
(previously called Type I PACAP receptor), VPAC1 (Type II), and VPAC2 (Type
III) [138]. These receptors belong to a family of seven-transmembrane GPCRs.
PAC1 receptors are coupled to both adenylate cyclase and phospholipase C
[139, 140], and VPAC receptors are mostly coupled to adenylyl cyclase. PAC1
and VPAC receptors play an important role in the transmission of sensory infor-
mation [141]. PACAP, vasoactive intestinal peptide, and other neuropeptides
like CCK and NPY and their receptors are up-regulated after nerve injury
[142, 143].
Other Mediators
Nitric Oxide (NO)

NO, a free radical gas acts as a messenger molecule and plays a role in
synaptic transmission both in the CNS and PNS. Immunohistochemical data
suggests that NO synthase, the enzyme that synthesizes NO from L-arginine,
is present in the CNS and PNS. Recent studies have suggested a role of NO
in nociceptive processing [144]. NO modulates spinal and sensory neuron
excitability through multiple mechanisms [145]. The activation of excitatory
amino acid receptors such as NMDA receptors causes intraneuronal elevation
of calcium, which stimulates NO synthase and production of NO [145, 146].
This formation of NO due to the activation of NMDA receptor indicates that
NO may act as a mediator of NMDA-induced nociceptive effects. NO has also
been implicated in the development of hyperexcitability resulting in hyperalge-
sia by increasing nociceptive transmitters at the central terminals. NO biosyn-
thesis inhibitors like NG-nitroarginine-L-methyl ester produce anti-nociceptive
effects.
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Signal Transduction
PKC
Activation of PKC has been implicated in the induction and/or main-
tenance of neuropathic pain behaviors [147–149]. Spinal cord administration
of GM1 ganglioside, an intracellular inhibitor of PKC translocation/activa-
tion, reverses both increased levels of membrane-bound PKC and pain-
related behaviors [150]. Many different groups have used nonspecific PKC
blockers, but these studies have not identified which of the ten isoforms of
PKC are involved in maintaining hyperalgesia. Malmberg et al. [151] created
a mouse lacking PKC gamma (PKC-␥). Mice that lacked PKC-␥ displayed
normal responses to acute pain stimuli, but they failed to develop a neuro-
pathic pain syndrome after partial sciatic nerve section. Thus, selective
inhibitors of PKC-␥ may help to alleviate nerve injury-induced neuropathic
pain states. Since acute pain responses in PKC-␥ null mice were not affected,
the added advantage of using selective PKC-␥ inhibitors is that the acute
pain responses, which have an important role in detecting injury, are left
untouched.
PKC-␧ has also been known to play a role in nociception. PKC-␧ is
activated by NGF and regulates the responses to NGF including activation of
extracellular signal-regulated kinases (ERK1, ERK2), isoforms of mitogen-
activated protein (MAP) kinases, and neurite outgrowth [152]. Studies on
PKC-␧ null mice indicate that PKC-␧ is required for the full expression of
carrageenan-induced hyperalgesia [153], suggesting a role in pain due to
inflammation. Inhibitors of PKC-␥ will help in reducing pain without affecting
normal nociceptive responses.
Gene Therapy for Pain?
The explosion in research into understanding the neural mechanisms

involved in pain has led to a search for more effective molecular treatment
options, compared to traditional pharmacological approaches. Establishing and
understanding various mediators and modulators involved in the pathophysiol-
ogy of pain will help in designing novel therapeutic agents. Gene therapy offers
a reasonable and physiological methodology to treat pain. Gene therapy can be
attempted at various levels: transcription, mRNA stability, and/or translation.
Gene transfer therapy to treat pain can focus on a combination of pharmaco-
logical, cellular and molecular approaches [154]. Pain therapy can be delivered
in the CNS or PNS, or both sites simultaneously. Targets in the PNS offer
advantages since pain information can be blocked before it reaches the spinal
Table 2. Examples of potential targets for gene therapy [156]
Target protein Classification
NK-1 (SP) receptor G-protein coupled receptor

Protein kinase C kinase
Vanilloid receptors nonselective cation channels
PN3/Nav1.8 sodium channel
Cannabinoids G-protein coupled receptors
Acetylcholine receptors G-protein coupled receptors
NMDA subtype of glutamate receptors ligand-gated ion channels
Table 3. Loss of function strategies

[182] Knock down or antisense technology
Small molecule inhibitors
Ribozymes
Aptamers
RNA interference (RNAi)
Inhibitory peptides
Antibodies
cord, and also because delivery in the PNS is not confounded by the problems
of CNS administration [155, 156] (table 2).
A molecule is a good target in the treatment of pain if it satisfies the fol-
lowing considerations: it should have a major role in pain sensation; interac-
tions with other molecules (agonists and antagonists) should have been studied
extensively; it should not disturb other normal physiological functions; and it
should be expressed in specific neuronal types so that the shut-off of therapy
is possible. Viral vectors, antisense oligonucleotide technology, and RNA
interference (RNAi) might all be useful techniques in controlling pain by
up-regulating anti-nociceptive and down-regulating pronociceptive targets
(table 3).
Viral Vectors
Viral-derived vectors have a natural ability to penetrate cells and deliver a
transgene into the host nucleus. The viral vector has the ability to attach, trans-
fer its genome and the transgene into the host, but is incapable of replication
(fig. 4). The most widely used viral vectors are derived from adenoviruses,
adeno-associated virus, herpes simplex viruses (HSV), or retroviruses. The
properties of various virus vector systems are described below (table 4). An
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36kb
Sequence Inverted Packaging Locus Ligand cDNA Poly A Heterologous Inverted

terminal sequence control response sequence terminal
repeats region element repeats
Function Viral Tissue Drug Therapeutic RNA Removal of

packaging specificity regulation gene processing viral genes
Fig. 4. Features of an optimized adenovirus gene therapy vector. Schematic diagram

of a gutted adenoviral vector with an adenoviral packaging sequence and terminal repeats
(ITR), containing a minimum of adenoviral genome sequences [179].
Table 4. Comparison of properties of various vector systems [183]
Features Retroviral Lentiviral Adenoviral AAV Naked/lipid

DNA
Maximum insert 7–7.5 kb 7–7.5 kb ⬃30 kb 3.5–4.0 kb Unlimited size

size
Concentrations ⬎108 ⬎108 ⬎1011 ⬎1012 No limitation
(viral particles
per ml)
Route of gene Ex vivo Ex/in vivo Ex/in vivo Ex/in vivo Ex/in vivo
delivery
Integration Yes Yes No Yes/No Very poor
Duration of Short Long Short Long Short
expression in vivo
Stability Good Not tested Good Good Very good
Ease of preparation Pilot scale-up, Not known Easy to Difficult to Easy to
(scale-up) up to 20–50 scale-up purify, difficult scale-up
liters to scale-up
Immunological Few Few Extensive Not known None
problems
Preexisting host Unlikely Unlikely, Yes Yes No
immunity except maybe
AIDS patients
Safety Insertional Insertional Inflammatory Inflammatory None
problems mutagenesis? mutagenesis? response, response, toxicity
toxicity
⫹ ⫹ Foreign
ITR E1 ITR ITR ITR
gene
LoxP LoxP Helper virus Vector

Cre-recombinase
expressing 293 cells
Reamplify in Cre-recombinase
expressing cells ⫹ Helper virus
⫺ E1 ⫹
ITR ITR ITR ITR
LoxP
Not
packaged Packaged
Fig. 5. The replication defective but still infective virus is dependent on the use of Cre-
recombinase expressing 293 cell line and a helper virus containing loxP-flanked packaging
sequence. The Cre-recombinase enzyme excises any segment of DNA flanked by a loxP
sequence (30 bp). Infection of the 293 cells with the helper virus with its ␺ sequence flanked
by loxP site results in excision of the viral packaging sequence, rendering the helper virus
DNA unpackagable. The helper virus provides all the functions for the packaging of the gut-
less virus [180].
ideal viral vector for gene therapy should be stable, have tissue-specific gene
expression, and should not elicit a host immune response. A recently described
helper-dependent gutless adenovirus is devoid of all viral genome. This vector
is designed by deletion of all of the viral genome except for the inverted
terminal repeat and the packaging (␺) sequence essential for viral packaging.
This virus can carry up to 30 kb of transgene (fig. 5), making it a useful size for
gene transfer. HSV, an enveloped double-stranded DNA virus, is another
commonly used gene transfer virus. The wild-type virus is responsible for the
common cold sore. The wild-type virus replicates initially in the skin or mucous
membranes and is then taken up by sensory nerve terminals. It can then estab-
lish a life long latent state in the nucleus of the sensory ganglion neurons. This
unique feature of the HSV can be exploited for applications directed towards
conditions that affect the PNS [157]. Peripheral inoculation of HSV vectors on
m e d w e d i . r u
abraded skin [158] or scarified cornea [159] allows the introduction of a trans-
gene to the sensory ganglia neurons without the need for other sophisticated
methods of viral vector delivery. Three weeks after infection with HSV encod-
ing rat preproenkephalin A via the hind footpads, strong expression of
preproenkephalin A mRNA was detected in the rat lumbar DRG [158, 160].
Wilson and Yeomans and colleagues [161, 162] used a similar approach and
demonstrated the anti-hyperalgesic effect of preproenkephalin overexpression
in primary sensory neurons on sensitization of sensory afferents by dimethyl-
sulfoxide or capsaicin application. Hao et al. [163] have used HSV vector-
mediated expression of proenkephalin in the DRG and demonstrated an
anti-allodynic effect in neuropathic pain. They also showed that the enkephalin
release enhances the effect of morphine, reducing ED50 of morphine 10-fold
and the animals also did not develop tolerance to the continued production of
vector-mediated enkephalin over a period of several weeks. Taken together, sev-
eral of the above studies suggest that viral vector-mediated expression of
proenkephalin may be a novel way to treat patients with neuropathic pain.
Production of site-specific peptides is of great interest in the field of gene
therapy, but these may require various modifications in order to facilitate secre-
tion or activity in vivo. Addition of N-terminal signal peptide is not always
sufficient to achieve this goal. To overcome this problem, addition of the prepro-
sequence of mouse nerve growth factor to ␤-EP was tested [164]. Retrovirus-
mediated expression of a hybrid construct of the preprosequence of NGF and
human ␤-EP in primary fibroblasts resulted in the secretion of ␤-EP.
Transplantation of such ␤-EP-secreting cells into the brain or spinal cord could
provide an ex vivo gene therapy approach for the treatment of chronic, opioid-
sensitive pain states [164].
Concerns about viral vector distribution in the CNS have limited current
gene therapy efforts (table 5). The problem of CNS distribution might be over-
come by transferring genes to the meninges surrounding the spinal cord. For
example, a recombinant adenovirus encoding a secreted form of ␤-EP was
delivered by intrathecal infusion and the resulting increase in ␤-EP secretion
by the meningeal piamater cells attenuated inflammatory hyperalgesia in a
carrageenan-injection model of persistent pain. This method can be adapted to
treat pain in neurodegenerative disorders in which broad spatial distribution of
therapeutic effect is critical [165].
Viral vectors have also been used to deliver antisense molecules to
control the expression of specific genes in vivo [166, 167]. Thus, viral vector
approaches can be used to treat chronic pain states in which plastic changes
occur in the neuronal systems. However, in spite of attempts in carefully design-
ing viral vectors, they have not been widely accepted for the use in the treat-
ment of pain.
Table 5. Comparison of viral vectors and antisense oligonucleotides [155]
Advantages Disadvantages
Viral vectors • effective delivery of large • immunogenicity prevents

exogenous DNA repeated administration
• longer duration of action of viral vector
• possibility of specific • direct cellular toxicity
targeting • inability to target
• effective in nondividing specific subset of cells
cells • difficulty in penetrating
blood brain barrier
• may cause insertional
mutagenesis (due to
integration of viral
vector into host genome)
• possibility of viral
replication
• possibility of creation
of a new recombinant
virus in vivo (for retrovirus)
ODN • specificity of gene • difficulty in generating
inhibition and isolating an active
• minimal immunogenicity oligomer
• effective in nondividing • difficulty in gaining
cells intracellular access
• not integrated into host • toxicity may be caused
genome by nonspecific effects
• short duration of action of the ODN
• difficulty in penetrating
blood brain barrier
Antisense Oligonucleotide Technology

Antisense oligonucleotides are complementary to a portion of target
mRNA [168] and have advantages over viral vectors (table 5). Binding of the
antisense molecule to target mRNA disturbs the ability of the mRNA to be read
by the translational machinery, and thus blocks the synthesis of the encoded
protein (fig. 6). Antisense oligonucleotides are modified to enhance entry into
cells and are made to be resistant to nucleases within the cell. Three regions in
a DNA sequence are considered best for standard antisense design; the 5⬘ cap
region, the AUG initiation codon, and the 3⬘ untranslated region of mRNA.
Most antisense molecules are 15–20 bases long. This length is sufficient to pick
out an unique sequence from others. The oligonucleotides enter the cells by
fluid phase pinocytosis, receptor-mediated endocytosis or both.
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Stimuli
Pain
Pronociceptive
protein
mRNA DNA mRNA DNA
Antisense oligo
Antisense oligo
Nucleus Nucleus
Normal gene activity Block of gene activity
Fig. 6. Antisense oligonucleotide and potential sites of action [155].
Antisense technology might be used to knock down targets involved in

nociception such as NMDA receptors, PKC, neurokinin 1 receptor, and sodium
channels [155] (table 6). Although this seems to be a feasible technology, there
are practical difficulties in designing a perfect oligomer with greatest
specificity and determining that the significant effects observed are due to the
antisense oligomer, and not due to other nonantisense effects.
RNAi
Post-transcriptional gene silencing and RNAi involve the specific suppres-
sion of genes by complementary dsRNA [169]. RNAi provides a powerful
method of gene silencing in eukaryotic cells. Specific genetic interference by
double-stranded RNA in Caenorhabditis elegans was first discovered by Fire
et al. [170] in 1998. Double-stranded RNA rather than single-stranded antisense
RNA is introduced within cells. Once inside the cell, the double-stranded RNA
molecules are cleaved by ribonuclease III into twenty-one to twenty-two
nucleotide short interfering RNAs which are replicated by an RNA-dependent
RNA polymerase. The short interfering RNA duplexes bind to a nuclease com-
plex to form the RNA-induced silencing complex, which then targets the homol-
ogous endogenous mRNA sequence, thus blocking further protein synthesis
[171]. However, not many experiments have been performed to determine the
utility of RNAi as a method of gene knockdown in postmitotic mammalian neu-
rons. Only recently, Krichevsky and Kosik [172] have applied the RNAi to post-
mitotic primary neuronal cultures. Thus, in the future one would potentially be
able to specifically block target molecules in DRG neurons to control pain.
Table 6. Effects of antisense oligonucleotide sequence in animal pain models [177]
mRNA Species Effects References
c-fos Rat ↓ c-fos protein immunoreactivity [184]

DOR Mice ↓ DOR protein but not mRNA and [185]
anti-nociception produced by DOR-selective
agonist (D-Ala2) deltorphin II
GAL Rat ↓ axotomy-induced upregulation of Gal [186]
protein, but no change in GAL mRNA
NK-1 R Rat ↓ behavioral response to formalin and NK-1 [187]
receptor protein immunoreactivity in
spinally SP-treated rats
Nav1.8 Rat ↓ SNS/PN3 protein immunoreactivity [188]
SNS/PN3 decreased in DRG and chronic nerve or [189]
tissue injury-induced hyperalgesia [190]
and allodynia
↓ TTX-R Na⫹ current density in cultured
sensory neurons and PGE2-induced
hyperalgesia
PKC ␣ Human Inhibition of phorbol ester-induced reduction [191]
(in vitro) of bradykinin-evoked calcium mobilization
NMDAR1 Mouse ↓ pain behavior and decreased receptor [192]
binding
Rat ↓ immunoreactive staining for NMDA-R1 [193]
and ↓ formalin-evoked behaviors
PSD- Rat Delayed onset of mechanical and thermal [194]
95/SAP90 hyperalgesia in chronic neuropathic
pain model
Other Gene Therapy Methods

Ribozymes are RNA molecules, which also act as enzymes. These cat-
alytic RNA molecules can be designed to recognize and bind to a specific
mRNA and cause cleavage of mRNA, thus preventing its translation into pro-
tein. While they represent an alternative to RNAi, achieving specificity and
delivery of these enzymes within the living tissue is difficult.
Neural stem cells are self-renewing precursors of neurons and glia with
numerous potential ex vivo gene therapy applications. The advantages of using
these precursors include their theoretically limitless clonal expansion in tissue
culture [173]. Neural progenitor cells could be genetically modified to express
exogenous genes for neurotransmitters, neurotrophic factors, or various ion
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channels. Other ex vivo transfected (iso- or xenogenic) cells can also be used to
treat nervous system disorders. Several drawbacks using such methods include
risk of toxicity, possible tumor formation, instability of transgenes, and lack of
cell specificity. In spite of these challenges, Ishii et al. [174] have successfully
used a combination of cell transplantation and gene transfer for the delivery of
␤-EP into the subarachanoid space in rats. The rats that received ␤-EP produc-
ing cells showed prominent analgesic effects for up to a month after transplan-
tation. Another study used xenogenic tumor cells secreting ␤-EP and
immunologically isolated in polymer capsules (microcapsules) to reduce pain
when transplanted into the CSF of rats [175].
Synthetic DNA delivery systems like liposomes have become increasingly
popular methods of gene transfer. Introduction of DNA into cells can now be
safely achieved by complexing DNA with cationic lipids. These complexes are
endocytosed into the cells, which involves binding, internalization, formation
of endosomes, fusion with lysosomes and lysis. Finally, the DNA which sur-
vives endocytotic processing and degradation by nucleases reaches the nucleus
[176]. Liposomes have been successfully used to inject DNA complexes into
rodent brains, but gene expression is transient. These methods could be used in
the treatment of various neurological diseases including the treatment of
intractable pain, where transient expression of the transgene is needed.
Future Directions in Pain Research
Pain sensation is complex and involves integrative mechanisms at the PNS

and CNS. Neuropathic pain is unresponsive to most conventional therapy. In
recent years, much has been elucidated concerning neuroanatomical circuits,
mediators of pain and transduction pathways involved in pain processing. This
information has led to the development of new and unconventional therapeutic
options for the treatment of pain. Ion channels (e.g., Nav1.8, Nav1.9), neuro-
transmitters, neuropeptides and their receptors present on pain-sensing neurons
are important potential targets for therapy. PKC and other kinases also offer as
targets for analgesic development.
Among gene therapy options, the use of antisense oligonucleotides seems
promising but delivery to specific cell types remains problematic. Viral vectors
are attractive candidates but safety and targeting issues remain. Modulation of
proteins involved in hyperalgesia by understanding the proteome will lead to
more effective therapies for pain relief. Focal drug delivery via microinfusion
systems may be necessary adjuncts to analgesic design. In the future, pain
management will be a multidisciplinary approach that will include pharmaco-
logical intervention, minimally invasive procedures, and gene therapy targeted to
specific cell types or specific neuron class with high efficacy and minimum side
effects. It is difficult to predict which of these approaches may lead to a clini-
cally applicable means of producing analgesia. What is certain is that each of
these therapies must be precisely regulated for optimal clinical effects with opti-
mal pharmacological specificity. However this field evolves, future analgesics
depend on a growing knowledge of the nociceptive system and its aberrations.
References
1 Waldmann R, Lazdunski M: H(⫹)-gated cation channels: Neuronal acid sensors in the NaC/DEG
family of ion channels. Curr Opin Neurobiol 1998;8:418–424.
2 McCleskey EW, Gold MS: Ion channels of nociception. Annu Rev Physiol 1999;61:835–856.
3 Basbaum AI, Fields HL: Endogenous pain control systems: Brainstem spinal pathways and endorphin
circuitry. Annu Rev Neurosci 1984;7:309–338.
4 Millan MJ: The induction of pain: An integrative review. Prog Neurobiol 1999;57:1–164.
5 Caterina MJ, Schumacher MA, Tominaga M, Rosen TA, Levine JD, Julius D: The capsaicin receptor:
A heat-activated ion channel in the pain pathway. Nature 1997;389:816–824.
6 Gunthorpe MJ, Benham CD, Randall A, Davis JB: The diversity in the vanilloid (TRPV) receptor
family of ion channels. Trends Pharmacol Sci 2002;23:183–191.
7 Ahluwalia J, Urban L, Capogna M, Bevan S, Nagy I: Cannabinoid 1 receptors are expressed in
nociceptive primary sensory neurons. Neuroscience 2000;100:685–688.
8 Tominaga M, Caterina MJ, Malmberg AB, Rosen TA, Gilbert H, Skinner K, Raumann BE,
Basbaum AI, Julius D: The cloned capsaicin receptor integrates multiple pain-producing stimuli.
Neuron 1998;21:531–543.
9 Reichling DB, Levine JD: Heat transduction in rat sensory neurons by calcium-dependent activa-
tion of a cation channel. Proc Natl Acad Sci USA 1997;94:7006–7011.
10 Cesare P, McNaughton P: A novel heat-activated current in nociceptive neurons and its sensitiza-
tion by bradykinin. Proc Natl Acad Sci USA 1996;93:15435–15439.
11 Caterina MJ, Leffler A, Malmberg AB, Martin WJ, Trafton J, Petersen-Zeitz KR, Koltzenburg M,
Basbaum AI, Julius D: Impaired nociception and pain sensation in mice lacking the capsaicin
receptor. Science 2000;288:306–313.
12 Davis JB, Gray J, Gunthorpe MJ, Hatcher JP, Davey PT, Overend P, Harries MH, Latcham J,
Clapham C, Atkinson K, Hughes SA, Rance K, Grau E, Harper AJ, Pugh PL, Rogers DC,
Bingham S, Randall A, Sheardown SA: Vanilloid receptor-1 is essential for inflammatory thermal
hyperalgesia. Nature 2000;405:183–187.
13 Szallasi A, Blumberg PM: Vanilloid (Capsaicin) receptors and mechanisms. Pharmacol Rev
1999;51:159–212.
14 Kwak J, Wang MH, Hwang SW, Kim TY, Lee SY, Oh U: Intracellular ATP increases capsaicin-
activated channel activity by interacting with nucleotide-binding domains. J Neurosci 2000;20:
8298–8304.
15 Premkumar LS, Ahern GP: Induction of vanilloid receptor channel activity by protein kinase C.
Nature 2000;408:985–990.
16 De Petrocellis L, Harrison S, Bisogno T, Tognetto M, Brandi I, Smith GD, Creminon C, Davis JB,
Geppetti P, Di Marzo V: The vanilloid receptor (VR1)-mediated effects of anandamide are
potently enhanced by the cAMP-dependent protein kinase. J Neurochem 2001;77:1660–1663.
17 Jordt SE, Tominaga M, Julius D: Acid potentiation of the capsaicin receptor determined by a key
extracellular site. Proc Natl Acad Sci USA 2000;97:8134–8139.
18 Smart D, Gunthorpe MJ, Jerman JC, Nasir S, Gray J, Muir AI, Chambers JK, Randall AD, Davis JB:
The endogenous lipid anandamide is a full agonist at the human vanilloid receptor (hVR1). Br J
Pharmacol 2000;129: 227–230.
medwedi.ru
19 Richardson JD, Kilo S, Hargreaves KM: Cannabinoids reduce hyperalgesia and inflammation via
interaction with peripheral CB1 receptors. Pain 1998;75:111–119.
20 Richardson JD, Aanonsen L, Hargreaves KM: Antihyperalgesic effects of spinal cannabinoids.
Eur J Pharmacol 1998;345:145–153.
21 Martin WJ, Loo CM, Basbaum AI: Spinal cannabinoids are anti-allodynic in rats with persistent
inflammation. Pain 1999;82:199–205.
22 Herzberg U, Eliav E, Bennett GJ, Kopin IJ: The analgesic effects of R(⫹)-WIN 55,212–2 mesy-
late, a high affinity cannabinoid agonist, in a rat model of neuropathic pain. Neurosci Lett 1997;
221:157–160.
23 Zygmunt PM, Petersson J, Andersson DA, Chuang H, Sorgard M, Di Marzo V, Julius D, Hogestatt
ED: Vanilloid receptors on sensory nerves mediate the vasodilator action of anandamide. Nature
1999;400: 452–457.
24 Di Marzo V, Blumberg PM, Szallasi A: Endovanilloid signaling in pain. Curr Opin Neurobiol
2002;12:372–379.
25 McKemy DD, Neuhausser WM, Julius D: Identification of a cold receptor reveals a general role
for TRP channels in thermosensation. Nature 2002;416:52–58.
26 Story GM, Peier AM, Reeve AJ, Eid SR, Mosbacher J, Hricik TR, Earley TJ, Hergarden AC,
Andersson DA, Hwang SW, McIntyre P, Jegla T, Bevan S, Patapoutian A: ANKTM1, a TRP-like
channel expressed in nociceptive neurons, is activated by cold temperatures. Cell 2003;112:
819–829.
27 Vergnolle N, Bunnett NW, Sharkey KA, Brussee V, Compton SJ, Grady EF, Cirino G, Gerard N,
Basbaum AI, Andrade-Gordon P, Hollenberg MD, Wallace JL: Proteinase-activated receptor-2 and
hyperalgesia: A novel pain pathway. Nat Med 2001;7:821–826.
28 Vergnolle N, Wallace JL, Bunnett NW, Hollenberg MD: Protease-activated receptors in inflam-
mation, neuronal signaling and pain. Trends Pharmacol Sci 2001;22:146–152.
29 Hollenberg MD: Protease-activated receptors: PAR4 and counting: How long is the course?
Trends Pharmacol Sci 1999;20:271–273.
30 Mantyh PW, Yaksh TL: Sensory neurons are PARtial to pain. Nat Med 2001;7:772–773.
31 Furst S: Transmitters involved in antinociception in the spinal cord. Brain Res Bull 1999;48:
129–141.
32 Flores CM: The promise and pitfalls of a nicotinic cholinergic approach to pain management. Pain
2000;88:1–6.
33 Bannon AW, Decker MW, Holladay MW, Curzon P, Donnelly-Roberts D, Puttfarcken PS, Bitner
RS, Diaz A, Dickenson AH, Porsolt RD, Williams M, Arneric SP: Broad-spectrum, non-opioid
analgesic activity by selective modulation of neuronal nicotinic acetylcholine receptors. Science
1998;279:77–81.
34 Burnstock G: A unifying purinergic hypothesis for the initiation of pain. Lancet 1996;347:
1604–1605.
35 Burnstock G: P2X receptors in sensory neurones. Br J Anaesth 2000;84:476–488.
36 North RA: Molecular physiology of P2X receptors. Physiol Rev 2002;82:1013–1067.
37 Bradbury EJ, Burnstock G, McMahon SB: The expression of P2X3 purinoreceptors in sensory
neurons: Effects of axotomy and glial-derived neurotrophic factor. Mol Cell Neurosci
1998;12:256–268.
38 Eriksson J, Bongenhielm U, Kidd E, Matthews B, Fried K: Distribution of P2X3 receptors in the
rat trigeminal ganglion after inferior alveolar nerve injury. Neurosci Lett 1998;254:37–40.
39 Stoop R, Surprenant A, North RA: Different sensitivities to pH of ATP-induced currents at four
cloned P2X receptors. J Neurophysiol 1997;78:1837–1840.
40 North RA, Barnard EA: Nucleotide receptors. Curr Opin Neurobiol 1997;7:346–357.
41 Thomas S, Virginio C, North RA, Surprenant A: The antagonist trinitrophenyl-ATP reveals
co-existence of distinct P2X receptor channels in rat nodose neurones. J Physiol 1998;509:411–417.
42 Taiwo YO, Levine JD: Direct cutaneous hyperalgesia induced by adenosine. Neuroscience 1990;
38:757–762.
43 Sawynok J: Adenosine receptor activation and nociception. Eur J Pharmacol 1998;347:1–11.
44 Waldmann R: Proton-gated cation channels – Neuronal acid sensors in the central and peripheral
nervous system. Adv Exp Med Biol 2001;502:293–304.
45 Escoubas P, De Weille JR, Lecoq A, Diochot S, Waldmann R, Champigny G, Moinier D, Menez A,
Lazdunski M: Isolation of a tarantula toxin specific for a class of proton-gated Na⫹ channels.
J Biol Chem 2000;275: 25116–25121.
46 Patel AJ, Honore E: Properties and modulation of mammalian 2P domain K⫹ channels. Trends
Neurosci 2001;24:339–346.
47 Yost CS: Potassium channels: Basic aspects, functional roles, and medical significance.
Anesthesiology 1999;90:1186–1203.
48 Patel AJ, Honore E, Maingret F, Lesage F, Fink M, Duprat F, Lazdunski M: A mammalian two pore
domain mechano-gated S-like K⫹ channel. Embo J 1998;17:4283–4290.
49 Maingret F, Patel AJ, Lesage F, Lazdunski M, Honore E: Mechano- or acid stimulation, two interac-
tive modes of activation of the TREK-1 potassium channel. J Biol Chem 1999;274:26691–26696.
50 Maingret F, Lauritzen I, Patel AJ, Heurteaux C, Reyes R, Lesage F, Lazdunski M, Honore E:
TREK-1 is a heat-activated background K(⫹) channel. Embo J 2000;19:2483–2491.
51 Kim Y, Bang H, Kim D: TASK-3, a new member of the tandem pore K(⫹) channel family. J Biol
Chem 2000;275:9340–9347.
52 Lesage F, Lazdunski M: Molecular and functional properties of two-pore-domain potassium chan-
nels. Am J Physiol Renal Physiol 2000;279:F793–F801.
53 Waxman SG, Dib-Hajj S, Cummins TR, Black JA: Sodium channels and pain. Proc Natl Acad Sci
USA 1999;96:7635–7639.
54 Gold MS, Reichling DB, Shuster MJ, Levine JD: Hyperalgesic agents increase a tetrodotoxin-
resistant Na⫹ current in nociceptors. Proc Natl Acad Sci USA 1996;93:1108–1112.
55 Baker MD, Wood JN: Involvement of Na⫹ channels in pain pathways. Trends Pharmacol Sci
2001;22:27–31.
56 Eglen RM, Hunter JC, Dray A: Ions in the fire: Recent ion-channel research and approaches to
pain therapy. Trends Pharmacol Sci 1999;20:337–342.
57 Boucher TJ, Okuse K, Bennett DL, Munson JB, Wood JN, McMahon SB: Potent analgesic effects
of GDNF in neuropathic pain states. Science 2000;290:124–127.
58 Cummins TR, Waxman SG: Downregulation of tetrodotoxin-resistant sodium currents and upreg-
ulation of a rapidly repriming tetrodotoxin-sensitive sodium current in small spinal sensory neu-
rons after nerve injury. J Neurosci 1997;17:3503–3514.
59 Dib-Hajj S, Black JA, Felts P, Waxman SG: Down-regulation of transcripts for Na channel alpha-
SNS in spinal sensory neurons following axotomy. Proc Natl Acad Sci USA 1996;93:14950–14954.
60 Dib-Hajj SD, Tyrrell L, Black JA, Waxman SG: NaN, a novel voltage-gated Na channel, is
expressed preferentially in peripheral sensory neurons and down-regulated after axotomy. Proc
Natl Acad Sci USA 1998;95:8963–8968.
61 Omote K, Kawamata T, Kawamata M, Namiki A: Formalin-induced release of excitatory amino
acids in the skin of the rat hindpaw. Brain Res 1998;787:161–164.
62 Carlton SM, Hargett GL, Coggeshall RE: Localization and activation of glutamate receptors in
unmyelinated axons of rat glabrous skin. Neurosci Lett 1995;197:25–28.
63 deGroot J, Zhou S, Carlton SM: Peripheral glutamate release in the hindpaw following low and
high intensity sciatic stimulation. Neuroreport 2000;11:497–502.
64 Parsons CG: NMDA receptors as targets for drug action in neuropathic pain. Eur J Pharmacol
2001;429:71–78.
65 Conn PJ, Pin JP: Pharmacology and functions of metabotropic glutamate receptors. Annu Rev
Pharmacol Toxicol 1997;37:205–237.
66 Dogrul A, Ossipov MH, Lai J, Malan TP Jr, Porreca F: Peripheral and spinal antihyperalgesic
activity of SIB-1757, a metabotropic glutamate receptor (mGLUR(5)) antagonist, in experimen-
tal neuropathic pain in rats. Neurosci Lett 2000;292:115–118.
67 Walker K, Reeve A, Bowes M, Winter J, Wotherspoon G, Davis A, Schmid P, Gasparini F, Kuhn R,
Urban L: mGlu5 receptors and nociceptive function. II. mGlu5 receptors functionally expressed on
peripheral sensory neurones mediate inflammatory hyperalgesia. Neuropharmacology 2001;40:9–10.
68 Bhave G, Karim F, Carlton SM, Gereau RW4th: Peripheral group I metabotropic glutamate recep-
tors modulate nociception in mice. Nat Neurosci 2001;4:417–423.
69 Carlton SM, Neugebauer V: Peripheral metabotropic glutamate receptors as drug targets for pain
relief. Expert Opin Ther Targets 2002;6:349–361.
medwedi.ru
70 Karim F, Bhave G, Gereau RW4th: Metabotropic glutamate receptors on peripheral sensory neu-
ron terminals as targets for the development of novel analgesics. Mol Psychiatry 2001;6:615–617.
71 Karim F, Wang CC, Gereau RW4th: Metabotropic glutamate receptor subtypes 1 and 5 are acti-
vators of extracellular signal-regulated kinase signaling required for inflammatory pain in mice.
J Neurosci 2001;21:3771–3779.
72 Malcangio M, Bowery NG: GABA and its receptors in the spinal cord. Trends Pharmacol Sci
1996;17:457–462.
73 Carlton SM, Zhou S, Coggeshall RE: Peripheral GABA(A) receptors: Evidence for peripheral
primary afferent depolarization. Neuroscience 1999;93:713–722.
74 Sawynok J: GABAergic mechanisms of analgesia: An update. Pharmacol Biochem Behav
1987;26:463–474.
75 Morello CM, Leckband SG, Stoner CP, Moorhouse DF, Sahagian GA: Randomized double-blind
study comparing the efficacy of gabapentin with amitriptyline on diabetic peripheral neuropathy
pain. Arch Intern Med 1999;159:1931–1937.
76 Mao J, Chen LL: Gabapentin in pain management. Anesth Analg 2000;91:680–687.
77 Taylor CP: Mechanisms of action of gabapentin. Rev Neurol (Paris) 1997;153(suppl 1):S39–S45.
78 DeLeo JA, Yezierski RP: The role of neuroinflammation and neuroimmune activation in persistent
pain. Pain 2001;90:1–6.
79 Ferreira SH, Lorenzetti BB, Bristow AF, Poole S: Interleukin-1 beta as a potent hyperalgesic agent
antagonized by a tripeptide analogue. Nature 1988;334:698–700.
80 Watkins LR, Martin D, Ulrich P, Tracey KJ, Maier SF: Evidence for the involvement of spinal cord
glia in subcutaneous formalin induced hyperalgesia in the rat. Pain 1997;71:225–235.
81 Watkins LR, Wiertelak EP, Goehler LE, Smith KP, Martin D, Maier SF: Characterization of
cytokine-induced hyperalgesia. Brain Res 1994;654:15–26.
82 Wagner R, Myers RR: Endoneurial injection of TNF-alpha produces neuropathic pain behaviors.
Neuroreport 1996;7:2897–2901.
83 Sorkin LS, Doom CM: Epineurial application of TNF elicits an acute mechanical hyperalgesia in
the awake rat. J Peripher Nerv Syst 2000;5:96–100.
84 Copray JC, Mantingh I, Brouwer N, Biber K, Kust BM, Liem RS, Huitinga I, Tilders FJ, Van Dam
AM, Boddeke HW: Expression of interleukin-1 beta in rat dorsal root ganglia. J Neuroimmunol
2001;118:203–211.
85 Schafers M, Geis C, Svensson CI, Luo ZD, Sommer C: Selective increase of tumour necrosis
factor-alpha in injured and spared myelinated primary afferents after chronic constrictive injury of
rat sciatic nerve. Eur J Neurosci 2003;17:791–804.
86 Shamash S, Reichert F, Rotshenker S: The cytokine network of Wallerian degeneration: Tumor
necrosis factor-alpha, interleukin-1alpha, and interleukin-1beta. J Neurosci 2002;22:3052–3060.
87 Lindholm D, Heumann R, Meyer M, Thoenen H: Interleukin-1 regulates synthesis of nerve growth
factor in non-neuronal cells of rat sciatic nerve. Nature 1987;330:658–659.
88 Winkelstein BA, Rutkowski MD, Sweitzer SM, Pahl JL, DeLeo JA: Nerve injury proximal or dis-
tal to the DRG induces similar spinal glial activation and selective cytokine expression but differ-
ential behavioral responses to pharmacologic treatment. J Comp Neurol 2001;439:127–139.
89 Sweitzer SM, Colburn RW, Rutkowski M, DeLeo JA: Acute peripheral inflammation induces
moderate glial activation and spinal IL-1beta expression that correlates with pain behavior in the
rat. Brain Res 1999;829:209–221.
90 Wang CX, Olschowka JA, Wrathall JR: Increase of interleukin-1beta mRNA and protein in the
spinal cord following experimental traumatic injury in the rat. Brain Res 1997;759:190–196.
91 Wagner R, Janjigian M, Myers RR: Anti-inflammatory interleukin-10 therapy in CCI neuropathy
decreases thermal hyperalgesia, macrophage recruitment, and endoneurial TNF-alpha expression.
Pain 1998;74:35–42.
92 Johansson A, Bennett GJ: Effect of local methylprednisolone on pain in a nerve injury model.
A pilot study. Reg Anesth 1997;22:59–65.
93 Zadina JE, Hackler L, Ge LJ, Kastin AJ: A potent and selective endogenous agonist for the
mu-opiate receptor. Nature 1997;386:499–502.
94 Przewlocki R, Labuz D, Mika J, Przewlocka B, Tomboly C, Toth G: Pain inhibition by endomor-
phins. Ann NY Acad Sci 1999;897:154–164.
95 Stein C, Machelska H, Binder W, Schafer M: Peripheral opioid analgesia. Curr Opin Pharmacol
2001;1:62–65.
96 Przewlocki R, Przewlocka B: Opioids in chronic pain. Eur J Pharmacol 2001;429:79–91.
97 Ueda H, Inoue M: Peripheral morphine analgesia resistant to tolerance in chronic morphine-
treated mice. Neurosci Lett 1999;266:105–108.
98 Yashpal K, Dam TV, Quirion R: Quantitative autoradiographic distribution of multiple neurokinin
binding sites in rat spinal cord. Brain Res 1990;506:259–266.
99 Yashpal K, Dam TV, Quirion R: Effects of dorsal rhizotomy on neurokinin receptor sub-types in
the rat spinal cord: A quantitative autoradiographic study. Brain Res 1991;552:240–247.
100 Mantyh PW, DeMaster E, Malhotra A, Ghilardi JR, Rogers SD, Mantyh CR, Liu H, Basbaum AI,
Vigna SR, Maggio JE: Receptor endocytosis and dendrite reshaping in spinal neurons after
somatosensory stimulation. Science 1995;268:1629–1632.
101 Mantyh PW, Rogers SD, Honore P, Allen BJ, Ghilardi JR, Li J, Daughters RS, Lappi DA, Wiley RG,
Simone DA: Inhibition of hyperalgesia by ablation of lamina I spinal neurons expressing the sub-
stance P receptor. Science 1997;278:275–279.
102 Levine JD, Fields HL, Basbaum AI: Peptides and the primary afferent nociceptor. J Neurosci
1993;13:2273–2286.
103 Kuraishi Y, Nanayama T, Ohno H, Minami M, Satoh M: Antinociception induced in rats by
intrathecal administration of antiserum against calcitonin gene-related peptide. Neurosci Lett
1988;92:325–329.
104 Zhang X, Xu ZQ, Bao L, Dagerlind A, Hokfelt T: Complementary distribution of receptors for
neurotensin and NPY in small neurons in rat lumbar DRGs and regulation of the receptors and
peptides after peripheral axotomy. J Neurosci 1995;15:2733–2747.
105 Zhang X, Ji RR, Nilsson S, Villar M, Ubink R, Ju G, Wiesenfeld-Hallin Z, Hokfelt T: Neuropeptide
Y and galanin binding sites in rat and monkey lumbar dorsal root ganglia and spinal cord and effect
of peripheral axotomy. Eur J Neurosci 1995;7:367–380.
106 Yaksh TL: Spinal opiate analgesia: Characteristics and principles of action. Pain 1981;11:293–346.
107 Epelbaum J, Dournaud P, Fodor M, Viollet C: The neurobiology of somatostatin. Crit Rev
Neurobiol 1994;8:25–44.
108 Patel YC: Molecular pharmacology of somatostatin receptor subtypes. J Endocrinol Invest
1997;20:348–367.
109 Selmer I, Schindler M, Allen JP, Humphrey PP, Emson PC: Advances in understanding neuronal
somatostatin receptors. Regul Pept 2000;90:1–18.
110 Wettstein JG, Earley B, Junien JL: Central nervous system pharmacology of neuropeptide Y.
Pharmacol Ther 1995;65:397–414.
111 Hua XY, Boublik JH, Spicer MA, Rivier JE, Brown MR, Yaksh TL: The antinociceptive effects of
spinally administered neuropeptide Y in the rat: Systematic studies on structure-activity relation-
ship. J Pharmacol Exp Ther 1991;258:243–248.
112 Cridland RA, Henry JL: Effects of intrathecal administration of neuropeptides on a spinal noci-
ceptive reflex in the rat: VIP, galanin, CGRP, TRH, somatostatin and angiotensin II. Neuropeptides
1988;11:23–32.
113 Wiesenfeld-Hallin Z, Villar MJ, Hokfelt T: Intrathecal galanin at low doses increases spinal reflex
excitability in rats more to thermal than mechanical stimuli. Exp Brain Res 1988;71:663–666.
114 Post C, Alari L, Hokfelt T: Intrathecal galanin increases the latency in the tail-flick and hot-plate
test in mouse. Acta Physiol Scand 1988;132:583–584.
115 Wynick D, Thompson SW, McMahon SB: The role of galanin as a multi-functional neuropeptide
in the nervous system. Curr Opin Pharmacol 2001;1:73–77.
116 Liu HX, Hokfelt T: The participation of galanin in pain processing at the spinal level. Trends
Pharmacol Sci 2002;23:468–474.
117 Zhang X, Dagerlind A, Elde RP, Castel MN, Broberger C, Wiesenfeld-Hallin Z, Hokfelt T:
Marked increase in cholecystokinin B receptor messenger RNA levels in rat dorsal root ganglia
after peripheral axotomy. Neuroscience 1993;57:227–233.
118 Verge VM, Wiesenfeld-Hallin Z, Hokfelt T: Cholecystokinin in mammalian primary sensory
neurons and spinal cord: In situ hybridization studies in rat and monkey. Eur J Neurosci 1993;
5:240–250.
medwedi.ru
119 Marceau F: Kinin B1 receptors: A review. Immunopharmacology 1995;30:1–26.
120 Calixto JB, Cabrini DA, Ferreira J, Campos MM: Kinins in pain and inflammation. Pain 2000;
87:1–5.
121 Dray A, Urban L: New pharmacological strategies for pain relief. Annu Rev Pharmacol Toxicol
1996;36:253–280.
122 Shin J, Cho H, Hwang SW, Jung J, Shin CY, Lee SY, Kim SH, Lee MG, Choi YH, Kim J,
Haber NA, Reichling DB, Khasar S, Levine JD, Oh U: Bradykinin-12-lipoxygenase-VR1 signal-
ing pathway for inflammatory hyperalgesia. Proc Natl Acad Sci USA 2002;99:10150–10155.
123 Narumiya S, FitzGerald GA: Genetic and pharmacological analysis of prostanoid receptor func-
tion. J Clin Invest 2001;108:25–30.
124 Narumiya S, Sugimoto Y, Ushikubi F: Prostanoid receptors: Structures, properties, and functions.
Physiol Rev 1999;79:1193–1226.
125 Funk CD: Prostaglandins and leukotrienes: Advances in eicosanoid biology. Science 2001;294:
1871–1875.
126 FitzGerald GA, Patrono C: The coxibs, selective inhibitors of cyclooxygenase-2. N Engl J Med
2001;345:433–442.
127 Raymond JR, Mukhin YV, Gelasco A, Turner J, Collinsworth G, Gettys TW, Grewal JS, Garnovskaya
MN: Multiplicity of mechanisms of serotonin receptor signal transduction. Pharmacol Ther 2001;
92:179–212.
128 Kroeze WK, Kristiansen K, Roth BL: Molecular biology of serotonin receptors structure and
function at the molecular level. Curr Top Med Chem 2002;2:507–528.
129 Hill SJ, Ganellin CR, Timmerman H, Schwartz JC, Shankley NP, Young JM, Schunack W, Levi R,
Haas HL: International Union of Pharmacology. XIII. Classification of histamine receptors.
Pharmacol Rev 1997;49:253–278.
130 Li L, Deng YS, Zhou XF: Downregulation of TrkA expression in primary sensory neurons after
unilateral lumbar spinal nerve transection and some rescuing effects of nerve growth factor infu-
sion. Neurosci Res 2000;38:183–191.
131 Verge VM, Gratto KA, Karchewski LA, Richardson PM: Neurotrophins and nerve injury in the
adult. Philos Trans R Soc Lond B Biol Sci 1996;351:423–430.
132 Lewin GR, Ritter AM, Mendell LM: Nerve growth factor-induced hyperalgesia in the neonatal
and adult rat. J Neurosci 1993;13:2136–2148.
133 Apfel SC: Neurotrophic factors and pain. Clin J Pain 2000;16(suppl 2):S7–S11.
134 Boucher TJ, McMahon SB: Neurotrophic factors and neuropathic pain. Curr Opin Pharmacol
2001;1:66–72.
135 Bennett DL, Michael GJ, Ramachandran N, Munson JB, Averill S, Yan Q, McMahon SB, Priestley
JV: A distinct subgroup of small DRG cells express GDNF receptor components and GDNF is
protective for these neurons after nerve injury. J Neurosci 1998;18:3059–3072.
136 Ghatei MA, Takahashi K, Suzuki Y, Gardiner J, Jones PM, Bloom SR: Distribution, molecular
characterization of pituitary adenylate cyclase-activating polypeptide and its precursor encoding
messenger RNA in human and rat tissues. J Endocrinol 1993;136:159–166.
137 Kausz M, Arimura A, Koves K: Distribution and somatotopical localization of pituitary adenylate
cyclase activating polypeptide (PACAP) in the trigeminal ganglion of cats and rats. Ann NY Acad
Sci 1998;865:529–532.
138 Harmar AJ, Arimura A, Gozes I, Journot L, Laburthe M, Pisegna JR, Rawlings SR, Robberecht P,
Said SI, Sreedharan SP, Wank SA, Waschek JA: International Union of Pharmacology. XVIII.
Nomenclature of receptors for vasoactive intestinal peptide and pituitary adenylate cyclase-
activating polypeptide. Pharmacol Rev 1998;50:265–270.
139 Spengler D, Waeber C, Pantaloni C, Holsboer F, Bockaert J, Seeburg PH, Journot L: Differential
signal transduction by five splice variants of the PACAP receptor. Nature 1993;365:170–175.
140 Vaudry D, Gonzalez BJ, Basille M, Yon L, Fournier A, Vaudry H: Pituitary adenylate cyclase-
activating polypeptide and its receptors: From structure to functions. Pharmacol Rev 2000;
52:269–324.
141 Dickinson T, Fleetwood-Walker SM: VIP and PACAP: Very important in pain? Trends Pharmacol
Sci 1999;20:324–329.
142 Fristad I, Heyeraas KJ, Kvinnsland IH: Neuropeptide Y expression in the trigeminal ganglion and
mandibular division of the trigeminal nerve after inferior alveolar nerve axotomy in young rats.
Exp Neurol 1996;142:276–286.
143 Noguchi K, Senba E, Morita Y, Sato M, Tohyama M: Prepro-VIP and preprotachykinin mRNAs
in the rat dorsal root ganglion cells following peripheral axotomy. Brain Res Mol Brain Res
1989;6:327–330.
144 Cizkova D, Lukacova N, Marsala M, Marsala J: Neuropathic pain is associated with alterations
of nitric oxide synthase immunoreactivity and catalytic activity in dorsal root ganglia and spinal
dorsal horn. Brain Res Bull 2002;58:161–171.
145 Luo ZD, Cizkova D: The role of nitric oxide in nociception. Curr Rev Pain 2000;4:459–466.
146 Riedel W, Neeck G: Nociception, pain, and antinociception: Current concepts. Z Rheumatol 2001;
60:404–415.
147 Palecek J, Paleckova V, Dougherty PM, Willis WD: The effect of phorbol esters on the responses
of primate spinothalamic neurons to mechanical and thermal stimuli. J Neurophysiol 1994;71:
529–537.
148 Mao J, Price DD, Phillips LL, Lu J, Mayer DJ: Increases in protein kinase C gamma immunore-
activity in the spinal cord dorsal horn of rats with painful mononeuropathy. Neurosci Lett 1995;
198:75–78.
149 Lin Q, Peng YB, Willis WD: Possible role of protein kinase C in the sensitization of primate
spinothalamic tract neurons. J Neurosci 1996;16:3026–3034.
150 Mao J, Price DD, Mayer DJ, Hayes RL: Pain-related increases in spinal cord membrane-bound
protein kinase C following peripheral nerve injury. Brain Res 1992;588:144–149.
151 Malmberg AB, Chen C, Tonegawa S, Basbaum AI: Preserved acute pain and reduced neuropathic
pain in mice lacking PKCgamma. Science 1997;278:279–283.
152 Hundle B, McMahon T, Dadgar J, Messing RO: Overexpression of epsilon-protein kinase C
enhances nerve growth factor-induced phosphorylation of mitogen-activated protein kinases and
neurite outgrowth. J Biol Chem 1995;270:30134–30140.
153 Khasar SG, Lin YH, Martin A, Dadgar J, McMahon T, Wang D, Hundle B, Aley KO, Isenberg W,
McCarter G, Green PG, Hodge CW, Levine JD, Messing RO: A novel nociceptor signaling path-
way revealed in protein kinase C epsilon mutant mice. Neuron 1999;24:253–260.
154 Eaton MJ: Emerging cell and molecular strategies for the study and treatment of painful periph-
eral neuropathies. J Peripher Nerv Syst 2000;5:59–74.
155 Wu CL, Garry MG, Zollo RA, Yang J: Gene therapy for the management of pain. I. Methods and
strategies. Anesthesiology 2001;94:1119–1132.
156 Wu CL, Garry MG, Zollo RA, Yang J: Gene therapy for the management of pain. II. Molecular
targets. Anesthesiology 2001;95:216–240.
157 Mata M, Glorioso JC, Fink DJ: Targeted gene delivery to the nervous system using herpes simplex
virus vectors. Physiol Behav 2002;77:483–488.
158 Antunes Bras JM, Epstein AL, Bourgoin S, Hamon M, Cesselin F, Pohl M: Herpes simplex virus
1-mediated transfer of preproenkephalin A in rat dorsal root ganglia. J Neurochem 1998;70:
1299–1303.
159 Shimeld C, Whiteland JL, Nicholls SM, Grinfeld E, Easty DL, Gao H, Hill TJ: Immune cell infil-
tration and persistence in the mouse trigeminal ganglion after infection of the cornea with herpes
simplex virus type 1. J Neuroimmunol 1995;61:7–16.
160 Pohl M, Braz J: Gene therapy of pain: Emerging strategies and future directions. Eur J Pharmacol
2001;429:39–48.
161 Wilson SP, Yeomans DC, Bender MA, Lu Y, Goins WF, Glorioso JC: Antihyperalgesic effects of
infection with a preproenkephalin-encoding herpes virus. Proc Natl Acad Sci USA 1999;96:
3211–3216.
162 Wilson SP, Yeomans DC: Virally mediated delivery of enkephalin and other neuropeptide trans-
genes in experimental pain models. Ann NY Acad Sci 2002;971:515–521.
163 Hao S, Mata M, Goins W, Glorioso JC, Fink DJ: Transgene-mediated enkephalin release enhances
the effect of morphine and evades tolerance to produce a sustained antiallodynic effect in neuro-
pathic pain. Pain 2003;102:135–142.
medwedi.ru
164 Beutler AS, Banck MS, Bach FW, Gage FH, Porreca F, Bilsky EJ, Yaksh TL: Retrovirus-mediated
expression of an artificial beta-endorphin precursor in primary fibroblasts. J Neurochem 1995;
64:475–481.
165 Finegold AA, Mannes AJ, Iadarola MJ: A paracrine paradigm for in vivo gene therapy in the
central nervous system: Treatment of chronic pain. Hum Gene Ther 1999;10:1251–1257.
166 Finegold AA, Perez FM, Iadarola MJ: In vivo control of NMDA receptor transcript level in
motoneurons by viral transduction of a short antisense gene. Brain Res Mol Brain Res 2001;
90:17–25.
167 Collin E, Mantelet S, Frechilla D, Pohl M, Bourgoin S, Hamon M, Cesselin F: Increased in vivo
release of calcitonin gene-related peptide-like material from the spinal cord in arthritic rats. Pain
1993;54:203–211.
168 Myers KJ, Dean NM: Sensible use of antisense: How to use oligonucleotides as research tools.
Trends Pharmacol Sci 2000;21:19–23.
169 McManus MT, Sharp PA: Gene silencing in mammals by small interfering RNAs. Nat Rev Genet
2002;3:737–747.
170 Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC: Potent and specific genetic
interference by double-stranded RNA in Caenorhabditis elegans. Nature 1998;391:806–811.
171 Hammond SM, Caudy AA, Hannon GJ: Post-transcriptional gene silencing by double-stranded
RNA. Nat Rev Genet 2001;2:110–119.
172 Krichevsky AM, Kosik KS: RNAi functions in cultured mammalian neurons. Proc Natl Acad Sci
USA 2002;99:11926–11929.
173 Zlokovic BV, Apuzzo ML: Cellular and molecular neurosurgery: Pathways from concept to real-
ity. II. Vector systems and delivery methodologies for gene therapy of the central nervous system.
Neurosurgery 1997;40:805–812; discussion 812–813.
174 Ishii K, Isono M, Inoue R, Hori S: Attempted gene therapy for intractable pain: Dexamethasone-
mediated exogenous control of beta-endorphin secretion in genetically modified cells and
intrathecal transplantation. Exp Neurol 2000;166:90–98.
175 Saitoh Y, Taki T, Arita N, Ohnishi T, Hayakawa T: Analgesia induced by transplantation of encap-
sulated tumor cells secreting beta-endorphin. J Neurosurg 1995;82:630–634.
176 Luo D, Saltzman WM: Synthetic DNA delivery systems. Nat Biotechnol 2000;18:33–37.
177 Luo Z: Molecular dissection of pain mediators. Pain Reviews 2000;7:37–64.
178 Sawynok J: Topical and peripherally acting analgesics. Pharmacol Rev 2003;55:1–20.
179 Nabel GJ: Development of optimized vectors for gene therapy. Proc Natl Acad Sci USA 1999;
96:324–326.
180 Parks RJ, Chen L, Anton M, Sankar U, Rudnicki MA, Graham FL: A helper-dependent adenovirus
181 Besson JM: The neurobiology of pain. Lancet 1999;353:1610–1615.
182 Henning SW, Beste G: Loss-of-function strategies in drug target validation. Curr Drug Discov
2002;May:17–21.
183 Verma IM, Somia N: Gene therapy – Promises, problems and prospects. Nature 1997;389:239–242.
184 Huang W, Simpson RK Jr: Antisense of c-fos gene attenuates Fos expression in the spinal cord
induced by unilateral constriction of the sciatic nerve in the rat. Neurosci Lett 1999;263:61–64.
185 Lee CE, Kest B, Jenab S, Inturrisi CE: Effect of supraspinal antisense oligodeoxynucleotide
treatment on delta-opioid receptor mRNA levels in mice. Brain Res Mol Brain Res 1997;
48:17–22.
186 Ji RR, Zhang X, Wiesenfeld-Hallin Z, Hokfelt T: Expression of neuropeptide Y and neuropep-
tide Y (Y1) receptor mRNA in rat spinal cord and dorsal root ganglia following peripheral tissue
inflammation. J Neurosci 1994;14:6423–6434.
187 Hua XY, Chen P, Polgar E, Nagy I, Marsala M, Phillips E, Wollaston L, Urban L, Yaksh TL, Webb M:
Spinal neurokinin NK1 receptor down-regulation and antinociception: Effects of spinal NK1
receptor antisense oligonucleotides and NK1 receptor occupancy. J Neurochem 1998;70:688–698.
188 Lai J, Gold MS, Kim CS, Bian D, Ossipov MH, Hunter JC, Porreca F: Inhibition of neuropathic
pain by decreased expression of the tetrodotoxin-resistant sodium channel, NaV1.8. Pain 2002;
95:143–152.
189 Porreca F, Lai J, Bian D, Wegert S, Ossipov MH, Eglen RM, Kassotakis L, Novakovic S, Rabert DK,
Sangameswaran L, Hunter JC: A comparison of the potential role of the tetrodotoxin-insensitive
sodium channels, PN3/SNS and NaN/SNS2, in rat models of chronic pain. Proc Natl Acad Sci USA
1999;96:7640–7644.
190 Khasar SG, Gold MS, Levine JD: A tetrodotoxin-resistant sodium current mediates inflammatory
pain in the rat. Neurosci Lett 1998;256:17–20.
191 Levesque L, Dean NM, Sasmor H, Crooke ST: Antisense oligonucleotides targeting human
protein kinase C-alpha inhibit phorbol ester-induced reduction of bradykinin-evoked calcium
mobilization in A549 cells. Mol Pharmacol 1997;51:209–216.
192 Rydh-Rinder M, Berge OG, Hokfelt T: Antinociceptive effects after intrathecal administration of
phosphodiester-, 2⬘-O-allyl-, and C-5-propyne-modified antisense oligodeoxynucleotides target-
ing the NMDAR1 subunit in mouse. Brain Res Mol Brain Res 2001;86:23–33.
193 Garry MG, Malik S, Yu J, Davis MA, Yang J: Knock down of spinal NMDA receptors reduces
NMDA and formalin evoked behaviors in rat. Neuroreport 2000;11:49–55.
194 Tao F, Tao YX, Gonzalez JA, Fang M, Mao P, Johns RA: Knockdown of PSD-95/SAP90 delays
the development of neuropathic pain in rats. Neuroreport 2001;12:3251–3255.
Kim Burchiel, MD
Chairman, Department of Neurological Surgery, L 472
Oregon Health & Science University, 3181 SW Sam Jackson Park Road
Portland, OR 97239 (USA)
Tel. ⫹1 503 494 4173, Fax ⫹1 503 494 7161, E-Mail burchiek@ohsu.edu
medwedi.ru
Gene Transfer in the Treatment of Pain

David Fink a, Marina Mataa, Joseph C. Gloriosob
a
Department of Neurology, University of Michigan and VA Ann Arbor
Healthcare System, Ann Arbor, Mich., and bDepartment of Molecular
Genetics and Biochemistry, University of Pittsburgh, Pittsburgh, Pa., USA
Introduction
The Physiology of Pain

Pain is an unpleasant sensory and affective experience that serves an essen-
tial biological role in alerting an organism to tissue damage. The perception of
acute pain is essential for survival in a potentially hostile environment, and an
elaborate set of specialized high threshold sensory transducers, nociceptors that
respond to painful heat, cold, pressure, and alterations in the peripheral micro-
environment are designed to detect these acute stimuli. In the setting of chronic
tissue damage, changes in pH and ionic composition of the peripheral micro-
environment and the release of bioactive peptides such as cytokines, growth
factors, and kinins, all act to sensitize peripheral nociceptors. In addition, con-
tinued neural transmission through pain pathways leads to central changes at the
level of the spinal cord and higher centers that together result in a heightened
pain experience. While these changes may serve an adaptive role in preventing
the use of an injured body part, thus promoting recovery and repair, the same
processes lead to spontaneous or exaggerated pain that does not serve any func-
tional biological purpose. Pain that persists beyond the course of the acute insult
or pain that accompanies a chronic primary process that cannot be cured repre-
sents a major medical and social problem resulting in enormous cost to the indi-
vidual and to society. Such chronic pain may result from continued peripheral
injury (inflammatory or ‘nociceptive’ pain) or from damage to neural structures
in the absence of peripheral tissue damage (neuropathic or central pain).
Derivatives of the active agents extracted from the poppy seed (opiate drugs)
and willow bark (nonsteroidal anti-inflammatory drugs) remain among our
most effective and widely used analgesic drugs, but increased understanding of
the pathways involved in acute pain perception and the modifications that occur
in chronic pain have now set the stage for the rational design of novel thera-
peutic agents to treat chronic pain. Peripheral nociceptors consisting of cells
with unmyelinated (C-fibers) or thinly myelinated (A␦ fibers) axons represent
a subclass of neurons whose cell bodies are located in the dorsal root ganglion
(DRG). The central projection of the bipolar axons of the primary nociceptors
synapse on ‘second order’ neurons in the dorsal horn of spinal cord in a regional
and anatomically defined manner. Second order neurons located in the dorsal
horn project rostrally to the thalamus and the parabrachial nucleus in the brain-
stem. Pain-related neurons in the thalamus project primarily to somatosensory
cortex, conveying the discriminative aspects of the pain sensation; neurons in
the parabrachial nucleus project to the hippocampus and amygdala (among
other brain regions) to mediate the affective components of the pain experience.
Descending pathways, integrated in the periaqueductal gray of the midbrain and
relayed through the nucleus raphe magnus, project caudally to the dorsal horn
of spinal cord to synapse with inhibitory interneurons.
The principal neurotransmitter released from axons of the primary nocicep-
tor at the dorsal horn is glutamate, although corelease of peptides including sub-
stance P, neuropeptide Y, dynorphin and galanin from the primary afferent serve
to modulate the pain response. In the dorsal horn, intrinsic inhibitory interneurons
may modulate the transmission of nociceptive information through the release of
neurotransmitters such as ␥-aminobutyric acid (GABA) acting on GABAA and
GABAB receptors, enkephalin acting through ␦ opioid receptors, and endomor-
phin 1 and 2 acting at the ␮ opioid receptor. Descending projections from brain-
stem nuclei control the activity of the inhibitory interneurons through the release
of monoamine neurotransmitters including norepinephrine and serotonin.
In states of chronic pain, there are post-translational or transcriptional
changes in primary nociceptors that alter the threshold, excitability, or trans-
mission properties of these neurons. A prolonged increase in the activity of
peripheral nociceptors also results in the sensitization of second order neurons,
through phosphorylation of ion channels and receptors as well as transcrip-
tional changes in second order neurons. In addition, there may be remodeling
in the dorsal horn, including sprouting of primary afferents after injury and loss
of inhibitory interneurons. Imaging studies have also identified complex central
changes in the activity of subcortical and cortical structures that occur in states
characterized by chronic pain.
The most direct approach to treating pain is to alleviate the primary incit-
ing cause of the pain. But when the primary disease process cannot be cured,
or the pain persists after the identified inciting cause has been treated, other
approaches are required. Many of the neurotransmitters or neurotransmitter
receptors that are the targets of pharmacological therapy are widely distributed
Gene Transfer in the Treatment of Pain 323
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through the nervous system and may be present in other organs as well. As a result,
the dose of many of the drugs that may be used to treat pain are limited by side
effects resulting from the action of these agents on neural pathways unrelated
to pain processing, or from the action of these agents directly on non-neural tis-
sues. Opioid receptors, for example, are present on peripheral nociceptors, on
the second order projection neurons in the dorsal horn of spinal cord, and in
brainstem and brain centers involved in pain processing and other cognitive and
affective functions as well as in the urinary bladder and the intestine. As a
result, high-dose treatment with opiate drugs may be complicated by alterations
in mood and/or cognition, urinary retention, and constipation that limit the dose
that may be administered. Gene transfer offers the possibility to achieve local
release of analgesic substances to act at the spinal or peripheral level to maxi-
mize the analgesic effectiveness while minimizing side effects.
Cell Transplantation for Pain Relief
One method of gene transfer involves the transplantation of cells that carry
and express the gene of interest. Chromaffin cells of the adrenal medulla natu-
rally express and release a number of neuroactive substances, many of which are
involved in the pain-processing pathway at the spinal level. These include sero-
tonin, GABA, galanin, and met-enkephalin [1, 2]. Accordingly, the injection of
chromaffin cells into the lumbar subarachnoid space reduces pain-related
behavior in models of neuropathic pain, and in rodent models, inflammatory
pain induced by subcutaneous injection of a dilute solution of formalin [3, 4].
Such grafts reduce touch-induced elevation of c-fos expression in spinal cord
[5] and prevent the loss of endogenous GABA synthesis in the dorsal horn [6].
Several different mechanisms have been implicated in the analgesic effect of
chromaffin cell grafts. Chromaffin cell grafts release met-enkephalin, and the
level of met-enkephalin in CSF is increased following grafting [7], but the grafts
also elevate CSF catecholamine levels [8]. It is possible that indirect effects,
such as catecholamine stimulation by the release of inhibitory neurotransmitters
from dorsal horn interneurons might account for some of the effects of the graft.
In a neurophysiological study Hentall et al. [9] demonstrated that the intrathe-
cal transplantation of chromaffin cells prevented the normal development of
‘windup’, a phenomenon of electrophysiological potentiation that is character-
istic of chronic pain, in second-order wide dynamic range neurons of the dorsal
horn. It appears from those studies that interference with potentiation is due to
the release of molecules that persistently block the NMDA receptor (or block
cellular events mediated by these receptors), separate from the possible effect of
inhibitory neurotransmitters from the graft [9].
Fink/Mata/Glorioso 324
Cells also can be modified to produce specific desired gene products.
Wu et al. [10] showed that AtT-20 cells, a cell line which produces and releases
␤-endorphin, and AtT-20/hENK cells, an AtT-20 cell line transfected with the
human proenkephalin gene (PE) and secreting enkephalin and ␤-endorphin,
implanted into the mouse subarachnoid space produced an isoproterenol-
stimulated anti-nociceptive effect that was dose related and could be blocked by
naloxone. Mice receiving AtT-20 cell implants developed tolerance to ␤-endor-
phin and the ␮-opioid agonist DAMGO, whereas mice receiving genetically
modified AtT-20/hENK cell implants developed tolerance to the ␦-opioid ago-
nist DPDPE. Genetically modified AtT-20/hENK cell implants, but not AtT-20
cell implants, reduced the development of acute morphine tolerance in the host
mice [10].
Transplants of other cell lines modified to secrete substances that might act
as inhibitory neurotransmitters at the spinal level to block nociceptive neuro-
transmission have included the demonstration that a neuronal cell line geneti-
cally modified to secrete galanin [11] or engineered to secrete GABA [12] are
anti-nociceptive in models of chronic neuropathic pain. A cell line engineered
to produce and release brain-derived neurotrophic factor has been shown to
reduce allodynia and hyperalgesia in the chronic constriction injury model of
neuropathic pain [13].
The mechanical alternative to cell transplantation is peptide delivery using
an intrathecal pump. Both approaches target the pharmcological agent to the
lumbar spinal cord in order to minimize the effect of these agents on the brain
and on peripheral organs. A theoretical advantage of cell transplantation is the
ability of the cells to deliver peptide neurotransmitter (such as enkephalin) in
their natural conformation and not in a derivative state. Intrathecal pumps may
be used to deliver opioid analgesic drugs such as morphine. Intrathecal deliv-
ery reduces the dose requirement and thus limits side effects, but tolerance may
develop. Even intrathecal administration of the modified derivatives is limited
by the very short half-life of these agents. While cell transplants produce a
continuous release of the native peptide, release of additional substances (e.g.,
␤-endorphin, serotonin) which may add to the effectiveness of these transplants
in the animal models may complicate the practical implementation of the cells
as agents for human treatment. In addition, the possibility of an immune reaction
or nonspecific scar formation resulting from the injection of foreign material
into the subarachnoid space is not fully known. Nonetheless, transplantation of
encapsulated bovine chromaffin into the subarachnoid space has been tested in
patients with severe chronic pain not satisfactorily managed with conventional
therapies. The patients received no pharmacological immunosuppression. Histo-
logical analysis, immunostaining, and analysis of secretory function all confirmed
survival and biochemical function of the encapsulated cells up to 6 months after
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implantation. Reductions in morphine intake and improvement in pain ratings
were observed in several patients [14]. Similar results were observed in an open
phase II trial of patients with intractable pain from cancer who received adrenal
medullary allografts [15]. While analgesic efficacy was suggested by a reduc-
tion or stabilization in opioid use, a controlled trial has not yet been reported.
Adenoviral Gene Transfer to Meningeal Cells

in the Treatment of Pain
Some of the problems attendant on injection of foreign cells into the sub-
arachnoid space can be avoided by the direct transfer of the gene of interest into
meningeal cells lining the subarachnoid space. For example, Finegold et al. [16]
used a first-generation replication deficient adenoviral (Ad5) vector containing
the coding sequence for human ␤-endorphin to transduce cells of the pia mater.
Ad5-mediated gene transfer resulted in the release of ␤-endorphin into the CSF
and attenuated inflammatory hyperalgesia, measured as the thermal withdrawal
latency in the carageenen model of inflammatory pain in the rodent, without
affecting basal nociceptive responses. Although the inflammatory response
elicited by the first-generation adenoviral vectors employed in this study lim-
ited the duration of transgene expression, a similar approach using later-gener-
ation vectors might be appropriate for patients with severe refractory pain in the
terminal stages of a disease process.
Herpes-Mediated Gene Transfer in the Treatment of Pain
An alternative but related approach uses vector-mediated gene transfer to

transduce neurons, rather than meningeal cells. For this purpose, herpes sim-
plex virus (HSV) has proven to be a useful gene transfer vector. HSV is a
neurotropic virus that naturally infects skin and mucous membranes. Following
the initial epithelial infection, HSV is taken up by nerve terminals in the skin
and carried by retrograde axonal transport to the cell bodies of sensory neurons
in the DRG where the viral DNA is inserted through a nuclear pore into the
nucleus. The uptake and transport of the virion from the skin is an efficient
process mediated first by interactions between specific viral envelope glyco-
proteins and high-affinity receptors in the sensory nerve terminals in the skin
[17, 18], followed by specific interactions of capsid and tegument proteins with
dynein molecules in the axoplasm to mediate the retrograde axonal transport
along microtubules to the cell body [19]. The highly efficient delivery of viral
DNA to the DRG neuronal nucleus from an original infection in the skin coupled
with the natural ability of the viral genome to establish a life-long latent state
as an intranuclear episomal element makes HSV a very effective gene transfer
vector for the peripheral nervous system.
Pohl et al. demonstrated that an HSV-based vector in which the HSV thymi-
dine kinase sequence is replaced by the cDNA coding for human proenkephalin,
injected under the skin in the paw of a rat, transduced DRG neurons to produce
enkephalin [20]. Immunoreactive met-enkephalin is transported anterogradely
(i.e., away from the cell body) in both directions in the bipolar axon from DRG
neurons, towards the spinal cord and back towards the skin with a larger amount
moving peripherally than centrally [21]. Electrically stimulated release of met-
enkephalin from nerve terminals can be demonstrated in an in vitro preparation
[Pohl, pers. commun.].
Wilson et al. [22] then showed that subcutaneous inoculation of a similar
tk-deleted HSV vector expressing PE reduces hyperalgesia measured by the
sensitization of the foot withdrawal response after application of capsaicin
(C fibers) or dimethyl sulfoxide (A␦ fibers). The effect of the vector persisted
for at least 7 weeks after the inoculation of the vector subcutaneously into the
dorsum of the foot. Baseline foot withdrawal responses to noxious radiant heat
mediated by A␦ and C fibers were similar in animals infected with PE-encod-
ing and ␤-galactosidase-encoding vectors, demonstrating that the PE-express-
ing vector selectively blocked hyperalgesia without disrupting the baseline
sensory neurotransmission. This blockade of sensitization was reversed by the
administration of the opioid antagonist naloxone, apparently acting in the spinal
cord [22].
Deletion of HSV-TK impairs the ability of the virus to replicate in neurons
while leaving replication characteristics in non-neuronal cells intact. HSV gene
expression occurs in a rigid temporal sequence and only five of the more than
eighty HSV genes that are expressed during the lytic replication cycle are char-
acterized as ‘immediate early’ (IE) genes. The expression of IE genes begins
immediately after the viral entry into the nucleus, activated by a viral protein
(VP16) contained in the tegument, and does not require the de novo expression
of other viral proteins. Deletion of even one essential IE gene from the HSV
genome creates a recombinant that can be propagated in a complementing cell
line that provides the essential IE gene product in trans. These IE gene-deleted
HSV vectors are incapable of replication in noncomplementing cells [23].
Introduced into animals, such replication-incompetent vectors do not replicate,
but instead traffic to DRG neurons and establish a persistent state in a fashion
identical to that observed for the replication-competent recombinants. We have
examined the pain-relieving properties of a replication-incompetent HSV vector-
expressing human PE in rodent models of inflammatory pain, neuropathic pain,
and pain resulting from cancer in bone.
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10
Cumulative pain score

8
*
6
**
**
4
2
SHZ 7d 14 d 28 d 28 d IT
⫹7 d naltrexone
SHPE
Fig. 1. Time course of the anti-nociceptive effect of SHPE inoculation in inflamma-

tory pain (formalin test). The cumulative pain score during the delayed phase of the forma-
lin test (10 min to one hour after the inoculation of formalin) was significantly reduced in
animals inoculated with the PE-expressing vector SHPE. The vector-mediated effect was
maximal one week after the inoculation of the vector, and was no longer statistically signifi-
cant in animals tested 4 weeks after vector inoculation. However reinoculation of the vector
at 4 weeks reestablished the analgesic effect (28 ⫹ 7 day group). Intrathecal administration
of the ␦ opioid receptor antagonist naltrexone in animals tested one week after vector inocu-
lation (IT naltrexone) blocked the vector-mediated analgesic effect. From [31].
Injected under the skin of the foot, the PE-expressing HSV vector was
detected in the DRG by PCR using primers specific for the human PE
sequence, and the expression of PE mRNA was detected by RT-PCR using the
same sequences [24]. In the formalin test of inflammatory pain, injection of the
PE-expresssing HSV vector reduced spontaneous pain behavior during
the delayed phase (10–60 min after the injection of formalin) without affecting
the acute pain score. This effect was reversed by the intrathecal administration
of naltrexone [24], suggesting in agreement with Wilson et al. that the site of
action of the released transgene product is in the dorsal horn of spinal cord. The
analgesic effect was limited to the injected limb; formalin testing on the limb
contralateral to the injection showed no analgesic effect [Glorioso, unpubl.
observations], further suggesting that release of enkephalin from primary affer-
ent terminals in the dorsal horn was limited to the region of their projections in
dorsal horn.
HSV vector-mediated analgesic effects persisted for several weeks and then
waned. Animals tested 4 weeks after vector inoculation showed no significant
reduction in pain-related behavior during the delayed phase of the formalin
SHPE SHZ Vehicle
12 Reinoculation ** ** **
Inoculation **
10 **
Threshold (gms)
8 *
* *
6
*
0 5 10 15
Weeks after spinal nerve ligation
Fig. 2. Time course of the anti-allodynic effect of SHPE inoculation in neuropathic

pain (spinal nerve ligation model). Injection of SHPE (but not control vector SHZ) resulted
in a sustained anti-allodynic effect in neuropathic pain that lasted for several weeks.
Reinoculation of the vector 6 weeks after the initial inoculation re-established the anti-
allodynic effect. The vector-mediated pain-relieving effect was reversed by intraperitoneal
administration of naloxone (data not shown). From [26].
test [24]. However, in animals reinoculated with the vector 4 weeks after the ini-
tial inoculation and then tested with formalin injection at 5 weeks, a substantial
and significant anti-nociceptive effect was demonstrated, which was at least as
great as the initial effect [24]. The time course of the vector-mediated effect is
consistent with the known time course of the human cytomegalovirus immedi-
ate-early promoter that was used to drive transgene expression in these experi-
ments. The fact that the reinjection could re-establish the initial analgesic effect
suggests, but does not prove, that the animals had not developed tolerance to the
vector-mediated release of enkephalin. The effectiveness of reinoculation also
suggests that the exposure of animals to the HSV vector does not elicit a neu-
tralizing immune response that would be capable of attenuating gene transfer
from the vector inoculation.
We have also examined the effect of transgene-mediated enkephalin
release in the spinal nerve ligation model of neuropathic pain [25]. Isolated L5
spinal nerve ligation distal to the DRG results in a painful state that can be
quantified by measures of mechanical and thermal hypersensitivity.
Subcutaneous injection of the PE-expressing vector into the foot one week after
spinal nerve ligation resulted in an anti-allodynic effect that lasted for several
weeks [26]. The characteristics of this anti-allodynic effect were similar to
those observed in the formalin model. Reinoculation of the vector at 6 weeks
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Sham Vehicle
SHZ SHPE
60 Sham
Ligation
Vehicle
50 SHZ
SHPE
40
fos-LI neurons
30 *
20 ** *
**
10
0
I-II III-IV V-VI VIII-X
Laminae
b
Fig. 3. Transduction with SHPE blocks touch-induced elevation in c-fos expression in

the dorsal horn. Animals with neuropathic pain from spinal nerve ligation stimulated with
gentle rubbing of the paw show a characteristic increase in c-fos expression in dorsal horn
neurons seen in vehicle-treated animals (a, top right panel). Subcutaneous inoculation of
SHPE one week after spinal nerve ligation substantially blocked this induction in c-fos
expression (a, bottom right panel). The quantitative data are shown in b. From [26].
re-established the anti-allodynic effect; the magnitude of the effect produced by

reinoculation was at least as great as that produced by the initial injection and
the effect persisted for a longer time after the reinoculation than that produced
by the initial inoculation. Intraperitoneal naloxone reversed the anti-allodynic
effect.
Spontaneous ambulatory pain score 3
2.5
1.5
*
1
0.5
(11) (10) (9) (4)

0
SHPE SHZ SHPE
⫹ NTX
tumor
Fig. 4. Transduction with SHPE reduces pain-related behavior in a model of pain result-
ing from cancer in bone. Rats with experimental osteogenic sarcoma of the distal femur
(shown in right part of radiograph) demonstrate a spontaneous pain behavior that is reduced
significantly in those animals that were inoculated subcutaneously in the foot with SHPE one
week after tumor implantation. The analgesic effect of the vector was reversed by intrathecal
naltrexone. From [28].
The effect of this vector-mediated anti-allodynic effect in neuropathic pain

was continuous throughout the day. Animals tested repeatedly at different times
through the day showed a similar elevation in threshold at all times tested [26].
Intraperitoneal morphine produced a greater anti-allodynic effect than the vec-
tor alone, but the inoculation of the maximum dose of morphine produced an
anti-allodynic effect that persisted for only 1–2 h before waning. The effect of
vector-mediated enkephalin (acting predominantly at ␦ opioid receptors) and
morphine (acting predominantly at ␮ opioid receptors) was additive. The ED50
of morphine was shifted from 1.8 ␮g/kg in animals with neuropathic pain from
spinal nerve ligation treated with PBS or inoculated with a control vector
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expressing lacZ, to 0.15 ␮g/kg in animals that had been injected with the
PE-expressing vector one week after spinal nerve ligation. Twice daily inocula-
tion of morphine (10 mg/kg IP) in spinal nerve ligated animals resulted in the
development of tolerance by one week; beyond that timepoint, the continued
twice-daily administration of morphine had no anti-allodynic effect. Animals
that had been inoculated with the PE-expressing vector one week after spinal
nerve ligation continued to demonstrate the anti-allodynic effect of the vector
despite the induction of tolerance to morphine [26].
We also examined the effect of vector-mediated expression of PE in a
rodent model of pain resulting from cancer in bone [27]. Implantation of NTCT
2,472 cells into the distal femur resulted in a significant spontaneous pain-related
behavior that increased between 2 and 3 weeks after tumor injection. Animals
that received a subcutaneous inoculation of the PE-expressing vector into the
plantar surface of the foot one week after tumor injection showed a significant
reduction in the ambulatory pain score when compared to control vector-inocu-
lated tumor-bearing animals at both 2 and 3 weeks after tumor injection, an
effect that was reversed by intrathecal naltrexone [28]. Radiographical analysis
of tumor-bearing mice inoculated with SHZ or SHPE demonstrated bone loss
indicative of the presence of the osteolytic tumor, and there was no evidence that
transgene expression had any effect on tumor growth [28].
Similar effects have been demonstrated in adjuvant-induced polyarthritis
in the rat [29]. Using a replication-competent HSV vector expressing PE, Pohl
et al. showed that subcutaneous inoculation of the vector not only markedly
improved the locomotion and reduced hyperalgesia, but also that the release of
enkephalin from the peripheral terminals of the DRG axons resulted in a slow-
ing of the progression of bone destruction. In that model both the slowing of
joint destruction as well as reversal of the analgesic effect by a peripherally
acting substituted naloxone analog suggest that the site of action of transgene-
mediated enkephalin released from transduced neurons is at the peripheral pro-
jection rather than in the spinal cord. However, the effect on joint destruction
appears to be unique to the model of arthritis employed [Pohl, pers. commun.].
Whether this approach will be effective in the treatment of human pain
should be determined quite soon. A proposal for a phase I human trial to exam-
ine the safety and tolerability of subcutaneous inoculation of an HSV vector
deleted for the IE genes ICP4, ICP22, ICP27 and ICP41, and expressing human
PE under the control of the human cytomegalovirus immediate-early promoter
was presented to the Recombinant Advisory Committee at the NIH (for details,
see RAC Protocol #0201–529 at http://www.webconferences.com/nihoba/
20–21_june_02.htm). The study protocol describes the enrollment of 18 patients
with cancer metastatic to a vertebral body resulting in pain unresponsive to
maximal conventional management, who will receive an inoculation of the
HSV vector subcutaneously in the dermatome corresponding to the radicular
distribution of the pain. In this dose-escalation trial, 3 patients will be enrolled
at each dose, increasing at half-log intervals.
Pain treatment by transduction of DRG neurons using an HSV vector will
be limited to pain syndromes that result in regional or focal pain. The noninva-
sive means of delivery of the vector, the focal effect of transgene expression,
and the synergy with opiate drug treatment are chief advantages of this
approach. However, once the transgene is introduced into the DRG neuron it
cannot be removed, and using the current vectors, transgene expression is not
regulated. This should not prove a problem for the use of enkephalin, and the
transgene expression driven by the human cytomegalovirus promoter is tran-
sient. But in the use of other transgenes, or promoters designed to drive long
term gene expression, these issues will resume further consideration and vector
engineering. The fact that these patients are facing a fatal course of the disease
with severe pain that is frequently not responsive even to high-dose systemic
opioids strengthens the rationale for local, targeted gene transfer for pain relief.
Gene Transfer to the Brain in Models of Pain
There are other sites within the neuraxis where pain transmission may be
interrupted by vector-mediated neurotransmitter expression. Injection of a
replication-competent (tk-deleted) HSV vector expressing proenkepahlin into
the amygdala bilaterally has been shown to reduce pain-related behavior in the
delayed phase of the formalin test (animals tested 4 days after vector inocula-
tion) [30], and injection of an HSV amplicon vector expressing glutamic acid
decarboxylase to result in the release GABA in brain nuclei also reduces pain-
related behaviors [Jasmin and Rabkin, pers. commun.]. While these experi-
ments demonstrate that focal neurotransmitter effects can be achieved by gene
transfer to the brain as well as the peripheral nervous system, applications to
human clinical therapies are likely to take longer to develop, given the compli-
cated neuropharmacology of CNS function.
Conclusion
Cell transplantation or vector-mediated gene transfer, by providing a

means to target expression focally in the nervous system, may allow the use
of short-lived macromolecules identical to the endogenous substances to
enhance pain relief in specific situations. In the future, other macromolecules
acting to interrupt the process responsible for the development of the pain
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(such as anti-inflammatory cytokines or specific neurotrophic factors in neu-
ropathic pain) may be delivered in a similar fashion.
Acknowledgments
This work was supported by grants from the NIH (JCG and DJF), the Department of
Veterans Affairs (MM and DJF), and the Juvenile Diabetes Foundation Research International
(DJF).
References
1 Wilson SP, Chang KJ, Viveros OH: Opioid peptide synthesis in bovine and human adrenal chro-
maffin cells. Peptides 1981;2(suppl 1):83–88.
2 Unsicker K: The trophic cocktail made by adrenal chromaffin cells. Exp Neurol 1993;123:167–173.
3 Hama AT, Sagen J: Alleviation of neuropathic pain symptoms by xenogeneic chromaffin cell
grafts in the spinal subarachnoid space. Brain Res 1994;651:183–193.
4 Siegan JB, Sagen J: Attenuation of formalin pain responses in the rat by adrenal medullary trans-
plants in the spinal subarachnoid space. Pain 1997;70:279–285.
5 Sagen J, Wang H: Adrenal medullary grafts suppress c-fos induction in spinal neurons of arthritic
rats. Neurosci Lett 1995;192:181–184.
6 Ibuki T, et al: Loss of GABA-immunoreactivity in the spinal dorsal horn of rats with peripheral
nerve injury and promotion of recovery by adrenal medullary grafts. Neuroscience 1997;76:
845–858.
7 Sagen J, Kemmler JE: Increased levels of Met-enkephalin-like immunoreactivity in the spinal cord
CSF of rats with adrenal medullary transplants. Brain Res 1989;502:1–10.
8 Sagen J, Kemmler JE, Wang H: Adrenal medullary transplants increase spinal cord cerebrospinal
fluid catecholamine levels and reduce pain sensitivity. J Neurochem 1991;56:623–627.
9 Hentall ID, Noga BR, Sagen J: Spinal allografts of adrenal medulla block nociceptive facilitation
in the dorsal horn. J Neurophysiol 2001;85:1788–1792.
10 Wu HH, Wilcox GL, McLoon SC: Implantation of AtT-20 or genetically modified AtT-20/hENK
cells in mouse spinal cord induced antinociception and opioid tolerance. J Neurosci 1994;14:
4806–4814.
11 Eaton MJ, et al: Lumbar transplant of neurons genetically modified to secrete galanin reverse
pain-like behaviors after partial sciatic nerve injury. J Peripher Nerv Syst 1999;4:245–257.
12 Eaton MJ, et al: Transplants of neuronal cells bioengineered to synthesize GABA alleviate chronic
neuropathic pain. Cell Transplant 1999;8:87–101.
13 Cejas PJ, et al: Lumbar transplant of neurons genetically modified to secrete brain-derived neuro-
trophic factor attenuates allodynia and hyperalgesia after sciatic nerve constriction Pain. 2000;
86:195–210.
14 Buchser E, et al: Immunoisolated xenogenic chromaffin cell therapy for chronic pain. Initial clin-
ical experience. Anesthesiology 1996;85:1005–1012; discussion A29–A30.
15 Lazorthes Y, et al: Human chromaffin cell graft into the CSF for cancer pain management:
A prospective phase II clinical study. Pain 2000;87:19–32.
16 Finegold AA, Mannes AJ, Iadarola MJ: A paracrine paradigm for in vivo gene therapy in the cen-
tral nervous system: treatment of chronic pain. Hum Gene Ther 1999;10:1251–1257.
17 Spear PG, Eisenberg RJ, Cohen GH: Three classes of cell surface receptors for alphaherpesvirus
entry. Virology 2000;275:1–8.
18 Shukla D, Spear PG: Herpesviruses and heparan sulfate: An intimate relationship in aid of viral
entry. J Clin Invest 2001;108:503–510.
19 Smith GA, Enquist, LW: BREAK INS AND BREAK OUTS: Viral interactions with the cytoskele-
ton of mammalian cells. Annu Rev Cell Dev Biol 2002;18:135–161.
20 Antunes Bras JM, et al: Herpes simplex virus 1-mediated transfer of preproenkephalin A in rat
dorsal root ganglia. J Neurochem 1998;70:1299–1303.
21 Antunes Bras J, et al: Met-enkephalin is preferentially transported into the peripheral processes of
primary afferent fibres in both control and HSV1-driven proenkephalin A overexpressing rats.
Neuroscience 2001;103:1073–1083.
22 Wilson SP, et al: Antihyperalgesic effects of infection with a preproenkephalin-encoding herpes
virus. Proc Natl Acad Sci USA 1999;96:3211–3216.
23 DeLuca NA, McCarthy AM, Schaffer PA: Isolation and characterization of deletion mutants of
herpes simplex virus type 1 in the gene encoding immediate-early regulatory protein ICP4. J Virol
1985;56:558–570.
24 Goss JR, et al: Antinociceptive effect of a genomic herpes simplex virus-based vector expressing
human proenkephalin in rat dorsal root ganglion. Gene Ther 2001;8:551–556.
25 Kim SH, Chung JM: An experimental model for peripheral neuropathy produced by segmental
spinal nerve ligation in the rat. Pain 1992;50:355–363.
26 Hao S, et al: Transgene-mediated enkephalin release enhances the effect of morphine and evades
tolerance to produce a sustained antiallodynic effect. Pain 2003;102:135–142.
27 Schwei MJ, et al: Neurochemical and cellular reorganization of the spinal cord in a murine model
of bone cancer pain. J Neurosci 1999;19:10886–10897.
28 Goss JR, et al: Herpes vector-mediated expression of proenkephalin reduces pain-related behav-
ior in a model of bone cancer. pain. Ann Neurol 2002;52:662–665.
29 Braz J, et al: Therapeutic efficacy in experimental polyarthritis of viral-driven enkephalin over-
production in sensory neurons. J Neurosci 2001;21:7881–7888.
30 Kang W, et al: Herpes virus-mediated preproenkephalin gene transfer to the amygdala is antinoci-
ceptive. Brain Res 1998;792:133–135.
31 Chen X, et al: Herpes simplex virus type 1 ICP0 protein does not accumulate in the nucleus of
primary neurons in culture. J Virol 2000;74:10132–10141.
David Fink, MD
1914 Taubman Center/0316, 1500 E Medical Center Drive
Ann Arbor, MI 48109-0316 USA
Tel. ⫹1 734 936 9070, Fax ⫹1 734 763 5059, E-Mail djfink@umich.edu
medwedi.ru
Neurovascular Disorders

Gene Discovery Underlying Stroke

Frank C. Baronea, Simon J. Read b
a
High Throughput Biology, GlaxoSmithKline, King of Prussia, Pa., USA,
b
RIRA, Astra Zeneca, Mereside, Alderley Park, Macclesfield, Cheshire, UK
Introduction
As first-pass efforts to complete sequencing of the entire human genome

are now concluded [1], there is an increased interest in the application of
genomic approaches to aid in the discovery, development, and rationale use of
drugs. As databases of differential gene expression have expanded, so has the
expectation of identifying novel drug targets for disease intervention. Indeed,
significant work has already been carried out to understand gene expression
changes in many diseased organ systems, including the ischemic brain [2–6].
Early epidemiological studies of the 1970s provided initial evidence for a
genetic influence in stroke. The Framingham study was one of the first studies
to suggest that a positive parental history of stroke contributed significant risk
to the offspring [7]. Thirty years later, stroke remains an area of substantial
unmet medical need. The complexity of stroke undoubtedly reflects the hetero-
geneity of the human stroke population, the contribution of monogenic and
polygenic disorders to this disease process, and the interactions of these with a
multitude of environmental factors.
This chapter focuses on genetics of risk and sensitivity to ischemic stroke.
It will discuss how inheritance relates to the broader stroke population and pro-
vide a detailed discussion of the stroke genomics literature. It will describe how
pre-clinical models of spontaneous stroke can be applied to humans to identify
the chromosomal loci of risk, and how the changes in gene expression associated
with stroke are associated with poststroke brain injury, resolution of brain injury,
and brain recovery processes. In addition, it will provide a detailed discussion of
several differential gene expression analyses techniques. This will include a
detailed discussion of genes identified using different techniques and the impor-
tance of a stroke model that has been well characterized and representative of the
type of stroke most often observed in man. Issues of validation of potential stroke
targets, the relevance of the expression of neuroprotective and neurodestructive
genes and their specific timings, genes involved in endogenous brain protection
and in brain recovery of function/plasticity, and the emerging problems with han-
dling novel/unknown genes that may be discovered from these analyses of dif-
ferential gene expression also will be addressed.
Ischemic Stroke
Stroke is the third largest cause of death in the USA, ranking only behind
heart disease and cancer. It is the leading cause of disability in the USA and has
the highest disease burden cost. Ischemic strokes comprise the majority of
strokes, between 70–80% of all strokes. No medical treatment is approved for
the treatment of acute ischemic stroke other than thrombolytic agents such as
tPA, which for optimum results must be administered within 3 h after stroke
onset. At most centers, only 1–2% of the stroke patients meet the criteria for
treatment with this thrombolytic agent. Aspirin and anticoagulants (where
embolic phenomena are documented) also are utilized as preventative therapy.
Estimates indicate that there are about 775,000 new stroke cases per year in the
USA, with a prevalence of about 4 million surviving, but at an increased risk of
a secondary cardiovascular event. In the USA, stroke is costly, with an annual
health care cost of $30–50 billion. Estimates indicate that stroke is responsible
for half of all the patients hospitalized for acute neurological disease [8, 9].
Stroke risk factors include both genetic and environmental factors. Stroke
risk factors that can be treated include high blood pressure, heart disease, ciga-
rette smoking, transient ischemic attacks, and high red blood cell count. Risk
factors for stroke that cannot be changed include age, gender (men have ⬃20%
greater risk of stroke than women), race (African-Americans have a much
higher risk of death and disability from stroke), diabetes mellitus, prior stroke,
and family history of strokes. Other controllable risk factors, secondary risk
factors, for stroke that also contribute to heart disease include high blood
LDL-cholesterol and lipids, physical inactivity, and obesity [10].
Genetics of Increased Stroke Risk
The strongest evidence for a genetic risk to stroke comes from twin studies.
Proband concordance rates have long been used to identify the heritability of a
trait or disorder. The concept of concordance is that for a disorder of genetic pre-
disposition, the rate will be higher for monozygotic twins than dizygotic twins.
Gene Expression in Stroke 337
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Aside from genetic influence, it is assumed that other factors, such as environ-
mental exposure will be approximately similar for both types of twins [11]. An
elevated proband-wise concordance rate for stroke risk in monozygotic twins
over dizygotic twins (17.7 vs. 3.6%) has confirmed a genetic predisposition to
stroke with a role of environmental factors [12]. A more recent twin study has
refined cohort analysis to stroke risk by assessing individual stroke phenotypes
that may be influenced by genetic factors [13]. In this study, the phenotype of
white matter hyperintensity volumes using magnetic resonance imaging (MRI)
was applied and genetic factors accounted for 71% of the variation in this end-
point [13].
A large number of familial studies have verified that a history of paternal
or maternal stroke is associated with an occurrence of stroke in offspring, and
that a positive paternal history of stroke was an independent prognostic predic-
tor of stroke [14–16]. In a cohort of men studied since 1913, maternal history
of stroke increased relative stroke risk by 3-fold [17]. Similarly, it has been
reported that a positive family history of stroke in any first-degree relative was
an independent predictor of stroke mortality in women aged 50–79, but not in
men [18]. Moreover, a family history of stroke in men was an independent pre-
dictor of coronary heart disease aged 50–64 years, indicating that genetic risk
factors for stroke may be shared with other cardiovascular disorders that have a
high genetic component [18]. Indeed, studies of the relative risk of other cere-
brovascular diseases with less heterogeneous phenotypes are able to document
strong patterns of inheritance. Subarachnoid hemorrhage occurs with a relative
risk of 6.6 in first-degree relatives compared to second-degree relatives [19].
Defining specific stroke subtypes may be the key to elucidating the exact
degree of genetic contribution to any particular phenotype. From twin studies,
it appears that the extent to which genetic factors may contribute to stroke risk
varies with age. These factors are caveats to the identification of therapeutic tar-
gets from candidate gene strategies, and one must remember that a candidate
gene approach for inheritance of risk factors may only be relevant to a highly
limited stroke subpopulation.
Simple Stroke-Like Diseases: Single Gene Mutations
Identification of possible genetic determinants of stroke risk has been

hampered by the lack of similar patient populations. Mendelian disorders with
specific stroke-like phenotypes have been explored as genetic models of the
more general population. These disorders include CADASIL (cerebral autoso-
mal dominant arteriopathy with subcortical infarcts and leukoencephalopathy)
[20], MELAS (mitochondrial encephalopathy, lactic acidosis-and stroke-like
Barone/Read 338
syndrome) [21], Sneddon’s syndrome [22], familial hemiplegic migraine [23]
and hereditary coagulopathies [24]. Although these subgroups contribute little
to the overall prevalence of stroke, genes identified from them are hoped to
highlight potential commonalities in the wider patient population. Studies on
CADASIL and MELAS are examples of such approaches.
CADASIL was originally described as an inherited, autosomally dominant
dementia with multiple infarcts [25]. Epidemiologically, CADASIL is limited
to sporadic cases in Europe [26, 27] and North America [28, 29]. The principal
symptoms of the CADASIL are migraine with aura, ischemic stroke, and psy-
chiatric symptoms including dementia [30]. In these patients, T2-weighted MRI
reveals small periventricular white matter hyperintensities often involving the
internal capsule [31]. The CADASIL gene, identified as Notch 3, is located at
the chromosomal loci 19p13.1–13.2 [27, 32]. The Notch genes regulate the lin-
12/sel-12 signaling pathway that is known to be important in development,
although the normal adult function of Notch genes remains unknown [33]. An
interesting association of the Notch 3 gene with Alzheimer’s disease has also
been discovered. Notch gene products interact with the presenilin 1 pathway as
substrates for ␥-secretase. This enzyme is known to have a key pathological role
in the production of A␤ peptide, although the modulatory role that Notch 3 may
have in this disease process is undefined [34, 35]. The Notch 3 gene encodes
a transmembrane protein composed of 2,321 amino acids, presumed to have a
receptor function and located primarily on smooth muscle cells [30]. In
CADASIL, approximately 90% of patients have missense mutations in extra-
cellular domains of the protein product, whilst in about 70% of patients, the
mutation is located within exons 3 and 4 [35]. All known mutations associated
with CADASIL result in the removal or addition of cysteine residues and it is
proposed that the expression of these mutated Notch 3 proteins results in cere-
bral vascular smooth muscle dysfunction [36].
Whether abnormalities in Notch signaling impact on the broader stroke pop-
ulation is at present unknown, although the pathogenesis of CADASIL, character-
ized by the progressive disruption of vascular endothelium with secondary fibrosis
and thrombosis is typical of some stroke subpopulations [37]. Anticoagulant ther-
apy has been tried in CADASIL without positive results [30]. More broadly,
CADASIL also has close relationships to Alzheimer’s disease; signaling compo-
nents of the presenilin pathway are shared with the Notch pathway [38]. The
presenilin-1-regulated ␥-secretase cleaves both the Notch intracellular domain and
␤-amyloid precursor protein for subsequent translocation to the nucleus and bind-
ing to DNA [38]. Therefore, whilst the pathology of CADASIL may bear similar-
ity to stroke, the cell biology also has potential connections with Alzheimer’s
disease. Since vascular risk factors and/or disease can impact on both vascular
dementia and Alzheimer’s disease, these relationships are intriguing [39–41].
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MELAS are characterized by migraine-like headache, nausea, seizures,
and stroke-like episodes. Lesions are most commonly found in the occipital and
parietal regions, with high lactate levels found within lesions using proton
nuclear magnetic resonance [42]. Patients typically have mutations of mito-
chondrial DNA for the tRNA-leu gene at an A-G transition mutation at
nucleotide position 3243 [42, 43] and at a T-C transition at position 3271 [44].
It has been speculated that as mutations accumulate, a gradual mitochondrial
dysfunction develops [45]. It is unclear how widespread such mutations are in
the broader stroke population. Indeed, cases of MELAS have been reported
without a family history, suggesting that these point mutations may be sponta-
neous [46]. Pharmacological interventions have reflected a unique nature of
MELAS within stroke/cardiovascular disease subpopulations. Antithrombotic
therapy has been employed in MELAS patients for cardiac complications asso-
ciated with left ventricular dysfunction [47].
CADASIL and MELAS demonstrate that several relatively rare ‘stroke-
like’ syndromes can be used to explore potential genetic determinants of stroke.
Parallel strategies have been adopted with similar success in other more com-
plex, multifactorial polygenic traits such as hypertension [48–50]. Genes such
as 11␤-hydroxylase in glucocorticoid-remediable aldosteronism have been
shown to mediate hereditary hypertension in these patients [51]. However, as in
stroke genetics, narrowing heterogeneity and studying single gene or Mendelian
disorders may have limited application to the broader patient population.
Ischemic Stroke: Complex Genetic Associations
In common with many diseases, there are individuals with complex genetic
profiles which confer vulnerability to stroke, as well as poststroke gene expres-
sion, which can contribute to increased cerebral ischemic stroke effects.
Candidate gene studies in heterogeneous stroke populations minimize issues of
limited patient population by the choice of a functionally relevant gene and its
relationship with a particular phenotype. This is termed ‘association’ and is a sta-
tistical measure of the dependence of a particular phenotype (e.g., ischemic
stroke with the presence of a particular candidate gene/allele). Therefore, associ-
ation can be positive (i.e., significant statistical relationship/association between
the gene of choice and phenotype), or negative (i.e., absence of significant rela-
tionship/association between gene/allele and phenotype). Candidate gene poly-
morphisms with a positive association with stroke include: apolipoprotein E
[52–54], ACE ([55–58], fibrinogen [59], Factor V [60] and Prothrombin [61].
Those gene polymorphisms with a negative association with stroke include:
eNOS [62], methyl-tetrahydrofolate [61, 63], angiotensinogen [55], Factor V
Barone/Read 340
[64, 65], Factor VII [65], Factor VIII [66] and prothrombin [67]. Atrial natriuretic
peptide (ANP) has a particularly strong positive association with stroke [68–70].
Candidate gene choice is frequently driven by accepted stroke risk factors
(e.g., hypertension, hemostasis and abnormalities in lipid metabolism) and
indeed significant positive associations of numerous markers with ischemic
stroke have been identified. At present, however, it is difficult to identify can-
didate gene associations with ischemic stroke [4]. Reproducibility of these gene
expression associations in different patient populations (e.g., different race or
genetic backgrounds) also is not known. The bewildering combination of pos-
sible outcomes for candidate gene association studies is related to the genomic
and phenotypic heterogeneity of the global stroke population. Studies are typi-
cally designed with case controls or by cohorts to enable close approximation
of phenotype between affected and nonaffected individuals. Superimposed
upon these levels of variation are issues in the timing of stroke onset, in the
variability of environmental influences and penetrance (i.e., not all individuals
of a given genotype will express the phenotype). Finally, although the human
genome project has been completed [1], identifying functionality of gene prod-
ucts lags significantly behind. Currently, it is estimated that only approximately
10% of the human genome has been ascribed function [24]. Certainly more
work needs to be done in this area, and issues related to stroke genomics that
include risk and the expression of genes underlying brain vulnerability and
ischemic sensitivity must be considered.
Preclinical Models of Spontaneous Stroke
It is with these caveats in mind that studies have focused on animal mod-
els of spontaneous stroke, where environmental and genetic variability can be
controlled. Bioinformatic approaches using synteny can facilitate the matching
of ‘stroke loci’ found in stroke-prone rats to candidate genes on the human
chromosome. Heterogeneity of risk factors and life events in humans has made
it advantageous to study rodent models. Highly homogeneous populations of
stroke-prone rats have been isolated from the incompletely inbred, sponta-
neously hypertensive rat (SHR) and then inbred further for this phenotype.
Initial studies using this stroke-prone rat indicated that the degree of functional
collateral blood flow after occlusion of the middle cerebral artery (MCAo) was
inherited in an autosomally recessive manner [71–73]. The authors studied
luminal diameters in vascular anastomoses between middle and anterior cere-
bral arteries and hypothesized that a single gene not directly linked to hyper-
tension determined the collateral flow phenotype. Further genetic comparisons
between strains were hampered by heterogeneity.
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Narrowing the genotype by further crossing SHR rats with stroke-prone ani-
mals allowed cosegregation of genes defining various stroke phenotypes and for
homogeneity of alleles for hypertension [74]. Two separate groups have utilized
these inbred populations for identification of genes associated with manifestation
of specific stroke phenotypes. A genome-wide screen was performed in an F2
cross-obtained by mating stroke-prone and SHR rats, in which latency to stroke
was used as a phenotype [75]. This study identified three major quantitative trait
loci (QTLs) designated, STR-1, STR-2, and STR-3. Of these, STR-2 and STR-3
conferred a protective effect against stroke in the presence of stroke-prone alleles
and STR-2 colocalized with the candidate gene encoding ANP and brain natri-
uretic peptide (BNP). Furthermore, interactions between alleles from within
STR-1 and STR-2 suggested that this phenotype was a reasonable model of the
polygenicity of stroke in man. Follow-up sequencing to characterize ANP and
BNP as candidates for stroke revealed point mutations in ANP and no differences
in BNP. In vitro functional studies indicated lower ANP promoter activation in
endothelial cells from stroke-prone rats versus SHR, with significantly lower
ANP expression in the brain and no difference in BNP expression [68]. To deter-
mine the in vivo significance of the STR-2 lowered ANP promoter activation in
stroke-prone animals, in comparison to stroke-resistant animals, a cosegregation
analysis of stroke occurrence in SHR stroke-prone rats/SHR stroke-resistant F2
descendants and ANP expression was performed [69]. It was found that reduced
expression of ANP did cosegregate with the appearance of early strokes in F2
animals [70]. Therefore, although lowered ANP expression may be part of the
phenotype of the protective STR-2 QTL, it is unlikely that this is the primary pro-
tective mechanism in these animals. Parallel human studies of the role of ANP in
cerebrovascular disease have confirmed that variation in the ANP gene may rep-
resent an independent risk factor for stroke in humans [4, 75] and emphasizes the
utility of this cohort of animals as a model of ANP dysfunction in multiple
subtypes of stroke.
Two other groups have utilized a modified model of the stroke-prone ani-
mal, employing F2 hybrids derived from crossing the stroke-prone SHR with
Wistar-Kyoto rats [76, 77]. One group [76] used brain weight poststroke as the
phenotype for linkage analysis, after the discovery that F2 animals had higher
levels of brain edema formation poststroke. This group found clear evidence of
the linkage of phenotype to a gene on chromosome 4, which contributed to the
severity of brain edema and was independent of blood pressure and STR3 iden-
tified by others [75]. The other group [77] designed studies to identify the
genetic component responsible for large infarct volumes in the stroke-prone rat
in response to a focal ischemic insult. To do this, they performed a genome
scan in an F2 cross-derived from the stroke-prone rat and the normotensive
Wistar-Kyoto rat [77]. Unlike others [75], they were only able to identify one
Barone/Read 342
major QTL responsible for large infarct volumes. This QTL was located on rat
chromosome 5, and like STR-2, it colocalized with ANP and BNP, and was
blood pressure independent. Unlike STR-2, this locus showed a much higher
significance (lod 16.6) and accounted for greater (i.e., 67%) of phenotypic
variance [77]. Subsequent studies identified that infarct volumes in the F1 rats
were approximately identical to those of the stroke-prone animals, suggesting
a dominant mode of inheritance [78].
Authors have argued over the significance of the overlap of STR-2 identi-
fied by some [55] with the QTL identified by others [77] on chromosome 5. It
is unclear how the two phenotypes studied, latency to stroke (i.e., relative risk)
[75] and size of infarct after occlusion (i.e., sensitivity to focal ischemia) [77],
should physiologically relate to each other. However, this may only become
apparent when individual genes are cosegregated with each phenotype. At pre-
sent, altered ANP expression appears to play a role in the phenotype described
by one group [75], but has been excluded from a role in the colony used by the
other group [77, 79].
What can be concluded from each of these stroke-prone rat models?
Certainly, each represents a unique and valid model of stroke for the study of
inheritance, and for a role of candidate genes, in particular, stroke phenotypes.
Neither colony represents a definitive model of human stroke, although linking
identified candidate genes in these stroke-prone colonies to the human popula-
tion has made progress [70]. One such research strategy that we have used is the
analysis of genomic synteny between the rat and human genome. This bioinfor-
matic approach seeks to align regions of homology using evolutionary con-
served markers and has been applied with some success in relating animal
models to human genetics in other disease paradigms such as non-insulin-depen-
dent diabetes [80]. Relating identified loci from stroke-prone animals to the
human genome offers a strategy for potential identification of candidate genes.
For example, the STR-2 region of rat chromosome 5 shows well-conserved gene
order and synteny with the human chromosome region 1p35–36. The high level
of synteny between these regions makes this region ideal for rat-human com-
parative analysis. Sequence tagged sites localized to this region have been iden-
tified and mapped to human transcript clusters. As many as 132 transcripts have
been identified in this region. The main candidates with some rationale for
involvement in stroke are shown in table 1.
Interestingly, only a few candidate genes identified at 1p35–36 have been
examined in association studies. ANP has recently been assessed for associa-
tion with multiple subtypes of stroke [70]. The polymorphism G664A, respon-
sible for a valine-methionine substitution in proANP peptide was found to be
positively associated with the occurrence of stroke [70]. In contrast, methylene-
tetrahydrofolate, another marker located at 1p35–36, was negatively associated
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Table 1. 1p36–p35 positional candidates with a biological rationale in stroke*
Homology Rationale in stroke
CD30L receptor precursor Nerve growth factor receptor

superfamily
Tumor necrosis factor receptor 2 Mouse KO increases neuronal
damage in response to insults
Human MASP-2 serine protease protein – Depletion of complement system
complement processing protease improves outcome following cerebral
ischemia
gp40 mucin – putative influenza virus receptor Role in cell adhesion
Human Tropomyosin-related protein- May be related to sustained
exclusively neuronal/brain expression contraction during cerebral
vasospasm
Human protease proMch6 (Mch6)/CASPASE-9 Critical upstream activator of
the caspase cascade in vivo
EPHRIN RECEPTOR EphA2/Tyrosine-protein Role in neuronal development
kinase receptor ECK
Human PDGF-associated protein Unknown
Endothelin-converting enzyme E1 Enzyme that produces potent
vasoconstriction
WNT4 protein precursor Possible role in synaptic plasticity.
Linked to JNK signaling and
indirectly to Notch (CADASIL)
EPHRIN RECEPTOR EphB2/Tyrosine-protein Role in neuronal development
kinase receptor ERK
Stathmin – v high brain expression Phosphorylated by CAM kinase II
Corticosteriod-binding protein – yeast Stress response
putative bicistronic heat shock proteins
Platelet-activating factor receptor Unknown
Dishevelled-1 Possible role in synaptic plasticity.
Linked to JNK signaling and
indirectly to Notch
Atrial natriuretic peptide A Localized with LOD peak
hypertension, see text
Brain natriuretic peptide B Localized with LOD peak
hypertension, see text
Complement component 1, q subcomponent, Depletion of complement system
alpha polypeptide (C1QA) improves outcome following cerebral
ischemia
5,10-Methylene-tetrahydrofolate reductase Heterozygous mutations are
significant cause of stroke in
general population, see text
Brain-specific angiogenesis inhibitor (BAI2) Regulator of angiogenesis
Platelet phospholipase A2, group IIA Antiplatelet agents modify stroke risk
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Table 1 (continued)
Homology Rationale in stroke
Sodium hydrogen exchanger-1 pH regulator of acidity associated

with postischemic damage
Platelet-activating factor receptor (PTAFR) PAF involved in arterial thrombosis;
Antiplatelet agents modify stroke risk
*This table documents the identification of human candidate genes that are syntenic to the
STR2 region of rat chromosome 5 identified in a cohort of SHR-stroke prone animals. STR2
shows well-conserved gene order and synteny with the human chromosome region 1p35–36
(between D1S503–D1S2667). The high level of synteny between these regions makes this
region ideal for rat-human comparative analysis. Sequence tagged sites localized to this
region have been identified and mapped to human transcript clusters. Many transcripts,
specifically 132 of them, have been identified in this region, with the main candidates listed
above.
with occurrence of stroke [79]. Further studies may elucidate the predictability
of markers of 1p35–36 and association with stroke for those genes listed in
table 1.
In contrast, rat-human synteny in the regions of the rat STR-1 and STR-3
loci are not well conserved, as several disruptions of synteny appear to have
been introduced during evolution. It may be difficult to determine the exact
regions of synteny between these rat loci and human chromosomal loci, and
thus to extrapolate the candidate genes from rat to human. Human chromoso-
mal regions syntenic with STR-1 span regions of two human chromosomes,
around 16p11 and 19q13. Human synteny with the STR-3 region also appears
to be disrupted, with regions of synteny mapping telomerically to opposite
arms of chromosome 7 (7p21 and 7q35). Of course, this is a problem of ani-
mal modeling of human diseases in general and is not restricted only to
ischemic stroke.
Stroke-Associated Gene Expression in the

Evolution of Brain Injury
Cerebral ischemia is a powerful stimulus for the de novo expression and

up-regulation of numerous genes [2, 4, 5, 81, 82]. In terms of isolation of gene
candidates for a neuroprotective strategy, interpretation of expression changes
has proven difficult. The multitude of animal models of ischemia with varying
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genetic heterogeneity and infarct pathophysiology, is also complicated by spa-
tial and temporal variations that have largely confounded interpretation.
Furthermore, assays of differential expression have varying sensitivity to the
relative fold-increase or -decrease in mRNA expression. As a result, ‘fishing’
exercises (i.e., differential expression detection studies) will often result in
‘catches’ (i.e., hits) of differential gene expression that vary depending on the
assay employed.
Bearing in mind this bewildering array of complexity, the next section
addresses animal model(s) that might be utilized with differential gene expres-
sion analysis, target confirmation methodology that is necessary following the
identification and confirmation of a differentially expressed gene (i.e., a ‘hit’),
and the functional assessment of these genes in the disease process. A hierar-
chical critical path that depicts the path from target identification to target
confirmation/validation is depicted schematically in figure 1.
Clinical Relevance of Ischemic Stroke Models
The failure of several putative neuroprotective agents in recent large multi-

centered clinical trials [83, 84] has led to critical re-examination of preclinical
models of ischemia [85]. Heterogeneity in the human stroke population and the
multitude of well-defined animal models of ischemia have led to attempts to
refine model choice as related to patient subgroups [86, 87]. In an effort to
stratify patient groups that can be predicted using specific animal models,
authors have focused on the use of MRI signatures, particularly perfusion-
weighted or diffusion-weighted imaging (PWI, DWI) mismatches. Two main
groups of acute stroke patients are identifiable: those with evolving infarcts in
which lesion PWI ⬎ DWI, or those with a stabilized infarct where PWI ⱕ DWI
[88, 89]. Such PWI/DWI assessments have been proposed to correlate to the
extent of salvageable tissue, with approximately 70% of patients exhibiting
PWI lesions ⬎ DWI at 6 h poststroke, and about 50% of patients exhibiting this
mismatch at 24 h poststroke [89].
Applying the same imaging paradigms to animal models of focal ischemia
should enable translation of preclinical pathophysiology into predictive out-
comes in the appropriate patient population. However, detailed comparisons of
the development of PWI/DWI signatures between animal models of ischemia
are difficult to establish due to the use of various rat strains, anesthetics, and
modes of ischemia induction. However, broad comparisons are possible by
exploring the development of DWI signal as a marker of lesion volume with
respect to time. Data in certain animal models of focal stroke can show a delay
in the development of DWI hyperintensity (i.e., brain lesion size) that lags
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Critical path from expression to target validation
RDA, Microarrays,
Animal model mRNA differential expression
SAGE, SSH, DD
Reproducibility/commonality of ‘hits’?
Confirm differential expression Northern analysis
Expression analysis Quantitative RT-PCR
Expression studies
mRNA localization In situ

hybridization (ISH)
Confirmation and localization ELISA, Western analysis,

of protein expression Immunohistochemistry (IHC)
Targeted gene KO
Genetic/transcriptional
Antisense
modification
Adenoviral transfection
Functional studies
Appropriate PK
In-Vivo pharmacological studies tool compound
treatment
Fig. 1. Critical pathway for target identification and confirmation. Following selection
of an animal model appropriate to clinical subpopulation, broad mRNA differential expres-
sion strategies are adopted employing differential expression assays such as RDA, microar-
rays, subtractive hybridization (SSH), serial analysis of gene expression (SAGE) and/or
differential display (DD). Across these assays, reproducibility in identified hits are explored
as a technique of prioritizing subsequent studies to confirm differential expression.
Comprehensive expression analysis using RT-PCR or Northern analysis allows confirmation
of identified hits and fully quantified temporal profiling. This can also include RNA local-
ization using in-situ hybridization (ISH). Protein confirmation by ELISA, Western analysis
and immunohistochemistry (IHC) follows mRNA profiling, to confirm translation. In an
ischemic brain, pooling of mRNA and uncoupling of translation can occur as indicated in the
text. Finally, functional studies encompassing target gene knockout, adenoviral transfection
and in vivo pharmacology complete validation of a potential target gene. Appropriate chem-
istry directed against the verified biological target could result in the eventual discovery of a
drug for stroke.
behind a perfusion deficit (i.e., PWI changes associated with stroke or focal
ischemia). This delay is attributable, in part, to the relative contribution of
insufficient collateral flow and the peri-infarct depolarization that injures the
poorly perfused, ischemic brain during infarct evolution [87, 90].
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Direct MCAo by proximal electrocoagulation of the middle cerebral artery
produces an expanding DWI lesion, with an initial marked expansion at 4 h
followed by a small increase from 4 to 24 h [91]. Electrocoagulation occlusion
of the distal MCA produces a much more rapidly evolving infarct with a near
maximal DWI lesion observed by 1–2 h [92, 93]. Available embolic models of
focal stroke using intra-arterial injection of thrombin [94] or aged [95] or
fibrin-rich [96] clots have reported similar expansion of DWI hyperintensity.
For example, following thrombin injection, DWI hyperintensity is apparent at
80 min postadministration, with the volume gradually expanding up to 24 h
[94]. Intraluminal suture occlusion produces a range of DWI lesion progression
dependent on whether the filament is introduced via the common carotid artery
[97] or the external carotid artery [98]. Permanent MCAo via common carotid
artery suture produces a rapid evolution of DWI hyperintensity within minutes,
followed by maximal expansion by 2 h [99, 100]. In comparison, permanent
MCAo without occluding the common carotid artery (i.e., via the external com-
mon carotid artery) [98], evokes an initial rapid expansion of DWI hyperinten-
sity over the first 30 min followed by final infarct volume reached at 7 h
[101–103]. A close inspection of this model identifies it as exhibiting a flow
mismatch similar to that in man, i.e., it represents the type of evolving infarct
that should provide information relevant to human stroke [87]. For this reason
a number of groups [6, 104, 105] have decided to use this model for differen-
tial gene expression studies.
Differential Gene Expression Methodologies
The detection of genes that are differentially expressed due to stroke can
be identified using simpler techniques such as Northern blotting, RT-PCR, or
in situ hybridization. These techniques involve the selection and study of a
specific gene of interest based on previous data that provides a biological
rationale for study in stroke or another specific disease. However, more
sophisticated screening techniques are now available that can identify groups
of differentially expressed genes, both known and unknown. These screening
techniques include subtractive hybridization, differential hybridization, repre-
sentational differential analysis (RDA), serial analysis of gene expression
(SAGE), and differential display [106]. The identification of differentially
expressed genes in stroke has employed the simpler techniques as well as these
newer techniques.
Variations in assay and threshold of detection can result in the isolation of
gene sets that differ according to assay selection. Therefore, to ensure maxi-
mum confidence in the detection of adaptive up-regulation of gene expression,
Barone/Read 348
reproducibility of genes and pathways should be verified across several dif-
ferential expression techniques and independent cross-validation of a gene’s
up-regulation should be emphasized. A summary of stroke-associated gene
expression discovered by all the techniques is listed in table 2. Only those
gene/message expression changes in the rat following MCAo were confirmed
using more than one expression detection technique. A brief summary of the
more complex techniques is provided below.
Differential Display
Differential display is a means of comparing all poly-A mRNA between
experimental and control populations. In this technique, mRNA is converted into
first strand cDNA with reverse transcription followed by polymerase chain reac-
tion (PCR) with multiple sets of primers. The PCR products are then displayed
with control and experimental samples side-by-side on high-resolution denatur-
ing gel. In this way, differential gene expression is apparent. This technique has
been applied with success to isolate differentially expressed products following
experimental MCAo. For example, differential display was used after rat MCAo
to discover a gene that encodes adrenomedullin, a member of the calcitonin
gene-related peptide family [113]. This analysis was followed by temporal stud-
ies using Northern analysis, which confirmed that expression of mRNA levels
increased in the ischemic cortex at 3 and 6 h after MCAo and levels remained
elevated for up to 15 days. Immunohistochemical studies to confirm protein
expression then localized adrenomedullin to ischemic neuronal processes. In
functional studies, synthetic adrenomedullin microinjected into the precon-
stricted rat pial arteries produced dose-dependent relaxation of the vessels. In
addition, intracerebroventricular administration of adrenomedullin, prior to and
after MCAo, increased the degree of focal ischemic injury. Other groups have
also used this technique in ischemia to identify differentially expressed mRNAs
such as a zinc transporter gene [144] and an ADP-ribosylation factor like gene
[145]. Other examples include the transcription factor SEF-2 [146], proteosome
p112 [146], and ST-38 chemokine [147] following rat MCAo. Differential dis-
play is useful but very labor intensive. It is most useful for examining several
RNA samples simultaneously and has been used extensively for temporal, dose-
response, and multiple treatment studies. Although differential display is ‘semi-
quantitative,’ only relatively a small amount of total RNA (approximately 15 ␮g)
are required. Some problems include high false positive rates that cannot be con-
firmed by RT-PCR or Northern blotting. Modifications such as subtracted dif-
ferential display, which removes unregulated cDNA by mRNA subtraction prior
to differential display [148], represent improvements. Confidence in an isolated
candidate gene can be improved by using independent follow-up assays of gene
expression in parallel.
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Table 2. Summary of differential gene expression changes determined by techniques that measure transcription differences following focal
ischemia stroke in the rat*
Barone/Read
Gene Stroke Early RNA Later RNA RNA/Protein Protein Reference(s)

pMCAo ⫽ p expression ⱕ24 h expression ⱖ24 h cross-validation demonstrated/
tMCAo ⫽ t techniques verified
Immediate early genes

NGFI-A p Yes No ISH No 107
NGFI-B p Yes No ISH No 107
NGFI-C p Yes No ISH No 107
NGFI-C p Yes No ISH No 3
Nurr-1 p Yes No ISH No 107
erg-2 p Yes No ISH No 107
erg-3 p Yes No ISH No 107
Zif 268, c-fos p Yes No Northern analysis No 108
NF-␬B t Yes No IHC Yes 109
Gel shift analysis
NF-␬B t Yes (subunit Yes (subunit Western blotting Yes 110
specific) specific) IHC
Gel shift analysis
Activating transcription t Yes No IHC Yes 111
factor (decrease) Western analysis
c-fos, c-jun, zif 268 p Yes No Northern analysis No 108
ATF-3 p, t Yes ND RT-PCR, ISH, IHC No 105
Cytokines
IL-1 receptor p Yes (subunit No RT-PCR No 113
specific)
IL-1RA p Yes Yes RT-PCR No 113
350
IL-1RA p Yes ND RT-PCR No 114
Gene Expression in Stroke
IL-1B p Yes Yes Northern analysis No 115

IL-1B p Yes No ISH No 116
IL-1␤ p Yes No RT-PCR No 117
IL-1␤ t Yes Yes Northern analysis No 118
IL-2 p No change No change RT-PCR No 117
IL6, Zif 268, c-fos p Yes No Northern analysis No 108
CINC/IL-2 p Yes ND ISH No 119
IL-10 p Yes (6 h) No RT-PCR No 117
TNF-␣ p Yes Yes ISH, IHC, RT-PCR Yes 120
TNF-␣ t Yes Yes Northern analysis No 108
TNF-␣ p Yes No RT-PCR No 117
TNF-␣ p Yes Yes Northern analysis Yes 121
IHC
LIF t Yes Yes RT-PCR Yes 122
Western blot
IHC
LIF p Yes Yes RT-PCR No 104
SOCS-3 p Yes Yes RT-PCR No 104
IL-1␤ p, t Yes No RT-PCR, ISH No 105
TNF␣ p, t Yes No RT-PCR, ISH No 105
Inflammation
COX-1 t No change No change RT-PCR No 123
COX-2 t Yes Yes RT-PCR, IHC Yes 123
COX-2 p Yes RT-PCR No 104
MCP-1 t Yes Yes ELISA Yes 124
MCP-1 p Yes Yes Nothern analysis Yes 125
RT-PCR, IHC
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Table 2 (continued)
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MCP-1 p No Yes Northern analysis No 126

MCP-1 p Yes Yes RT-PCR No 104
iNOS t Yes No RT-PCR No 123
iNOS t Yes Yes RT-PCR, IHC Yes 127
MCP-3 p, t Yes Yes Northern analysis No 128
IP10 p Yes Yes Northern analysis No 129
CXCR3 p Yes Yes Northern analysis No 130
Heme oxygenase p, t Yes ND RT-PCR, ISH ND 105
LPS-binding protein p No Yes RT-PCR ND 6
Apoptosis
Bax t Yes No ISH No 131
Caspase 1,6,7,8,11 p Yes ND RT-PCR No 132
Caspase 2,9 p No change ND RT-PCR No 133
Caspase 3 t No Yes ISH No 131
Caspase-3 p Yes No RT-PCR No 133
Fas, Fas-L p Yes ND RT-PCR No 133
TR3-death receptor p Yes ND RT-PCR No 133
p75 NGF-R p Yes ND RT-PCR No 133
Arc p Yes No ISH Yes 3
BIS p, t Yes ND RT-PCR, ISH No 105
Arc p Yes RT-PCR No 104
Growth factors
VEGF p Yes No ISH, IHC Yes 134
352
VEGF t Yes Yes Northern analysis Yes 135
Western blotting
VEGF receptor p No Yes ISH, IHC Yes 134

VGF p Yes Yes RT-PCR No 104
BDNF t Yes No ISH No 136
TGF␤-1 p No Yes Northern analysis No 143
TGF-␤1 p ND Yes RT-PCR No 104
Narp p, t Yes ND RT-PCR, ISH No 105
Cathepsin C p No Yes RT-PCR No 6
Cystatin B p No Yes RT-PCR No 6
Agrin p Yes No RT-PCR No 6
JAK-2 p Yes No RT-PCR No 6
cpg-21 p Yes No RT-PCR No 6
Narp p Yes No RT-PCR No 6
Other genes
Adrenomedullin p Yes Yes Northern analysis Yes 112
IHC
HIF-1 p No Yes ISH, IHC Yes 134
Hsp-27 p ND Yes RT-PCR No 104
Hsp-70 p ND Yes RT-PCR No 104
Hsp-70 t Yes No In situ No 137
autoradiography
GADD 45 t Yes No In situ No 137
autoradiography
MIP-1␣ p Yes Yes Northern analysis Yes 125
RT-PCR, IHC
MIP-1␣ p, t Yes No In situ No 138
MIP-3␣ p Yes RT-PCR No 104
CRH p Yes No ISH No 139
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Table 2 (continued)
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NT-3 t Yes (decrease) No ISH No 136

Trk-B t Yes No ISH No 136
Osteopontin p Yes Yes ISH, IHC Yes 140
Osteopontin p No Yes Northern analysis Yes 141
IHC
Osteoactivin p ND Yes RT-PCR No 104
TIMP-1 p Yes No Northern analysis No 142
TIMP-1 p Yes Yes Subtractive No 142
cDNA libraries
Southern analysis
TIMP-1 p ND Yes RT-PCR No 104
CD14 p ND Yes RT-PCR No 104
CD44 p ND Yes RT-PCR No 104
GADD45␥ p ND Yes RT-PCR No 104
Xin p Yes RT-PCR No 104
Hsp-70 p, t Yes ND RT-PCR, ISH No 105
Cyr61 p, t Yes ND RT-PCR, ISH No 105
Lox-1 p, t Yes ND RT-PCR, ISH No 105
Rad p Yes No RT-PCR No 6
G33A p Yes No RT-PCR No 6
HYCP2 p Yes No RT-PCR No 6
Mim-3 p Yes No RT-PCR No 6
CELF p Yes No RT-PCR No 6
354
Tenascin p Yes No RT-PCR No 6
DAF p Yes No RT-PCR No 6
Cip-26 p No Yes RT-PCR No 6

PHAS-I p No Yes RT-PCR No 6
TBFII p No RT-PCR No 6
Spr p No Yes RT-PCR No 6
Glycerol-3 phosphate p No Yes RT-PCR No 6
dehydrogenase
PRG1 p No Yes RT-PCR No 6
*This table only lists increased message expression that has been validated within or between labs independently. The importance of this
validation in addition to the verification of translated protein for candidate genes is emphasized in the text. Permanent MCAo ⫽ pMCAo;
transient MCAO ⫽ tMCAo; ND ⫽ not determined; ISH ⫽ in situ hybridization; IHC ⫽ immunohistochemistry.
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Subtractive Hybridization
Subtractive hybridization compares qualitative differences in gene expres-
sion between two experimental paradigms. This is usually achieved by
hybridization of biotinylated ‘driver’ cDNAs to the mRNA pool from the tar-
get tissue. Duplexes of driver cDNAs and target mRNAs are then removed,
resulting in a pool of target mRNAs expressed only by the target tissue [149].
Down-regulated mRNAs are determined by carrying out the reaction in
reverse. Modifications to the assay include suppression subtractive hybridiza-
tion and RDA, where the polymerase chain reaction replaces physical subtrac-
tion methods to enrich for differentially expressed transcripts. Such
modifications emphasize differential mRNA of both low and high abundance,
rather than biasing selection of only highly expressed genes as is the case with
the more basic subtractive hybridization methodology. Suppression subtractive
hybridization has been used to identify candidate genes with putative roles in
experimental cerebral ischemia. For example, the induced expression of a rat
homolog to human monocyte chemotactic protein-3 (MCP-3) was identified in
the ischemic brain [128]. Independent Northern analysis identified increases in
MCP-3 mRNA observed at 12 h postischemia, with 49-fold and 17-fold increases
over control in permanent and temporary MCAo, respectively. Significant
induction of MCP-3 in the ischemic cortex was sustained up to 5 days after
ischemic injury. In other models, subtractive hybridization has been less
widely used to identify candidate genes, perhaps due to the technique demands
of the subtraction approach, although false positives are less frequent. The sub-
tractive hybridization approach also has been used to successfully identify a
novel cDNA clone (pGSH3), expressed only after ischemia in the gerbil cortex
[149], which turned out to be a homolog of a 72-kilodalton human heat-shock
protein (hsp70) gene. Basal cortical levels were found to be low, but 8 h after
a 10-min transient forebrain ischemia, gene expression became prominent in
the cerebral cortex.
RDA
RDA is a relatively novel PCR-coupled, genome subtractive process [150]
that until recently [104, 105] had not been used to assay differential expression
in models of cerebral ischemia. RDA is conceptually similar to subtractive
hybridization, but the unavailability of a commercially produced kit for RDA
has meant that it has been less broadly exploited. RDA was originally estab-
lished to monitor differences in genomic DNA content between individuals, it
was later modified to identify differences in gene expression [150]. The robust
gene expression changes that characterize the MCAo model are detectable with
RDA, as we have recently been able to show [104]. Subtracting ischemic cor-
tex from rats 24 h following permanent MCAo from similarly treated tissue
Barone/Read 356
from sham-operated animals, we identified candidate ischemia-regulated tran-
scripts. Primary confirmation of the accumulation of these gene products in the
ischemic cortex was confirmed using SYBR Green RT-PCR, followed by the
more comprehensive time course analysis using TaqMan RT-PCR in selected
cases. Several genes identified through this approach had previously been
reported to increase following MCAo, such as heat shock proteins (hsp 27 and
hsp 70) and others (MCP-1, MIP3␣, COX-2, TGF-␤1, tissue inhibitor of matrix
metalloproteinase (TIMP-1) and Arc), but some were newly identified as
MCAo-induced genes in this study (LIF, SOCS-3, VGF, CD44, CD14, CD81,
osteoactivin, GADD45␥ and Xin) [104]. In another study [105] using this tech-
nique, 128 unique gene fragments were isolated and 13 were selected for
RT-PCR analysis. Many transcripts were verified to be differentially expressed
by RT-PCR, including four genes not previously implicated in stroke: neuronal
activity-regulated pentraxin (Narp), cysteine rich protein 61 (cyr61), Bcl2-
binding protein Bis (Bcl-2-interacting death suppressor), and lectin-like
ox-LDL receptor (Lox-1).
Microarray Analysis
All the above strategies identify relatively small numbers of differentially
expressed genes. Large numbers of DNA fragments (110–450 bp) are produced
in the process, which need to be confirmed and frequently extended to full
lengths to obtain gene identity. Although all of these technologies are useful for
isolating candidate genes, they are of limited utility in providing a broad char-
acterization of the expression of large number of genes within a particular
model. Array-based technology, on the other hand, allows large-scale and
prospective analysis of gene expression as well as time-response profiling and
drug treatment analysis (pharmacogenomics). Whether using arrays of oligo-
nucleotides [151, 152] or gene fragments [153], the array technology allows par-
allel expression monitoring of numerous genes at the same time [154]. The
limitations and biases of the technique are obviously in the selection of genes
to study on the array. The power and quality of microarrays has continued to
improve significantly, and now thousands of genes can be evaluated at a time.
The pioneer study [3] using this technique in the context of stroke was applied
to studying gene expression in a proximal MCAo electrocautery model [155].
Oligonucleotide probe arrays were employed with 750 predetermined genes
optimized for gene expression in rat bone and cartilage. The gene chip (Roche,
ROEZ002) was used to monitor gene expression after 3 h of permanent focal
ischemia. To determine genes differentially expressed as a consequence of
ischemia, the authors took tissue from the ipsilateral frontal and parietal cortices
and compared their expression to corresponding regions on the contralateral
side. A significant change in transcription was defined as a 2-fold or greater
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increase or decrease in expression compared to the contralateral hemisphere. The
authors described a significant up-regulation of 24 genes in the parietal and
frontal cortices and striatum, with particularly robust changes in c-fos, NGFI-A,
NGFI-B, NGFI-C, Krox20, Nor-1, cyclooxygenase-2, and Arc. This study
clearly demonstrated the utility of array technology in the analysis of gene reg-
ulation following experimental cerebral ischemia. The use of arrays optimized
for bone and cartilage genes unfortunately limited the usefulness to 15% of the
total gene representation on the array. Nevertheless, key gene families such as
the phosphatases (MKP-1 and MKP-3) and the chemokines (MCP-1 and MIP-1␣)
were represented and expression profiles agreed with previous findings
[156, 157]. No change in ‘housekeeping’ genes GAPDH and ␤-actin were found
(ipsilateral vs. contralateral).
Another outstanding study [6] used the rat Affymetrix U34A oligo-
nucleotide array to assess 8740 transcripts in the peri-infarction rat cortex 24 h
after thread MCAo, a model representing slow stroke evolution, as in man [98].
Using strict analysis criteria, less than 4% of transcripts were regulated (e.g.,
264 were up-regulated and 64 were down-regulated), of which 163 had not been
reported to be modified in stroke previously. In terms of functional groups,
G-protein-related genes were least variable, while cytokines, chemokines,
stress proteins and cell adhesion and immune molecules were most modulated.
Quantitative RT-PCR of selected genes identified early up-regulated genes
including Narp, Rad, G33A, HYCP2, Pim-3, Cpg21, Jak2, CELF, Tenascin, and
DAF. Late up-regulated genes (⬎24 h) included cathepsin C, Cip-26, cystatin B,
PHAS-I, TBFII, Spr, PRG1, and LPS-binding protein [6]. Implications of
up-regulated glycerol 3-phosphate dehydrogenase, plasticity-related transcripts,
gene regulation related to cell survival, death and tissue repair and functional
recovery, and biochemical pathways related to gene changes were evaluated [6].
SAGE
SAGE yields information about absolute transcript numbers of many, if not
all, genes expressed in a given tissue and therefore allows for the identification
of differentially expressed genes when applied to tissues in different conditions
[158–160]. The technique is based on the reduction of each expressed transcript
sequence to short (14–15 bp), yet representative, sequences (tags) at a defined
position, which are concatenated into long molecules. Sequencing these mole-
cules reveals the identity of multiple transcripts simultaneously. The number of
times a particular tag is detected in a SAGE library, therefore, provides a quan-
titative and digital measure of gene expression [158–160]. In a recent and an ele-
gant study, differentially expressed genes in mouse brain 14 h after the induction
of focal cerebral ischemia were determined using SAGE [160]. From the esti-
mated 30,000 genes of the mouse genome, at least 24,590 genes were detected
Barone/Read 358
by SAGE. Analysis of ⬎60,000 transcripts revealed 83 up-regulated and 94
down-regulated transcripts, defined as greater than or equal to 8-fold difference
from baseline. Up-regulated genes were classified as transport and secretion
(4%), ribosomal proteins (4%), DNA/RNA metabolism (4%), cytoskeletal (6%),
protein folding and degradation (12%), intermediary metabolism (16%), signal
transduction (22%), or unclassified (32%). Metallothionein-II (MT-II) was
found to be the most significantly up-regulated transcript in the ischemic hemi-
sphere. MT-I and MT-II both appear to be induced by metals, glucocorticoids,
and inflammatory signals in a coordinated manner, yet their function remains
unknown. Up-regulation of both MT-I and MT-II was confirmed by Northern
blotting. MT-I and MT-II mRNA expression increased immediately after 2 h of
transient ischemia, with a maximum after 16 h. Western blotting and immuno-
histochemistry revealed MT-I/-II up-regulation in the ischemic hemisphere,
whereas double-labeling demonstrated colocalization of MT with markers for
astrocytes as well as for monocytes/macrophages. The completeness of this
study (fig. 1) was demonstrated by the use of MT-I- and MT-II-deficient mice,
which developed an approximately 3-fold larger infarcts than wild-type mice
and a significantly worse neurological outcome [160].
Assay Variation and Confidence in Identified Gene Expression
In the preceding sections we discussed many of the techniques available

for the detection of differential gene expression and some of the ‘within assay’
issues associated with each technique. Next, we will discuss in more detail
model-to-model differences, the importance of the poststroke timings of RNA
sampling, and different experimental paradigms in stroke that might help us
discover genes that have roles in brain protection or tolerance, and studies that
can be used to look for genes that might contribute to the recovery/plasticity of
the brain postinjury.
There are several issues which warrant discussion including the significant
variability between techniques, and the identification of false positive and false
negative results. Assays of differential expression have an inherent variability
dependent on assay methodology, sensitivity, and reaction efficiency. When
exploring disease paradigms which are powerful stimulators of gene expression
such as cerebral ischemia, the usual tendency is to highlight gene sets or func-
tional groups that are up-regulated and differentially expressed. Given the large
numbers of genes identified, it is difficult to confirm all the differentially
expressed genes and false positive and false negative differential gene expres-
sion becomes an issue. False positives can broadly be defined as genes whose
differential expression is not subsequently confirmed by an independent study
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(i.e., RT-PCR, Northern analysis, in situ hybridization, etc.). False negatives, in
contrast, are genes that are, in fact, differentially expressed, but not detected as
such by the complex assay employed (e.g., subtractive hybridization, RDA,
DNA microarray).
To manage these issues, we have employed the strategy of using multiple
assays of differential expression on the same RNA pool and then cross-validating
all of the numerous differentially expressed products between assays. Identifying
commonalities in expression across assays increases confidence in particular
results, and genes identified across two or more assays receive a higher priority
for confirmatory studies. A table of descending confidence in ‘hits’ can then be
constructed. This technique for handling large numbers of ‘hits’ avoids issues of
biasing the identification of differential expression to a single assay and also
interassay variability. Subsequent analysis by Taqman RT-PCR has confirmed
that robust differential gene expression identified across all assays had particu-
larly high-fold increases in differential expression. This strategy is also useful for
identifying false negatives (i.e., products differentially expressed but not detected
as such in assays) [4]. The ‘complimentary-techniques’ approach at target vali-
dation maximizes the coverage of differential gene expression by minimizing the
losses due to the technical vagaries of any single technique.
Confirmation: An Integral Part of Differential

Gene Expression
The techniques cited above for the identification of differentially

expressed mRNAs represent starting points for the study of gene expression
following stroke. All data derived by these methods require confirmation from
an independent study to remove false positives, and this usually forms part of a
broader analysis of expression of the gene that has been identified [3–6,
104–106, 160]. Traditional methods for analyzing gene expression include
techniques such as Northern blotting, RNAse protection, in situ hybridization,
and semi-quantitative RT-PCR. All of these methodologies have been used to
study the expression of individual genes or small groups of genes in stroke
models. Differential screening methodologies ideally generate large numbers of
‘hits’ which require rapid confirmation in a high throughput system.
Recently, real-time quantitative RT-PCR techniques, such as ‘Taqman’
probes or SYBR green [161] to monitor an accumulating PCR product in real
time, allow an accurate comparison of initial PCR template numbers. These
assays can be carried out in 96 or 384 well formats and can utilize robots,
reducing operator time and error. With these techniques, it is possible to carry
out rapid confirmation of many differentially expressed genes simultaneously
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or to undertake a more detailed expression analysis of a single ‘hit’ [142]. For
example, Taqman RT-PCR has been extensively applied to temporal profiling
of caspase expression following MCAo in rats [132, 133] and SYBR green has
been used to confirm differentially expressed genes identified by RDA [104].
Taqman is a PCR-based technique that is more sensitive than other confirma-
tory technologies such as Northern blotting. Typically, Taqman RT-PCR uses
approximately 50 ng of total RNA per gene, while Northern blotting uses
10–20 ␮g. Additionally, Taqman RT-PCR is as sensitive as in situ hybridization,
with the added advantage of higher throughput. Perhaps most importantly for
paradigms such as MCAo, where gene expression can exceed 600-fold over that
observed in naïve animals, Taqman PCR can quantitate gene expression over
five to six orders of magnitude without multiple dilution series, as necessitated
by other assays [115]. Clearly, PCR-based technologies such as Taqman
RT-PCR and SYBR Green RT-PCR, whilst in their infancy in application to the
study of cerebral ischemia [104, 132, 133], offer advantages for confirmation
and expansion of data on differential gene expression.
These techniques are also of value in testing hypotheses about genes
already known to be regulated in stroke models, where differential expression
is suggested by other biological evidence/data. The sensitivity of PCR-based
methodologies suggests that sufficient RNA can be isolated from a single ani-
mal to allow the simultaneous assessment of several hundred genes. A large
body of data can be amassed and the expression of many different genes com-
pared in a single study, without drawbacks such as variation between studies,
operators and cohorts of animals. The major drawback of high-throughput
quantitative RT-PCR is that while it allows for the rapid assessment of changes
in gene expression at the level of mRNA, it is not able to provide information
on the precise cellular localization of such changes. A detailed understanding
of stroke models utilized for differential gene expression is essential (i.e., the
models have to be adequately characterized over time for cellular changes). The
cell-type and intracellular locations of changes in gene expression are impor-
tant. For example, neurons and oligodendrocytes die within the ischemic infarct
[162, 163] particularly after 12 h of ischemia. Astrocytes and microglia are
decreased in number in the core region of the lesion and proliferation of both
of these cell types occurs in the marginal areas [164]. Polymorphonuclear
leukocytes, and later macrophages, invade the lesion after around 12 h and for
days after [163–165]. Changes in gene expression have to be understood in the
context of these evolving cell types present at any given time after stroke.
Ultimately, expression profiling must involve techniques such as in situ
hybridization and immunohistochemistry, which allow the localization of
expression to be viewed in relation to the structure of the evolving lesion, and
the identification of the types of cells in which expression is occurring.
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Confirmation of expression and time course of protein translation is essen-
tial for verification that up-regulated gene transcription has proceeded to the
formation of protein. This point is especially pertinent given the severe energy
perturbations in the ischemic brain. During this state, transcription and transla-
tion can become uncoupled due to energetic demands of assembling protein. This
effect is temporally and spatially dependent and has been extensively reviewed
[5, 81], including the uncoupling of mRNA transcription and protein translation
following MCAo [4]. Issues associated with the sensitivity of protein-detection
assays must be considered for some proteins. Ultimately, it is proteins which are
pivotal in cellular function, and thus proteomic analysis in addition to mRNA
analysis will point the way ahead.
Functional Studies,Transgenic Studies, and

in vivo Pharmacology
There are already many examples from the literature where transgenic animal
studies and/or pharmacological studies have coincided with gene expression stud-
ies to demonstrate the involvement of specific gene expression in focal stroke
injury or protection. For example, cyclooxygenase-2-deficient mice are known to
exhibit reduced susceptibility to brain injury [166]. IL-1ra was shown to be neu-
roprotective in brain injury [167] well before the altered expression of the IL-1 sys-
tem in stroke was demonstrated [114]. IL-6 also has been shown to be
neuroprotective in stroke [168, 169]. In addition, it has been shown that blocking
thyrotropin-releasing hormone provides a significant protection against ischemic
brain damage and associated neurological deficits [170, 171]. Following stroke,
treatment with BDNF reduces brain injury in the MCAo model [172]. Genes for
all these proteins have been shown to be up-regulated in stroke models. One recent
example is Metallothionene-II as a major neuroprotective gene in mouse focal
cerebral ischemia [160]. In this study, changes in the metallothionene-II gene were
verified using multiple methods including immunohistochemistry, in situ
hybridization and Western blot. In addition, stroke in the metallothionene knock-
out mouse resulted in stroke three times larger than in wild-type mice [160].
Additional Models for Discovering Gene Targets in Brain Injury
Neuroprotective or Neurodestructive Gene Expression

As pointed out previously [2], focal ischemia stimulates multiple gene
expression changes. Focal ischemia is a very powerful reformatting and repro-
gramming stimulus for the brain. There are broad and robust gene expression
Barone/Read 362
responses that occur following the focal stroke that are exhibited as temporal
episodes or ‘waves’ of expression of different groups of genes [2]. These
waves are largely comprised of increased expression of inflammatory
cytokines including IL-6 and IL-1ra. In addition, growth factors (e.g., BDNF)
that might be expected to play a neuroprotective role following stroke also
increase in this time frame. The increased cytokine gene expression appears to
drive leukocyte infiltration, a poststroke brain response to injury, and is asso-
ciated with secondary brain injury and repair processes following stroke. Later
waves of new gene expression include mediators, which appears to be impor-
tant in tissue remodeling (i.e., resolution of ischemic tissue injury) and per-
haps recovery of function. These issues are important in relation to the models
suggested below that may provide new directions in future differential gene
expression analysis.
Preconditioning Stress in Brain Tolerance Strategies

Certain stimuli that can cause injury will protect the brain against subse-
quent, severely injuring stimuli if applied at a low intensity (i.e., subthreshold
for injury) prior to that severe injury. This phenomenon involves complex
processes involved in endogenous organ protection. For ischemic stimuli, this
phenomenon has been termed ischemic preconditioning (PC) or ischemic tol-
erance (IT). PC is a reaction to a potentially noxious stimulus such as hypoxia,
ischemia, or inflammation. A short ischemic preconditioning event can result in
a resistance to severe ischemic tissue injury. This phenomenon has been
described in brain and heart, and may represent a fundamental cell response to
certain types or levels of injury [173–176]. PC or IT in the brain is associated
with a protected state that develops over hours, persists for days or longer and
involves de novo protein synthesis [177, 178]. MRI can demonstrate the evolu-
tion of infarction and its reduction by tolerance induction [179–181].
Functional or motor effects are protected by PC [177]. It is interesting that the
stroke-prone rat (discussed earlier in the chapter as a model of spontaneous
stroke and used as an experimental model for gene associations) are signifi-
cantly more sensitive to cerebral ischemia [92] and exhibit greater brain injury
to cerebral ischemia and also exhibit a significantly reduced degree of IT to PC
[179–181].
The brain changes associated with brain ischemia involve a progression of
both injurious and protective processes as brain injury evolves and is then
repaired following an insult such as a stroke. Focal cerebral ischemia induces a
complex series of mechanisms [2, 173, 176] that result in infarcted tissue, a sit-
uation in which neurodestruction has overwhelmed neuroprotection. The major
pathophysiological mechanisms of tissue destruction in stroke involve acute
mechanisms of excitotoxicity and delayed mechanisms of inflammation and
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apoptosis. The corresponding protective tissue responses include up-regulation
of neurotrophic factors and protective or ‘stress’ genes/proteins. Later regener-
ative or restorative processes may allow recovery of some brain function post-
stroke [2], but generally are not sufficient to correct the initial damage. Because
IT paradigms also provide insight into the mechanisms of endogenous brain
neuroprotection in the absence of any destructive processes, it is believed that
understanding the signaling and mechanisms involved in precondition-induced
IT can discover new targets and approaches to protect the brain and other end
organs from the injury/disease. IT has been demonstrated in the human brain
[182, 183], and so differential gene expression strategies in IT experimental
models have physiological relevance for studying apoptosis and other post-
stroke complications [184].
This endogenous brain protection phenomenon appears to represent a
fundamental protective response to injury following prior stress [173, 176,
185, 186]. Models of brain protection provide an opportunity to identify novel
protective gene expression associated with the development of brain tolerance.
Data from differential gene expression suggests that neurotrophic factors,
stress proteins, and cytokines contribute to the tolerance response to ischemia
and other forms of stress in the brain [173, 174, 176–178, 187–189]. For
example, IT is associated with an increased expression of the neuroprotective
protein IL-1ra [177] and heat shock proteins [178] and a reduced postischemic
expression of the early response genes, c-fos and zif268 [177]. A number of
techniques including suppressive subtractive hybridization methodology have
been applied to discover genes responsible for IT following PC [190]. With
suppressive subtractive hybridization, tissue inhibitor of matrix metallopro-
teinase (TIMP-1) was identified as one candidate molecule in the stroke
response and IT. Northern analysis confirmed that TIMP-1 mRNA was signif-
icantly elevated at 24 h and 2 days after PC, which corresponded well to the
onset of IT [190].
Strategies in Brain Recovery, Plasticity and Recovery of Function

Gene changes occurring one or more days after stroke might provide
insight into reparative and recovery processes. While neurological functional
deficits occur following stroke, there may be a recovery of brain function that
occurs spontaneously or improves with training following stroke [2, 191–193].
Sampling tissue during functional brain recovery in animal models (i.e., at later
time periods poststroke) might be expected to provide an opportunity to iden-
tify novel genes important for long-term brain regeneration or plasticity. These
studies would be amenable to differential gene expression analyses, but would
be profiled at later poststroke timepoints or under treatment conditions shown
to facilitate such brain regeneration/recovery, for example after the introduction
Barone/Read 364
of putative neuroprotective or neuroregenerative agents. It is again interesting
that stroke-prone rats that are more sensitive to and exhibit greater brain injury
to cerebral ischemia also exhibit a greater degree of neurological deficits with
significantly less spontaneous recovery of neurological deficits poststroke
[92, 180, 181]. Recent data indicating complete recovery of neurological func-
tion in the SHR, and by comparison the lack of improvement/recovery of
neurological function in the stroke-prone rat (i.e., with similar degrees of
absolute brain injury) suggests that the stroke-prone rat might be a valuable
model for the evaluation of neurodegenerative drugs poststroke [181]. From the
same point of view, differential gene expression studies could be used in
the future to compare SHR-SP and SHR in order to elucidate mechanisms and
to discover new targets that facilitate neurobehavioral recovery of the injured
brain.
Gene Therapy and Gene Target Validation

In recent years, clinical progress in gene therapy has proceeded in parallel
with in vivo gene transfer for physiological studies and gene validation.
Therapeutic neovascularization for ischemic diseases has been one particularly
encouraging area of study. For example, animal models involving intramuscu-
lar injection of naked plasmid or adenoviral carried DNA encoding vascular
endothelial growth factor (VEGF) have shown promotion of angiogenesis in
ischemic limbs [194, 195]. Similarly, in clinical trials, VEGF gene transfer aug-
ments the population of circulating endothelial progenitor cells and transiently
increases plasma levels of VEGF [196]. Furthermore, myocardial transfer of
naked plasmid DNA phVEGF(165) has been found to augment perfusion of
ischemic myocardium and reduces the size of defects documented at rest by sin-
gle-photon emission CT-imaging [197]. Ex vivo gene transfer, employing the
modification of cultured cells and subsequent implantation into a host organ-
ism, is a proven strategy for recovery from CNS injury and could incorporate
cells expressing genes that confer protection from stroke. An analogous area of
research has been in recovery from long-term rodent hemiparkinsonism by
implantation of cells following 6-OHDA lesioning, which has been found to
improve behavioral deficit for up to 13 months [198].
In vivo gene transfer, the delivery of a gene directly to recipient somatic
cells, has also been explored for neuroprotection and recovery from CNS
injury. The delivery of the proto-oncogene bcl-2 has been examined in gerbil
models using adeno-associated virus vectors. Transduction of both pre- and
postforebrain ischemia was found to prevent DNA fragmentation in hippocam-
pal CA1 neurons, commonly associated with cell death induced by ischemia
[199]. Adenoviral transfection of the endogenous cytokine antagonist IL-1ra
also has demonstrated neuroprotection in transient focal cerebral ischemia and
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reperfusion models in the mouse [200, 201]. In addition, the neuroprotective
potential of heat shock protein-70 on brain injury, including that produced by
brain ischemia, has been tested with viral vectors. Use of transgenic animals
and gene transfer technology to overexpress heat shock protein-70 clearly pro-
tects the brain [202, 203] in the context of stroke.
The Elusiveness of Novel Gene Targets and the Future
Initially, the movement to discover differential gene expression in brain

destruction or protection was driven by the hope of discovering novel genes that
would provide pathways to therapeutics. The complexity of understanding and
applying resources to gene fragments in the hopes of ultimately reaching this
‘therapeutic nirvana’ has not yet occurred. We have identified several genes that
have been differentially expressed in ischemic or tolerant brain tissue, and
which appear to be important, but there still exists a therapeutic void. In phar-
maceutical development, the odds of developing a successful drug are generally
better for the pursuit of known genes as therapeutic targets, although the new
science of pharmacogenetics as a lead optimization tool may change this man-
ner of operation. Many other factors can make investing resources into work on
unknown genes costly, risky, and difficult to pursue. Some of these include the
absence of any understanding of an identifiable function for the unknown pro-
tein, and if it is in fact associated with a novel gene, and lack of any concrete,
supporting information that the novel gene/protein has any involvement in the
pathophysiology of stroke rather than being an epiphenomenon. In spite of all
this, it is clear that the therapeutic targets of the future exist in the complex pat-
terns of gene expression underlying stroke. As such, unknown or novel genes
represent a potential source of collaboration between academic and industrial
laboratories. Potential novel gene products could be evaluated for tissue distri-
bution, function and relevance in tissue injury and protection, in a collaborative
and productive setting.
Clearly, the methodology is available to identify differential gene expres-
sion in stroke in addition to other conditions of brain disease. However, the
methodology needs to be developed with the caveats discussed above. If one
operates as we have suggested, using cross-validating technologies in animal
models and human tissue to validate ‘hits,’ there is potential for many signifi-
cant opportunities for the biological discovery, not only related to stroke, but in
many other conditions such as end organ failure in various cardiovascular dis-
eases, in areas such as oncology, and perhaps extending further to other very
complex problems such as substance abuse, tolerance, addiction and drug
dependency.
Barone/Read 366
Acknowledgements
The authors would like to thank their collaborators in this research, especially Dr. Andy
Parsons, Dr. David Harrison, Dr. Karen Philopia, Dr. Karen Kabnick, Dr. Shawn O’Bien,
Dr. Steve Clark, Dr. Mary Brawner, Dr. Giora Feuerstein, Dr. Xinkang Wang, Dr. Ray White,
Dr. Stewart Bates, Dr. Jeff Legos and Dr. Israel Gloger. It has been a pleasure working with
all of them in this exciting area.
References
1 Stanton LW, Garrard LJ, Damm D, Garrick BL, Lam A, Kapoun AM, Zheng Q, Protter AA,
Schreiner GF, White RT: Altered patterns of gene expression in response to myocardial infarction.
Circ Res 2000;86:939–945.
2 Barone FC, Feuerstein GZ: Inflammatory mediators and stroke: New opportunities for novel ther-
apeutics (Review). J Cereb Blood Flow Metab 1999;15:819–834.
3 Soriano MA, Tessier M, Certa U, Gill R: Parallel gene expression monitoring using oligonu-
cleotide probe arrays of multiple transcripts with an animal model of focal ischemia. J Cereb
Blood Flow Metab 2000;20:1045–1055.
4 Read SJ, Parsons AA, Harrison DC, Philpott K, Kabnick K, Brawner M, O’Brien S, Clark S,
Bates S, Gloger I, Legos JJ, Barone FC: Stroke genomics: Approaches to identify, validate and
understand adaptive gene expression changes in ischemic stroke (Review). J Cereb Blood Flow
Metab 2001;21:755–778.
5 Sharp FR, Lu A, Tang Y, Millhorn DE: Multiple molecular penumbras after focal cerebral
ischemia. J Cereb Blood Flow Metab 2000;20:1011–1032.
6 Lu A, Tang Y, Ran R, Clark JF, Aronow BJ, Sharp FR: Genomics of the periinfarction cortex after
focal cerebral ischemia. J Cereb Blood Flow Metab 2003;23:786–810.
7 Kannel WB, Wolf PA, Verter J, McNamara PM: Epidemiologic assessment of the role of blood
pressure in stroke. The Framingham study. JAMA 1970;214:301–310.
8 Stephenson J: Rising stroke rates spur efforts to identify risks, prevent disease. JAMA 1998;
279:1239–1240.
9 Fisher M, Bogousslavsky J: Further evolution toward effective therapy for acute ischemic stroke.
JAMA 1998;279:1298–1303.
10 Pancioli AM, Broderick J, Kothari R, Brott T, Tuchfarber A, Miller R, Khoury J, Jauch E: Public
perception of stroke warning signs and knowledge of potential risk factors. JAMA 1998;279:
1288–1292.
11 Hrubec Z, Robinette CD: The study of human twins in medical research. N Engl J Med 1984;
310:435–441.
12 Brass LM, Isaacsohn JL, Merikangas KR, Robinette CD: A study of twins and stroke. Stroke
1992;23:221–223.
13 Carmelli D, DeCarli C, Swan GE, Jack LM, Reed T, Wolf PA, Miller BL: Evidence for genetic
variance in white matter hyperintensity volume in normal elderly male twins. Stroke 1998;29:
1177–1181.
14 Jousilahti P, Rastenyte D, Tuomilehto J, Sarti C, Vartiainen E: Parental history of cardiovascular
disease and risk of stroke. A prospective follow-up of 14371 middle-aged men and women in
Finland. Stroke 1997;28:1361–1366.
15 Kiely DK, Wolf PA, Cupples LA, Beiser AS, Myers RH: Familial aggregation of stroke. The
Framingham Study. Stroke 1993;24:1366–1371.
16 Liao D, Myers R, Hunt S, Shahar E, Paton C, Burke G, Province M, Heiss G: Familial history of
stroke and stroke risk. The Family Heart Study. Stroke 1997;28:1908–1912.
17 Welin L, Svardsudd K, Wilhelmsen L, Larsson B, Tibblin G: Analysis of risk factors for stroke in
a cohort of men born in 1913. N Engl J Med 1987;317:521–526.
medwedi.ru
18 Khaw KT, Barrett-Connor E: Family history of stroke as an independent predictor of ischemic
heart disease in men and stroke in women. Am J Epidemiol 1986;123:59–66.
19 Bromberg JE, Rinkel GJ, Algra A, Greebe P, van Duyn CM, Hasan D, Limburg M, ter Berg HW,
Wijdicks EF, van Gijn J: Subarachnoid haemorrhage in first and second degree relatives of
patients with subarachnoid. Brit Med J 1995;311:288–289.
20 Tournier-Lasserve E, Joutel A, Melki J, Weissenbach J, Lathrop GM, Chabriat H, Mas JL,
Cabanis EA, Baudrimont M, Maciazek J: Cerebral autosomal dominant arteriopathy with sub-
cortical infarcts and leukoencephalopathy maps to chromosome 19q12. Nat Genet 1993;3:
256–259.
21 Hirano M, Pavlakis SG: Mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like
episodes (MELAS): Current concepts. J Child Neurol 1994;9:4–13.
22 Lossos A, Ben Hur T, Ben Nariah Z, Enk C, Gomori M, Soffer D: Familial Sneddon’s syndrome.
J Neurol 1995;242:164–168.
23 Joutel A, Bousser MG, Biousse V, Labauge P, Chabriat H, Nibbio A, Maciazek J, Meyer B, Bach MA,
Weissenbach J: A gene for familial hemiplegic migraine maps to chromosome 19. Nat Genet 1993;
5:40–45.
24 Hassan A, Markus HS: Genetics and ischemic stroke. Brain 2000;123:1784–1812.
25 Sourander P, Walinder J: Hereditary multi-infarct dementia. Lancet 1977;1:1015.
26 Chabriat H, Tournier-Lasserve E, Vahedi K, Leys D, Joutel A, Nibbio A, Escaillas JP, Iba-Zizen MT,
Bracard S, Tehindrazanarivelo A: Autosomal dominant migraine with MRI white-matter abnor-
malities mapping to the CADASIL locus. Neurology 1995;45:1086–1091.
27 Dichgans M, Mayer M, Muller-Myhsok B, Straube A, Gasser T: Identification of a key recombi-
nant narrows the CADASIL gene region to 8 cM and argues against allelism of CADASIL and
familial hemiplegic migraine. Genomics 1996;32:151–154.
28 Hedera P, Friedland RP: Cerebral autosomal dominant arteriopathy with subcortical infarcts and
leukoencephalopathy: Study of two American families with predominant dementia. J Neurol Sci
1997;146:27–33.
29 Desmond DW, Moroney JT, Lynch T, Chan S, Chin SS, Shungu DC, Naini AB, Mohr JP:
CADASIL in a North American family: Clinical, pathologic, and radiologic findings. Neurology
1998;51:844–849.
30 Viitanen M, Kalimo H: CADASIL: Hereditary arteriopathy leading to multiple brain infarcts and
dementia. Ann NY Acad Sci 2000;903:273–284.
31 Chabriat H, Levy C, Taillia H, Iba-Zizen MT, Vahedi K, Joutel A, Tournier-Lasserve E, Bousser MG:
Patterns of MRI lesions in CADASIL. Neurology 1998;51:452–457.
32 Joutel A, Corpechot C, Ducros A, Vahedi K, Chabriat H, Mouton P, Alamowitch S, Domenga V,
Cecillion M, Marechal E, Maciazek J, Vayssiere C, Cruaud C, Cabanis EA, Ruchoux MM,
Weissenbach J, Bach JF, Bousser MG, Tournier-Lasserve E: Notch3 mutations in CADASIL,
a hereditary adult-onset condition causing stroke and dementia. Nature 1996;383:707–710.
33 Artavanis-Tsakonas S, Rand MD, Lake RJ: Notch signaling: Cell fate control and signal integra-
tion in development. Science 1999;284:770–776.
34 Levitan D, Greenwald I: Facilitation of lin-12-mediated signalling by sel-12, a Caenorhabditis ele-
gans S182 Alzheimer’s disease gene. Nature 1995;377:351–354.
35 Joutel A, Vahedi K, Corpechot C, Troesch A, Chabriat H, Vayssiere C, Cruaud C, Maciazek J,
Weissenbach J, Bousser MG, Bach JF, Tournier-Lasserve E: Strong clustering and stereotyped
nature of Notch3 mutations in CADASIL patients. Lancet 1997;350:1511–1515.
36 Joutel A, Tournier-Lasserve E: Notch signalling pathway and human diseases. Semin Cell Dev
Biol 1998;9:619–625.
37 Ruchoux MM, Maurage CA: Endothelial changes in muscle and skin biopsies in patients with
CADASIL. Neuropathol Appl Neurobiol 1998;24:60–65.
38 De Strooper B, Annaert W, Cupers P, Saftig P, Craessaerts K, Mumm JS, Schroeter EH,
Schrijvers V, Wolfe MS, Ray WJ, Goate A, Kopan R: A presenilin-1-dependent gamma-secretase-
like protease mediates release of Notch intracellular domain. Nature 1999;398:518–522.
39 Kudo T, Imaizumi K, Tanimukai H, Katayama T, Sato N, Nakamura Y, Tanaka T, Kashiwagi Y,
Jinno Y, Tohyama M, Takeda M: Are cerebrovascular factors involved in Alzheimer’s disease?
Neurobiol Aging 2000;21:215–224.
Barone/Read 368
40 Schmidt R, Schmidt H, Fazekas F: Vascular risk factors in dementia. J Neurol 2000;247:81–87.
41 Skoog I: Vascular aspects in Alzheimer’s disease. J Neural Transm Suppl 2000;59:37–43.
42 Ciafaloni E, Ricci E, Shanske S, Moraes CT, Silvestri G, Hirano M, Simonetti S, Angelini C,
Donati MA, Garcia C: MELAS: Clinical features, biochemistry, and molecular genetics. Ann
Neurol 1992;31:391–398.
43 Macmillan C, Lach B, Shoubridge EA: Variable distribution of mutant mitochondrial DNAs
(tRNA(Leu[3243])) in tissues of symptomatic relatives with MELAS: The role of mitotic segre-
gation. Neurology 1993;43:1586–1590.
44 Sakuta R, Goto Y, Horai S, Nonaka I: Mitochondrial DNA mutations at nucleotide positions 3243
and 3271 in mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes:
A comparative study. J Neurol Sci 1993;115:158–160.
45 Kovalenko SA, Tanaka M, Yoneda M, Iakovlev AF, Ozawa T: Accumulation of somatic nucleotide
substitutions in mitochondrial DNA associated with the 3243 A-to-G tRNA(leu)(UUR) mutation
in encephalomyopathy and cardiomyopathy. Biochem Biophys Res Commun 1996;222:201–220.
46 Rastenyte D, Tuomilehto J, Sarti C: Genetics of stroke – A review. J Neurol Sci 1998;153:132–145.
47 Kosinski C, Mull M, Lethen H, Topper R: Evidence for cardioembolic stroke in a case of Kearns-
Sayre syndrome. Stroke 1995;26:1950–1952.
48 Dominiczak AF, Negrin DC, Clark JS, Brosnan MJ, McBride MW, Alexander MY: Genes and
hypertension: From gene mapping in experimental models to vascular gene transfer strategies.
Hypertension 2000;35:164–172.
49 Jeffs B, Clark JS, Anderson NH, Gratton J, Brosnan MJ, Gauguier D, Reid JL, Macrae IM,
Dominiczak AF: Sensitivity to cerebral ischaemic insult in a rat model of stroke is determined by
a single genetic locus. Nat Genet 1997;16:364–367.
50 Brosnan MJ, Clark JS, Jeffs B, Negrin CD, Van Vooren P, Arribas SM, Carswell H, Aitman TJ,
Szpirer C, Macrae IM, Dominiczak AF: Genes encoding atrial and brain natriuretic peptides as
candidates for sensitivity to brain ischemia in stroke-prone hypertensive rats. Hypertension 1999;
33:290–297.
51 Lifton RP, Dluhy RG, Powers M, Ulick S, Lalouel JM: The molecular basis of glucocorticoid-
remediable aldosteronism, a Mendelian cause of human hypertension. Trans Assoc Am Physicians
1992;105:64–71.
52 Couderc R, Mahieux F, Bailleul S, Fenelon G, Mary R, Fermanian J: Prevalence of apolipoprotein
E phenotypes in ischemic cerebrovascular disease. A case-control study. Stroke 1993;24:661–664.
53 Pedro-Botet J, Senti M, Nogues X, Rubies-Prat J, Roquer J, D’Olhaberriague L, Olive J:
Lipoprotein and apolipoprotein profile in men with ischemic stroke. Role of lipoprotein(a),
triglyceride-rich lipoproteins, and apolipoprotein E polymorphism. Stroke 1992;23:1556–1562.
54 Margaglione M, Seripa D, Gravina C, Grandone E, Vecchione G, Cappucci G, Merla G, Papa S,
Postiglione A, Di Minno G, Fazio VM: Prevalence of apolipoprotein E alleles in healthy subjects
and survivors of ischemic stroke: An Italian Case-Control Study. Stroke 1998;29:399–403.
55 Nakata Y, Katsuya T, Rakugi H, Takami S, Sato N, Kamide K, Ohishi M, Miki T, Higaki J,
Ogihara T: Polymorphism of angiotensin converting enzyme, angiotensinogen, and apolipopro-
tein E genes in a Japanese population with cerebrovascular disease. Am J Hypertens 1997;10:
1391–1395.
56 Sharma P, Carter ND, Barley J, Brown MM: Molecular approach to assessing the genetic risk of
cerebral infarction: Deletion polymorphism in the gene encoding angiotensin 1-converting
enzyme. J Hum Hypertens 1994;8:645–648.
57 Elbaz A, Amarenco P: Genetic susceptibility and ischemic stroke. Curr Opin Neurol 1999;12:47–55.
58 Margaglione M, Celentano E, Grandone E, Vecchione G, Cappucci G, Giuliani N, Colaizzo D,
Panico S, Mancini FP, Di Minno G: Deletion polymorphism in the angiotensin-converting enzyme
gene in patients with a history of ischemic stroke. Arterioscler Thromb Vasc Biol 1996;16:304–309.
59 Kessler C, Spitzer C, Stauske D, Mende S, Stadlmuller J, Walther R, Rettig R: The apolipoprotein E
and beta-fibrinogen G/A-455 gene polymorphisms are associated with ischemic stroke involving
large-vessel disease. Arterioscler Thromb Vasc Biol 1997;17:2880–2884.
60 Albucher JF, Guiraud-Chaumeil B, Chollet F Cadroy Y, Sie P: Frequency of resistance to activated
protein C due to factor V mutation in young patients with ischemic stroke. Stroke 1996;27(4):
766–767.
medwedi.ru
61 DeStefano V, Chiusolo P, Paciaroni K, Casorelli I, Rossi E, Molinari M, Servidei S, Tonali PA,
Leone G: Prothrombin G20210A mutant genotype is a risk factor for cerebrovascular ischemic
disease in young patients. Blood 1998;91:3562–3565.
62 MacLeod MJ, Dahiyat MT, Cumming A, Meiklejohn D, Shaw D, St Clair D: No association
between Glu/Asp polymorphism of NOS3 gene and ischemic stroke. Neurology 1999;53: 418–420.
63 Nakata Y, Katsuya T, Takami S, Sato N, Fu Y, Ishikawa K, Takiuchi S, Rakugi H, Miki T, Higaki J,
Ogihara T: Methylenetetrahydrofolate reductase gene polymorphism: Relation to blood pressure
and cerebrovascular disease. Am J Hypertens 1998;11:1019–1023.
64 Kontula K, Ylikorkala A, Miettinen H, Vuorio A, Kauppinen-Makelin R, Hamalainen L, Palomaki H,
Kaste M: Arg506Gln factor V mutation (factor V Leiden) in patients with ischemic cerebrovascular
disease and survivors of myocardial infarction. Thromb Haemost 1995;73:558–560.
65 Corral J, Gonzalez-Conejero R, Lozano ML, Rivera J, Vicente V: Genetic polymorphisms of
factor VII are not associated with arterial thrombosis. Blood Coag Fibrinolysis 1998;9:
267–272.
66 Catto AJ, Kohler HP, Bannan S, Stickland M, Carter A, Grant PJ: Factor XIII Val 34 Leu: A novel
association with primary intracerebral hemorrhage. Stroke 1998;29:813–816.
67 Poort SR, Rosendaal FR, Reitsma PH, Bertina RM: A common genetic variation in the 3⬘-untrans-
lated region of the prothrombin gene is associated with elevated plasma prothrombin levels and
an increase in venous thrombosis. Blood 1996;88:3698–3703.
68 Rubattu S, Ridker P, Stampfer MJ, Volpe M, Hennekens CH, Lindpaintner K: The gene encoding
atrial natriuretic peptide and the risk of human stroke. Circulation 1999;100:1722–1726.
69 Rubattu S, Giliberti R, Ganten U, Volpe M: Differential brain atrial natriuretic peptide expression
co-segregates with occurrence of early stroke in the stroke-prone phenotype of the spontaneously
hypertensive rat. J Hypertens 1999;17:1849–1852.
70 Rubattu S, Lee-Kirsch MA, DePaolis P, Giliberti R, Gigante B, Lombardi A, Volpe M,
Lindpaintner K: Altered structure, regulation, and function of the gene encoding the atrial natri-
uretic peptide in the stroke-prone spontaneously hypertensive rat. Circ Res 1999;85:900–905.
71 Coyle P, Odenheimer DJ, Sing CF: Cerebral infarction after middle cerebral artery occlusion in
progenies of spontaneously stroke-prone and normal rats. Stroke 1984;15:711–716.
72 Coyle P: Outcomes to middle cerebral artery occlusion in hypertensive and normotensive rats.
Hypertension 1984;6:169–174.
73 Coyle P, Heistad DD: Development of collaterals in the cerebral circulation. Blood Vessels
1991;28: 183–189.
74 Nagaoka A, Iwatsuka H, Suzuoki Z, Okamoto K: Genetic predisposition to stroke in sponta-
neously hypertensive rats. Am J Physiol 1976;230:1354–1359.
75 Rubattu S, Volpe M, Kreutz R, Ganten U, Ganten D, Lindpaintner K: Chromosomal mapping of
quantitative trait loci contributing to stroke in a rat model of complex human disease. Nature
Genet 1996;13:429–434.
76 Ikeda K, Nara Y, Matumoto C, Mashimo T, Tamada T, Sawamura M, Nabika T, Yamori Y: The
region responsible for stroke on chromosome 4 in the stroke-prone spontaneously hypertensive
rat. Biochem Biophys Res Commun 1996;229:658–662.
77 Jeffs B, Clark JS, Anderson NH, Gratton J, Brosnan MJ, Gauguier D, Reid JL, Macrae IM,
Dominiczak AF: Sensitivity to cerebral ischemic insult in a rat model of stroke is determined by
a single genetic locus. Nat Genet 1997;16:364–367.
78 Gratton JA, Sauter A, Rudin M, Lees KR, McColl J, Reid JL, Dominiczak AF, Macrae IM:
Susceptibility to cerebral infarction in the stroke-prone spontaneously hypertensive rat is inherited
as a dominant trait. Stroke 1998;29:690–694.
79 Brosnan MJ, Clark JS, Jeffs B, Negrin CD, Van Vooren P, Arribas SM, Carswell H, Aitman TJ,
Szpirer C, Macrae IM, Dominiczak AF: Genes encoding atrial and brain natriuretic peptides as
candidates for sensitivity to brain ischemia in stroke-prone hypertensive rats. Hypertension 1999;
33:290–297.
80 Ktorza A, Bernard C, Parent V, Penicaud L, Froguel P, Lathrop M, Gauguier D: Are animal mod-
els of diabetes relevant to the study of the genetics of non-insulin-dependent diabetes in humans?
Diabetes Metab 1997;23(suppl 2):38–46.
81 Koistinaho J, Hokfelt T: Altered gene expression in brain ischemia. Neuroreport 1997;8:1–8.
Barone/Read 370
82 Iadecola C, Ross ME: Molecular pathology of cerebral ischemia: Delayed gene expression and
strategies for neuroprotection. Ann NY Acad Sci 1997;835:203–217.
83 Clark WM, Raps EC, Tong DC, Kelly RE: Cervene (Nalmefene) in acute ischemic stroke: Final
results of a phase III efficacy study. The Cervene Stroke Study Investigators. Stroke 2000;31:
1234–1239.
84 Lees KR, Asplund K, Carolei A, Davis SM, Diener HC, Kaste M, Orgogozo JM, Whitehead J:
Glycine antagonist (gavestinel) in neuroprotection (GAIN International) in patients with acute stroke:
A randomised controlled trial. GAIN International Investigators. Lancet 2000;355:1949–1954.
85 Feinklestein SP, Fisher M, Furland AJ, Goldstein LB, Gorelick PB, Kaste M, Lees KR, Traystman RJ,
Albers GW, Anwer UE, Ashwood T, Barone FC, et al: Recommendations for standards regarding pre-
clinical neuroprotective and restorative drug development. Stroke 1999;30:2752–2758.
86 DeGraba TJ, Pettigrew LC: Why do neuroprotective drugs work in animals but not humans?
Neurol Clin 2000;18:475–493.
87 Parsons AA, Irving EA, Legos JJ, Lenhard SC, Chandra S, Schaeffer TR, Haimback RE, White RF,
Hunter AJ, Barone FC: Acute stroke therapy: Issues for translating pre-clinical neuroprotection to
therapeutic reality. Curr Opin Investig Drugs 2000;1:452–463.
88 Baird AE, Warach S: Magnetic resonance imaging of acute stroke. J Cereb Blood Flow Metab
1998;18:583–609.
89 Albers GW: Expanding the window for thrombolytic therapy in acute stroke. The potential role of
acute MRI for patient selection. Stroke 1999;30:2230–2237.
90 Hossmann KA: Periinfarct depolarizations. Cerebrovas Brain Metab Rev 1996;8:195–208.
91 Gill R, Sibson NR, Hatfield RH, Burdett NG, Carpenter TA, Hall LD, Pickard JD: A comparison
of the early development of ischemic damage following permanent middle cerebral artery occlu-
sion in rats as assessed using magnetic resonance imaging and histology. J Cereb Blood Flow
Metab 1995;15:1–11.
92 Barone FC, Price WJ, White RF, Willette RN, Feuerstein GZ: Genetic hypertension and increased
susceptibility to cerebral ischemia. Neurosci Biobehav Rev 1992;16:219–233.
93 Chandra S, White RF, Everding D, Feuerstein GZ, Coatney RW, Sarkar SK, Barone FC: Use of
diffusion-weighted MRI and neurological deficit scores to demonstrate beneficial effects of
isradipine in a rat model of focal ischemia. Pharmacology 1999;58:292–299.
94 Zhang Z, Zhang RL, Jiang Q, Raman SB, Cantwell L, Chopp M: A new rat model of thrombotic
focal cerebral ischemia. J Cereb Blood Flow Metab 1997;17:123–135.
95 Jiang Q, Zhang RL, Zhang ZG, Ewing JR, Jiang P, Divine GW, Knight RA, Chopp M: Magnetic
resonance imaging indexes of therapeutic efficacy of recombinant tissue plasminogen activator
treatment of rat at 1 and 4 hours after embolic stroke. J Cereb Blood Flow Metab 2000;20:21–27.
96 Busch E, Kruger K, Allegrini PR, Kerskens CM, Gyngell ML, Hoehn-Berlage M, Hossmann KA:
Reperfusion after thrombolytic therapy of embolic stroke in the rat: Magnetic resonance and bio-
chemical imaging. J Cereb Blood Flow Metab 1998;18:407–418.
97 Koizumi J, Yoshida Y, Nakaazawa T, Ooneda G: Experimental studies of ischemic brain edema.
I. A new experimental model of cerebral embolism in rats which recirculation can be introduced
in the ischemic area. Jpn J Stroke 1986;8:1–8.
98 Longa EZ, Weinstein PR, Carlson S, Cummins R: Reversible middle cerebral artery occlusion
without craniectomy in rats. Stroke 1989;20:84–91.
99 Neumann-Haefelin T, Kastrup A, de Crespigny A, Yenari MA, Ringer T, Sun GH, Moseley ME:
Serial MRI after transient focal cerebral ischemia in rats: Dynamics of tissue injury, blood-brain
barrier damage, and edema formation. Stroke 2000;31:1965–1972.
100 Li FH, Liu KF, Silva MD, Omae T, Sotak CH, Fenstermacher JD, Fisher M: Transient and perma-
nent resolution of ischemic lesions on diffusion-weighted imaging after brief periods of focal
ischemia in rats – Correlation with histopathology. Stroke 2000;31:946–953.
101 Gyngell ML, Busch E, Schmitz B, Kohno K, Back T, Hoehn-Berlage M, Hossmann KA:
Evolution of acute focal cerebral ischaemia in rats observed by localized 1H MRS, diffusion-
weighted MRI, and electrophysiological monitoring. NMR Biomed 1995;8:206–214.
102 Kohno K, Hoehn-Berlage M, Mies G, Back T, Hossmann KA: Relationship between diffusion-
weighted MR images, cerebral blood flow, and energy state in experimental brain infarction. Magn
Reson Imaging 1995;13:73–80.
medwedi.ru
103 Kohno K, Back T, Hoehn-Berlage M, Hossmann KA: A modified rat model of middle cerebral
artery thread occlusion under electrophysiological control for magnetic resonance investigations.
Magn Reson Imaging 1995;13:65–71.
104 Bates S, Read SJ, Harrison DC, Topp S, Morrow R, Gale D, Murdock P, Barone FC, Parsons AA,
Gloger IS: Molecular characterisation by subtractive hybridisation of the Zea Longa model of per-
manent middle cerebral artery occlusion in the rat. Identification of novel gene expression fol-
lowing experimental ischemia. Mol Brain Res 2001;93:70–80.
105 Schwarz DA, Barry G, Mackay KB, Manu F, Naeve GS, Vana AM, Verge G, Conlon PJ, Foster AC,
Maki RA: Identification of differentially expressed genes induced by ischemic stroke. Mol Brain
Res 2002;101:12–22.
106 Feuerstein GZ, Barone FC, Wang X: Role of Molecular Biology in strategies to discover novel tar-
gets for drug discovery; in Levy A, Grauer E, Ben-Nathan D, deKloet R (eds): New Frontiers in
Stress Research; Modulation of Brain Function, New York, Hardwood Academic Publishers,
1996, pp 285–293.
107 Honkaniemi J, States BA, Weinstein PR, Espinoza J, Sharp FR: Expression of zinc finger imme-
diate early genes in rat brain after permanent middle cerebral artery occlusion. J Cereb Blood
Flow Metab 1997;17:636–646.
108 Wang XK, Yue TL, Young PR, Barone FC, Feuerstein GZ: Expression of interleukin 6, c-fos and
zif268 mRNA in rat ischemic cortex. J Cereb Blood Flow Metab 1995;15:166–171.
109 Stephenson D, Yin T, Smalstig EB, Hsu MA, Panetta J, Little S, Clemens J: Transcription factor
nuclear factor-kappa B is activated in neurons after focal cerebral ischemia. J Cereb Blood Flow
Metab 2000;20:592–603.
110 Gabriel C, Justicia C, Camins A, Planas AM: Activation of nuclear factor-kappaB in the rat brain
after transient focal ischemia. Mol Brain Res 1999;65:61–69.
111 Martin-Villalba A, Winter C, Brecht S, Buschmann T, Zimmermann M, Herdegen T: Rapid and
long-lasting suppression of the ATF-2 transcription factor is a common response to neuronal
injury. Mol Brain Res 1998;20:158–166.
112 Wang XK, Yue TL, Barone FC, White RF, Clark RK, Willette RN, Sulpizio AC, Aiyar NV,
Ruffolo RR Jr, Feuerstein GZ: Discovery of adrenomedullin in rat ischemic cortex and evidence
for its role in exacerbating focal brain ischemic damage. PNAS (USA) 1995;92:11480–11484.
113 Wang XK, Barone FC, Aiyar NV, Feuerstein GZ: Interleukin-1 receptor and receptor antagonist
gene expression after focal stroke in rats. Stroke 1997;28:155–161.
114 Medhurst AD, Harrison DC, Read SJ, Campbell CA, Robbins MJ, Pangalos MN: The use of
TaqMan RT-PCR assays for semiquantitative analysis of gene expression in CNS tissues and dis-
ease models. J Neurosci Methods 2000;98:9–20.
115 Liu T, McDonnell PC, Young PR, White RF, Siren AL, Barone FC, Feuerstein GZ: Interleukin-1␤
expression in ischemic rat cortex. Stroke 1993;24:1746–1751.
116 Buttini M, Sauter A, Boddeke HW: Induction of interleukin-1 beta mRNA after focal cerebral
ischaemia in the rat. Brain Res Mol Brain Res 1994;23:126–134.
117 Zhai QH, Futrell N, Chen FJ: Gene expression of IL-10 in relationship to TNF alpha, IL-1beta and
IL-2 in the rat brain following middle cerebral artery occlusion. J Neurol Sci 1997;152:119–124.
118 Wang XK, Yue TL, Barone FC, White RF, Gagnon RC, Feuerstein GZ: Concomitant cortical
expression of TNF-alpha and IL-1 beta mRNAs follows early response gene expression in tran-
sient focal ischemia. Molec Chem Neuropathol 1994;23:103–114.
119 Liu T, Young PR, McDonnell PC, White RF, Barone FC, Feuerstein GZ: Cytokine-induced neutrophil
chemoattractant mRNA expressed in cerebral ischemia. Neurosci Lett Suppl 1993;164:125–128.
120 Buttini M, Appel K, Sauter A, Gebicke-Haerter PJ, Boddeke HW: Expression of tumor necrosis
factor alpha after focal cerebral ischaemia in the rat. Neuroscience 1996;71:1–16.
121 Liu T, Clark RK, McDonnell PC, Young PR, White RF, Barone FC, Feuerstein GZ: Tumor necro-
sis factor-alpha expression in ischemic neurons. Stroke 1994;25:1481–1488.
122 Suzuki S, Tanaka K, Nogawa S, Ito D, Dembo T, Kosakai A, Fukuuchi Y: Immunohistochemical
detection of leukemia inhibitory factor after focal cerebral ischemia in rats. J Cereb Blood Flow
Metab 2000;20:661–668.
123 Nogawa S, Zhang F, Ross ME, Iadecola C: Cyclo-oxygenase-2 gene expression in neurons con-
tributes to ischemic brain damage. J Neurosci 1997;15:2746–2755.
Barone/Read 372
124 Yamagami S, Tamura M, Hayashi M, Endo N, Tanabe H, Katsuura Y, Komoriya K: Differential
production of MCP-1 and cytokine-induced neutrophil chemoattractant in the ischemic brain after
transient focal ischemia in rats. J Leukoc Biol 1999;65:744–749.
125 Kim JS, Gautam SC, Chopp M, Zaloga C, Jones ML, Ward PA, Welch KM: Expression of mono-
cyte chemoattractant protein-1 and macrophage inflammatory protein-1 after focal cerebral
ischemia in the rat. J Neuroimmunol 1995;56:127–134.
126 Wang XK, Yue TL, Barone FC, Feuerstein GZ: Monocyte chemoattractant protein-1 (MCP-1)
mRNA expression in rat ischemic cortex. Stroke 1995;26:661–665.
127 Iadecola C, Zhang F, Casey R, Clark HB, Ross ME: Inducible nitric oxide synthase gene expres-
sion in vascular cells after transient focal cerebral ischemia. Stroke 1996;27:1373–1380.
128 Wang XK, Li X, Yaish-Ohad S, Sarau HM, Barone FC, Feuerstein GZ: Molecular cloning and
expression of the rat monocyte chemotactic protein-3 gene: A possible role in stroke. Mol Brain
Res 1999;71:304–312.
129 Wang XK, Ellison J, Siren AL, Lysko PG, Yue TL, Barone FC, Shatzman A, Feuerstein GZ:
Prolonged expression of interferon inducible protein-10 in ischemic cortex after permanent occlu-
sion of the middle cerebral artery in the rat. J Neurochem 1998;71:1194–1204.
130 Wang XK, Li X, Schmidt D, Foley JJ, Barone FC, Ames RS, Sarau HM: Identification and mol-
ecular characterization of rat CXCR3: Discovery of increased expression and interferon-inducible
protein-10 binding are increased in focal stroke. Mol Pharmacol 2000;57:1190–1198.
131 Schmidt-Kastner R, Truettner J, Zhao W, Belayev L, Krieger C, Busto R, Ginsberg MD:
Differential changes of bax, caspase-3 and p21 mRNA expression after transient focal brain
ischemia in the rat. Mol Brain Res 2000;79:88–101.
132 Harrison DC, Davis RP, Bond BC, Campbell CA, James MF, Parsons AA, Philpott KL: Caspase
mRNA expression in a rat model of focal cerebral ischemia. Mol Brain Res 2001;89:133–146.
133 Harrison DC, Medhurst AD, Bond BC, Campbell CA, Davis RP, Philpott KL: The use of quanti-
tative RT-PCR to measure mRNA expression in a rat model of focal ischemia – Caspase-3 as a
case study. Mol-Brain Res 2000;75:143–149.
134 Marti HJ, Bernaudin M, Bellail A, Schoch H, Euler M, Petit E, Risau W: Hypoxia-induced vas-
cular endothelial growth factor expression precedes neovascularization after cerebral ischemia.
Am J Pathol 2000;156:965–976.
135 Hayashi T, Abe K, Suzuki H, Itoyama Y: Rapid induction of vascular endothelial growth factor
gene expression after transient middle cerebral artery occlusion in rats. Stroke 1997;28:
2039–2044.
136 Kokaia Z, Zhao Q, Kokaia M, Elmer E, Metsis M, Smith ML, Siesjo BK, Lindvall O: Regulation
of brain-derived neurotrophic factor gene expression after transient middle cerebral artery occlu-
sion with and without brain damage. Exp Neurol 1995;136:73–88.
137 Schmidt-Kastner R, Zhao W, Truettner J, Belayev L, Busto R, Ginsberg MD: Pixel-based image
analysis of HSP70, GADD45 and MAP2 mRNA expression after focal cerebral ischemia:
Hemodynamic and histological correlates. Mol Brain Res 1998;63:79–97.
138 Takami S, Nishikawa H, Minami M, Nishiyori A, Sato M, Akaike A, Satoh M: Induction of
macrophage inflammatory protein MIP-1alpha mRNA on glial cells after focal cerebral ischemia
in the rat. Neurosci Lett 1997;227:173–176.
139 Wong ML, Loddick SA, Bongiorno PB, Gold PW, Rothwell NJ, Licinio J: Focal cerebral ischemia
induces CRH mRNA in rat cerebral cortex and amygdala. Neuroreport 1995;6:1785–1788.
140 Ellison JA, Velier JJ, Spera P, Jonak ZL, Wang X, Barone FC, Feuerstein GZ: Osteopontin and its
integrin receptor alpha(v)beta3 are upregulated during formation of the glial scar after focal
stroke. Stroke 1998;29:1698–1706.
141 Wang XK, Louden C, Yue TL, Ellison JA, Barone FC, Solleveld H, Feuerstein GZ: Delayed
expression of osteopontin after focal stroke in the rat. J Neurosci 1998;18:2075–2083.
142 Wang XK, Barone FC, White RF, Feuerstein GZ: Subtractive cloning identifies tissue inhibitor of
matrix metalloproteinase-1 (TIMP-1) increased gene expression in focal stroke. Stroke
1998;29:516–520.
143 Wang XK, Yue TL, White RF, Barone FC, Feuerstein GZ: Transforming growth factor ß1,
exhibits delayed gene expression following focal cerebral ischemia. Brain Res Bull 1995;36:
607–609.
medwedi.ru
144 Tsuda M, Imaizumi K, Katayama T, Kitagawa K, Wanaka A, Tohyama M, Takagi T: Expression of
zinc transporter gene, ZnT-1, is induced after transient forebrain ischemia in the gerbil. J Neurosci
1997;17:6678–6684.
145 Katayama T, Imaizumi K, Tsuda M, Mori Y, Takagi T, Tohyama M: Expression of an ADP-
ribosylation factor like gene, ARF4L, is induced after transient forebrain ischemia in the gerbil.
Mol Brain Res 1998;56:66–75.
146 Wigle D, Ho W, Lo D, Francis J, Eubanks JH, Wallace MC: Altered expression levels of SEF-2
and p112 in the rat hippocampus after transient cerebral ischemia: Identification by mRNA dif-
ferential display. J Cereb Blood Flow Metab 1999;19:435–442.
147 Utans-Schneitz U, Lorez H, Klinkert WE, da Silva J, Lesslauer W: A novel rat CC chemokine,
identified by targeted differential display, is upregulated in brain inflammation. J Neuroimmunol
1998;92:179–190.
148 Wang XB, Uhl GR: Subtracted differential display: Genes with amphetamine-altered expression
patterns include calcineurin. Mol Brain Res 1998;53:344–347.
149 Barr FG, Emanuel BS: Application of a subtraction hybridization technique involving photoacti-
vatable biotin and organic extraction to solution hybridization analysis of genomic DNA. Analyt
Biochem 1990;186:369–373.
150 Lisitsyn NA: Representational difference analysis: Finding the differences between genomes.
Trends Genet 1995;11:303–307.
151 Lockhart DJ, Dong H, Byrne MC, Follettie MT, Gallo MV, Chee MS, Mittmann M, Wang C,
Kobayashi M, Horton H, Brown EL: Expression monitoring by hybridization to high-density
oligonucleotide arrays. Nat Biotechnol 1996;14:1675–1680.
152 Lipshutz RJ, Fodor SP, Gingeras TR, Lockhart DJ: High density synthetic oligonucleotide arrays.
Nat Genet 1999;21:20–24.
153 Schena M, Shalon D, Davis RW, Brown PO: Quantitative monitoring of gene expression patterns
with a complementary DNA microarray. Science 1995;270:467–470.
154 Young RA: Biomedical discovery with DNA arrays. Cell 2000;102:9–15.
155 Tamura A, Graham DI, McCulloch J, Teasdale GM: Focal cerebral ischaemia in the rat.
I. Description of technique and early neuropathological consequences following middle cerebral
artery occlusion. J Cereb Blood Flow Metab 1981;1:53–60.
156 Wiessner C, Neumann-Haefelin T, Vogel P, Back T, Hossmann KA: Transient forebrain ischemia
induces an immediate-early gene encoding the mitogen-activated protein kinase phosphatase
3CH134 in the adult rat brain. Neuroscience 1995;64:959–966.
157 Gass P, Eckhardt A, Schroder H, Bravo R, Herdegen T: Transient expression of the mitogen-
activated protein kinase phosphatase MKP-1 (3CH134/ERP1) in the rat brain after limbic
epilepsy. Mol Brain Res 1996;41:74–80.
158 Velculescu VE, Zhang L, Vogelstein B, Kinzler KW: Serial analysis of gene expression. Science
1995;270:484–487.
159 Velculescu VE, Vogelstein B, Kinzler KW: Analyzing uncharted transcriptomes with SAGE.
Trends Genet 2000;16:423–425.
160 Trendelenburg G, Prass K, Priller J, Kapinya K, Polley A, Muselmann C, Ruscher K, Kannbley U,
Schmitt AO, Castell S, Wiegand F, Meisel A, Rosenthal A, Dirnagl U: Serial analysis of gene
expression identifies Metallothionein-II as major neuroprotective gene in mouse focal cerebral
ischemia. J Neurosci 2002;22:5879–5888.
161 Gibson UE, Heid CA, Williams PM: A novel method for real time quantitative RT-PCR. Genome
Res 1996;6:995–1001.
162 Mandai K, Matsumoto M, Kitagawa K, Matsushita K, Ohtsuki T, Mabuchi T, Colman DR,
Kamada T, Yanagihara T: Ischemic damage and subsequent proliferation of oligodendrocytes in
focal cerebral ischemia. Neuroscience 1997;77:849–861.
163 Clark RK, Lee EV, Fish CJ, White RF, Price WJ, Jonak ZL, Feuerstein GZ, Barone FC:
Development of tissue damage, inflammation and resolution following stroke: An immunohisto-
chemical and quantitative planimetric study. Brain Res Bull 1993;31:565–572.
164 Davies CA, Loddick SA, Stroemer RP, Hunt J, Rothwell NJ: An integrated analysis of the pro-
gression of cell responses induced by permanent focal middle cerebral artery occlusion in the rat.
Exp Neurol 1998;154:199–212.
Barone/Read 374
165 Kochanek PM, Hallenbeck JM: Polymorphonuclear leukocytes and monocytes/macrophages in
the pathogenesis of cerebral ischemia and stroke. Stroke 1992;23:1367–1379.
166 Iadecola C, Niwa K, Nogawa S, Zhao X, Nagayama M, Araki E, Morham S, Ross ME: Reduced
susceptibility to ischemic brain injury and N-methyl-D-aspartate-mediated neurotoxicity in
cyclooxygenase-2-deficient mice. Proc Natl Acad Sci USA 2001;98:1294–1299.
167 Relton JK, Rothwell NJ: Interleukin-1 receptor antagonist inhibits ischemic and excitotoxic neu-
ronal damage in the rat. Brain Res Bull 1992;29:243–246.
168 Ali C, Nicole O, Docagne F, Lesne S, MacKenzie ET, Nouvelot A, Buisson A, Vivien D: Ischemia-
induced interleukin-6 as a potential endogenous neuroprotective cytokine against NMDA receptor-
mediated excitotoxicity in the brain. J Cereb Blood Flow Metab 2000;20:956–966.
169 Loddick SA, Turnbull AV, Rothwell NJ: Cerebral interleukin-6 is neuroprotective during perma-
nent focal cerebral ischemia in the rat. J Cereb Blood Flow Metab 1998;18:176–179.
170 Borlongan CV, Stahl CE, Redei E, Wang Y: Prepro-thyrotropin-releasing hormone 178–199 exerts
partial protection against cerebral ischemia in adult rats. Neuroreport 1999;10:3501–3505.
171 Yonemori F, Yamaguchi T, Nakayama H, Narita K, Hojo S, Tamura A: Effect of JTP-2942, a novel
thyrotropin-releasing hormone analog, on motor deficits after chronic focal cerebral ischemia in
rats. J Cereb Blood Flow Metab 2000;20:74–81.
172 Yamashita K, Wiessner C, Lindholm D, Thoenen H, Hossmann KA: Post-occlusion treatment with
BDNF reduces infarct size in a model of permanent occlusion of the middle cerebral artery in rat.
Metab Brain Dis 1997;12:271–280.
173 Dirnagl U, Simon RP, Hallenbeck JM: Ischemic tolerance and endogenous neuroprotection.
Trends Neurosci 2003;26:248–254.
174 Chen J, Graham SH, Zhu RL, Simon RP: Stress proteins and tolerance to focal cerebral ischemia.
J Cereb Blood Flow Metab 1996;16:566–577.
175 Dawson VI, Dawson TE: Neuronal ischaemic preconditioning. Trends Pharmacol Sci 2000;21:
423–424.
176 Kirino T: Ischemic tolerance. J Cereb Blood Flow Metab 2002;22:1283–1296.
177 Barone FC, White RF, Spera PA, Ellison J, Currie RW, Wang X, Feuerstein GZ: Ischemic precon-
ditioning and brain tolerance: Temporal histological and functional outcomes, protein synthesis
requirement, and interleukin-1 receptor antagonist and early gene expression. Stroke 1998;
29:1937–1950.
178 Currie RW, Ellison JA, White RF, Feuerstein GZ, Wang XK, Barone FC: Benign focal ischemic
preconditioning induces neuronal Hsp70 and prolonged astrogliosis with expression of Hsp27.
Brain Res 2000;863:169–181.
179 Purcell JE, Lenhard SC, White RF, Schaeffer T, Barone FC, Chandra S: Strain-dependent response
to cerebral ischemic preconditioning: Differences between spontaneously hypertensive and
stroke-prone spontaneously hypertensive rats. Neurosci Lett 2003;339:151–155.
180 Barone FC, Maguire S, Strittmatter R, White RF, Schaeffer T, Lenhard SC, Purcell JE, Chandra S:
Longitudinal MRI measures brain injury and its resolution: Reduced neurological recovery post-
stroke and decreased brain tolerance following ischemic preconditioning in stroke-prone rats.
J Cereb Blood Flow Metab 2001;21(suppl 1):S230.
181 Maguire S, Stritmatter R, Chandra S, Barone FC: Stroke prone rats exhibit prolonged neurologi-
cal deficits indicating disruption of post-stroke brain recovery. Neuroscience 2003;71:1–16.
182 Weih M, Kallenberg K, Bergk A, Dirnagl U, Harms L, Wernecke KD, Einhaupl KM: Attenuated
stroke severity after prodromal TIA: A role for ischemic tolerance in the brain? Stroke 1999;30:
1851–1854.
183 Moncayo J, de Freitas GR, Bogousslavsky J, Altieri M, van Melle G: Do transient ischemic attacks
have a neuroprotective effect? Neurology 2000;54:2089–2094.
184 McLaughlin BA, Hartnett KA, Erhardt JA, Legos JJ, White RF, Barone FC, Aizenman E:
Caspase-3 activation is essential for neuroprotection in preconditioning. Proc Natl Acad Sci USA
2003;100:715–720.
185 Yellon DM, Baxter GF, Garcia-Dorado D, Heusch G, Sumeray MS: Ischemic preconditioning:
Present position and future directions. Cardiovasc Res 1998;37:21–33.
186 Lawson CS, Downey JM: Preconditioning: State of the art myocardial protection (Review).
Cadiovasc Res 1993;27:542–550.
medwedi.ru
187 Chen J, Simon RP: Ischemic tolerance in the brain. Neurology 1997;48:306–311.
188 Wang XK, Li X, Currie RW, Willette RN, Barone FC, Feuerstein GZ: Application of real-time
polymerase chain reaction to quantitate induced expression of interleukin-1beta mRNA in
ischemic brain tolerance. J Neurosci Res 2000;59:238–246.
189 Wang XK, Li X, Erhardt JA, Barone FC, Feuerstein GZ: Detection of tumor necrosis factor-alpha
mRNA induction in ischemic brain tolerance by means of real-time polymerase chain reaction.
190 Wang XK, Yaish-Ohad S, Li X, Barone FC, Feuerstein GZ: Use of suppression subtractive
hybridization strategy for discovery of increased tissue inhibitor of matrix metalloproteinase-1
gene expression in brain ischemic tolerance. J Cereb Blood Flow Metab 1998;18:1173–1177.
191 Rossini PM, Pauri F, Cicinelli P, Pasqualetti P, Traversa R, Tecchio F: Neuromagnetic recordings
and magnetic brain stimulation in the evaluation of sensorimotor hand area interhemispheric dif-
ferences: Normative, experimental and patients’ data. Electroencephal Clin Neurophysiol 1999;
50(suppl):210–220.
192 Dobkin BH: Functional rewiring of brain and spinal cord after injury: The three Rs of neural repair
and neurological rehabilitation. Curr Opin Neurol 2000;13:655–659.
193 Hunter AJ, Hatcher J, Virley D, Nelson P, Irving E, Hadingham SJ, Parsons AA: Functional assess-
ments in mice and rats after focal stroke. Neuropharmacology 2000;39:806–816.
194 Lee LY, Patel SR, Hackett NR, Mack CA, Polce DR, El-Sawy T, Hachamovitch R, Zanzonico P,
Sanborn TA, Parikh M, Isom OW, Crystal RG, Rosengart TK: Focal angiogen therapy using
intramyocardial delivery of an adenovirus vector coding for vascular endothelial growth factor
121. Ann Thorac Surg 2000;69:14–24.
195 Gowdak LH, Poliakova L, Wang X, Kovesdi I, Fishbein KW, Zacheo A, Palumbo R, Straino S,
Emanueli C, Marrocco-Trischitta M, Lakatta EG, Anversa P, Spencer RG, Talan M, Capogrossi MC:
Adenovirus-mediated VEGF(121) gene transfer stimulates angiogenesis in normoperfused skeletal
muscle and preserves tissue perfusion after induction of ischemia. Circulation 2000;102:565–571.
196 Kalka C, Masuda H, Takahashi T, Gordon R, Tepper O, Gravereaux E, Pieczek A, Iwaguro H,
Hayashi SI, Isner JM, Asahara T: Vascular endothelial growth factor(165) gene transfer augments
circulating endothelial progenitor cells in human subjects. Circ Res 2000;86:1198–1202.
197 Vale PR, Losordo DW, Milliken CE, Maysky M, Esakof DD, Symes JF, Isner JM: Left ventricu-
lar electromechanical mapping to assess efficacy of phVEGF(165) gene transfer for therapeutic
angiogenesis in chronic myocardial ischemia. Circulation 2000;102:965–974.
198 Cao L, Zhao YC, Jiang ZH, Xu DH, Liu ZG, Chen SD, Liu XY, Zheng ZC: Long-term phenotypic
correction of rodent hemiparkinsonism by gene therapy using genetically modified myoblasts.
Gene Ther 2000;7:445–449.
199 Shimazaki K, Urabe M, Monahan J, Ozawa K, Kawai N: Adeno-associated virus vector-mediated
bcl-2 gene transfer into post-ischemic gerbil brain in vivo: Prospects for gene therapy of ischemia-
induced neuronal death. Gene Ther 2000;7:1244–1249.
200 Yang GY, Zhao YJ, Davidson BL, Betz AL: Overexpression of interleukin-1 receptor antagonist
in the mouse brain reduces ischemic brain injury. Brain Res 1997;751:181–188.
201 Yang GY, Mao Y, Zhou LF, Ye W, Liu XH, Gong C, Lorris BA: Attenuation of temporary focal
cerebral ischemic injury in the mouse following transfection with interleukin-1 receptor antago-
nist. Brain Res Mol Brain Res 1999;72:129–137.
202 Yenari MA, Fink SL, Sun GH, Chang LK, Patel MK, Kunis DM, Onley D, Ho DY, Sapolsky RM,
Steinberg GK: Gene therapy with HSP 72 is neuroprotective in rat models of stroke and epilepsy.
Ann Neurol 1998;44:584–591.
203 Yenari MA, Giffard RG, Sapolsky RM, Steinberg GK: The neuroprotective potential of heat shock
protein 70 (HSP 70). Mol Med Today 1999;5:525–531.
Frank C. Barone, PhD

High Throughput Biology, UW2108, GlaxoSmithKline
709 Swedeland Road, King of Prussia, PA 19406 (USA)
Tel. ⫹1 610 270 6824, Fax ⫹1 610 270 6505, E-Mail frank.c.barone@gsk.com
Barone/Read 376
Molecular Mediators of
Hemorrhagic Stroke
R. Loch Macdonald
Department of Neurosurgery, University of Chicago Medical Center, Chicago,
Ill., USA
Introduction
This chapter will discuss events mediating hemorrhagic stroke at a molec-

ular level and underlying diseases that increase the risk of developing such
stroke. Hemorrhagic stroke encompasses intracerebral hemorrhage (ICH) that
may be secondary to hypertension; congenital and acquired disorders of blood
coagulation; intracranial vascular diseases such as vascular malformations,
aneurysms, and amyloid angiopathies; and subarachnoid hemorrhage (SAH).
Information on the etiological and pathophysiological mechanisms involved in
underlying causes of ICH or SAH (e.g., hypertension) will be presented. The
treatment of spontaneous ICH is currently limited to surgical evacuation of the
hematoma, ventricular drainage, medical measures to reduce intracranial pres-
sure, and supportive care; biologically based therapies that have been tested in
experimental settings will be discussed.
Etiology of ICH
Hypertension
Most large series have found that the most common disease associated
with ICH is hypertension [163]. Additional risk factors for ICH include age,
race, and severe hypocholesterolemia. Some cases of ICH may be related to
various congenital coagulopathies, including more specifically for the brain
mutations in the genes for the A subunit of factor 13, which is involved in cross-
linking fibrin [18]. The mechanism of hemorrhage is unclear, but studies
demonstrate an association between ICH associated with hypertension (most
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commonly in the putamen, thalamus, pons, and deep cerebellar nuclei) and
lipohyalinosis of small penetrating arteries and arterioles. A population-based
study [209] of 188 cases of ICH in the Cincinnati area found that 121 (64%) of
hemorrhages were nonlobar and that 54% of the nonlobar hemorrhages were
attributable to hypertension. Hypertension causes numerous detrimental effects
on the circulation including decreasing endothelium-dependent relaxations via
effects on the endothelium and accelerating atherosclerosis. Charcot-Bouchard
aneurysms [21] of the penetrating arterioles (i.e., chronically dilated arterioles
secondary to hypertension) were traditionally thought to be the source of hyper-
tensive ICH. This assumption has been questioned by autopsy studies, which
found these aneurysms difficult to identify [19]. Alternative explanations are
that hypertension causes lipohyalinosis of penetrating arterioles, which dam-
ages the arteriolar wall leading to dissection and hemorrhage. It is also possi-
ble that lipohyalinosis occludes arterioles and there is hemorrhage into the area
of brain infarcted by the occlusion or from the occluded arteriole due to an
increased upstream pressure.
The molecular basis of hypertension is complex. Adoption and twin stud-
ies suggest that more than half the variability in blood pressure between indi-
viduals in Western populations is genetically determined [195]. Blood pressure
is not determined by a single gene, and animal models suggest the mode of
inheritance is governed by at least 6–10 genes. In addition, genes that encode
proteins in the second messenger pathways for the primary regulatory proteins
may be involved, enhancing the complexity of the task involved in identifying
the genes. Hypertension is a heterogeneous condition in which underlying
genotypes may give rise to different diseases associated with hypertension.
Polygenic inheritance, variable penetrance, gene-environment interactions,
genetic and pathological heterogeneity, and late age of onset make the molec-
ular genetics of diseases like hypertension and stroke difficult to study, espe-
cially using traditional linkage-based genetic analysis [195]. Candidate genes
involved in the renin-angiotensin-aldosterone system and in the regulation of
intracellular ion homeostasis have been most extensively investigated, but
many other genes are likely to be involved, including those regulating vascular
tone and cardiovascular function. There have been reports of associations
between angiotensin-converting enzyme alleles and hypertension [221] and the
sodium-potassium ATPase and hypertension [184].
Amyloid Angiopathies
Amyloid angiopathy (i.e., cerebrovascular amyloidosis) is an important
cause of lobar ICH [202]. In amyloid angiopathy there is deposition of
eosinophilic material in the walls of the cerebral and cerebellar arteries, includ-
ing leptomeningeal arteries, arterioles, venules and capillaries. These patients
Macdonald 378
develop lobar ICH late in life, usually after the age of 70. There may be multi-
ple hemorrhages over time and rarely simultaneous hemorrhages in multiple
areas of the brain. The coexistence of brain amyloid deposition that may be
associated with Alzheimer’s disease (AD) has been noted pathologically and
correlates with the clinical evidence of dementia in approximately 30% of
patients with amyloid-related ICH [202]. The exact relationship between spo-
radic amyloid angiopathy and AD pathogenesis is unclear. Amyloid angiopathy
can occur in AD along with amyloid plaques, neurofibrillary tangles, and gran-
ulovacuolar degeneration, and is found in 75–100% of AD cases, whereas amy-
loid angiopathy can occur in the absence of dementia and the other
neuropathological changes of AD. This pattern suggests the underlying genetic
and etiological variability in the disease. Individuals with the apolipoprotein-E4
allele have been found to be at increased risk of developing late-onset AD [27].
This observation led to interest in whether patients with amyloid angiopathy-
related ICH were more likely to have this allele. Woo et al. [209] reported that
there is an association between the ␧2 allele (apoE2) or ␧4 allele (apoE4)
and ICH from amyloid angiopathy, consistent with other findings [46, 126].
Polymorphisms in the endoglin gene may also be associated with spontaneous
ICH [4]. Endoglin is a membrane glycoprotein primarily associated with human
vascular endothelium; a component of the transforming growth factor-␤
(TGF-␤) receptor complex, it binds TGF-␤1 with high affinity and mutations
are associated with hereditary hemorrhagic telangiectasia (HHT1). A number
of inherited forms of amyloid angiopathy have been described. Dutch families
living in a small area of the Netherlands were described with hereditary cere-
bral hemorrhage with amyloidosis, known as ‘Dutch-type’ amyloidosis [13].
These patients develop ICH due to amyloid angiopathy of meningeal and corti-
cal penetrating arteries and arterioles. Some patients develop dementia that is
thought to be secondary to multiple ICH; typical AD does not develop and the
neuropathological characteristics of the changes in the brain appear different
(e.g., neurofibrillary tangles are absent). The inheritance of ‘Dutch-type’ cere-
bral hemorrhage with amyloidosis is autosomal dominant and is due to a muta-
tion of the amyloid precursor protein. Hereditary cerebral hemorrhage with
amyloidosis of the Icelandic type is an autosomal dominant disorder causing
ICH [140]. This amyloidosis is due to a mutation in the gene encoding the cys-
teine protease inhibitor cystatin C (gamma-trace) found on chromosome 20.
The mutation causes the protein to form amyloid deposits in cerebral vessels.
This cause of ICH is quite rare; screening of patients with sporadic ICH for
mutations in specific exons of cystatin C and the amyloid precursor protein did
not reveal mutations [45].
Gerstmann-Sträussler-Scheinker disease is a spongiform encephalopathy
that results from a mutation in the prion protein. Brain deposition of amyloid
Molecular Mediators of Hemorrhagic Stroke 379
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occurs in this disease as well as other spongiform encephalopathies, although
ICH is not an usual feature. A prion protein cerebral amyloid angiopathy, how-
ever, has been described in which a mutation in the prion protein is associated
with its deposition as amyloid in the brain and cerebral blood vessels [44].
Again, ICH is not common in this disease. However, patients with oculo-
leptomeningeal amyloidosis can present with ICH [165]. This condition is asso-
ciated with amyloid deposited in the cerebral vasculature that is composed of
transthyretin or prealbumin. Additional mutations in transthryetin leading to
amyloid angiopathy are described in type 1 familial amyloidotic polyneuropathy
[2]. Understanding why some forms of amyloid angiopathy such as ‘Dutch-
type’ amyloidosis lead to ICH, whereas others in which another protein is
deposited do not cause ICH will be important in determining the pathogenesis
of amyloid-related ICH.
Arteriovenous Malformations and Other Vascular Malformations

Intracranial vascular malformations are classified as arteriovenous (AVM),
cavernous, venous, capillary, or mixed. The first three types are the most impor-
tant clinically. Most AVMs are sporadic and there are no underlying genetic fac-
tors predisposing to their occurrence. They may be associated with the
Osler-Weber-Rendu and Wyburn-Mason syndromes. Familial occurrence of
AVMs is documented, but these cases make up less than 1% of sporadic single
AVMs [35, 217]. Some of these patients have multiple AVMs and inheritance
may be related to Osler-Weber-Rendu syndrome. Roger Wyburn-Mason [210]
described the association of retinal and midbrain AVMs and occasionally facial
nevi in a 1943 monograph. The defining features of this disorder and its genetic
basis are still uncertain. Additional rare familial disorders with cerebrovascular
manifestations include multiple systemic hemangiomatosis which is character-
ized by cavernous malformations of the skin, viscera, central nervous system
and peripheral nerves [35]; blue rubber bleb nevus syndrome which consists of
bluish cavernous malformations of the skin and various cerebrovascular mal-
formations; cutaneomeningospinal angiomatosis (Cobb’s disease or dermato-
spinal angioma) which is characterized by skin port-wine stain associated with
spinal AVM; and hereditary neurocutaneous angiomatosis, which is character-
ized by multiple skin cavernous malformations or AVMs along with brain
AVMs and venous anomalies.
Osler-Weber-Rendu syndrome (hereditary hemorrhagic telangiectasia) is
inherited in an autosomal dominant fashion and is characterized by capillary
telangiectases of the skin, mucosa, and viscera [50]. Penetrance is high but the
phenotype is variable. Several mutations have been identified. HHT1 is defined
by a mutation in the gene for endoglin on chromosome 9q, a transforming
growth factor-␤ binding protein on endothelial cells. A second mutation,
Macdonald 380
termed HHT2, was identified on chromosome 12q and encodes activin-1 like
receptor, which is also a cell-surface receptor for the TGF-␤ family of growth
factors. The phenotype varies, but there may be multiple systemic telangiec-
tasias as well as AVMs in the brain. These patients also tend to have pulmonary
AVMs. The incidence ranges from one in 2351 among the French population of
Ain to one in 40,000 in England. The most common clinical manifestation is
epistaxis.
Cavernous malformations are known to have a genetic basis. Kufs [91]
may have been the first to suggest that cavernous malformations might be
inherited. A large Mexican-American family was reported in 1982, in whom
a pattern of autosomal dominant inheritance with variable penetrance seemed
present [60]. Rigamonti et al. [120, 166] described 24 patients with cavernous
malformations, of which 13 were members of 6 unrelated Mexican-American
families. The familial cavernous malformations differed from sporadic cases
in that 75% of familial lesions were multiple, whereas only 10–25% of spo-
radic ones were. They estimated that 30–50% of cavernous malformations
were familial although more conservative estimates are as low as 6% [70].
The location of genes for familial cavernous malformations were mapped to
chromosomes 7q (CCM1) [31, 48, 104], 7p (CCM2) and 3q (CCM3) [29].
One of the genes identified as mutated in CCM1 in French and Mexican-
American families is KRIT1 [94, 169]. The function of this protein and how
mutations in it lead to the formation of cavernous malformations are
unknown. Denier et al. [30] noted that KRIT1 messenger ribonucleic acid
(mRNA) was expressed ubiquitously during mouse development from E7.5 to
E9.5, after which expression became restricted to neurons and epithelia.
Vascular expression was restricted to large embryonic blood vessels. KRIT1
protein colocalized with microtubules in endothelial cells and has been
reported to interact with Krev1 and integrin cytoplasmic domain-associated
protein-1 [49]. Based on these findings, it was hypothesized that KRIT1 is
involved in the determination of endothelial cell shape and function and dis-
ruption of KRIT1 leads to abnormal endothelial tube formation and
cavernous malformations.
Developmental venous anomalies were previously called venous malfor-
mations, but now are believed to be congenital variations in venous drainage.
Vikkula et al. [201] described a mutation in the receptor tyrosine kinase TIE2
gene on chromosome 9p21 in two families with multiple venous malformations.
This mutation was inherited in an autosomal dominant manner. The mutation
results in an increased activity of TIE2, an endothelial cell-specific receptor
tyrosine kinase expressed in developing endothelial cells and important in blood
vessel development. The venous malformations in these families were in the
skin and mucosa but not the brain.
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Intracranial Aneurysms
The common saccular intracranial aneurysm is an acquired lesion that usu-
ally occurs at an arterial bifurcation and is secondary to hemodynamic stress-
mediated degeneration of the internal elastic lamina. Factors predisposing to the
development and the rupture of intracranial aneurysms include age, gender, cig-
arette smoking, hypertension and familial predisposition. There is some geo-
graphical variation in the incidence of SAH with higher rates reported in Japan
and Finland [6, 73, 206]. The risk of SAH in African-Americans in the Greater
Cincinnati area was twice that of Caucasians [14]. Such geographical and racial
differences could reflect genetically based population differences that, if iden-
tified, could lead to insight into the etiology and pathogenesis of intracranial
aneurysms. On the other hand, they could result from variations in the age dis-
tribution, availability of medical care and prevalence of smoking, alcohol
intake, atherosclerosis and hypertension in the populations studied. Of course,
some of these risk factors themselves may have underlying genetic bases.
Some intracranial aneurysms are ‘familial’ in that they appear to be inher-
ited by an autosomal dominant or multifactorial mechanism. It is difficult to
exclude the presence of shared environmental factors in these cases. Studies of
identical twins reared apart have been used to examine the genetic basis of dis-
eases and stroke [5], but such studies have not been conducted for intracranial
aneurysms. Familial aneurysms are usually defined by aneurysms occurring in
two or more first- to third-degree relatives. First-degree relatives of patients
with SAH had a 3-fold elevated risk of developing SAH compared to the gen-
eral population of Denmark [41]. In a series of 485 patients with SAH, 16 of
237 (7%) of respondents to a questionnaire reported having blood relatives with
the same condition [133]. The genetic basis for familial aneurysms is unknown
at this time.
Several investigators have searched for genes associated with sporadic
intracranial aneurysms. Alleles of the ␣1 antitrypsin gene that are associated
with reduced enzymatic activity were found to be more common in patients
with aneurysms than controls in one study [175]. Further work is needed to
confirm this relationship. A study in a Japanese population identified a genetic
locus associated with intracranial aneurysms on chromosome 7q11 [142], and
later refined that locus to the gene for collagen type I ␣-2 (COL1A2) at 7q22.1.
Experimental studies of intracranial aneurysms in animals have focused on
induced hypertension, inhibition of collagen cross-linking with ␤-aminopropio-
nitrile, a toxin which causes lathryism, and carotid artery occlusion to induce
flow changes in the cerebral circulation. To examine the molecular basis for
aneurysm formation, Peters et al. [152] used global gene expression analysis
(SAGE-Lite) to examine gene expression in an aneurysm from a 3-year-old
girl. There was significant overexpression of genes encoding extracellular
Macdonald 382
matrix components including various collagen isoforms and elastin, and genes
involved in extracellular matrix turnover (TIMP-3, OSF-2), cell adhesion and
antiadhesion (SPARC, hevin), cytokinesis (PNUTL2), and cell migration
(tetraspanin-5). Their interpretation from this single gene expression analysis
was that the aneurysm was characterized by typical wound healing and tissue
remodeling responses, but the underlying basis for its formation could not be
established.
Inducible nitric oxide synthase (iNOS) has been detected at the orifices of
human and rat aneurysms by immunohistochemical staining, whereas it is nor-
mally absent from cerebral arteries [39]. Aminoguanidine, an inhibitor of NOS,
decreased both early aneurysmal changes and the incidence of aneurysms in
rats. The defibrinogenic agent, batroxobin, which may decrease shear stress by
reducing blood viscosity, prevented the induction of iNOS as well as early
aneurysmal changes in their model. These data suggest that NO formed by
iNOS may contribute to the formation of cerebral aneurysms.
Other diseases that may be associated with intracranial aneurysms include
fibromuscular dysplasia, coarctation of the aorta, autosomal dominant poly-
cystic kidney disease (PKD), and Ehlers-Danlos (ED) syndrome [174]. There are
other genetic syndromes in the literature in which aneurysms have occurred but
the relationship in these cases is more speculative. The most common disease in
which cerebral aneurysms occur may be fibromuscular dysplasia or FMD [117].
FMD was reported in the renal arteries of 1.1% of 819 autopsies [61]. In a
review of five series of patients undergoing angiography (approximately 22,000
patients), FMD was noted in about 0.5% of cases (0.25–0.61%). About 85% of
FMD cases are in women. Mettinger et al. [117] has reported that 21% of 284
patients with FMD had intracranial aneurysms. Hypertension from renal artery
involvement was not necessarily present. The incidence of FMD in patients pre-
senting with intracranial aneurysms and SAH is uncertain. Clinical presentation
may be with cerebral infarction, intracranial aneurysm, or as an incidental find-
ing. Pathologically, FMD is characterized by the intimal proliferation of smooth
muscle cells that have a phenotype altered to resemble myofibroblasts. There is
an associated proliferation of extracellular matrix and collagen. Genetic factors
may play a role in some cases, although well-documented familial cases are rare.
The inheritance pattern appears to be autosomal dominant with reduced pene-
trance in males. There is no conclusive genetic basis, but Bofinger et al. [12]
studied polymorphisms of angiotensin converting enzyme and found that one
allele was associated with FMD. Autosomal dominant PKD is also associated
with intracranial aneurysms. The relationship between these diseases has been
studied in 88 patients selected from 378 known cases of PKD [20]. Cranial CT
was performed in 60 patients, cerebral angiography in 21, and both procedures
in 11. Four patients had aneurysms (4% incidence, 95% confidence interval
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0.1–9%). These results suggest an increased risk of aneurysms in patients with
PKD. Other reports have detected intracranial aneurysms in upto 40% of patients
with PKD. At least two genetic defects are identified in PKD [3]. About 85% of
cases (PKD1) are due to mutations in the gene for polycystin located on chro-
mosome 16p, which is a large glycoprotein that is postulated to be important in
cell-cell and cell-extracellular matrix interactions. PKD2 is due to mutations in
a gene on chromosome 4, which encodes a protein that is postulated to be a
membrane spanning protein, perhaps an ion channel. PKD is also associated
with hypertension secondary to kidney failure, and it has been suggested that the
latter leads to aneurysms in these cases, although there are cases reported with-
out well-documented hypertension.
ED syndrome is another collagen disease that is characterized by hyper-
elasticity of the skin, joint hypermobility, bruising, and abnormal scarring. The
skin may be relatively normal and the joints not excessively mobile in ED Type 4,
which is of interest in stroke. These patients develop spontaneous dissections of
large extracranial arteries as well as intestinal ruptures. Carotid cavernous fistu-
las are the most common cerebrovascular lesion although aneurysms also are
reported [3, 173]. The molecular basis of ED syndrome is mutations in the gene
for type III procollagen. Type III collagen is a major extracellular structural col-
lagen in the walls of blood vessels and is composed of three ␣-helix polypeptide
chains that form a triple helix. The type III collagen gene is located on chromo-
some 2 and numerous mutations causing ED Type 4 have been reported. Alberts
[2, 3] found only one well-documented case of a patient with ED Type 4 and a
true saccular intracranial aneurysm, suggesting that simple defects in type III
collagen are not an usual cause of these aneurysms. Studies of humans with
familial and sporadic intracranial aneurysms have not found mutations in type III
collagen to be responsible [92].
Marfan’s syndrome may be associated with saccular, fusiform, or dissec-
ting aneurysms [3]. This disease results from mutations in fibrillin, a gene on
chromosome 15q. Fibrillin is a glycoprotein that is a part of elastic and nonelas-
tic microfibrils. Most aneurysms described are extracranial or in the cavernous
internal carotid artery. Few true saccular aneurysms have been identified.
These patients are prone to dissections of the large elastic and muscular arter-
ies and dissections could underlie some of the reported cases of aneurysmal
disease.
Patients with coarctation of the aorta were suggested to be at increased
risk of having intracranial aneurysms and of SAH at a young age. In early
reports, many of the patients had hypertension which may have increased the
incidence of aneurysmal disease. Early diagnosis and treatment of the coarc-
tation was reported in 182 patients who were followed for a total of 3288
patient-years. There were no deaths from intracranial hemorrhage, suggesting
Macdonald 384
that early repair and prevention of chronic hypertension in these patients
reduces or eliminates the risk of intracranial aneurysm [11]. More study is
needed to ascertain whether aneurysms in these patients are due to arterial
hypertension or a common vascular defect.
Pathophysiology of ICH
Direct and Indirect Molecular Effects

Pathophysiological mechanisms of brain damage from ICH are hypothe-
sized to include direct damage to the brain due to physical disruption by the
hematoma and secondary injury due to the enlargement of the hematoma,
increased intracranial pressure, brain edema, toxic effects of substances released
from the blood clot, and possibly ischemia around the clot. Two studies in which
computed tomography was performed sequentially after ICH documented that
enlargement occurs in about one quarter of patients if the first CT scan is
obtained within one hour of the ictus [15, 37]. The risk of the enlargement
decreases with time. Microscopically, there is extracellular edema appearing
around ICH in humans within a time course of hours. Polymorphonuclear lym-
phocytes appear around the ICH by 2 days and peak at 4 days. Microglia are seen
at this time and ingest ICH and damaged brain tissue. Gliosis, characterized by
gemistocytic astrocytes appears after days and may persist for months to years.
The pathogenesis of brain injury secondary to SAH is multifactorial and
has not been extensively investigated. Processes involved include global and
focal ischemia from increased intracranial pressure and decreased cerebral per-
fusion pressure, cerebral herniation, ICH, subdural hemorrhage, hydro-
cephalus, and aneurysm rebleeding. It is assumed that most of the initial
damage in patients who are neurologically impaired after SAH is due to global
ischemia since intracranial pressure rises with bleeding and reduces cerebral
perfusion pressure.
Whether or not there are additional direct effects of the SAH itself on the
brain is open to question, but the possibility has been raised based on experi-
mental and clinical studies demonstrating that cerebral blood flow (CBF) is
reduced after SAH in proportion to the clinical grade and that this often is
accompanied by reduced cerebral metabolic demand. Mitochondrial respira-
tion, sodium-potassium ATPase activity, extracellular potassium and calcium
are altered in brain tissue of experimental animals exposed to subarachnoid
blood, although the relationship of these changes to the alterations in CBF and
metabolism has not been studied [36, 72, 105, 218].
Molecular changes may occur in the brain after SAH as a result of the SAH
itself or due to secondary processes listed above that often result in ischemia.
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SAH increases c-fos, c-jun and HSP70 mRNA in a number of brain regions in
rats [55, 135]. Neonatal capsaicin treatment which destroys unmyelinated
c fibers, or sectioning of meningeal afferents, results in reduced c-fos in the
trigeminal nuclear complex, parabrachial nucleus, and medullary lateral reticu-
lar nucleus, but not in nucleus of tractus solitarius, area postrema, ependyma,
piamater and arachnoid. This pattern suggests that SAH directly induces c-fos
protein expression in the nucleus of the tractus solitarius, area postrema,
ependyma, pia mater and arachnoid and that in other areas (trigeminal nuclear
complex, parabrachial nucleus, and medullary lateral reticular nucleus) induc-
tion may be secondary to trigeminovascular nerve fibers. Creation of SAH in
rats by endovascular perforation of the intracranial anterior cerebral arteries
causes an SAH that may resemble that occurring in man since intracranial pres-
sure is elevated for some time [9, 200]. This pattern differs from other models
of SAH, where blood is injected slowly into the cerebrospinal fluid cisterns or
a blood clot is placed surgically around the arteries. SAH caused by endovas-
cular perforation increases HSP70 protein in multiple brain areas bilaterally for
up to 5 days, whereas cisternal blood injections do not [115]. HSP70 is known
to be induced by ischemia, which was postulated to be the cause in these experi-
ments although CBF was not measured. Other experiments were conducted in
which HSP70 induction in the hippocampus occurred when SAH in rats was
induced by blood injections that increased intracranial pressure, whereas saline
injections produced only transient elevations in intracranial pressure and tran-
sient HSP70 expression. These data are consistent with a role for ischemia in
these models and in acute changes after SAH. Acute vasoconstriction with
reduction in CBF has been reported to occur in this model and it has been
reported to be reversible with NO donors, suggesting that decreased NO avail-
ability contributes to the reduced blood flow [10, 178].
Erythrocyte lysis and hemoglobin are probably involved in the delayed
cerebral vasospasm that is discussed below. Interest in their role earlier after
SAH also has been raised based on animal experiments performed by Matz
et al. [111] Hemoglobin metabolism is catalyzed initially by heme oxygenase
[HO], an enzyme that exists in three different forms. The inducible form,
HO-1, was induced diffusely in microglia throughout the brain of rats after cis-
ternal injection of whole blood, erythrocyte hemolysate, or oxyhemoglobin.
Focal areas of expression of HO-1 and HSP70 and of DNA fragmentation were
also noted after the injection of hemolysate [111, 112]. Since HSP70 is induced
in areas of ischemia, two conclusions can be drawn. The lack of widespread
increase in HSP70 suggests that there was no overall stress response or
ischemia in the brain in this model, whereas focal expression is probably due
to ischemia secondary to acute vasospasm or direct toxic effects of the blood
products. The focal areas were blocked by the pretreatment of rats with
Macdonald 386
tirilazad-like anti-oxidants, U101033E and U74389G [197]. Induction of HO-1
expression in the brain after SAH in rats occurred after injection of erythrocyte
hemolysate, but injection of the vasoconstrictor endothelin did not [93]. Since
the degree of vasospasm of the basilar artery was similar in both cases, induc-
tion of HO-1 may be due to effects of blood and not vasospasm. Evidence for
direct effects of SAH on the brain was presented by Matz et al. [114]. Injection
of erythrocyte hemolysate into the cortical subarachnoid space of mice pro-
duced DNA fragmentation and laddering and terminal deoxyuridine nick end-
labeling of cells, consistent with apoptotic cell death in cortex after SAH. In
this model, transgenic mice overexpressing copper-zinc superoxide dismutase,
an antioxidant enzyme, exhibited less cell death [113]. iNOS protein was
increased in microglia, astrocytes and neurons of the brains of rats 7 days after
SAH and vasospasm induced by two injections of blood into the cisterna
magna 2 days apart [208].
Brain Edema
Most research has focused on brain edema and ischemia around intra-
cerebral hematomas. Cerebral edema is defined as an increase in the brain
water content. The most common classifications are vasogenic, cytotoxic, or
interstitial. Edema develops within hours of hemorrhage and peaks at 4–10
days in humans before resolving [194]. Edema after ICH is probably a combi-
nation of vasogenic edema due to blood brain barrier breakdown and cytotoxic
edema due to cellular injury. The time course is shorter in animal models, with
a peak at 3–4 days. Animal models of ICH suggest that different processes
contribute to edema formation at different times. Pathophysiological mecha-
nisms involved in edema may include hydrostatic pressure from clot forma-
tion, clot retraction, thrombin release during coagulation, erythrocyte lysis
with hemoglobin release, complement activation, disruption of the blood brain
barrier, and ischemia reperfusion [211]. Edema developing within the first
hours probably is secondary to hydrostatic pressure changes and clot retrac-
tion. This conclusion is based on studies demonstrating that the blood brain
barrier is intact within 8 h of ICH in pigs [204] and blood clotting extrudes
serum from the clot, which then appears on CT scan as a hypodense rim
around the ICH.
A series of experiments using a rat model of ICH have provided impor-
tant information about the pathogenesis of edema developing after this initial
phase of clot retraction. Injection of pure thrombin into the rat brain caused
cerebral edema 24 h later that was prevented by the selective thrombin
inhibitors hirudin and ␣-NAPAP [95]. Injection of plasma with prothrombi-
nase to activate the coagulation cascade led to edema formation after 24 h,
whereas injection of pure serum, plasma or washed, intact erythrocytes alone
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did not cause edema [96]. Edema 24 h after ICH was also reduced by hirudin,
further supporting a role for thrombin generated by coagulation in the genera-
tion of edema at this time after ICH in rats. Thrombin-induced edema was sug-
gested to be secondary to the disruption of the blood brain barrier and to
cytotoxic effects of thrombin on glia based on studies showing normal CBF
but disruption of the blood brain barrier at the time of thrombin-induced
edema as well as studies in vitro documenting toxic effects of thrombin on C6
glioma cells [97]. Additional evidence supporting a role for blood coagulation
in generating brain edema was that injection of heparinized whole blood into
rats produced less edema than injection of unheparinized blood that subse-
quently clotted [212]. These findings are consistent with human studies report-
ing that edema around thrombolysis-related ICH is less than that around ICH
occurring in patients with normal clotting [42]. In patients who are anticoag-
ulated or have had intravascular thrombolytics, there would be expected to be
less clot retraction and thrombin generation.
Activation of complement also contributes to edema formation at both
24 and 72 h after ICH in rats [71]. Possible mechanisms are the formation of
membrane attack complexes by complement that facilitate erythrocyte lysis,
which is known to release hemoglobin and possibly other substances from the
erythrocyte that can produce edema. The membrane attack complexes also may
damage cells in the brain itself, leading to neuronal injury and edema as well as
leading to further breakdown of the blood brain barrier. When various blood
fractions were injected into the brain of rats, edema occurred after exposure to
lysed erythrocytes and hemoglobin but not immediately after the injection of
intact erythrocytes [212]. Hemoglobin, its breakdown products and/or reactions
induced by them are numerous and may be toxic to a variety of cell types
including neurons [32, 116]. Hemoglobin, hemin, bilirubin, or FeCl2 injections
into the basal ganglia of rats all increased brain water content 24 h after injec-
tion. Hemoglobin is metabolized by HO enzymes, the inducible form of which
is called HO-1. This enzyme was up-regulated after hemoglobin injection.
Interestingly, inhibition of HO with tin protoporphyrin-9 or scavenging of iron
by deferoxamine reduced hemoglobin-mediated edema.
These animal studies suggest that within the first hours of ICH, clot retrac-
tion and plasma proteins derived from the hematoma initiate the development
of cerebral edema. Initially, the blood brain barrier is intact but it breaks down
to some extent, possibly secondary to thrombin generated by clotting in the
hematoma and by cytotoxic effects of thrombin, resulting in vasogenic edema.
There may also be leakage of additional thrombin, clotting factors, and com-
plement proteins that produce further edema by mechanisms discussed above.
These processes are believed to be important in edema formation within
24–72 h of ICH. With time there is lysis of erythrocytes and edema formation
Macdonald 388
secondary to effects of the released erythrocyte contents and this contributes to
edema at longer times after ICH.
Ischemia
Numerous studies have measured regional CBF around ICH in animal
models and in humans [164]. Animal experiments have produced conflicting
results as to whether ICH reduces CBF and for how long. This ambiguity is
because the rate of development of the ICH, as well as its location, final vol-
ume, and possibly the animal species studied affect changes in CBF that may
be observed. Many early studies modeled ICH by inflating a balloon in the
brain, which may produce more marked changes in the CBF and less edema
than blood. Recent studies of ICH in dogs and pigs found no reduction in CBF
and no evidence of ischemia around the ICH [164, 204]. Qureshi et al. [162]
have reviewed studies of CBF after ICH in humans, with similar findings as
in recent animal studies. Within 48 h of ICH there may be some reduction in
CBF around the ICH, secondary to the reduced cerebral metabolic demand.
Positron emission tomography studies suggest that regional oxygen extrac-
tion is normal or reduced. CBF subsequently may normalize, increase above
normal, or remain depressed before returning to normal after 14 days.
Overall, it appears that the pathogenesis of edema and brain injury around
ICH is multifactorial but is related to factors other than mass effect from the
hematoma and that ischemia and reperfusion play a relatively minor role in
most cases [211].
Apoptosis and Inflammation

Additional processes documented to occur around experimental ICH
include apoptosis and inflammation. Neurons and astrocytes around ICH in rats
may demonstrate DNA fragmentation and changes characteristic of apoptosis
[63, 110]. Histological studies demonstrate areas of brain necrosis in these
models, but these studies suggest that brain damage occurs by active processes
such as apoptosis as well. Inflammation is observed around experimental ICH
and its contribution to the formation of edema is suggested by studies demon-
strating that the depletion of leukocytes and platelets by whole-body irradiation
reduces edema around ICH in rats [77]. Early attempts to inhibit inflammation
may demonstrate some benefit, but inflammation probably evolved to maintain
the life of the organism in the face of injury and may have beneficial effects as
well. Whether selection pressures or whatever mechanisms of evolution
selected for favorable responses specifically to ICH is open to question, but it
is reasonable to assume that there are favorable aspects to inflammation and
inhibition is of unproven clinical benefit. The detailed nature of the inflamma-
tory response to ICH is unknown but as more selective pharmacological and
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biological methods to manipulate inflammation become available, this would
seem to be an area worthy of further investigation.
Microvascular Vasospasm
Most of the delayed deterioration that can complicate SAH has been attrib-
uted to the delayed cerebral vasospasm in large arteries, discussed below.
Whether or not the arteries and arterioles are affected after they enter the brain
substance has been studied in several experimental models and using positron
emission tomography in humans. Experimental models have generally not
found evidence that SAH constricts the penetrating arteries [128] or that they
are abnormal pharmacologically [80]. Ohkuma et al. [136] reported that corro-
sion casts of cerebral arteries after SAH in dogs did demonstrate microvascular
spasm [139]. Yet the potential for fixation artifacts and postmortem changes
renders these studies virtually impossible to interpret in the opinion of this
author. Clinical studies have usually been consistent with dilation of small
arteries during vasospasm [47, 64] although recent data have been interpreted
as suggesting that there is small artery spasm after SAH [137, 219]. These stud-
ies did not measure the arterial diameter however, so the hypothesis of
microvessel spasm remains unproven.
Cerebral Vasospasm
Most work on SAH has focused on cerebral vasospasm. Vasospasm is
associated with changes in the endothelial, smooth muscle, and adventitial
fibroblasts of the affected major subarachnoid cerebral arteries. These changes
can be observed grossly, histologically, ultrastructurally, or as changes in
expression of mRNA and protein or function of the artery, measured at the sin-
gle cell, excised artery or in vivo level.
Global Changes in Gene Expression in Vasospasm

Differential display, cDNA arrays and serial analysis of gene expression
can be used to ascertain differences in mRNA expression levels between two or
more sets of samples. A large number of differences may be produced that can
be used to generate hypotheses about disease pathogenesis. Several investiga-
tors have reported the analysis of vasospastic arteries using high-throughput
molecular techniques. For example, fluorescent differential display was used to
screen for changes in gene expression after SAH in rats [192]. Onda et al. [141]
reported that eleven known genes were up-regulated after SAH in dogs, which
included growth factor genes (VEGF, BiP protein; glucose-regulated protein
78), growth-arrest and DNA damage-inducible protein (gadd45), neuromod-
ulin, protein disulfide isomerase-related protein P5, acid sphingomyelinase-like
phosphodiesterase) and inflammation genes (monocyte chemotactic protein 1,
Macdonald 390
cystatin B, inter-␣-trypsin inhibitor family heavy chain-related protein, serum
amyloid A protein, glycoprotein 130). They found that Frizzled-6, the gene for
a human WNT receptor (involved in development and carcinogenesis), was
down-regulated. The middle cerebral arteries of monkeys with or without
vasospasm after SAH were analyzed using a cDNA array containing oligonuc-
leotides for 5184 genes [102]. Five hundred and thirty seven of the genes (10%)
were expressed in the arteries. One hundred and sixty four did not change sig-
nificantly (31%) and 373 genes (69%) were differentially expressed at 3, 7 or
14 days after SAH. Many showed increasing expression with time. Functions of
differentially-expressed genes included regulation of gene expression, cell pro-
liferation, inflammation, membrane proteins and receptors, and kinases and
phosphatases.
Smooth Muscle Contraction

Smooth muscle contracts in response to electrical, chemical, and mechan-
ical stimulation. These stimuli cause an increase in intracellular free Ca2⫹. The
source of Ca2⫹ may be influx from the extracellular space, release from intra-
cellular stores, or a combination. Ca2⫹ influx may occur through voltage-gated
or possibly receptor-operated Ca2⫹ channels. Release from intracellular stores
is via the inositol triphosphate pathway or Ca2⫹-induced Ca2⫹ release. The
increased Ca2⫹ binds calmodulin and activates myosin light chain kinase, which
phosphorylates myosin light chain. This increases actin-activated myosin
ATPase activity, cross-bridge cycling, and contracts the muscle. There is a vari-
able relationship between intracellular Ca2⫹ concentration, contraction velocity,
force maintenance and the level of phosphorylation of myosin light chain.
Contraction results in all cases from an interaction between actin and myosin.
After the initial phase, the contraction or force development may persist with
lower rates of cross-bridge cycling. This usually is associated with the return of
intracellular Ca2⫹ to basal or near basal levels, reduced myosin light chain
phosphorylation and decreased cross-bridge cycling. The persistence of con-
traction in the absence of the processes that mediate it initially involves various
secondary processes that may include a latch bridge state, thin-filament (actin)
regulatory processes and/or changes in Ca2⫹ sensitivity of the contractile appa-
ratus. Second messenger systems that involve protein kinase C, tyrosine
kinases, and mitogen-activated protein (MAP) kinases also regulate smooth
muscle contraction and have been the subject of some study in vasospasm.
Kinases are enzymes that phosphorylate proteins. The regulation of smooth
muscle contraction is complex and is reviewed elsewhere [7, 99].
Several investigators have measured the levels of contractile proteins in
vasospastic arteries [56, 119]. Most have reported reductions in the actin and
myosin proteins during vasospasm and this was postulated to be due to proteolytic
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degradation secondary to calpains that are activated by increased intracellular
Ca2⫹ in vasospastic smooth muscle cells. A role for calpains in vasospasm was
supported by the evidence of calpain activation in cerebral arteries for days after
SAH in dogs and rabbits [98, 215] and the fact that topical application of calpain
inhibitors to the dog basilar artery reduced the degree of vasospasm [118]. The
results of assays of myosin light chain phosphorylation and of amounts of
caldesmon and calponin in vasospastic arteries, however, have been inconsistent
[16, 38, 56, 139, 190]. In addition, detailed studies in a nonhuman primate model
of vasospasm demonstrated that vasospasm could be maintained for up to
2 weeks by repeated stimulation with blood clot and relaxation of the vasospastic
arteries became progressively slower with increasing duration of exposure to
perivascular blood clot [189, 222]. These authors concluded that contractile
processes were relatively preserved after SAH, whereas there was a reduction in
the ability of arteries to relax after SAH. Reductions, if any, in contractile pro-
teins, did not impair the ability of the artery to remain contracted.
Numerous intracellular signal transduction mechanisms mediating smooth
muscle contraction have been investigated in vasospasm [99]. The complexity
of these pathways consisting of numerous enzyme isoforms, interactions
between parallel cascades as well as differences between species and in the bio-
chemistry of the cerebral arteries at different times after SAH, and use of non-
specific pharmacological agents to draw conclusions about relative roles of the
various pathways, all have complicated interpretation of these studies. The pro-
tein kinase C pathway is perhaps the most extensively investigated. This family
of kinases consists of at least eleven isoforms with differing requirements for
Ca2⫹, phospholipids, and diacylglycerol for activation [83]. Inhibitors of pro-
tein kinase C such as H-7 were reported to reverse established vasospasm in
dogs [108]. In addition, vasospastic dog basilar artery contains increased levels
of diacylglycerol, an endogenous activator of protein kinase C and demon-
strates increased protein kinase C activity [99, 109]. Peterson and colleagues
[216] reported, however, that diacylglycerol concentrations were not elevated
after SAH in dogs and that protein kinase C activity was not significantly ele-
vated. This observation can be reconciled with their studies in vitro, which
showed that minor changes observed in protein kinase C activity and membrane
translocation could significantly alter smooth muscle contraction [81]. A major
limitation of those studies is the lack of specificity of the pharmacological drug
protein kinase inhibitors that were used. H-7, staurosporine and similar drugs
such as fasudil (AT877, HA1077) are kinase inhibitors with varying degrees of
specificity for multiple kinases involved in smooth muscle contraction. In the
absence of assays for each kinase in treated tissue and/or of the concentration
of drug achieved in the tissue, it is difficult to be certain as to the mechanism
of the effect of these drugs.
Macdonald 392
Nishizawa et al. [131, 132] have conducted a series of studies to test their
hypothesis that NO, which is tonically released from cerebral arteries, inhibits or
reduces the activation of protein kinase C. Therefore, a reduction in NO would
increase protein kinase C activity and contribute to vasospasm. A correlation was
found between vasospasm, reductions in arterial cyclic guanosine monophos-
phate which is increased by NO, and increased protein kinase C activity.
Vasospastic arteries also showed an increase in protein kinase C-dependent tone
when studied under isometric tension in vitro [130]. Protein kinase C may pro-
duce vasospasm by other mechanisms. Fujikawa et al. [38] noted that the pattern
of myosin light chain phosphorylation in vasospastic dog arteries was consistent
with phosphorylation by myosin light chain kinase but not protein kinase C.
Phosphorylation of calponin was detected in these arteries, which was suggested
to contribute to vasospasm indirectly through PKC.
Tyrosine kinases are another class of kinases that are involved in smooth
muscle contraction and that have been postulated to be involved in vasospasm.
They are classified as nonreceptor, membrane-bound (such as pp60c-src), mem-
brane-spanning receptor (typically growth factor receptors such as insulin,
platelet-derived growth factor and epidermal growth factor receptors), and
cytosolic tyrosine kinases (e.g., c-abl, c-fes). The receptor tyrosine kinases acti-
vated by growth factors in some cases act through signal transduction pathways
such as the ERK/MAP kinase pathway, discussed below. Several in vitro studies
where solutions of erythrocyte hemolysate were added to cerebral arteries sus-
pended under isometric tension were purported to support a role for tyrosine
kinase activation in the contractions induced by hemolysate, since tyrosine
kinase inhibitors reduced the contractions [85]. These studies are supportive but
provide no direct evidence for a role of tyrosine kinase activation in vasospasm.
More direct evidence was the observation that vasospasm after SAH in dogs was
associated with phosphorylation of known intracellular substrates of tyrosine
kinases such as Shc, Raf1, and MAP kinases [38]. Furthermore, topical applica-
tion of the tyrosine kinase inhibitor, genistein, reversed vasospasm.
Vascular smooth muscle contains several types of MAP kinases [1]. MAP
kinases were identified and named based on their activation in response to stim-
ulation of cells by mitogens. Growth factors (epidermal, platelet-derived,
fibroblast, nerve, insulin and insulin-like growth factors) and other agonists,
some of which contract smooth muscle, including vasopressin, angiotensin II,
platelet-activating factor, endothelins(ETs) and muscarinic agonists, all act on
various cell membrane receptors and result in the activation of intracellular
MAP kinases. There are at least three interacting MAP kinase pathways, each
of which consists of a kinase that activates the MAP kinase and a kinase that
activates MEK (MEK kinase). One postulated function of MAP kinases in
smooth muscle is to phosphorylate caldesmon, which would remove the
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inhibitory effect of caldesmon on contraction. Zubkov et al. [224] reported that
PD-98059, an inhibitor of the MAP kinase pathway, reduced contractions and
prevented MAP kinase immunoreactivity increase in rabbit basilar artery
exposed to erythrocyte hemolysate. In vivo investigations of the MAP kinase
pathway in vasospasm are limited to one study showing that the ERK pathway
was activated during vasospasm after SAH in dogs [38].
The most recent interest in kinase involvement in vasospasm has centered
on the role of Rho kinase. Rho proteins are low-molecular-weight G proteins
belonging to the Ras superfamily of 20 to 30 kDa GTP-binding proteins [199].
This class of G proteins is distinct from the heterotrimeric G proteins [177]. The
Rho G proteins are involved in cytoskeletal organization, membrane traffick-
ing, regulation of transcription, control of cell growth and development and
smooth muscle contraction. They function in a complex pathway that includes
numerous other regulatory proteins, but the main effect of interest is the acti-
vation of the Rho kinases such as p160ROCK (ROCKII). Rho kinase phospho-
rylates the myosin-binding subunit of myosin light chain phosphatase. This
reduces the phosphatase activity and maintains myosin light chain phosphory-
lation and smooth muscle contraction. Other actions may occur as well.
Interestingly, fasudil (HA1077 or AT877), which was reported to decrease
vasospasm and improve outcome in patients with aneurysmal SAH [182], is a
protein kinase inhibitor with order of potency p160ROCK ⬎ cAMP-dependent
protein kinase ⬎ protein kinase C ⬎ myosin light chain kinase. Biochemical
studies of vascular smooth muscle during vasospasm suggest that the activation
of Rho kinase and the resulting Ca2⫹ sensitization may contribute to vasospasm
[170]. Most of the actions of fasudil that inhibit kinase activity should reduce
vasospasm, making it a candidate drug for the treatment of vasospasm. The rel-
atively modest effects observed clinically would need to be reconciled with this,
perhaps being related to the inability to administer adequate doses without pro-
ducing hypotension or other undesirable cardiovascular effects.
Smooth Muscle Relaxation

Multiple mechanisms can produce smooth muscle relaxation including
removal of the contractile stimulus which reduces intracellular Ca2⫹. There may
be dephosphorylation of myosin light chain by myosin light chain phosphatase
mediated by cAMP-protein kinase A, reduction in intracellular Ca2⫹ by activa-
tion of K⫹ channels and relaxation mediated by the guanylate cyclase-cGMP-
protein kinase G system. Vasodilatory pathways may be selectively impaired
during vasospasm [186, 222].
There is little evidence for impairment in the cAMP-protein kinase A relax-
ation pathway after SAH [147, 153]. More evidence points to abnormalities in
K⫹ channels and the cGMP pathway in vasospasm. Vascular smooth muscle is
Macdonald 394
depolarized during vasospasm in dogs and nicroandil, a K⫹ channel opener that
also may activate soluble guanylate cyclase, reversed vasospasm in this model
[57]. Vasodilation of the rat basilar artery after SAH was impaired in response
to agents that produce relaxation by the cGMP pathway such as acetylcholine
and sodium nitroprusside [186]. Relaxation to papaverine, 8-bromoguanosine
3⬘,5⬘-cyclic monophosphate and brain natriuretic peptide (an activator of par-
ticulate guanylate cyclase) were not affected and relaxation to aprikalim and
calcitonin-gene related peptide, which activate ATP-sensitive K⫹ channels, were
augmented.
Endothelial Dysfunction and Vasospasm

Endothelial dysfunction is widely believed to be important in the genesis
of vasospasm. Questions remaining include the mechanism by which perivas-
cular blood clot injures the endothelium and whether or not such changes are a
cause or consequence of vasospasm. Cerebrovascular endothelium synthesizes
vasoconstrictors such as ETs and prostaglandin F2␣ and vasodilators such as
NO, prostacyclin, superoxide anion radical, and endothelium-derived hyperpo-
larizing factor [33]. Perivascular nerves and astrocytes also may synthesize
some of these compounds. Any reduction in vasodilators and increase in vaso-
constrictors could contribute to vasospasm. As mentioned above, prostacyclin
and the cAMP system do not seem to be substantially altered after SAH. On the
other hand, much interest has focused on NO which is synthesized by NOS.
Endothelial NOS is localized primarily to endothelial cells and neuronal NOS
to perivascular nerves [220]. There is a basal release of NO from these sources
that reduces the tone of cerebral arteries and the removal or impairment of
response could contribute to vasospasm.
Endothelium-dependent relaxation is impaired after SAH and different
pharmacological studies have demonstrated impairment at all of the possible
sites in the pathway including impaired synthesis and release of NO [68],
scavenging and destruction of NO before it reaches the smooth muscle
[76, 214], and impaired ability of smooth muscle to generate cGMP and relax
in response to NO [34, 86]. These studies are based mainly on the pharmaco-
logical study of excised arteries and on biochemical measurements of sub-
stances in the arteries. No comprehensive study has been carried out. The
level of soluble guanylate cyclase protein was reduced in vasospastic dog basi-
lar artery although there was no significant reduction in endothelial NOS [79].
In a monkey model of SAH, the reverse was found in which there was a sig-
nificant reduction in mRNA coding for endothelial NOS, whereas levels of
neuronal NOS and soluble guanylate cyclase were not reduced [66]. In the
same model, another group reported that the immunoreactivity for neuronal
NOS was reduced [158].
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iNOS usually is not detectable in unstimulated cells, but it may be induced
by inflammatory mediators such as lipopolysaccharide, cytokines (interferon-
gamma, IL-1␤, TNF-␣) and cerebral ischemia and decreased by IL-4, IL-10,
TGF-␤, basic FGF, aldosterone, HSP70, insulin-like growth factor, dexametha-
sone and NO. Increased iNOS expression has been documented to occur in
arteries in response to injuries such as endothelial removal and balloon angio-
plasty. iNOS produces larger amounts of NO. These may be protective under
these circumstances by reducing platelet and leukocyte adherence, decreasing
smooth muscle proliferation and acting as a compensatory mechanism in
response to endothelial dysfunction or loss [84]. On the other hand, the pro-
duction of NO by iNOS in inflammatory cells probably has physiologically
important antimicrobial and antitumor effect. NO may combine under specific
conditions with superoxide anion radical to produce peroxynitrite (ONOO⫺)
which can damage proteins and other biological molecules. A series of studies
by Sayama et al. [171, 172] reported that SAH in rats is associated with an
increase in iNOS mRNA in basal portions of the brain and that immunoreac-
tivity to iNOS was present in inflammatory cells in the subarachnoid space.
Vasospasm was reduced by the administration of the relatively selective iNOS
inhibitor, aminoguanidine.
Another pathway of smooth muscle relaxation involves membrane hyper-
polarization. There are many mechanisms involved, but one is mediated by
endothelial cells that may produce a diffusable substance, endothelium-
derived hyperpolarizing factor that acts on smooth muscle cells to hyperpolar-
ize them and thereby cause vascular relaxation [34]. Since the membrane
potential in smooth muscle cells is controlled in large part by K⫹ conductance,
this substance may activate K⫹ channels, leading to K⫹ efflux, membrane
hyperpolarization, closing of voltage-gated Ca2⫹ channels, reduction in intra-
cellular Ca2⫹ and thereby smooth muscle relaxation [58]. The precise nature of
the endothelium-derived hyperpolarizing factor is unclear, although in the
coronary arteries they may be cytochrome P450 metabolites produced from
metabolism of arachidonic acid such as epoxides (e.g., epoxy-eicosatrienoic
acid) formed by the epoxygenase pathway. There has been little specific inves-
tigation of endothelium-derived hyperpolarization in vasospasm, but it has
been suggested that of the naturally occurring vascular relaxation mechanisms,
those mediated by K⫹ channels may be the least affected by SAH and may thus
be a useful therapeutic target [147, 187]. Of the contractile substances released
by endothelial cells, the ET system is most widely studied. There are three
21-amino acid long ET peptides, ET-1,2,3 [34]. They show sequence homol-
ogy to sarafotoxin S6c, the venom of the snake Atraclaspis engadensis. ETs
are synthesized as preproETs of approximately 200 amino acids and then
cleaved by endopeptidases to big ETs and then to ETs by the ET converting
Macdonald 396
enzymes [34]. ET acts on ETA receptors found predominantly on smooth mus-
cle, leading to contraction, on ETB receptors located on smooth muscle where
they may mediate contraction (ETB1) and on endothelial cells (ETB2) where
they mediate relaxation by release of NO and/or prostacyclin.
The ETs have been suggested to be important in the pathogenesis of
vasospasm based on studies reporting that ET concentrations are elevated in
cerebrospinal fluid after SAH in humans and experimental animals [75, 106,
179, 193] and in arteries of animals with SAH [183], and that antagonists of ET
receptors prevent or reduce vasospasm in animals [17, 129, 138, 205, 225].
Contradictory reports have appeared on ET antagonist effects [28, 40, 51, 89,
223] and most are consistent with the lack of demonstrated effect of TAK-044,
an ET receptor antagonist, on vasospasm in humans [181]. In data from exper-
imental models, expression of ETA receptor mRNA was increased after SAH in
dogs [74]. Vasospasm was prevented in this model by intrathecal administration
of the ETA receptor antagonist, BQ-123. In a monkey model of SAH, mRNA
levels of preproET-1, preproET-3, and ETA receptors were unchanged in
vasospastic arteries 7 days after SAH, whereas there was a significant increase
in the ETB [65]. These findings are consistent with other studies in rabbits and
dogs showing that SAH is associated with a shift from ETA to ETB receptor
expression in the vasospastic arteries [167].
The ET system has been used as a target for the antisense approach to reduc-
ing expression of genes. Antisense oligonucleotides are short DNA sequences
that are taken up by cells and contain a sequence that is complementary to the
gene of interest. There are several possible mechanisms of action which results
in blocking transcription and/or translation of the gene. Antisense
oligoncleotides directed at preproET-1 were incorporated into all layers of the
basilar artery after intracisternal injection into rats, and were shown to reduce
contractions of the basilar artery exposed in situ to whole blood hemolysate for
up to 72 h after treatment [145]. In dogs, intracisternal antisense oligoDNAs to
preproET-1 did not prevent vasospasm although they did when administered in
combination with tissue plasminogen activator [137]. The role of ETs in
vasospasm remains incompletely investigated. Interpretation of the results of
studies are complicated by the observation that cerebral ischemia may be associ-
ated with an increase in ET-1 in brain, arteries, and cerebrospinal fluid [157], that
contractions of arteries to various substances can be potentiated by subthreshold
concentrations of ET-1, and that receptor sensitivity may also be altered.
Gene Expression in Vasospasm

One of the central processes in vasospasm is the prolonged hemolysis of ery-
throcytes in the subarachnoid space after SAH, leading to the release of hemo-
globin and its breakdown products. Physiological metabolism of hemoglobin is
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mediated by the initial release of heme from the globin chain that is facilitated by
the oxidation of ferrous (Fe2⫹) iron in oxy- or deoxyhemoglobin to ferric (Fe3⫹)
iron in methemoglobin. The heme groups are enyzmatically broken down by HO
into biliverdin, free iron and carbon monoxide. HO-2 is a constitutively expressed
form that is found in many tissues, whereas HO-1 is an inducible enzyme that is
increased in response to heavy metals, oxidative stress, ultraviolet light, heme,
and numerous other stimuli. There is increased hemoglobin and oxidative stress
in the subarachnoid space after SAH and it is therefore perhaps not surprising that
HO-1 mRNA was found to be increased in the basilar artery of rats with SAH
[190]. More interesting was the finding that preventing the increase in HO-1 with
antisense oligodeoxynucleotides aggravated vasospasm. Ono et al. [144] reported
that HO-1 protein was increased in the cerebral arteries after SAH in monkeys. In
addition, the iron released by the HO metabolism of heme would be expected to
increase ferritin protein, as ferritin is an iron-sequestering protein. Indeed, an
increase in ferritin protein was found in this model. Effects of modulating these
responses were not reported.
Inf lammation
Inflammation is the response of a living tissue to injury. From an evolu-
tionary perspective, one would expect it to mediate reactions that would favor
survival of the organism. To some extent, however, detrimental effects have
been emphasized in the medical literature. Evidence suggests that inflammation
may have beneficial and detrimental effects. For example, administration of
proinflammatory cytokines early after spinal cord injury in mice exacerbated
injury, whereas administration later appeared to reduce injury [87]. Moreover,
activated macrophages and microglia have been shown to promote axonal
regrowth after spinal cord injury in rats [161].
The hypothesis that vasospasm is an inflammatory disease or that inflam-
mation in general worsens vasospasm is simplistic in that it considers inflamma-
tion to be a single process. Few studies, however, have attempted to assess roles
of specific aspects of the inflammatory reaction in vasospasm. A classic study by
Weir et al. [207] involved repeated blood injections given one week apart into
monkeys and suggests that a delayed-type hypersensitivity (Type IV) reaction
does not contribute to vasospasm since this did not produce worsening
vasospasm. Kubota et al. [90], however, studied the kinetics of lymphocyte sub-
sets in the cerebrospinal fluid of rats after SAH and found a pattern that resem-
bled such a reaction. It remains unknown as to whether this immune reaction is
an epiphenomenon since the former study did not perform immunological stud-
ies, and the latter did not modify the reaction to assess the effect on vasospasm.
Support for inflammatory mechanisms in vasospasm is provided by stud-
ies demonstrating immunoglobulin and complement components in the walls of
Macdonald 398
vasospastic arteries of animals [53] and man [69] and of elevated levels of cir-
culating immune complexes and activated complement in humans with SAH
[82, 149, 151, 196]. Outcome of patients after SAH is worse if there are ele-
vated levels of the proinflammatory cytokines IL-1 receptor antagonist and
TNF-␣ in the cerebrospinal fluid [107]. Anti-inflammatory drugs such as cor-
ticosteroids improved outcome after SAH in Phase 2 [25, 168] and Phase 3 tri-
als in humans [59] and reduced vasospasm in animal models [24, 26, 213].
Experimental evidence suggesting that cyclosporine A reduces vasospasm after
SAH in dogs [155] and monkeys [52] is not supported by subsequent studies in
humans [103], nor did a similar drug, FK506, prevent experimental vasospasm
[124, 129] in animal models.
One aspect of inflammation that seems to be important is the role of com-
plement in accelerating erythrocyte hemolysis in the subarachnoid space after
SAH [154]. Inhibition of complement-mediated reactions in rabbits reduces
vasospasm [43]. Another role of complement may be to form membrane attack
complexes [150]. Experiments using freshly isolated rat cerebral artery vascular
smooth muscle cells in vitro showed that these complexes caused large increases
in membrane conductance. These could be formed in the subarachnoid space
after SAH and could lead to smooth muscle cell damage and perhaps contraction.
One of the initial reactions in inflammation is the up-regulation of adhe-
sion molecules on the surface of endothelial cells. Several groups have
reported an increased immunoreactivity to intercellular adhesion molecule-1
or during vasospasm after SAH in rats and after perivascular blood placement
around the femoral artery in rats [54, 185]. Humans with aneurysmal SAH
also have elevated levels of immunoreactivity to intercellular adhesion mole-
cule-1 as well as other adhesion molecules (vascular cell adhesion molecule-1,
L-selectin) in cerebrospinal fluid [160]. Blockade of intercellular adhesion
molecule-1 with monoclonal antibodies reduced perivascular inflammation
and vasospasm in several vasospasm models [8, 148]. Ono et al. [143] admin-
istered antisense oligodeoxynucleotides to NF-␬B intrathecally to rabbits with
SAH. Vasospasm was reduced and importantly, unlike many other studies, evi-
dence that the oligonucleotides had the desired effect was provided by show-
ing reduced activity of NF-␬B as measured by gel-shift assay.
Remodeling, Fibrosis, Proliferation, and Phenotype Changes

Vasospastic arteries have reduced contractility and compliance compared
to normal arteries, and they develop histopathological changes within all layers
of the arterial wall [99, 101, 203]. The mechanism of these changes and
whether they cause or contribute to vasospasm or are only an epiphenomenon
is unknown. The characteristic response of an artery to injury is intimal prolif-
eration [176]. Interestingly, this can occur after injury to the tunica intima, but
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also to perivascular processes such as the placement of a cuff around an artery
[121]. There is an intimal proliferation in arteries that develop vasospasm after
SAH but this usually occurs after angiographic vasospasm and is rare enough a
cause of symptoms so as to be a subject of case reports [88, 159]. That it does
develop, however, proves that there has been some type of vascular injury.
Decreased arterial compliance in vasospastic arteries represents an
increased stiffness of the arterial wall and could be due to an increase in or an
alteration in the extracellular matrix or in the smooth muscle cells themselves
[78, 123, 125]. Increased elastin and collagen have been detected in vasospas-
tic arteries [123, 125]. Procollagen I and III mRNA were increased 7 and 14
days after clot placement around the rat femoral arteries, whereas TGF-␤
mRNA was increased at 3 days [78]. It was suggested that the growth factor
increase mediated the subsequent increase in collagen expression. Some inves-
tigators were unable to document an increase in collagen in arteries during
vasospasm [100]. A causative role of collagen synthesis in vasospasm was
demonstrated by Onoda et al. [146] who had reported the ability to reduce
femoral artery vasospasm in rats by the administration of antisense
oligodeoxynucleotides to procollagen type I [146].
Molecular Therapy for Vasospasm

Some gene therapy approaches for vasospasm have been mentioned above
where appropriate and the subject has been reviewed by other authors in this
volume [62]. As suggested by the plethora of pathways involved, many
approaches might be applicable to treating vasospasm. Other phenomena such
as cerebral ischemia also could be targeted and this is a situation where the time
taken for gene expression to be increased or decreased could be less of an issue
since vasospasm onset is delayed for days after SAH. Studies using marker
genes with adenovirus as a vector demonstrate that widespread transfection of
cells in the leptomeninges and the adventitia of large vessels can be achieved
after intrathecal injection into the normal subarachnoid space [23]. SAH did not
seem to affect gene transfer in vivo [122] in dogs, but SAH in humans might
obstruct cerebrospinal fluid circulation more substantially than in these animal
models. Injection of adenovirus expressing NOS into the cisterna magna of
dogs with SAH did not prevent vasospasm [22, 188]. Whether expression of
other genes might work awaits further study.
Biological Therapies for ICH
At present, few biological therapies have been tried for experimental ICH
and most therapy is strictly supportive. Some current treatments are of
Macdonald 400
unproven efficacy; the author considers it as important to consider that dexam-
ethasone may be neurotoxic and phenytoin also interacts by inducing liver
enzymes and may reduce the neuroprotective effects of folic acid [180, 191,
198]. Targets for such therapy might be reducing brain edema by inhibiting
some of the pathways discussed above. Identification of apoptosis in cells
around experimental ICH raises the possibility of neuroprotective strategies
aimed at preventing apoptosis. Because hypertension is an important risk fac-
tor for ICH and SAH, treatment of primary hypertension is important, and gene
therapy for experimental hypertension has met with some success [156]. These
treatments have not been tried clinically, but clearly there would be great advan-
tages to a gene therapy for hypertension that would eliminate the need for daily
administration of medications, often at substantial cost, and if properly modu-
lated, with fewer side effects. Some approaches taken have been either to
increase expression of genes mediating vasodilation or to decrease genes for
vasoconstriction. Overexpression of genes for proteins such as kallikrein,
adrenomedullin, atrial natriuretic peptide, and NOS have successfully reduced
blood pressure in rodent models using viral and nonviral methods. Reducing
vasoconstrictor gene expression utilizes an antisense approach and has led to
successful blood pressure reductions in rodent models with the inhibition of
angiotensinogen, angiotensin receptors, and adrenergic receptor subtypes.
Challenges remain, including maintaining duration of expression and achieving
physiological levels of blood pressure that are regulated in a normal manner.
Other approaches may target underlying causes of SAH such as aneurysms,
using molecular techniques; for example, fibrosis within experimental
aneurysms can be increased by the placement of gelatin-containing basic
fibroblast growth factor in the aneurysm [67], leading to the obliteration of the
aneurysm sac. This or other gene therapies to increase proliferation of fibrob-
lasts and/or arterial wall cells within the aneurysm sac could be used as
adjuncts to endovascular treatment of aneurysms. Similar strategies can be
envisioned for the treatment of AVM.
References
1 Adam LP: Mitogen-activated protein kinase; in Barany M (ed): Biochemistry of Smooth Muscle
Contraction. San Diego, Academic Press, 1996, pp 167–176.
2 Alberts MJ: Intracerebral hemorrhage and vascular malformations; in Alberts MJ (ed): Genetics
of Cerebrovascular Disease. Armonk, NY, Futura, 1999, pp 209–236.
3 Alberts MJ: Subarachnoid hemorrhage and intracranial aneurysms; in Alberts MJ (ed): Genetics
of Cerebrovascular Disease. Armonk, NY, Futura, 1999, pp 237–259.
4 Alberts MJ, Davis JP, Graffagnino C, McClenny C, Delong D, Granger C, Herbstreith MH,
Boteva K, Marchuk DA, Roses AD: Endoglin gene polymorphism as a risk factor for sporadic
intracerebral hemorrhage. Ann Neurol 1997;41:683–686.
medwedi.ru
5 Bak S, Gaist D, Sindrup SH: Genetic liability in stroke: A long-term follow-up study of Danish
twins. Stroke 2002;33:769–774.
6 Bamford J, Sandercock P, Dennis M, Burn J, Warlow C: A prospective study of acute cerebrovas-
cular disease in the community: The Oxfordshire Community Stroke Project – 1981–86.
II. Incidence, case fatality rates and overall outcome at one year of cerebral infarction, primary
intracerebral and subarachnoid haemorrhage. J Neurol Neurosurg Psychiatry 1990;53:16–22.
7 Bárány M: Biochemistry of Smooth Muscle Contraction. San Diego, Academic Press, 1996.
8 Bavbek M, Polin R, Kwan AL, Arthur AS, Kassell NF, Lee KS: Monoclonal antibodies against
ICAM-1 and CD18 attenuate cerebral vasospasm after experimental subarachnoid hemorrhage in
rabbits. Stroke 1998;29:1930–1935.
9 Bederson JB, Germano IM, Guarino L: Cortical blood flow and cerebral perfusion pressure
in a new noncraniotomy model of subarachnoid hemorrhage in the rat. Stroke 1995;26:
1086–1091.
10 Bederson JB, Levy AL, Ding WH, Kahn R, DiPerna CA, Jenkins AL III, Vallabhajosyula P: Acute
vasoconstriction after subarachnoid hemorrhage. Neurosurgery 1998;42:352–360.
11 Bobby JJ, Emami JM, Farmer RD, Newman CG: Operative survival and 40 year follow up of sur-
gical repair of aortic coarctation. Br Heart J 1991;65:271–276.
12 Bofinger A, Hawley C, Fisher P, Daunt N, Stowasser M, Gordon R: Polymorphisms of the renin-
angiotensin system in patients with multifocal renal arterial fibromuscular dysplasia. J Hum
Hypertens 2001;15:185–190.
13 Bornebroek M, Haan J, Maat-Schieman ML, van Duinen SG, Roos RA: Hereditary cerebral hem-
orrhage with amyloidosis-Dutch type (HCHWA-D). I. A review of clinical, radiologic and genetic
aspects. Brain Pathol 1996;6:111–114.
14 Broderick JP, Brott T, Tomsick T, Huster G, Miller R: The risk of subarachnoid and intracerebral
hemorrhages in blacks as compared with whites. N Engl J Med 1992;326:733–736.
15 Brott T, Broderick J, Kothari R, Barsan W, Tomsick T, Sauerbeck L, Spilker J, Duldner J, Khoury J:
Early hemorrhage growth in patients with intracerebral hemorrhage. Stroke 1997;28:1–5.
16 Butler WE, Barker FG, Crowell RM: Patients with polycystic kidney disease would benefit from
routine magnetic resonance angiographic screening for intracerebral aneurysms: A decision
analysis. Neurosurgery 1996;38:506–515.
17 Caner HH, Kwan AL, Arthur A, Jeng AY, Lappe RW, Kassell NF, Lee KS: Systemic administra-
tion of an inhibitor of endothelin-converting enzyme for attenuation of cerebral vasospasm fol-
lowing experimental subarachnoid hemorrhage. J Neurosurg 1996;85:917–922.
18 Catto AJ, Kohler HP, Bannan S, Stickland M, Carter A, Grant PJ: Factor XIII val 34 leu:
A novel association with primary intracerebral hemorrhage. Stroke 1998;29:813–816.
19 Challa VR, Moody DM, Bell MA: The Charcot-Bouchard aneurysm controversy: Impact of a new
histologic technique. J Neuropathol Exp Neurol 1992;51:264–271.
20 Chapman AB, Rubinstein D, Hughes R, Stears JC, Earnest MP, Johnson AM, Gabow PA,
Kaehny WD: Intracranial aneurysms in autosomal dominant polycystic kidney disease. N Engl
J Med 1992;327:916–920.
21 Charcot JM, Bouchard C: Nouvelles recherches sur la pathogénie de l’hémorragie cérébrale. Arch
Physiol Norm Pathol 1868;1:643–645.
22 Chen AF, Jiang SW, Crotty TB, Tsutsui M, Smith LA, O’Brien T, Katusic ZS: Effects of in vivo
adventitial expression of recombinant endothelial nitric oxide synthase gene in cerebral arteries.
23 Christenson SD, Lake KD, Ooboshi H, Faraci FM, Davidson BL, Heistad DD: Adenovirus-
mediated gene transfer in vivo to cerebral blood vessels and perivascular tissue in mice. Stroke
1998;29:1411–1416.
24 Chyatte D: Prevention of chronic cerebral vasospasm in dogs with ibuprofen and high-dose
methylprednisolone. Stroke 1989;20:1021–1026.
25 Chyatte D, Fode NC, Nichols DA, Sundt TM Jr: Preliminary report: Effects of high dose methyl-
prednisolone on delayed cerebral ischemia in patients at high risk for vasospasm after aneurysmal
subarachnoid hemorrhage. Neurosurgery 1987;21:157–160.
26 Chyatte D, Rusch N, Sundt TM Jr: Prevention of chronic experimental cerebral vasospasm with
ibuprofen and high-dose methylprednisolone. J Neurosurg 1983;59:925–932.
Macdonald 402
27 Corder EH, Saunders AM, Strittmatter WJ, Schmechel DE, Gaskell PC, Small GW, Roses AD,
Haines JL, Pericak-Vance MA: Gene dose of apolipoprotein E type 4 allele and the risk of
Alzheimer’s disease in late onset families. Science 1993;261:921–923.
28 Cosentino F, McMahon EG, Carter JS, Katusic ZS: Effect of endothelinA-receptor antagonist
BQ-123 and phosphoramidon on cerebral vasospasm. J Cardiovasc Pharmacol Ther 1993;22
(suppl 8):S332-S335.
29 Craig HD, Gunel M, Cepeda O, Johnson EW, Ptacek L, Steinberg GK, Ogilvy CS, Berg MJ,
Crawford SC, Scott RM, Steichen-Gersdorf E, Sabroe R, Kennedy CTC, Mettler G, Beis MJ,
Fryer A, Awad IA, Lifton RP: Multilocus linkage identifies two new loci for a Mendelian form of
stroke, cerebral cavernous malformation, at 7p15–13 and 3q25.2–27. Hum Mol Genet
1998;7:1851–1858.
30 Denier C, Gasc J, Chapon F, Domenga V, Lescoat C, Joutel A, Tournier-Lasserve E: Krit1/cerebral
cavernous malformation 1 mRNA is preferentially expressed in neurons and epithelial cells in
embryo and adult. Mech Dev 2002;117:363.
31 Dubovsky J, Zabramski JM, Kurth J, Spetzler RF, Rich SS, Orr HT, Weber JL: A gene responsi-
ble for cavernous malformations of the brain maps to chromosome 7q. Hum Mol Genet 1995;4:
453–458.
32 Everse J, Hsia N: The toxicities of native and modified hemoglobins. Free Radic Biol Med
1997;22:1075–1099.
33 Faraci FM: Endothelium-derived vasoactive factors and regulation of the cerebral circulation.
Neurosurgery 1993;33:648–659.
34 Faraci FM, Heistad DD: Regulation of the cerebral circulation: Role of endothelium and potas-
sium channels. [Review]. Physiol Rev 1998;78:53–97.
35 Fayad PB: Familial lesions and multiorgan vascular malformations; in Jafar JJ, Awad IA,
Rosenwasser RH (eds): Vascular Malformations of the Central Nervous System. Philadelphia,
Lippincott Williams & Wilkins, 1999, pp 161–167.
36 Fein JM: Brain energetics and circulatory control after subarachnoid hemorrhage. J Neurosurg
1976;45:498–507.
37 Fujii Y, Tanaka R, Takeuchi S, Koike T, Minakawa T, Sasaki O: Hematoma enlargement in spon-
taneous intracerebral hemorrhage. J Neurosurg 1994;80:51–57.
38 Fujikawa H, Tani E, Yamaura I, Ozaki I, Miyaji K, Sato M, Takahashi K, Imajoh-Ohmi S:
Activation of protein kinases in canine basilar artery in vasospasm. J Cereb Blood Flow Metab
1999;19:44–52.
39 Fukuda S, Hashimoto N, Naritomi H, Nagata I, Nozaki K, Kondo S, Kurino M, Kikuchi H:
Prevention of rat cerebral aneurysm formation by inhibition of nitric oxide synthase. Circulation
2000;101:2532–2538.
40 Gaetani P, Rodriguez y Baena R, Grignani G, Spanu G, Pacchiarini L, Paoletti P: Endothelin and
aneurysmal subarachnoid haemorrhage: A study of subarachnoid cisternal cerebrospinal fluid.
J Neurol Neurosurg Psychiatry 1994;57:66–72.
41 Gaist D, Vaeth M, Tsiropoulos I, Christensen K, Corder E, Olsen J, Sorensen HT: Risk of sub-
arachnoid haemorrhage in first degree relatives of patients with subarachnoid haemorrhage:
Follow up study based on national registries in Denmark. BMJ 2000;320:141–145.
42 Gebel JM, Brott TG, Sila CA, Tomsick TA, Jauch E, Salisbury S, Khoury J, Miller R, Pancioli A,
Duldner JE, Topol EJ, Broderick JP: Decreased perihematomal edema in thrombolysis-related
intracerebral hemorrhage compared with spontaneous intracerebral hemorrhage. Stroke 2000;31:
596–600.
43 German JW, Gross CE, Giclas P, Watral W, Bednar MM: Systemic complement depletion inhibits
experimental cerebral vasospasm. Neurosurgery 1996;39:141–145.
44 Ghetti B, Piccardo P, Frangione B, Bugiani O, Giaccone G, Young K, Prelli F, Farlow MR,
Dlouhy SR, Tagliavini F: Prion protein amyloidosis. Brain Pathol 1996;6:127–145.
45 Graffagnino C, Herbstreith MH, Roses AD, Alberts MJ: A molecular genetic study of intracere-
bral hemorrhage. Arch Neurol 1994;51:981–984.
46 Greenberg SM, Rebeck GW, Vonsattel JP, Gomez-Isla T, Hyman BT: Apolipoprotein E
epsilon 4 and cerebral hemorrhage associated with amyloid angiopathy. Ann Neurol 1995;38:
254–259.
medwedi.ru
47 Grubb RLJ, Raichle ME, Eichling JO, Gado MH: Effects of subarachnoid hemorrhage on cere-
bral blood volume, blood flow, and oxygen utilization in humans. J Neurosurg 1977;46:
446–453.
48 Gunel M, Awad IA, Anson J, Lifton RP: Mapping a gene causing cerebral cavernous malforma-
tion to 7q11.2-q21. Proc Natl Acad Sci USA 1995;92:6620–6624.
49 Gunel M, Laurans MS, Shin D, DiLuna ML, Voorhees J, Choate K, Nelson-Williams C, Lifton RP:
KRIT1, a gene mutated in cerebral cavernous malformation, encodes a microtubule-associated pro-
tein. Proc Natl Acad Sci USA 2002;99:10677–10682.
50 Guttmacher AE, Marchuk DA, White RI Jr: Hereditary hemorrhagic telangiectasia. N Engl J Med
1995;333:918–924.
51 Hamann G, Isenberg E, Strittmatter M, Schimrigk K: Absence of elevation of big endothelin in
subarachnoid hemorrhage. Stroke 1993;24:383–386.
52 Handa Y, Hayashi M, Takeuchi H, Kobayashi H, Kawano H, Kabuto M: Effect of cyclosporine on
the development of cerebral vasospasm in a primate model. Neurosurgery 1991;28:380–385.
53 Handa Y, Kabuto M, Kobayashi H, Kawano H, Takeuchi H, Hayashi M: The correlation between
immunological reaction in the arterial wall and the time course of the development of cerebral
vasospasm in a primate model. Neurosurgery 1991;28:542–549.
54 Handa Y, Kubota T, Kaneko M, Tsuchida A, Kobayashi H, Kawano H: Expression of intercellular
adhesion molecule 1 (ICAM-1) on the cerebral artery following subarachnoid haemorrhage in
rats. Acta Neurochir 1995;132(suppl):92–97.
55 Harada S, Kamiya K, Masago A, Iwata A, Yamada K: Subarachnoid hemorrhage induces c-fos,
c-jun and hsp70 mRNA expression in rat brain. Neuroreport 1997;8:3399–3404.
56 Harada T, Seto M, Sasaki Y, London S, Luo Z, Mayberg M: The time course of myosin light-chain
phosphorylation in blood-induced vasospasm. Neurosurgery 1995;36:1178–1182.
57 Harder DR, Dernbach P, Waters A: Possible cellular mechanism for cerebral vasospasm after
experimental subarachnoid hemorrhage in the dog. J Clin Invest 1987;80:875–880.
58 Harder DR, Roman RJ, Gebremedhin D, Birks EK, Lange AR: A common pathway for regulation
of nutritive blood flow to the brain: Arterial muscle membrane potential and cytochrome P450
metabolites. Acta Physiol Scand 1998;164:527–532.
59 Hashi K, Takakura K, Sano K, Ohta T, Saito I, Okada K: Intravenous hydrocortisone in large
doses for the treatment of delayed ischemic neurological deficits following subarachnoid hem-
orrhage – Results of a multi-center controlled double-blind clinical study. No to Shinkei 1988;
40:373–382.
60 Hayman LA, Evans RA, Ferrell RE, Fahr LM, Ostrow P, Riccardi VM: Familial cavernous
angiomas: Natural history and genetic study over a 5-year period. Am J Med Genet 1982;
11:147–160.
61 Healton EB, Mohr JP: Cerebrovascular fibromuscular dysplasia; in Barnett HJM, Mohr JP,
Stein BM, Yatsu FM (eds): Stroke. Pathophysiology, Diagnosis, and Management. New York,
Churchill Livingstone, 1998, pp 833–844.
62 Heistad DD, Faraci FM: Gene therapy for cerebral vascular disease. [Review]. Stroke 1996;27:
1688–1693.
63 Hickenbottom SL, Grotta JC, Strong R, Denner LA, Aronowski J: Nuclear factor-kappa B and cell
death after experimental intracerebral hemorrhage in rats. Stroke 1999;30:2472–2477.
64 Hino A, Mizukawa N, Tenjin H, Imahori Y, Taketomo S, Yano I, Nakahashi H, Hirakawa K:
Postoperative hemodynamic and metabolic changes in patients with subarachnoid hemorrhage.
Stroke 1989;20:1504–1510.
65 Hino A, Tokuyama Y, Weir B, Takeda J, Yano H, Bell GI, Macdonald RL: Changes in endothelial
nitric oxide synthase mRNA during vasospasm after subarachnoid hemorrhage in monkeys.
66 Hino A, Tokuyama Y, Weir B, Takeda J, Yano H, Bell GI, Macdonald RL: Changes in endothelial
nitric oxide synthase mRNA during vasospasm after subarachnoid hemorrhage in monkeys.
67 Hong L, Miyamoto S, Yamada K, Hashimoto N, Tabata Y: Enhanced formation of fibrosis in a rab-
bit aneurysm by gelatin hydrogel incorporating basic fibroblast growth factor. Neurosurgery
2001;49:954–960.
Macdonald 404
68 Hongo K, Kassell NF, Nakagomi T, Sasaki T, Tsukahara T, Ogawa H, Vollmer DG, Lehman RM:
Subarachnoid hemorrhage inhibition of endothelium-derived relaxing factor in rabbit basilar
artery. J Neurosurg 1988;69:247–253.
69 Hoshi T, Shimizu T, Kito K, Yamsaki N, Takahashi K, Takahashi M, Okada T, Kasuya H,
Kitamura K: Immunological study of late cerebral vasospasm in subarachnoid hemorrhage.
Detection of immunoglobulins, C3, and fibrinogen in cerebral arterial walls by immunofluores-
cence method. Neurol Med Chir 1984;24:647–654.
70 Hsu FPK, Rigamonti D, Huhn SL: Epidemiology of cavernous malformations; in Awad IA,
Barrow DL (eds): Cavernous Malformations. Park Ridge, American Association of Neurological
Surgeons, 1993, pp 13–23.
71 Hua Y, Xi G, Keep RF, Hoff JT: Complement activation in the brain after experimental intracere-
bral hemorrhage. J Neurosurg 2000;92:1016–1022.
72 Hubschmann OR, Nathanson DC: The role of calcium and cellular membrane dysfunction in
experimental trauma and subarachnoid hemorrhage. J Neurosurg 1985;62:698–703.
73 Inagawa T, Ishikawa S, Aoki H, Takahashi M, Yoshimoto H: Aneurysmal subarachnoid hemor-
rhage in Izumo City and Shimane Prefecture of Japan. Incidence. Stroke 1988;19:170–175.
74 Itoh S, Sasaki T, Asai A, Kuchino Y: Prevention of delayed vasospasm by an endothelin ETA
receptor antagonist, BQ-123: Change of ETA receptor mRNA expression in a canine subarach-
noid hemorrhage model. J Neurosurg 1994;81:759–764.
75 Juvela S: Plasma endothelin concentrations after aneurysmal subarachnoid hemorrhage. J Neurosurg
2000;92:390–400.
76 Kanamaru K, Weir BK, Findlay JM, Krueger CA, Cook DA: Pharmacological studies on relax-
ation of spastic primate cerebral arteries in subarachnoid hemorrhage. J Neurosurg 1989;71:
909–915.
77 Kane PJ, Modha P, Strachan RD, Cook S, Chambers IR, Clayton CB, Mendelow AD: The effect
of immunosuppression on the development of cerebral oedema in an experimental model of
intracerebral haemorrhage: Whole body and regional irradiation. J Neurol Neurosurg Psychiatry
1992;55:781–786.
78 Kasuya H, Weir B, Shen Y, Hariton G, Vollrath B, Ghahary A: Procollagen types I and III and
transforming growth factor-beta gene expression in the arterial wall after exposure to periarterial
blood. Neurosurgery 1993;33:716–721.
79 Kasuya H, Weir BK, Nakane M, Pollock JS, Johns L, Marton LS, Stefansson K: Nitric oxide syn-
thase and guanylate cyclase levels in canine basilar artery after subarachnoid hemorrhage.
J Neurosurg 1995;82:250–255.
80 Katusic ZS, Milde JH, Cosentino F, Mitrovic BS: Subarachnoid hemorrhage and endothelial
L-arginine pathway in small brain stem arteries in dogs. Stroke 1993;24:392–399.
81 Kawamata T, Peterson JW, Bun T, Zervas NT: Augmentation of both hemolysate-induced con-
traction and activation of protein kinase C by submaximum activation in canine cerebral arteries
in vitro. J Neurosurg 1997;87:908–915.
82 Kawano T, Yonekawa Y: Serum complements as indicator for predicting vasospasm and its sever-
ity after aneurysmal subarachnoid hemorrhage. Nippon Geka Hokan 1990;59:189–197.
83 Khalil RA, Morgan KG: Enzyme translocations during smooth muscle activation; in Barany M (ed):
Biochemistry of Smooth Muscle Contraction. San Diego, Academic Press, 1996, pp 307–318.
84 Kibbe M, Billiar T, Tzeng E: Inducible nitric oxide synthase and vascular injury. [Review].
Cardiovasc Res 1999;43:650–657.
85 Kim CJ, Kim KW, Park JW, Lee JC, Zhang JH: Role of tyrosine kinase in erythrocyte lysate-
induced contraction in rabbit cerebral arteries. J Neurosurg 1998;89:289–296.
86 Kim P, Schini VB, Sundt TM Jr, Vanhoutte PM: Reduced production of cGMP underlies the loss
of endothelium-dependent relaxations in the canine basilar artery after subarachnoid hemorrhage.
Circ Res 1992;70:248–256.
87 Klusman I, Schwab ME: Effects of pro-inflammatory cytokines in experimental spinal cord
injury. Brain Res 1997;762:173–184.
88 Kondziolka D, Bernstein M, Spiegel SM, ter Brugge K: Symptomatic arterial luminal narrowing
presenting months after subarachnoid hemorrhage and aneurysm clipping. J Neurosurg 1988;69:
494–499.
medwedi.ru
89 Kraus GE, Bucholz RD, Yoon KW, Knuepfer MM, Smith KR Jr: Cerebrospinal fluid endothelin-1
and endothelin-3 levels in normal and neurosurgical patients: A clinical study and literature review.
[Review]. Surg Neurol 1991;35:20–29.
90 Kubota T, Handa Y, Tsuchida A, Kaneko M, Kobayashi H: The kinetics of lymphocyte subsets and
macrophages in subarachnoid space after subarachnoid hemorrhage in rats. Stroke 1993;24:
1993–2000.
91 Kufs H: Über heredofamiliäre angiomatose des gehirns und der retina, ihre beziehungen zueinan-
der und zur angiomatose der haut. Z Neurol Psychiatry 1928;113:651–686.
92 Kuivaniemi H, Prockop D, Wu Y: Exclusion of mutations in the gene for type III collagen
(COL3A1) as a common cause of intracranial aneurysms or cervical artery dissections. Neurology
1993;43:2652–2658.
93 Kuroki M, Kanamaru K, Suzuki H, Waga S, Semba R: Effect of vasospasm on heme oxygenases
in a rat model of subarachnoid hemorrhage. Stroke 1998;29:683–688.
94 Laberge-le Couteulx S, Jung HH, Labauge P, Houtteville JP, Lescoat C, Cecillon M, Marechal E,
Joutel A, Bach JF, Tournier-Lasserve E: Truncating mutations in CCM1, encoding KRIT1, cause
hereditary cavernous malformations. Nat Genet 1999;23:189–193.
95 Lee KR, Betz AL, Keep RF, Chenevert TL, Kim S, Hoff JT: Intracerebral infusion of thrombin as
a cause of brain edema. J Neurosurg 1995;83:1045–1050.
96 Lee KR, Colon GP, Betz AL, Keep RF, Kim S, Hoff JT: Edema from intracerebral hemorrhage:
The role of thrombin. J Neurosurg 1996;84:91–96.
97 Lee KR, Kawai N, Kim S, Sagher O, Hoff JT: Mechanisms of edema formation after intracerebral
hemorrhage: Effects of thrombin on cerebral blood flow, blood-brain barrier permeability, and cell
survival in a rat model. J Neurosurg 1997;86:272–278.
98 Lee KS, Foley PL, Vanderklish P, Lynch G, Goto Y, Kassell FF: The role of calcium-activated pro-
teolysis in vasospasm after subarachnoid hemorrhage; in Findlay JM (ed): Cerebral Vasospasm.
Amsterdam, Elsevier, 1993, pp 85–88.
99 Macdonald RL, Weir B: Cerebral Vasospasm. San Diego, Academic, 2001.
100 Macdonald RL, Weir BK, Young JD, Grace MG: Cytoskeletal and extracellular matrix proteins in
cerebral arteries following subarachnoid hemorrhage in monkeys. J Neurosurg 1992;76:81–90.
101 Macdonald RL, Zhang J, Sima B, Johns L: Papaverine-sensitive vasospasm and arterial contrac-
tility and compliance after subarachnoid hemorrhage in dogs. Neurosurgery 1995;37:962–967.
102 Macdonald RL, Zhang ZD, Ono S, Komuro T: Up-regulation of parathyroid hormone receptor in
cerebral arteries after subarachnoid hemorrhage in monkeys. Neurosurgery 2002;50:1083–1091.
103 Manno EM, Gress DR, Ogilvy CS, Stone CM, Zervas NT: The safety and efficacy of cyclosporine
A in the prevention of vasospasm in patients with Fisher grade 3 subarachnoid hemorrhages: A
pilot study. Neurosurgery 1997;40:289–293.
104 Marchuk DA, Gallione CJ, Morrison LA, Clericuzio CL, Hart BL, Kosofsky BE, Louis DN,
Gusella JF, Davis LE, Prenger VL: A locus for cerebral cavernous malformations maps to chromo-
some 7q in two families. Genomics 1995;28:311–314.
105 Marzatico F, Gaetani P, Rodriguez y Baena R: Experimental subarachnoid hemorrhage: Lipid
peroxidation and Na⫹, K⫹-ATPase in different rat brain areas. Mol Chem Neuropathol 1989;
11:99.
106 Masaoka H, Suzuki R, Hirata Y, Emori T, Marumo F, Hirakawa K: Raised plasma endothelin in
aneurysmal subarachnoid haemorrhage. Lancet 1989;9:1402.
107 Mathiesen T, Edner G, Ulfarsson E, Andersson B: Cerebrospinal fluid interleukin-1 receptor
antagonist and tumor necrosis factor-␣ following subarachnoid hemorrhage. J Neurosurg 1997;
87:215–220.
108 Matsui T, Kaizu H, Itoh S, Asano T: The role of active smooth muscle contraction in the occur-
rence of chronic vasospasm in the canine two-hemorrhage model. J Neurosurg 1994;
80:276–282.
109 Matsui T, Sugawa M, Johshita H, Takuwa Y, Asano T: Activation of the protein kinase C-mediated
contractile system in canine basilar artery undergoing chronic vasospasm. Stroke 1991;22:
1183–1187.
110 Matsushita K, Meng W, Wang X, Asahi M, Asahi K, Moskowitz MA, Lo EH: Evidence for apop-
tosis after intercerebral hemorrhage in rat striatum. J Cereb Blood Flow Metab 2000;20:396–404.
Macdonald 406
111 Matz P, Turner C, Weinstein PR, Massa SM, Panter SS, Sharp FR: Heme-oxygenase-1 induction
in glia throughout rat brain following experimental subarachnoid hemorrhage. Brain Res 1996;
713:211–222.
112 Matz P, Weinstein P, States B, Honkaniemi J, Sharp FR: Subarachnoid injections of lysed blood
induce the hsp70 stress gene and produce DNA fragmentation in focal areas of the rat brain.
Stroke 1996;27:504–513.
113 Matz PG, Copin JC, Chan PH: Cell death after exposure to subarachnoid hemolysate correlates
inversely with expression of CuZn-superoxide dismutase. Stroke 2000;31:2450–2459.
114 Matz PG, Fujimura M, Chan PH: Subarachnoid hemolysate produces DNA fragmentation in a
pattern similar to apoptosis in mouse brain. Brain Res 2000;858:312–319.
115 Matz PG, Sundaresan S, Sharp FR, Weinstein PR: Induction of HSP70 in rat brain following sub-
arachnoid hemorrhage produced by endovascular perforation. J Neurosurg 1996;85:138–145.
116 Matz PG, Weinstein PR, Sharp FR: Heme oxygenase-1 and heat shock protein 70 induction in glia
and neurons throughout rat brain after experimental intracerebral hemorrhage. Neurosurgery 1997;
40:152–160.
117 Mettinger KL, Ericson K: Fibromuscular dysplasia and the brain. I. Observations on angiographic,
clinical and genetic characteristics. Stroke 1982;13:46–52.
118 Minami N, Tani E, Maeda Y, Yamaura I, Fukami M: Effects of inhibitors of protein kinase C and
calpain in experimental delayed cerebral vasospasm. J Neurosurg 1992;76:111–118.
119 Minami N, Tani E, Maeda Y, Yamaura I, Fukami M: Immunoblotting of contractile and cytoskele-
tal proteins of canine basilar artery in vasospasm. Neurosurgery 1993;33:698–706.
120 Moriarity JL, Wetzel M, Clatterbuck RE, Javedan S, Sheppard JM, Hoenig-Rigamonti K, Crone NE,
Breiter SN, Lee RR, Rigamonti D: The natural history of cavernous malformations: A prospective
study of 68 patients. Neurosurgery 1999;44:1166–1173.
121 Moroi M, Zhang L, Yasuda T, Virmani R, Gold HK, Fishman MC, Huang PL: Interaction of
genetic deficiency of endothelial nitric oxide, gender, and pregnancy in vascular response to
injury in mice. J Clin Invest 1998;101:1225–1232.
122 Muhonen MG, Ooboshi H, Welsh MJ, Davidson BL, Heistad DD: Gene transfer to cerebral blood
vessels after subarachnoid hemorrhage. Stroke 1997;28:822–828.
123 Nagasawa S, Handa H, Naruo Y, Moritake K, Hayashi K: Experimental cerebral vasospasm arte-
rial wall mechanics and connective tissue composition. Stroke 1982;13:595–600.
124 Nagata K, Sasaki T, Iwama J, Mori T, Iwamoto S, Nirei H, Hamada K, Kirino T: Failure of FK-506,
a new immunosuppressant, to prevent cerebral vasospasm in a canine two-hemorrhage model.
J Neurosurg 1993;79:710–715.
125 Nakamura S, Tsubokawa T, Yoshida K, Hirasawa T, Nakano M: Appearance of collagen fibers
in the cerebral vascular wall following subarachnoid hemorrhage. Neurol Med Chir 1992;32:
877–882.
126 Nicoll JA, Burnett C, Love S, Graham DI, Ironside JW, Vinters HV: High frequency of apolipopro-
tein E epsilon 2 in patients with cerebral hemorrhage due to cerebral amyloid angiopathy. Ann
Neurol 1996;39:682–683.
127 Nihei H, Kassell NF, Dougherty DA, Sasaki T: Does vasospasm occur in small pial arteries and
arterioles of rabbits? Stroke 1991;22:1419–1425.
128 Nirei H, Hamada K, Shoubo M, Sogabe K, Notsu Y, Ono T: An endothelin ETA receptor antago-
nist, FR139317, ameliorates cerebral vasospasm in dogs. Life Sci 1993;52:1869–1874.
129 Nishizawa S, Peterson JW, Shimoyama I, Iwasaki K, Uemura K: Therapeutic effect of a new
immunosuppressant, FK-506, on vasospasm after subarachnoid hemorrhage. Neurosurgery 1993;
32:986–991.
130 Nishizawa S, Yamamoto S, Uemura K: Interrelation between protein kinase C and nitric oxide in
the development of vasospasm after subarachnoid hemorrhage. Neurol Res 1996;18:89–95.
131 Nishizawa S, Yamamoto S, Yokoyama T, Ryu H, Uemura K: Chronological changes of arterial
diameter, cGMP, and protein kinase C in the development of vasospasm. Stroke 1995;26:
1916–1920.
132 Nishizawa S, Yamamoto S, Yokoyama T, Uemura K: Dysfunction of nitric oxide induces protein
kinase C activation resulting in vasospasm after subarachnoid hemorrhage. Neurol Res 1997;
19:558–562.
medwedi.ru
133 Norrgard O, Angquist KA, Fodstad H, Forsell A, Lindberg M: Intracranial aneurysms and here-
dity. Neurosurgery 1987;20:236–239.
134 Nozaki K, Boccalini P, Moskowitz MA: Expression of c-fos-like immunoreactivity in brainstem
after meningeal irritation by blood in the subarachnoid space. Neuroscience 1992;49:669–680.
135 Ohkuma H, Itoh K, Shibata S, Suzuki S: Morphological changes of intraparenchymal arterioles
after experimental subarachnoid hemorrhage in dogs. Neurosurgery 1997;41:230–235.
136 Ohkuma H, Manabe H, Tanaka M, Suzuki S: Impact of cerebral microcirculatory changes on cere-
bral blood flow during cerebral vasospasm after aneurysmal subarachnoid hemorrhage. Stroke
2000;31:1621–1627.
137 Ohkuma H, Parney I, Megyesi J, Ghahary A, Findlay JM: Antisense preproendothelin-oligoDNA
therapy for vasospasm in a canine model of subarachnoid hemorrhage. J Neurosurg 1999;90:
1105–1114.
138 Ohkuma H, Suzuki S: Histological dissociation between intra- and extraparenchymal portion of
perforating small arteries after experimental subarachnoid hemorrhage in dogs. Acta Neuropathol
1999;98:374–382.
139 Oka Y, Ohta S, Todo H, Kohno K, Kumon Y, Sakaki S: Protein synthesis and immunoreactivities
of contraction-related proteins in smooth muscle cells of canine basilar artery after experimental
subarachnoid hemorrhage. J Cereb Blood Flow Metab 1996;16:1335–1344.
140 Olafsson I, Thorsteinsson L, Jensson O: The molecular pathology of hereditary cystatin C amy-
loid angiopathy causing brain hemorrhage. Brain Pathol 1996;6:121–126.
141 Onda H, Kasuya H, Takakura K, Hori T, Imaizumi T, Takeuchi T, Inoue I, Takeda J: Identification
of genes differentially expressed in canine vasospastic cerebral arteries after subarachnoid hem-
orrhage. J Cereb Blood Flow Metab 1999;19:1279–1288.
142 Onda H, Kasuya H, Yoneyama T, Takakura K, Hori T, Takeda J, Nakajima T, Inoue I: Genomewide-
linkage and haplotype-association studies map intracranial aneurysm to chromosome 7q11.
Am J Hum Genet 2001;69:804–819.
143 Ono S, Date I, Onoda K, Shiota T, Ohmoto T, Ninomiya Y, Asari S, Morishita R: Decoy adminis-
tration of NF-kappaB into the subarachnoid space for cerebral angiopathy. Hum Gene Ther
1998;9:1003–1011.
144 Ono S, Zhang ZD, Marton LS, Yamini B, Windmeyer E, Johns L, Kowalczuk A, Lin G,
Macdonald RL: Heme oxygenase-1 and ferritin are increased in cerebral arteries after subarach-
noid hemorrhage in monkeys. J Cereb Blood Flow Metab 2000;20:1066–1076.
145 Onoda K, Ono S, Ogihara K, Shiota T, Asari S, Ohmoto T, Ninomiya Y: Inhibition of vascular con-
traction by intracisternal administration of preproendothelin-1 mRNA antisense oligoDNA in a rat
experimental vasospasm model. J Neurosurg 1996;85:846–852.
146 Onoda K, Ono S, Ogihara K, Shiota T, Asari S, Ohmoto T, Ninomiya Y: Role of extracellular
matrix in experimental vasospasm. Inhibitory effect of antisense oligonucleotide on collagen
induction. Stroke 1996;27:2102–2108.
147 Onoue H, Katusic ZS: The effect of subarachnoid hemorrhage on mechanisms of vasodilation
mediated by cyclic adenosine monophosphate. J Neurosurg 1998;89:111–117.
148 Oshiro EM, Hoffman PA, Dietsch GN, Watts MC, Pardoll DM, Tamargo RJ: Inhibition of experi-
mental vasospasm with anti-intercellular adhesion molecule-1 monoclonal antibody in rats. Stroke
1997;28:2031–2037.
149 Ostergaard JR, Kristensen BO, Svehag SE, Teisner B, Miletic T: Immune complexes and comple-
ment activation following rupture of intracranial saccular aneurysms. J Neurosurg 1987;66:
891–897.
150 Park CC, Shin ML, Simard JM: The complement membrane attack complex and the bystander
effect in cerebral vasospasm. J Neurosurg 1997;87:294–300.
151 Pellettieri L, Nilsson B, Carlsson CA, Nilsson U: Serum immunocomplexes in patients with sub-
arachnoid hemorrhage. Neurosurgery 1986;19:767–771.
152 Peters DG, Kassam AB, Feingold E, Heidrich-O’Hare E, Yonas H, Ferrell RE, Brufsky A:
Molecular anatomy of an intracranial aneurysm: Coordinated expression of genes involved in
wound healing and tissue remodeling. Stroke 2001;32:1036–1042.
153 Peterson EW, Searle R, Mandy FF, Leblanc R: The reversal of experimental vasospasm by
dibutyryl-3⬘,5⬘-adenosine monophosphate. J Neurosurg 1973;39:730–734.
Macdonald 408
154 Peterson JW, Kwun BD, Teramura A, Hackett JD, Morgan JA, Nishizawa S, Bun T, Zervas NT:
Immunological reaction against the aging human subarachnoid erythrocyte. A model for the onset
of cerebral vasospasm after subarachnoid hemorrhage. J Neurosurg 1989;71:718–726.
155 Peterson JW, Nishizawa S, Hackett JD, Bun T, Teramura A, Zervas NT: Cyclosporine A reduces
cerebral vasospasm after subarachnoid hemorrhage in dogs. Stroke 1990;21:133–137.
156 Phillips MI: Gene therapy for hypertension: The preclinical data. Methods Enzymol 2002;346:3–13.
157 Pluta RM, Boock RJ, Afshar JK, Clouse K, Bacic M, Ehrenreich H, Oldfield EH: Source and
cause of endothelin-1 release into cerebrospinal fluid after subarachnoid hemorrhage. J Neurosurg
1997;87:287–293.
158 Pluta RM, Thompson BG, Dawson TM, Snyder SH, Boock RJ, Oldfield EH: Loss of nitric oxide
synthase immunoreactivity in cerebral vasospasm. J Neurosurg 1996;84:648–654.
159 Pluta RM, Zauner A, Morgan JK, Muraszko KM, Oldfield EH: Is vasospasm related to prolifera-
tive arteriopathy? J Neurosurg 1992;77:740–748.
160 Polin RS, Bavbek M, Shaffrey ME, Billups K, Bogaev CA, Kassell NF, Lee KS: Detection of
soluble E-selectin, ICAM-1, VCAM-1, L-selectin in the cerebrospinal fluid of patients after
subarachnoid hemorrhage. J Neurosurg 1998;89:559–567.
161 Prewitt CM, Niesman IR, Kane CJ, Houle JD: Activated macrophage/microglial cells can promote
the regeneration of sensory axons into the injured spinal cord. Exp Neurol 1997;148:433–443.
162 Qureshi AI, Hanel RA, Kirmani JF, Yahia AM, Hopkins LN: Cerebral blood flow changes associ-
ated with intracerebral hemorrhage. Neurosurg Clin N Am 2002;13:355–370.
163 Qureshi AI, Tuhrim S, Broderick JP, Batjer HH, Hondo H, Hanley DF: Medical progress:
Spontaneous intracerebral hemorrhage. N Engl J Med 2001;344:1450–1460.
164 Qureshi AI, Wilson DA, Hanley DF, Traystman RJ: No evidence for an ischemic penumbra in
massive experimental intracerebral hemorrhage. Neurology 1999;52:266–272.
165 Revesz T, Holton JL, Lashley T, Plant G, Rostagno A, Ghiso J, Frangione B: Sporadic and famil-
ial cerebral amyloid angiopathies. Brain Pathol 2002;12:343–357.
166 Rigamonti D, Hadley MN, Drayer BP, Johnson PC, Hoenig-Rigamonti K, Knight JT, Spetzler RF:
Cerebral cavernous malformations. Incidence and familial occurrence. N Engl J Med 1988;319:
343–347.
167 Roux S, Loffler BM, Gray GA, Sprecher U, Clozel M, Clozel JP: The role of endothelin in exper-
imental cerebral vasospasm. Neurosurgery 1995;37:78–85.
168 Ryba M, Pastuszko M, Iwanska K, Bidzinski J, Dziewiecki C: Cyclosporine A prevents neuro-
logical deterioration of patients with SAH – A preliminary report. Acta Neurochir 1991;112:
25–27.
169 Sahoo T, Johnson EW, Thomas JW, Kuehl PM, Jones TL, Dokken CG, Touchman JW, Gallione CJ,
Lee-Lin SQ, Kosofsky B, Kurth JH, Louis DN, Mettler G, Morrison L, Gil-Nagel A, Rich SS,
Zabramski JM, Boguski MS, Green ED, Marchuk DA: Mutations in the gene encoding KRIT1,
a Krev1/rap1a binding protein, cause cerebral cavernous malformations (CCM1). Hum Mol Genet
1999;8:2325–2333.
170 Sato M, Tani E, Fujikawa H, Yamaura I, Kaibuchi K: Importance of Rho-kinase-mediated phos-
phorylation of myosin light chain in cerebral vasospasm. J Stroke Cerebrovasc Dis 2000;
9(suppl 1):223–224.
171 Sayama T, Suzuki S, Fukui M: Expression of inducible nitric oxide synthase in rats following sub-
arachnoid hemorrhage. Neurol Res 1998;20:79–84.
172 Sayama T, Suzuki S, Fukui M: Role of inducible nitric oxide synthase in the cerebral vasospasm
after subarachnoid hemorrhage in rats. Neurol Res 1999;21:293–298.
173 Schievink W, Michels VV, Piepgras DG: Neurovascular manifestations of heritable connective tis-
sue disorders. A review. Stroke 1994;25:889–903.
174 Schievink WI: Genetics of intracranial aneurysms. Neurosurgery 1997;40:651–662.
175 Schievink WI, Katzmann JA, Piepgras DG, Schaid DJ: Alpha-1-antitrypsin phenotypes among
patients with intracranial aneurysms. J Neurosurg 1996;84:781–784.
176 Schwartz SM, deBlois D, O’Brien ERM: The intima. Soil for atherosclerosis and restenosis. Circ
Res 1995;77:445–465.
177 Seasholtz TM, Majumdar M, Brown JH: Rho as a mediator of G protein-coupled receptor signal-
ing. Mol Pharmacol 1999;55:949–956.
medwedi.ru
178 Sehba FA, Ding WH, Chereshnev I, Bederson JB: Effects of S-nitrosoglutathione on acute vaso-
constriction and glutamate release after subarachnoid hemorrhage. Stroke 1999;30:1955–1961.
179 Seifert V, Loffler BM, Zimmermann M, Roux S, Stolke D: Endothelin concentrations in patients
with aneurysmal subarachnoid hemorrhage. Correlation with cerebral vasospasm, delayed
ischemic neurological deficits, and volume of hematoma. J Neurosurg 1995;82:55–62.
180 Seligmann H, Potasman I, Weller B, Schwartz M, Prokocimer M: Phenytoin-folic acid interaction:
A lesson to be learned. Clin Neuropharmacol 1999;22:268–272.
181 Shaw MD, Vermeulen M, Murray GD, Pickard JD, Bell BA, Teasdale GM: Efficacy and safety of
the endothelin, receptor antagonist TAK-044 in treating subarachnoid hemorrhage: A report by the
Steering Committee on behalf of the UK/Netherlands/Eire TAK-044 Subarachnoid Haemorrhage
Study Group. J Neurosurg 2000;93:992–997.
182 Shibuya M, Suzuki Y, Sugita K, Saito I, Sasaki T, Takakura K, Nagata, Kikuchi H, Takemae T,
Hidaka H: Effect of AT877 on cerebral vasospasm after aneurysmal subarachnoid hemorrhage.
Results of a prospective placebo-controlled double-blind trial. J Neurosurg 1992;76:571–577.
183 Shigeno T, Mima T, Yanagisawa M, Saito A, Goto K, Yamashita K, Takenouchi T, Matsuura N,
Yamasaki Y, Yamada K: Prevention of cerebral vasospasm by actinomycin D. J Neurosurg 1991;
74:940–943.
184 Shull MM, Hassenbein D, Loggie J, Daniels S, King A, Burton T, Lingrel JB: Discordant segre-
gation of Na⫹,K(⫹)-adenosine triphosphatase alleles and essential hypertension. J Hypertens
1992;10:1005–1010.
185 Sills AK Jr, Clatterbuck RE, Thompson RC, Cohen PL, Tamargo RJ: Endothelial cell expression
of intercellular adhesion molecule 1 in experimental posthemorrhagic vasospasm. Neurosurgery
1997;41:453–460.
186 Sobey CG, Heistad DD, Faraci FM: Effect of subarachnoid hemorrhage on dilatation of rat basi-
lar artery in vivo. Am J Physiol 1996;271:H126–H132.
187 Sobey CG, Heistad DD, Faraci FM: Effect of subarachnoid hemorrhage on cerebral vasodilatation
in response to activation of ATP-sensitive K⫹ channels in chronically hypertensive rats. Stroke
1997;28:392–396.
188 Stoodley M, Macdonald RL, Weihl CC, Zhang ZD, Lin G, Johns L, Kowalczuk A, Ghadge G,
Roos RP: Effect of adenoviral-mediated nitric oxide synthase gene transfer on vasospasm after
experimental subarachnoid hemorrhage. Neurosurgery 2000;46:1193–2003.
189 Stoodley M, Macdonald RL, Weir BK, Marton LS, Johns LM, Zhang ZD, Kowalczuk A:
Subarachnoid hemorrhage causes an adaptive response in cerebral arteries. J Neurosurg 2000;
93:463–470.
190 Sun H, Kanamaru K, Ito M, Suzuki H, Kojima T, Waga S, Kureishi Y, Nakano T: Myosin light
chain phosphorylation and contractile proteins in a canine two-hemorrhage model of subarach-
noid hemorrhage. Stroke 1998;29:2149–2154.
191 Supko DE, Johnston MV: Dexamethasone potentiates NMDA receptor-mediated neuronal injury
in the postnatal rat. Eur J Pharmacol 1994;270:105–113.
192 Suzuki H, Kanamaru K, Tsunoda H, Inada H, Kuroki M, Sun H, Waga S, Tanaka T: Heme oxy-
genase-1 gene induction as an intrinsic regulation against delayed cerebral vasospasm in rats.
J Clin Invest 1999;104:59–66.
193 Suzuki H, Sato S, Suzuki Y, Takekoshi K, Ishihara N, Shimoda S: Increased endothelin concen-
tration in CSF from patients with subarachnoid hemorrhage. Acta Neurol Scand 1990;81:
553–554.
194 Suzuki R, Ohno K, Hiratsuka H, Inaba Y: Chronological changes in brain edema in hypertensive
intracerebral hemorrhage observed by CT and xenon-enhanced CT; in Inaba Y, Klatzo I, Spatz M
(eds): Brain Edema. Berlin, Springer, 1985, pp 613–620.
195 Svetkey LP, O’Riordan E, Conlon PJ, Emovon O: Genetics of hypertension; in Alberts MJ (ed):
Genetics of Cerebrovascular Disease. Armonk, NY, Futura, 1999.
196 Tsementzis SA, Chao SW, Hitchcock ER, Gill JS, Beevers DG: Oligoclonal immunoglobulin G
in acute subarachnoid hemorrhage and stroke. Neurology 1986;36:395–397.
197 Turner CP, Panter SS, Sharp FR: Anti-oxidants prevent focal rat brain injury as assessed by induc-
tion of heat shock proteins (HSP70, HO-1/HSP32, HSP47) following subarachnoid injections of
lysed blood. Brain Res Mol Brain Res 1999;65:87–102.
Macdonald 410
198 UnoUno H, Eisele S, Sakai A, Shelton S, Baker E, DeJesus O, Holden J: Neurotoxicity of gluco-
corticoids in the primate brain. Horm Behav 1994;28:336–348.
199 Van Aelst L, D’Souza-Schorey C: Rho GTPases and signaling networks. Genes 1997;11:
2295–2322.
200 Veelken JA, Laing RJ, Jakubowski J: The Sheffield model of subarachnoid hemorrhage in rats.
Stroke 1995;26:1279–1283.
201 Vikkula M, Boon LM, Carraway KL III, Calvert JT, Diamonti AJ, Goumnerov B, Pasyk KA,
Marchuk DA, Warman ML, Cantley LC, Mulliken JB, Olsen BR: Vascular dysmorphogenesis
caused by an activating mutation in the receptor tyrosine kinase TIE2. Cell 1996;87:1181–1190.
202 Vinters HV: Cerebral amyloid angiopathy. A critical review. Stroke 1987;18:311–324.
203 Vorkapic P, Bevan RD, Bevan JA: Longitudinal time course of reversible and irreversible compo-
nents of chronic cerebrovasospasm of the rabbit basilar artery. J Neurosurg 1991;74:951–955.
204 Wagner KR, Xi G, Hua Y, Kleinholz M, de Court, Myers RE, Broderick JP, Brott TG: Lobar
intracerebral hemorrhage model in pigs: Rapid edema development in perihematomal white
matter. Stroke 1996;27:490–497.
205 Wanebo JE, Arthur AS, Louis HG, West K, Kassell NF, Lee KS, Helm GA: Systemic administra-
tion of the endothelin-A receptor antagonist TBC 11251 attenuates cerebral vasospasm after
experimental subarachnoid hemorrhage: Dose study and review of endothelin-based therapies in
the literature on cerebral vasospasm. [Review]. Neurosurgery 1998;43:1409–1417.
206 Weir B: Aneurysms Affecting the Nervous System. Baltimore, Williams & Wilkins, 1987.
207 Weir B, Erasmo R, Miller J, McIntyre J, Secord D, Mielke B: Vasospasm in response to repeated
subarachnoid hemorrhages in the monkey. J Neurosurg 1970;33:395–406.
208 Widenka DC, Medele RJ, Stummer W, Bise K, Steiger HJ: Inducible nitric oxide synthase: A pos-
sible key factor in the pathogenesis of chronic vasospasm after experimental subarachnoid hem-
orrhage. J Neurosurg 1999;90:1098–1104.
209 Woo D, Sauerbeck LR, Kissela BM, Khoury JC, Szaflarski JP, Gebel J, Shukla R, Pancioli AM,
Jauch EC, Menon AG, Deka R, Carrozzella JA, Moomaw CJ, Fontaine RN, Broderick JP: Genetic
and environmental risk factors for intracerebral hemorrhage: Preliminary results of a population-
based study. Stroke 2002;33:1190–1196.
210 Wyburn-Mason R: The Vascular Abnormalities and Tumors of the Spinal Cord and Its
Membranes. London, Henry Kimpton, 1943.
211 Xi G, Keep RF, Hoff JT: Pathophysiology of brain edema formation. Neurosurg Clin N Am
2002;13:371–383.
212 Xi G, Wagner KR, Keep RF, Hua Y, de Court, Broderick JP, Brott TG, Hoff JT, Muizelaar JP: Role
of blood clot formation on early edema development after experimental intracerebral hemorrhage.
Stroke 1998;29:2580–2586.
213 Yamakawa K, Sasaki T, Tsubaki S, Nakagomi T, Saito I, Takakura K: Effect of high-dose
methylprednisolone on vasospasm after subarachnoid hemorrhage. Neurol Med-Chir 1991;31:
24–31.
214 Yamamoto S, Nishizawa S, Yokoyama T, Ryu H, Uemura K: Subarachnoid hemorrhage impairs
cerebral blood flow response to nitric oxide but not to cyclic GMP in large cerebral arteries. Brain
Res 1997;757:1–9.
215 Yamaura I, Tani E, Maeda Y, Minami N, Saido T, Suzuki K: Calpain-calpastatin system of canine
basilar artery in vasospasm; in Findlay JM (ed): Cerebral Vasospasm. Amsterdam, Elsevier, 1993,
pp 137–140.
216 Yokota M, Peterson JW, Kaoutzanis MC, Yamakawa K, Sibilia R, Zervas NT: Protein kinase C and
diacylglycerol content in basilar arteries during experimental cerebral vasospasm in the dog.
J Neurosurg 1995;82:834–840.
217 Yokoyama K, Asano Y, Murakawa T, Takada M, Ando T, Sakai N, Yamada H, Iwata H: Familial
occurrence of arteriovenous malformation of the brain. J Neurosurg 1991;74:585–589.
218 Yufu K, Itoh T, Edamatsu R, Mori A, Hirakawa M: Effect of hyperbaric oxygenation on the Na⫹,
K(⫹)-ATPase and membrane fluidity of cerebrocortical membranes after experimental subarach-
noid hemorrhage. Neurochem Res 1993;18:1033–1039.
219 Yundt KD, Grubb RL Jr, Diringer MN, Powers WJ: Autoregulatory vasodilation of parenchymal
vessels is impaired during cerebral vasospasm. J Cereb Blood Flow Metab 1998;18:419–424.
medwedi.ru
220 Zanzinger J: Role of nitric oxide in the neural control of cardiovascular function. Cardiovasc Res
1999;43:639–649.
221 Zee RY, Lou YK, Griffiths LR, Morris BJ: Association of a polymorphism of the angiotensin I-
converting enzyme gene with essential hypertension. Biochem Biophys Res Commun 1992;
184:9–15.
222 Zhang ZD, Yamini B, Komuro T, Ono S, Johns L, Marton LS, Weir B, Macdonald RL: Vasospasm
in monkeys resolves because of loss of and encasement of subarachnoid blood clot. Stroke 2001;
32:1868–1874.
223 Zimmermann M, Seifert V: Endothelin and subarachnoid hemorrhage: An overview. [Review].
224 Zubkov AY, Ogihara K, Tumu P, Patlolla A, Lewis AI, Parent AD, Zhang J: Mitogen-activated pro-
tein kinase mediation of hemolysate-induced contraction in rabbit basilar artery. J Neurosurg
1999;90:1091–1097.
225 Zuccarello M, Boccaletti R, Romano A, Rapoport RM: Endothelin B receptor antagonists attenu-
ate subarachnoid hemorrhage-induced cerebral vasospasm. Stroke 1998;29:1924–1929.
R. Loch Macdonald, MD
Professor, Section of Neurosurgery
University of Chicago Medical Center
5841 South Maryland Avenue, Chicago, IL 60637 (USA)
Tel. ⫹1 773 702 2123, Fax ⫹1 773 702 3518, E-Mail rlmacdon@uchicago.edu
Macdonald 412
Advances towards Cerebrovascular

Gene Therapy
Yoshimasa Watanabe, Donald D. Heistad
Departments of Internal Medicine and Pharmacology, University of Iowa College
of Medicine, Cardiovascular Center, and Veterans Affairs Medical Center,
Iowa City, Iowa, USA
Introduction
More than a decade has passed, since in vivo gene transfer to blood vessels
was first reported [1]. At present, results from clinical trials indicate that gene
therapy holds promise for vascular diseases, including coronary artery disease
and limb ischemia [2–6]. Gene therapy has several potential advantages over
pharmacological therapies currently in use. First, disease-specific effects are
expected by introduction or manipulation of selected genes. Second, overex-
pression of therapeutic genes can provide prolonged production of therapeutic
molecules. Third, by local gene transfer, therapeutic molecules can be delivered
to a target tissue selectively, while minimizing systemic or nonspecific effects.
Previously, we suggested several strategies for treatment of cerebrovascular
diseases, which might be achieved by gene therapy [7]. These strategies include
the prevention of vasospasm after subarachnoid hemorrhage (SAH); stimulation
of collateral blood flow to ischemic regions of the brain by angiogenesis; and
treatment of atherosclerotic lesions of the extracranial cerebral arteries by
inhibiting thrombosis or proliferation of lesions, stabilizing plaques, and pre-
venting restenosis after angioplasty. Recent progress in studies using animal
models demonstrates the feasibility of gene therapy for several cerebrovascular
diseases, although some obstacles must be solved before clinical use is possible.
Here, we summarize progress in gene therapy targeted to cerebral arterial patho-
physiology under experimental settings, and discuss future directions towards
cerebrovascular gene therapy in humans.
Basic strategies for gene therapy may consist of (1) expression of
deficient genes or ‘reconstitution’; (2) overexpression of therapeutic genes;
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(3) down-regulation of pathogenic gene expression. Currently, these three
alternative strategies are achieved by the delivery of genes or nucleotides with
therapeutic potency into the target tissue or cells. In general, maintaining
long-term effects is the primary limitation of current gene therapy techniques,
especially for the treatment of chronic diseases. Nevertheless, a transient
therapeutic effect may be suitable for the treatment of several cardiovascular
disorders, such as prevention of restenosis after angioplasty and therapeutic
angiogenesis for ischemic tissue [8].
Basic Methods for Experimental Cerebrovascular

Gene Therapy
Choice of Gene Delivery System

Direct delivery of ‘naked’ plasmid DNA is effective in skeletal muscle [9].
However, because of the low efficiency of naked DNA-mediated gene transfer
into the vascular wall, viral or nonviral vectors have been used for vascular gene
transfer in most studies [10]. Replication-deficient recombinant adenoviruses
are widely used for the delivery of genes both in experimental gene transfer to
cerebral blood vessels and in some clinical trials of cardiovascular gene therapy
[8, 10]. Unlike retroviral vectors, both dividing and nondividing cells are
efficiently transfected with adenovirus Ad vectors, which is an advantage of Ad
vectors in transfecting quiescent vascular cells [11]. The vectors have a relatively
large capacity for cDNA inserts and are easily prepared in high viral titers [11].
Stimulation of immune and inflammatory responses, however, is a serious
limitation of Ad vectors. For example, Ad-mediated gene transfer into the
lumen of arteries provokes infiltration of T cells and up-regulation of adhesion
molecules in the vascular wall, intimal hyperplasia, and increased turnover of
endothelial cells, resulting in the early loss of transgene expression [12–14]. To
minimize adverse effects of Ad vectors, low titers of the virus could be used if
enhanced efficiency of transfection allows adequate expression of a transgene
[15]. Concomitant use of cationic polymer or lipids or calcium phosphate with
Ad vectors enhances transgene expression in cerebral arteries in vivo and
ex vivo [16–18].
Ad vectors have been altered to reduce immune responses in vascular
tissue, although they still have substantial immunogenicity [19–21]. A recent
technology for constructing a new generation of Ad vectors may help to
circumvent this problem [22]. Helper-dependent, or ‘gutless’ Ad vectors, which
do not contain most genes encoding viral proteins, are less toxic but maintain
efficient production of transgene expression in animals. However, difficulty in
purification (i.e., contamination by helper virus, which is required for the
Watanabe/Heistad 414
production of the helper-dependent vector) and great difficulty in large-scale
production are major limitations of this vector.
Adeno-associated viruses may be an alternative for Ad vectors. Adeno-
associated viral vectors have been shown to be useful in transducing vascular
cells in the carotid artery with reporter genes in vivo [23–25]. Adeno-associated
viruses represent useful vectors with low toxicity, but have limited transgene
capacity [22].
Cationic liposomes form a complex with DNA molecules and facilitate
uptake of DNA by cells. Nonviral methods have been used for gene transfer to
vascular tissue in vivo [10]; however, efficiency of gene transfer is a major lim-
itation of this method [26, 27]. Nevertheless, cationic liposome-mediated gene
transfer is attractive because of its potential for delivering large DNA molecules
without substantial toxicity [28, 29].
The oligo-deoxyribonucleotide (ODN)-based strategy is an alternative to
gene therapy based on gene transfer techniques [30]. This strategy includes
techniques utilizing specific interactions of antisense, ribozyme, or ‘decoy’
ODNs with gene expression machinery. Expression of a target gene is inhibited
by inactivation of the mRNA (antisense or ribozyme ODNs) or the transcription
factor (decoy ODNs). In addition, recent findings in cellular mechanisms of
gene silencing have been applied to the development of a new gene targeting
technology. Transfection of cells with a small interfering RNA (siRNA), an
RNA fragment, specifically recognizes and then degrades a target mRNA [31].
The siRNA technique is available for in vivo use by Ad vector-mediated delivery,
though it is not clear whether this method is applicable to gene therapy for
vascular diseases [32].
In vivo Gene Transfer to the Cerebral Circulation

The first successful in vivo gene transfer to blood vessels was achieved by
the intraluminal administration of a retroviral vector containing a reporter gene
into segments of the iliofemoral artery of pigs [1]. In rat carotid arteries,
efficient transgene expression was produced by this approach using Ad vectors
[26, 33]. In rabbit carotid arteries, functional overexpression of nitric oxide
synthase (NOS) in endothelium was also demonstrated [34, 35]. To date, the
intraluminal approach is the most common technique for in vivo gene transfer
to the carotid artery of experimental animals. However, in order to achieve
effective transfection of cells in the vessel wall by this approach, transient
interruption of blood flow is usually required for 10–30 min, which would
produce unacceptable cerebral ischemia in patients. In addition, delivery of
genes or nucleotides is limited to a short segment of the vessel, and medial
smooth muscle cells are not transfected unless endothelial integrity is
disrupted [36, 37].
Cerebrovascular Gene Therapy 415
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Fig. 1. Adenovirus-mediated gene transfer of bacterial ␤-galactosidase (␤Gal) to cerebral
arteries. Rat brain was examined histochemically after intracisternal injection of adenovirus
encoding ␤Gal. Expression of ␤Gal (blue staining) was seen on the ventral surface of the brain,
especially along major cerebral arteries. (Reprinted with permission from [38].)
To circumvent these problems, methods for perivascular delivery of

transgenes have been developed. For the intracranial circulation, transfection
of vascular and perivascular cells is achieved by the injection of vectors into
cerebrospinal fluid (CSF). Ooboshi et al. [38] injected an Ad vector containing
cDNA for reporter gene ␤-galactosidase (␤Gal), into the cisterna magna of rats.
One and 3 days after the injection of Ad, expression of ␤Gal was observed in
adventitial cells of large cerebral arteries and cells in perivascular tissue
(fig. 1). The same technique has been applied to Ad-mediated gene transfer in
other species including primates [39–42]. In several related studies, ODNs were
administered via the cisterna magna and distributed in all layers of intracranial
cerebral arteries [43, 44].
Fig. 2. Perivascular gene transfer to the carotid artery. Ad␤Gal was injected within the
sheath of the common carotid artery of atherosclerotic monkeys. Adventitial cells express-
ing ␤Gal were stained blue. (Reprinted with permission from [45].)
Functional expression of transgene products has been demonstrated by

in vivo gene transfer of vasoactive molecules using the perivascular approach
for intracranial arteries. After injection of Ad containing cDNA for endothelial
NOS (eNOS) into CSF, eNOS is overexpressed in adventitia of the basilar
artery of dogs [39]. Overexpressed eNOS in adventitia is functionally linked
to nitric oxide-dependent relaxation induced by bradykinin, which is normally
endothelium-dependent, in vessel rings denuded with endothelium. Gene
transfer of CGRP increases the level of cyclic adenosine monophosphate in the
basilar artery and attenuates contraction induced by histamine and serotonin
in vitro [42].
For the extracranial carotid artery, injection of vectors into the periarterial
sheath is a practical way of perivascular gene delivery [45] (fig. 2). In the rab-
bit carotid artery, functional overexpression of eNOS can be achieved using this
approach [46]. After Ad-mediated gene transfer to the periarterial sheath,
neointimal formation, which is commonly seen after intraluminal approach is
not induced, and inflammatory responses are confined to the adventitia [47].
As a less invasive method, local gene transfer of a secretory peptide to a
remote site may be an alternative strategy. For example, a vector can be injected
into skeletal muscle, and a therapeutic transgene product can be locally
overexpressed in the skeletal muscle and secreted into the systemic circulation
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[48, 49]. In addition to local gene transfer, intravenous administration of vectors
is another possible method. The liver is a major site of transgene overexpression
after intravenous gene transfer, and therapeutic gene products such as factor
VIII for hemophilia A are secreted into the systemic circulation [50]. Vascular
effects of circulating transgene products may be produced by binding to the
vessel wall. For example, extracellular superoxide dismutase (ECSOD), an
isoform of superoxide dismutase (SOD) that is released into the extracellular
space [51], is released from liver into the blood and binds to the vessel wall
after intravenous gene transfer [52]. Binding of ECSOD to vessel wall is
mediated by the interaction between heparan sulfate proteoglycans in vascular
tissue and the heparin-binding domain of the enzyme. Heparin-binding
ECSOD, but not a truncated form of the enzyme lacking the heparin-binding
domain, reduces arterial pressure and improves endothelial dysfunction after
intravenous gene transfer in hypertensive rats [52]. Thus, intravenous gene
transfer of secretory proteins can take advantage of endogenous extracellular
binding sites to localize effects in blood vessels.
Experimental Gene Therapy for Cerebrovascular Diseases
Cerebral Vasospasm after SAH

Vasospasm after SAH may be a good target for gene therapy because of the
unique characteristics of the syndrome. Delayed onset of vasospasm allows
a period of time for transgene expression or down-regulation of expression of a
specific gene, and the transiency of the risk of the syndrome will not require
prolonged effects of gene therapy. In addition, a perivascular approach via CSF
will allow widespread exposure of cerebral vessels to vectors at the base of the
brain, which are covered by subarachnoid hematoma after SAH. Existence of a
hematoma at the site of SAH does not attenuate transgene expression in cerebral
vessels mediated by an Ad vector [53, 54]. Furthermore, increase in the effi-
ciency of transfection or expression after SAH, by up-regulation of Ad receptor
expression or increased activity of the promotor encoded in an Ad vector, may
enhance transgene expression in vascular adventitia [54].
Vasospasm after SAH is probably caused by multiple, complex mechanisms
[55, 56]. Several strategies, including overexpression of molecules involved in
vasorelaxation or cytoprotective mechanisms and down-regulation of putatively
pathological genes, have been tested for experimental gene therapy for vasospasm
(table 1). Several studies have examined effects of local overexpression of nitric
oxide (NO) synthase, because there is substantial evidence that indicates impair-
ment of endothelium-dependent, NO-induced vasorelaxation after SAH [57]. In
rings of canine basilar arteries excised after experimental SAH, gene transfer of
Table 1. Gene therapy for vasospasm after experimental subarachnoid hemorrhage
Target genes Strategy Delivery Treatment protocol Reduction Animals Refs

method of spasm
eNOS GT Ad At SAH No D 58
eNOS GT Ad 24-hr pretreatment Yes D 59
ppCGRP GT Ad 30-min posttreatment Yes Rbt, D 69, 70
HO-1 GT Ad At SAH Yes R 75
ECSOD GT Ad 30-min posttreatment Yes Rbt 81
ppET-1 AS ODN 30-min posttreatment Yes* D 84
MAPK AS ODN At SAH Yes R 88
NF-␬B Decoy ODN At SAH Yes Rbt 44
eNOS, endothelial nitric oxide synthase; ppCGRP, preprocalcitonin gene-related peptide; HO-1, hemeoxyge-
nase-1; ECSOD, extracellular superoxide dismutase; ppET-1, preproendothelin-1; MAPK, mitogen-activated
protein kinase, NF-␬B, nuclear factor-␬B; GT, gene transfer; AS, antisense; Decoy, transcription factor decoy; Ad,
adenovirus; ODN, oligodeoxynucleotide; SAH, subarachnoid hemorrhage; D, dog; Rbt, rabbit; R, rat.
*In combination with recombinant tissue plasminogen activator.
eNOS partially restores bradykinin-induced, NO-dependent relaxation [54].

However, gene transfer of eNOS after SAH in dogs failed to protect against the
constriction of the basilar artery or decreased cerebral blood flow, despite the ele-
vation of local NOS activity and NO production [58]. Another study reported the
reduction of vasospasm by gene transfer of eNOS prior to induction of SAH,
although the study design is not clinically relevant [59]. Failure to prevent
vasospasm by eNOS gene transfer after SAH may be due in part to inactivation
of NO by oxyhemoglobin [58, 60]. In addition, imbalance between the activities
of soluble guanylate cyclase and phosphodiesterase, resulting in decreased levels
of cyclic guanosine monophosphate (cGMP), may contribute to impaired
responses of arterial smooth muscle to NO [61, 62].
Instead of altering NO-cGMP mediated mechanisms, we have employed the
vasodilator effects of calcitonin gene-related peptide (CGRP) for the prevention
of experimental cerebral vasospasm by gene transfer. In cerebral arteries after
experimental SAH, the smooth muscle cell membrane is depolarized, and relax-
ation in response to ATP-sensitive K⫹ channel (KATP channels) activators is pre-
served or enhanced [61, 63–65]. CGRP, a neuropeptide, which is contained in
cerebral sensory nerves, hyperpolarizes the membrane of arterial smooth mus-
cle cells and relaxes cerebral arteries at least in part via opening of K⫹ channels,
including KATP channels [66–68]. We constructed a recombinant Ad containing
prepro-CGRP gene, and demonstrated the prevention of vasospasm by intracis-
ternal gene transfer of CGRP after experimental SAH in rabbits [69] (fig. 3).
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a b c
Control AdCGRP
0
⫺10
⌬Diameter (%)
⫺20
⫺30
e
d
Fig. 3. Effects of gene transfer of preproCGRP on vasospasm following experimental

subarachnoid hemorrhage (SAH) in rabbits. Adenovirus-encoding preproCGRP (AdCGRP),
control virus (Ad␤Gal), or vehicle was injected into the cisterna magna 30 min after intracis-
ternal injection of autologous blood. a–d Angiograms of cerebral arteries of rabbits treated
with Ad␤Gal (a and b) or AdCGRP (c and d). Vertebral arteriography was performed before
(a and c), and 2 days after (b and d) SAH. e The percentage changes in the diameter
(⌬diameter) of the basilar artery 2 days after SAH. Values are mean ⫾ SEM.
This was, to our knowledge, the first successful transfer of vasoactive genes in
vivo for the prevention of vasospasm after SAH. Vasospasm is also reduced by
the same treatment in dogs after SAH [70].
Oxidative stress in cerebral arteries has been implicated in the pathogene-
sis of vasospasm after SAH, and several compounds that scavenge reactive
oxygen species are under clinical trials [71]. Heme oxygenase-1 (HO-1), an
inducible isoform of key enzymes for the catabolism of heme, appears to have
an important role in cellular protection against oxidative stress [72]. Transient
expression of HO-1 in cerebral arteries occurs at an early stage after experimen-
tal SAH [73, 74], and suppression of induction of HO-1 by an antisense ODN
for HO-1 exacerbates vasospasm after experimental SAH [73]. Vasospasm and
reduction of cerebral blood flow induced by hemoglobin was prevented, and
vasospasm after experimental SAH was ameliorated after intracisternal gene
transfer of HO-1 in rats [75].
As a defense mechanism against oxidative stress, we have examined
the effect of local overexpression of ECSOD on experimental vasospasm. We
speculated that increased extracellular superoxide (O2⭈⫺) plays a role in the
pathophysiology of vasospasm. Several previous studies in experimental animals
suggested the pathophysiological importance of superoxide O2⭈⫺ in cerebral
vasospasm after SAH [76–79]. We found that intracisternal gene transfer of
ECSOD increased local expression and activity of ECSOD after SAH in rabbits
and reduced vasospasm [80, 81]. Interestingly, gene transfer of a variant ECSOD
lacking tissue-binding ability produced high levels of ECSOD in CSF, but had no
effect on vasospasm, suggesting that binding of ECSOD to the vessel wall is
important for the reduction of vasospasm [81].
In addition to overexpression of therapeutic genes, ODN-based targeting of
genes putatively involved in pathophysiology of vasospasm has been tested in sev-
eral studies. The target genes of this strategy include preproendothelin-1 (ppET-1),
mitogen-activated protein kinase (MAPK), and nuclear factor-␬B (NF-␬B).
Evidence from several studies implicates endothelin-1 (ET-1) in the pathophysiol-
ogy of cerebral vasospasm after SAH [55, 57, 82, 83]. Pretreatment with an
intracisternal injection of an antisense ODN against ppET-1, a precursor of ET-1,
inhibits expression of mRNA of ppET-1 and attenuates hemolysate-induced cere-
bral vasospasm in rats [43]. In dogs with SAH, however, antisense ODN against
ppET-1 was found to have little effect, perhaps because a physical barrier of sub-
arachnoid clot prevented ODNs from entering into the basilar artery [84].
MAPK, a key molecule involved in the ras p21 signaling pathway in cells,
regulates the contraction of smooth muscle in some vascular tissues and may
be involved in abnormal contraction of cerebral arteries after SAH [85–87].
In rats, intracisternal injection of an antisense ODN against MAPK inhibits
MAPK expression in the basilar artery and reduces vasospasm after SAH [88].
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Increased expression of inflammatory cytokines and cell adhesion molecules
in cerebral arteries may be involved in the pathophysiology of vasospasm after
SAH [89–91]. Thus, NF-␬B, a key transcription factor in the regulation of expres-
sion of inflammatory cytokines and cell adhesion molecules [92], is a possible
target for the treatment of vasospasm. NF-␬B is activated in the basilar artery
of rabbits after SAH, and pretreatment with ‘decoy’ ODN for NF-␬B reduces
vasospasm [44].
Carotid Artery Stenosis

Restenosis of arteries is a major problem after coronary angioplasty and
stenting and is a potential target for gene therapy [93]. Carotid endarterec-
tomy and angioplasty with stenting are now in wide use for the treatment of
stenotic lesions of carotid arteries. Carotid restenosis is also a target for gene
therapy, although the incidence of restenosis in the carotid artery may not be as
high as that in coronary and iliofemoral arteries [94–96]. There are reports of
gene therapy for restenosis after balloon injury of the carotid artery in small
animals (table 2).
Inhibition of smooth muscle proliferation by blocking progression of the
cell cycle has been the most common approach to experimental gene therapy for
restenosis of the carotid artery. The first demonstration of an inhibitory effect of
antisense ODN examined the role of a proto-oncogene c-myb in neointima
formation in balloon-injured carotid artery of rats [97], but results were not sup-
ported by later studies [98, 99]. Local delivery of antisense ODN against c-myc,
another proto-oncogene, appears to be effective after carotid artery balloon injury
in rats [98, 100], and was tested in a clinical trial for the prevention of restenosis
after coronary angioplasty and stenting [101]. Antisense ODN-based gene target-
ing of essential components of the cell cycle, including cyclin-dependent kinases
(cdc2 and cdk2 kinases), proliferating-cell nuclear antigen, and cyclins B and
G1, has been shown to reduce the formation of neointima in rat carotid artery
[102–107]. Neointimal formation is inhibited also by transfecting smooth mus-
cle cells in injured vessel with cell cycle regulatory genes. This strategy includes
overexpression of cyclin-dependent kinase inhibitors (p21CIP1, p27KIP1 and a
fusion protein of p27KIP1 and p16INK4b), wild-type p53 (an activator of p21CIP),
cell cycle-associated transcription factors (GAX and GATA-6), and retinoblas-
toma proteins, which inhibit a key transcription factor E2F [108–119]. Gene
transfer of a fusion protein that consists of fragments of Rb protein and E2F
induces cell-cycle arrest in smooth muscle cells and reduces the neointimal
development after balloon injury [120]. Decoy administration of E2F is an
unique strategy to inhibit smooth muscle proliferation in injured carotid arteries
of rats [121]. This technique is applied to the prevention of vein-graft occlusion
after coronary bypass surgery in ongoing clinical trials [5, 122].
Table 2. Gene therapy for carotid neointimal formation after arterial injury
Target genes Strategy Delivery method Approach Animals Refs
c-myb, c-myc AS ODN PV, IV/US R, P 97–99, 100, 177

Cdc2 kinase, Cdk2 kinase AS ODN, HVJ-L PV, IL R 102–105
PCNA AS ODN PV R 102, 106
Cyclin B1, Cyclin G1 AS HVJ-L, IL R 105, 107
Retrovirus
p21CIP1, p27KIP1, p16INK4b GT Ad IL R, Rbt 108–112
p53 GT Pl, Ad, HVJ-L IL, IV/US R, Rbt 113, 114, 178
GAX, GATA-6 GT Ad IL R 115, 116
Rb proteins GT Ad IL R 117–120
E2F Decoy HVJ-L IL R 121
HSVtk (w/ganciclovir i.v.) GT Ad IL R 123
Fas-ligand GT Ad IL R 110, 124
Bcl-xL AS ODN IL Rbt 125
Hemeoxygenase-1 GT Ad IL R 126
bFGF GT (DN) Ad IL R 127
PDGFR (␤-chain) AS ODN PV R 128
PDGFR GT Ad IL R 129
(extracellular domain)
␤-ARK GT Ad IL R 130
(G␤␥-binding domain)
H-ras, MAPKK GT (DN) Pl, Ad PV, IL R 131–133
Activator protein-1 Decoy ODN IL Rbt 135
BMP-2 GT Ad IL R 136
Fortilin GT Ad IL R 137
Hirudin, TFPI GT Ad IL R, Rbt 138, 139
TIMP-1, TIMP-2 GT Ad IL R 141–143
TGF-␤1 AS Pl PV Rbt 144
MCP-1 GT (DN) Pl Intramuscular R, Rbt, M 48, 147
NF-␬B (p65) AS ODN PV R 148
NOS isoforms GT Ad, HVJ-L IL R, Rbt 152–154, 159
Protein kinase G GT Ad IL R 155
(catalytic domain)
CNP GT Ad IL R, Rbt 156, 157
Tissue kallikrein GT Ad IL R 158, 159
PGI2 synthase GT Pl, HVJ-L IL R 161–163
PCNA, proliferating cell nuclear antigen; HSVtk, herpes simplex virus thymidine kinase; bFGF, basic fibro-
blast growth factor; PDGFR, platelet-derived growth factor receptor; ␤-ARK, ␤-adrenergic receptor kinase;
MAPKK, mitogen-activated protein kinase kinase; BMP, bone morphogenetic protein; TFPI, tissue factor pathway
inhibitor; TIMP, tissue inhibitor of matrix metalloproteinase; TGF, transforming growth factor; MCP, monocyte
chemoattractant protein; NF-␬B, nuclear factor-␬B; NOS, nitric oxide synthase; PGI2, prostaglandin I2; AS, anti-
sense; GT, gene transfer; Decoy, transcription factor decoy; DN, dominant negative; ODN, oligodeoxynucleotide;
HVJ-L, hemagglutinating virus of Japan-liposome complex; Ad, adenovirus; Pl, plasmid; PV, perivascular; IV/US,
ultrasound-supported gene/nucleotide delivery; IL, intraluminal; R, rat; P, pig; Rbt, rabbit; M, monkey.
medwedi.ru
Besides the cytostatic strategy achieved by stasis of the cell cycle, another
strategy is the induction of cell death in proliferating smooth muscle cells. Gene
transfer of herpes simplex virus thymidine kinase followed by systemic adminis-
tration of ganciclovir, which is a cytotoxic combination for neoplastic cells, is
effective in reducing development of neointima [123]. Local overexpression of
Fas ligand, an activator of the extrinsic pathway of apoptosis, and down-regulation
of Bcl-xL, an anti-apoptotic regulator protein, facilitate apoptosis of neointimal
cells and inhibit neointimal formation [110, 124, 125]. In addition, overexpression
of Bcl-xL induces regression of established neointimal lesion [125]. Fas-ligand
overexpression may be advantageous in the context of Ad-mediated gene trans-
fer, because it inhibits infiltration of Fas-expressing T cells into the arterial wall
in response to the Ad vector treatment [110, 124]. After gene transfer of HO-1,
increase in the number of apoptotic cells and decrease in the number of prolifer-
ative cells were observed in the medial wall of injured carotid arteries, although
the mechanisms of these effects are not clear [126].
Mitogenic signal transduction in smooth muscle cells may also be a target
for inhibition of restenosis by gene therapy. In balloon-injured carotid arteries,
neointimal formation is inhibited by treatment with antisense ODNs for basic
fibroblast growth factor, or platelet-derived growth factor (PDGF) receptors
[127, 128]. Local gene transfer of the extracellular domain of PDGF receptors,
which binds PDGF-B chain and antagonizes the effect of PDGF, also inhibits
the formation of neointima [129].
Inhibition of intracellular signaling molecules of mitogenesis also reduces
neointimal hyperplasia after injury. This strategy includes overexpression of an
inhibitory peptide against the ␤␥ subunit of heterotrimeric G proteins, dominant-
negative mutant of ras p21, or MAPK kinase [130–133]. Decoy administration
against activator protein-1, a transcription factor activated through a signaling
pathway mediated by MAPKs in injured arterial wall [134], inhibits the formation
of neointima [135].
Bone morphogenetic protein-2, a member of the transforming growth factor
superfamily with bone-inducing activity, is discussed elsewhere in this volume
in the context of increasing bony fusion rates in spinal surgery. In the context of
cerebrovascular disease, bone morphogenetic protein-2 has growth-inhibitory
effects on smooth muscle cells, and limits the development of neointima after local
gene transfer to the injured rat carotid artery [136]. Thus bone morphogenetic pro-
tein might be useful as a gene transfer strategy to prevent restenosis. A recent study
also demonstrated that gene transfer of fortilin, a novel anti-apoptotic factor, inhib-
ited cellular proliferation in the vascular wall and reduced intimal thickening after
balloon injury [137]. Overexpression of hirudin or tissue factor pathway inhibitor
in balloon-injured carotid artery inhibits the activation of thrombin, a potent mito-
gen for vascular smooth muscle cells, and reduces neointima formation [138,
139]. This strategy may be advantageous, because the prevention and reduction of
thrombosis is also important for the treatment of arteriosclerotic lesions.
Both proliferation and migration of smooth muscle cells are major features
of neointima formation after arterial injury [140]. Effects of inhibition of cell
migration by tissue inhibitors of metalloproteinase, which inhibit matrix metallo-
proteinases, have been tested in injured carotid arteries. Matrix metalloproteinases
contribute to the digestion of extracellular matrix prior to the migration of smooth
muscle cells. Local or systemic gene transfer of tissue inhibitors of metallopro-
teinase inhibits (or delays) progression of intimal hyperplasia [141–143]. Inhibi-
tion of the deposition of extracellular matrix may also be effective in reduction of
neointimal formation after balloon injury. Antisense ODN against transforming
growth factor-␤1 inhibits the production of proteoglycans in injured vascular wall
and reduces the development of neointima in the rabbit carotid artery [144].
Recent studies suggest that inflammatory responses and oxidative stress in
the vascular wall contribute to neointima formation after arterial injury [145, 146].
Thus, suppression of inflammation and oxidative stress are possible strategies for
the treatment of restenosis. Gene transfer of a dominant negative mutant of mono-
cyte chemoattractant protein-1 or antisense ODN-mediated down-regulation of
transcription factor NF-␬B suppresses inflammatory changes in the vascular wall
and reduces intimal hyperplasia after arterial injury [48, 147, 148]. Enhancement
of cellular protection against oxidative stress by gene transfer of ECSOD inhibits
neointima formation after balloon injury of the rabbit aorta [149].
Gene transfer of NOS isoforms is a diverse strategy for anti-restenotic
therapy, because NO has anti-proliferative as well as anti-inflammatory and
anti-thrombotic properties, and is diffusible after release from its site of pro-
duction [150]. For example, von der Leyen et al. [151] demonstrated the inhi-
bition of neointima formation in balloon-injured rat carotid artery after gene
transfer of eNOS. Local overexpression of constitutive NOS isoforms inhibits
the proliferation of smooth muscle cells in balloon-injured carotid artery of
rats, and inflammatory response in the carotid artery of hyperlipidemic rabbits
[152, 153]. Reduced neointimal formation has been shown in rat carotid bal-
loon injury model after gene transfer of inducible NOS, at a relatively lower
titer of the Ad vector than after eNOS gene transfer [154].
In accordance with these studies, overexpression of a constitutively active
mutant of protein kinase G, an intracellular effector of the NO-cGMP system,
is also effective [155]. Locally overexpressed C-type natriuretic peptide, a
secreted peptide activator of particulate guanylate cyclase, reduces thrombus
formation, inflammatory response, and intimal thickening in injured carotid
arteries [156, 157]. Interestingly, these effects of C-type natriuretic peptide are
likely to depend on increased NO production through enhanced induction of
NOS in the vessel wall after injury [157].
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Gene transfer of human tissue kallikrein, which converts kininogen into
vasoactive kinin, inhibits neointima formation probably through activating
NO-cGMP system [158–160]. Increased tissue levels of cyclic adenosine mono-
phosphate may also contribute to this anti-proliferative effect of overexpressed
kallikrein [158, 160]. Increased production of prostacyclin (PGI2), which increases
cellular cyclic adenosine monophosphate level in injured carotid artery after gene
transfer of PGI2 synthase results in reduced intimal hyperplasia [161–163].
Because endothelium is important for maintaining physiological homeostasis
of the arterial wall by mechanisms including the production of NO and PGI2, facil-
itation of re-endothelialization is a reasonable strategy for the treatment of injured
arteries. Gene transfer of vascular endothelial growth factor (VEGF), a stimulator
of endothelial proliferation, accelerates re-endothelialization, suppresses intimal
hyperplasia, and reduces the frequency of thrombotic occlusion after balloon
injury of the femoral artery of rabbits [164]. VEGF gene transfer has been tested
for the prevention of coronary restenosis in patients undergoing angioplasty [4].
Local overexpression of PGI2 has also been reported to accelerate endothelial
regeneration after balloon injury [161].
Ischemia
Gene transfer of angiogenic growth factors is a promising strategy for the
development of collateral circulation in ischemic tissue. Therapeutic angiogene-
sis by gene transfer of angiogenic growth factors, including VEGF, hepatocyte
growth factor, and basic fibroblast growth factor, has been tried for the treatment
of myocardial and limb ischemia in clinical trials [2, 3, 6, 30]. In recent studies
using a cerebral hypoperfusion model in rats, intrathecal gene transfer of
hepatocyte growth factor or VEGF stimulated angiogenesis in the brain and
increased the cerebral blood flow [165]. Gene transfer of basic fibroblast growth
factor is also effective in inducing neovascularization in the brain of normal rats
[166]. Thus, this strategy may be applicable to the development of collateral ves-
sels in the brain for the prevention and treatment of cerebral ischemia. Other gene
transfer strategies may be useful for treating the effects of ischemic or repairing
ischemia-damaged tissue, but these fall outside the scope of this chapter.
Current Problems and Future Directions
Cerebral Vasospasm
Prevention of vasospasm after SAH is a promising target for the clinical
use of gene therapy techniques. Most studies, however, have been performed in
SAH models of small animals. Because the severity and temporal profile of
vasospasm in small animals are different from those in humans, findings must
be confirmed in larger animals, such as dogs and primates. Mechanisms of
development, maintenance, and resolution of cerebral vasospasm are not fully
understood, so there is no clear target for the treatment of vasospasm. Progress
in basic research addressing pathogenesis of vasospasm is important for the
establishment of effective gene therapy.
One of the major obstacles to the use of Ad-mediated gene transfer for treat-
ment of vasospasm after SAH is that Ad vectors induce inflammatory responses
in cerebral arteries [167]. Inflammation is also an important pathophysiological
change in cerebral vessels after SAH [90, 168, 169]. Another limitation of Ad
gene transfer is that transgene expression is achieved only in adventitial cells of
cerebral arteries by the perivascular approach [38, 40, 53]. Thus, it is difficult to
alter gene expression of smooth muscle cells and endothelial cells, where impor-
tant pathology occurs. ODNs produce little inflammation and diffuse deep into
all vessel layers [43, 44]. However, uptake of ODNs by cells in the vessel wall
may be insufficient to prevent vasospasm because subarachnoid blood can pre-
vent ODNs from reaching the arterial wall [84]. Efficacy of ODNs in prevention
of vasospasm, as summarized earlier, is unclear.
Carotid Stenosis
Before the clinical use of gene therapy for restenosis of the carotid artery,
a better delivery system for vectors or plasmids must be developed. Methods
used in most studies are not relevant to clinical settings, because vectors or
plasmids are placed in the lumen of the carotid artery with interruption of
blood flow for more than 10 min. Several studies suggest the feasibility of a
perivascular approach for prevention of neointimal formation in injured carotid
arteries [97–100, 106, 128, 133, 170]. Gene transfer into skeletal muscle, with
systemic release of a secretory peptide, is an alternative approach [48].
Recent developments in catheter- or stent-based vector delivery systems
may enable safe and effective intraluminal approaches for the carotid artery
[171–175]. In addition, a new method using intravenous ultrasound contrast
agents may be useful for intraluminal vascular gene delivery [176]. An ODN
that is bound to microbubbles prepared as a contrast agent is effectively
transduced into the wall of the extracranial carotid artery within the area of
insonation [177, 178]. There is considerable concern, however, that the
approach may produce additional endothelial damage.
In patients with carotid atherosclerotic lesions, prevention of thrombotic
events (both acute occlusion and distal embolism) and development of stenosis
is an important target for gene therapy. Established atherosclerotic lesions
are stabilized after gene transfer of a dominant negative mutant of monocyte
chemotactic protein-1 [49]. Progression of atherosclerotic lesions is also reduced
after gene transfer of the mutant monocyte chemotactic protein-1 [49] in the
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aorta of mice with deficiency of apolipoprotein E, which otherwise develop
spontaneous atherosclerosis. In an arterial thrombosis model in the femoral
artery of rabbits, local gene transfer of tissue plasminogen activator inhibited
the formation of thrombus in the vessel [179]. The concepts of these studies
may be useful for developing gene therapy for carotid atherosclerosis.
Ischemia and Collateral Circulation

It is not yet clear whether therapeutic gene transfer for angiogenesis is useful
for focal angiogenesis to regions of the brain that are at risk for ischemia. A recent
study has demonstrated that neovascularization in the ischemic limb of mice is
effectively achieved by intravenous administration of bone marrow-derived endo-
thelial progenitor cells transfected ex vivo with the VEGF gene [180]. This endo-
thelial progenitor cell-mediated gene therapy may be applicable to focal cerebral
ischemia, because endothelial progenitor cells incorporate into the vessels in the
ischemic area of the brain after occlusion of middle cerebral artery in mice [181].
Several issues have been raised regarding the safety of therapeutic gene
transfer for angiogenesis after accumulation of experience from clinical trials
[8]. For example, it is speculated that vascular malformations could develop at
the site of neovascularization and predispose to brain hemorrhage.
Conclusion
In general, safety is the paramount issue and obstacle for the establishment
of cerebrovascular gene therapy in the clinic. Development of safe and efficient
vectors for gene/nucleotide delivery in humans is urgently required. In addition,
tissue-specific targeting of vectors or transgene expression will be useful for
reducing the toxicity of the vectors or transgene products [120, 182, 183]. Data
will also be needed regarding pharmacokinetics and toxicity of vectors and
therapeutic genes or nucleotides [184]. It is not likely that gene therapy will be
clinically useful for cerebrovascular diseases in the near future, but we are opti-
mistic that the improvement of methods for gene therapy will ultimately result
in its clinical use.
Acknowledgments
We thank Drs. Yi Chu, Frank Faraci, and Carol Gunnett for their critical evaluation of
this manuscript. Original studies by authors were supported by funds from the Veterans
Administrations, NIH Grants HL16066, HL62984, NS24621, HL14388, DK54759, the
Carver Research Program of Excellence, the Wendy Hamilton Trust, and an award from the
American Heart Association 0120641Z.
References
1 Nabel EG, Plautz G, Nabel GJ: Site-specific gene expression in vivo by direct gene transfer into
the arterial wall. Science 1990;249:1285–1288.
2 Grines CL, Watkins MW, Helmer G, Penny W, Brinker J, Marmur JD, West A, Rade JJ, Marrott P,
Hammond HK, Engler RL: Angiogenic Gene Therapy (AGENT) trial in patients with stable
angina pectoris. Circulation 2002;105:1291–1297.
3 Losordo DW, Vale PR, Hendel RC, Milliken CE, Fortuin FD, Cummings N, Schatz RA, Asahara T,
Isner JM, Kuntz RE: Phase 1/2 placebo-controlled, double-blind, dose-escalating trial of myocardial
vascular endothelial growth factor 2 gene transfer by catheter delivery in patients with chronic
myocardial ischemia. Circulation 2002;105:2012–2018.
4 Laitinen M, Hartikainen J, Hiltunen MO, Eränen J, Kiviniemi M, Närvänen O, Mäkinen K,
Manninen H, Syvänne M, Martin JF, Laakso M, Ylä-Herttuala S: Catheter-mediated vascular
endothelial growth factor gene transfer to human coronary arteries after angioplasty. Hum Gene
Ther 2000;11:263–270.
5 Mann MJ, Whittemore AD, Donaldson MC, Belkin M, Conte MS, Polak JF, Orav EJ, Ehsan A,
Dell’Acqua G, Dzau VJ: Ex-vivo gene therapy of human vascular bypass grafts with E2F decoy:
The PREVENT single-centre, randomized, controlled trial. Lancet 1999;354:1493–1498.
6 Mäkinen K, Manninen H, Hedman M, Matsi P, Mussalo H, Alhava E, Yla-Herttuala S: Increased
vascularity detected by digital subtraction angiography after VEGF gene transfer to human lower
limb artery: A randomized, placebo-controlled, double-blinded phase II study. Mol Ther 2002;6:
127–133.
7 Heistad DD, Faraci FM: Gene therapy for cerebral vascular disease. Stroke 1996;27:1688–1693.
8 Isner JM, Vale PR, Symes JF, Losordo DW: Assessment of risks associated with cardiovascular
gene therapy in human subjects. Circ Res 2001;89:389–400.
9 Wolff JA, Malone RW, Williams P, Chong W, Acsadi G, Jani A, Felgner PL: Direct gene transfer
into mouse muscle in vivo. Science 1990;247:1465–1468.
10 Smith RC, Walsh K: Local gene delivery to the vessel wall. Acta Physiol Scand 2001;173:93–102.
11 Chu Y, Heistad DD: Gene transfer to blood vessels using adenoviral vectors. Methods Enzymol
2002;346:263–276.
12 Newman KD, Dunn PF, Owens JW, Schulick AH, Vrimani R, Sukhova G, Libby P, Dichek DA:
Adenovirus-mediated gene transfer into normal rabbit arteries results in prolonged vascular cell
activation, inflammation, and neointimal hyperplasia. J Clin Invest 1995;96:2955–2965.
13 Channon KM, Qian H, Youngblood SA, Olmez E, Shetty GA, Neplioueva V, Blazing MA, George SE:
Acute host-mediated endothelial injury after adenoviral gene transfer in normal rabbit arteries: Impact
on transgene expression and endothelial function. Circ Res 1998;82:1253–1262.
14 Vassalli G, Agah R, Qiao R, Aguilar C, Dichek DA: A mouse model of arterial gene transfer:
Antigen-specific immunity is a minor determinant of the early loss of adenovirus-mediated trans-
gene expression. Circ Res 1999;85:e25–e32.
15 Schulick AH, Newman KD, Virmani R, Dichek DA: In vivo gene transfer into injured carotid
arteries: Optimization and evaluation of acute toxicity. Circulation 1995;91:2407–2414.
16 Toyoda K, Ooboshi H, Chu Y, Fasbender A, Davidson BL, Welsh MJ, Heistad DD: Cationic poly-
mer and lipids enhance adenovirus-mediated gene transfer to rabbit carotid artery. Stroke
1998;29:2181–2188.
17 Toyoda K, Nakane H, Heistad DD: Cationic polymer and lipids augment adenovirus-mediated
gene transfer to cerebral arteries in vivo. J Cereb Blood Flow Metab 2001;21:1125–1131.
18 Toyoda K, Andresen JJ, Zabner J, Faraci FM, Heistad DD: Calcium phosphate precipitates augment
adenovirus-mediated gene transfer to blood vessels in vitro and in vivo. Gene Ther 2000;7:1284–1291.
19 Wen S, Schneider DB, Driscoll RM, Vassalli G, Sassani AB, Dichek DA: Second-generation
adenoviral vectors do not prevent rapid loss of transgene expression and vector DNA from the
arterial wall. Arterioscler Thromb Vasc Biol 2000;20:1452–1458.
20 Wen S, Driscoll RM, Schneider DB, Dichek DA: Inclusion of the E3 region in an adenoviral
vector decreases inflammation and neointima formation after arterial gene transfer. Arterioscler
Thromb Vasc Biol 2001;21:1777–1782.
medwedi.ru
21 Qian HS, Channon K, Neplioueva V, Wang Q, Finer M, Tsui L, George SE, McArthur J: Improved
adenoviral vector for vascular gene therapy: Beneficial effects on vascular function and inflam-
mation. Circ Res 2001;88:911–917.
22 Kay MA, Glorioso JC, Naldini L: Viral vectors for gene therapy: The art of turning infectious
agents into vehicles of therapeutics. Nat Med 2001;7:33–40.
23 Rolling F, Nong Z, Pisvin S, Collen D: Adeno-associated virus-mediated gene transfer into rat
carotid arteries. Gene Ther 1997;4:757–761.
24 Arnold TE, Gnatenko D, Bahou WF: In vivo gene transfer into rat arterial walls with novel adeno-
associated virus vectors. J Vasc Surg 1997;25:347–355.
25 Richter M, Iwata A, Nyhuis J, Nitta Y, Miller AD, Halbert CL, Allen MD: Adeno-associated
virus vector transduction of vascular smooth muscle cells in vivo. Physiol Genomics 2000;2:
117–127.
26 Lee SW, Trapnell BC, Rade JJ, Virmani R, Dichek DA: In vivo adenoviral vector-mediated gene
transfer into balloon-injured rat carotid arteries. Circ Res 1993;73:797–807.
27 Laitinen M, Pakkanen T, Donetti E, Baetta R, Luoma J, Lehtolainen P, Viita H, Agrawal R,
Miyanohara A, Friedmann T, Risau W, Soma M, Yla-Herttuala S: Gene transfer into the carotid
artery using an adventitial collar: Comparison of the effectiveness of the plasmid-liposome
complexes, retroviruses, pseudotyped retroviruses, and adenoviruses. Hum Gene Ther 1997;8:
1645–1650.
28 Nabel GJ, Nabel EG, Yang Z, Fox BA, Plautz GE, Gao X, Huang L, Shu S, Gordon D, Chang AE:
Direct gene transfer with DNA-liposome complexes in melanoma: Expression, biologic activity,
and lack of toxicity in humans. Proc Natl Acad Sci USA 1993;90:11307–11311.
29 Templeton NS, Lasic DD: New direction in liposome gene delivery. Mol Biotechnol 1999;11:
175–180.
30 Morishita R, Aoki M, Kaneda Y, Ogihara T: Gene therapy in vascular medicine: Recent advances
and future perspectives. Pharmacol Ther 2001;91:105–114.
31 Kitabwalla M, Ruprecht RM: RNA interference – A new weapon against HIV and beyond. N Engl
J Med 2002;347:1364–1367.
32 Xia H, Mao Q, Paulson HL, Davidson BL: siRNA-mediated gene silencing in vitro and in vivo.
Nat Biotechnol 2002;20:1006–1010.
33 Rome JJ, Shayani V, Newman KD, Farrell S, Lee SW, Virmani FA, Dichek DA: Adenoviral vector-
mediated gene transfer into sheep arteries using a double-balloon catheter. Hum Gene Ther 1994;5:
1249–1258.
34 Channon KM, Qian H, Neplioueva V, Blazing MA, Olmez E, Shetty GA, Youngblood SA,
Pawloski J, McMahon T, Stamler JS, George SE: In vivo gene transfer of nitric oxide synthase
enhances vasomotor function in carotid arteries from normal and cholesterol-fed rabbits. Circulation
1998;98:1905–1911.
35 Sato J, Mohácsi T, Noel A, Jost C, Gloviczki P, Mozes G, Katusic ZS, O’Brien T: In vivo gene
transfer of endothelial nitric oxide synthase to carotid arteries from hypercholesterolemic rabbits
enhances endothelium-dependent relaxations. Stroke 2000;31:968–975.
36 Rome JJ, Shayani V, Flugelman MY, Newman KD, Virmani FA, Dichek DA: Anatomic barriers
influence the distribution of in vivo gene transfer into the arterial wall. Modeling with microscopic
tracer particles and verification with a recombinant adenoviral vector. Arterioscler Thromb 1994;
14:148–161.
37 Schulick AH, Dong G, Newman KD, Virmani R, Dichek DA: Endothelium-specific in vivo gene
transfer. Circ Res 1995;77:475–485.
38 Ooboshi H, Welsh MJ, Rios CD, Davidson BL, Heistad DD: Adenovirus-mediated gene transfer
in vivo to cerebral blood vessels and perivascular tissue. Circ Res 1995;77:7–13.
39 Chen AFY, Jiang S-W, Crotty TB, Tsutsui M, Smith LA, O’Brien T, Katusic ZS: Effects of in vivo
adventitial expression of recombinant endothelial nitric oxide synthase gene in cerebral arteries.
40 Christenson SD, Lake KD, Ooboshi H, Faraci FM, Davidson BL, Heistad DD: Adenovirus-mediated
gene transfer in vivo to cerebral blood vessels and perivascular tissue in mice. Stroke 1998;29:
1411–1416.
41 Driesse MJ, Kros JM, Avezaat CJJ, Valerio D, Vecht CJ, Bout A, Sillevis Smitt PAE: Distribution
of recombinant adenovirus in the cerebrospinal fluid of nonhuman primates. Hum Gene Ther
1999;10:2347–2354.
42 Toyoda K, Faraci FM, Russo AF, Davidson BL, Heistad DD: Gene transfer of calcitonin gene-
related peptide to cerebral arteries. Am J Physiol 2000;278:H586–H594.
43 Onoda K, Ono S, Ogihara K, Shiota T, Asari S, Ohomoto T, Ninomiya Y: Inhibition of vascular
contraction of preproendothelin-1 mRNA antisense oligoDNA in a rat experimental vasospasm
model. J Neurosurg 1996;85:846–852.
44 Ono S, Date I, Onoda K, Shiota T, Ohomoto T, Ninomiya Y, Asari S, Morishita R: Decoy admin-
istration of NF-␬B into the subarachnoid space for cerebral angiopathy. Hum Gene Ther 1998;9:
1003–1011.
45 Rios CD, Ooboshi H, Piegors D, Davidson BL, Heistad DD: Adenovirus-mediated gene transfer
to normal and atherosclerotic arteries. A novel approach. Arterioscler Thromb Vasc Biol 1995;15:
2241–2245.
46 Kullo IJ, Mozes G, Schwartz RS, Gloviczki P, Katusic ZS, O’Brien T: Adventitial gene transfer of
recombinant endothelial nitric oxide synthase to rabbit carotid arteries alters vascular reactivity.
Circulation 1997;96:2254–2261.
47 Schneider DB, Sassani AB, Vassalli G, Driscoll RM, Dichek DA: Adventitial delivery minimizes
the proinflammatory effects of adenoviral vectors. J Vasc Surg 1999;29:543–550.
48 Mori E, Komori K, Yamaoka T, Tanii M, Kataoka C, Takeshita A, Usui M, Egashira K, Sugimachi K:
Essential role of monocyte chemoattractant protein-1 in development of restenotic changes (neointi-
mal hyperplasia and constrictive remodeling) after balloon angioplasty in hypercholesterolemic
rabbits. Circulation 2002;105:2905–2910.
49 Inoue S, Egashira K, Ni W, Kitamoto S, Usui M, Otani K, Ishibashi M, Hiasa K, Nishida K, Takeshita
A: Anti-monocyte chemoattractant protein-1 gene therapy limits progression and destabilization of
established atherosclerosis in apolipoprotein E-knockout mice. Circulation 2002;106:2700–2706.
50 Balagué C, Zhou J, Dai Y, Alemany R, Josephs SF, Anderson G, Hariharan M, Sethi E,
Prokopenko E, Jan H, Lou Y-C, Hubert-Leslie D, Ruiz L, Zhang W-W: Sustained high-level
expression of full-length human factor VIII and restoration of clotting activity in hemophiliac
mice using a minimal adenoviral vector. Blood 2000;95:820–828.
51 Oury TD, Day BJ, Crapo JD: Extracellular superoxide dismutase: A regulator of nitric oxide
bioavailability. Lab Invest 1996;75:617–636.
52 Chu Y, Iida S, Lund DD, Weiss RM, DiBona GF, Watanabe Y, Faraci FM, Heistad DD: Gene transfer
of extracellular superoxide dismutase reduces arterial pressure in spontaneously hypertensive rats:
Role of heparin-binding domain. Circ Res 2003;92:461–468.
53 Muhonen MG, Ooboshi H, Welsh MJ, Davidson BL, Heistad DD: Gene transfer to cerebral blood
vessels after subarachnoid hemorrhage. Stroke 1997;28:822–829.
54 Onoue H, Tsutsui M, Smith L, Stelter A, O’Brien T, Katusic ZS: Expression and function of
recombinant endothelial nitric oxide synthase gene in canine basilar artery after experimental
subarachnoid hemorrhage. Stroke 1998;29:1959–1966.
55 Sobey CG, Faraci FM: Subarachnoid haemorrhage: What happens to the cerebral arteries? Clin
Exp Pharmacol Physiol 1998;25:867–876.
56 Dietrich HH, Dacey RG Jr: Molecular keys to the problems of cerebral vasospasm. Neurosurgery
2000;46:517–530.
57 Faraci FM, Heistad DD: Regulation of the cerebral circulation: Role of endothelium and potas-
sium channels. Physiol Rev 1998;78:53–97.
58 Stoodley M, Weihl CC, Zhang Z, Lin G, Johns LM, Kowalczuk A, Ghadge G, Roos RP, Macdonald
RL: Effect of adenovirus-mediated nitric oxide synthase gene transfer on vasospasm after experi-
mental subarachnoid hemorrhage. Neurosurgery 2000;46:1193–1203.
59 Khurana VG, Smith LA, Baker TA, Eguchi D, O’Brien T, Katusic ZS: Protective vasomotor effects
of in vivo recombinant endothelial nitric oxide synthase gene expression in a canine model of
cerebral vasospasm. Stroke 2002;33:782–789.
60 Macdonald RL, Weir BKA: A review of hemoglobin and the pathogenesis of cerebral vasospasm.
Stroke 1991;22:971–982.
medwedi.ru
61 Sobey CG, Heistad DD, Faraci FM: Effect of subarachnoid hemorrhage on dilatation of rat basilar
artery in vivo. Am J Physiol 1996;271:H126–H132.
62 Sobey CG, Quan L: Impaired cerebral vasodilator responses to NO and PDE V inhibition after
subarachnoid hemorrhage. Am J Physiol 1999;277:H1718–H1724.
63 Harder DR, Dernbach P, Waters P: Possible cellular mechanism for cerebral vasospasm after
experimental subarachnoid hemorrhage in the dog. J Clin Invest 1987;80:875–880.
64 Zuccarello M, Banosso CL, Lewis AI, Sperelakis N, Rapoport RM: Relaxation of subarachnoid
hemorrhage-induced spasm of rabbit basilar artery by the K⫹ channel activator cromakalim.
Stroke 1996;27:311–316.
65 Sobey CG, Heistad DD, Faraci FM: Effect of subarachnoid hemorrhage on cerebral vasodilatation
in response to activation of ATP-sensitive K⫹ channels in chronically hypertensive rats. Stroke
1997; 28:392–397.
66 Nelson MT, Huang Y, Brayden JE, Hescheler J, Standen NB: Arteriolar dilatations in response to
CGRP involve activation of K⫹ channels. Nature 1990;344:770–773.
67 McCulloch J, Uddman R, Kingman TA, Edvinson L: Calcitonin gene-related peptide: Functional
role in cerebrovascular circulation. Proc Natl Acad Sci USA 1986;83:5731–5735.
68 Kitazono T, Heistad DD, Faraci FM: Role of ATP-sensitive K⫹ channels in CGRP-induced dilatation
of basilar artery in vivo. Am J Physiol 1993;265:H581–H585.
69 Toyoda K, Faraci FM, Watanabe Y, Ueda T, Andresen JJ, Chu Y, Otake S, Heistad DD: Gene transfer
of calcitonin gene-related peptide prevents vasoconstriction after subarachnoid hemorrhage. Circ Res
2000;87:818–824.
70 Satoh M, Perkins E, Kimura H, Tang J, Chu Y, Heistad DD, Zhang JH: Posttreatment with
adenovirus-mediated gene transfer of calcitonin gene-related peptide to reverse cerebral vasospasm
in dogs. J Neurosurg 2002;97:136–142.
71 Treggiari-Venzi MM, Suter PM, Romand J-A: Review of medical prevention of vasospasm after
aneurysmal subarachnoid hemorrhage: A problem of neurointensive care. Neurosurgery 2001;48:
249–262.
72 Morse D, Choi AMK: Heme oxygenase-1. The ‘emerging molecule’ has arrived. Am J Respir Cell
Mol Biol 2002;27:8–16.
73 Suzuki H, Kanamaru K, Tsunoda H, Inada H, Kuroki M, Sun H, Waga S, Tanaka T: Heme
oxygenase-1 gene induction as an intrinsic regulation against delayed cerebral vasospasm in rats.
J Clin Invest 1999;104:59–66.
74 Ono S, Zhang Z-D, Marton LS, Yamini B, Windmeyer E, Johns L, Kowalczuk A, Lin G,
Macdonald RL: Heme oxygenase-1 and ferritin are increased in cerebral arteries after subarach-
noid hemorrhage in monkeys. J Cereb Blood Flow Metab 2000;20:1066–1076.
75 Ono S, Komuro T, Macdonald RL: Heme oxygenase-1 gene therapy for prevention of vasospasm
in rats. J Neurosurg 2002;96:1094–1102.
76 Mori T, Nagata K, Town T, Tan J, Matsui T, Asano T: Intracisternal increase of superoxide anion
production in a canine subarachnoid hemorrhage model. Stroke 2001;32:636–642.
77 Shishido T, Suzuki R, Qian L, Hirakawa K: The role of superoxide anions in the pathogenesis of
cerebral vasospasm. Stroke 1994;25:864–868.
78 Kamii H, Kato I, Kinouchi H, Chan PH, Epstein CJ, Akabane A, Okamoto H, Yoshimoto T:
Amelioration of vasospasm after subarachnoid hemorrhage in transgenic mice overexpressing
CuZn-superoxide dismutase. Stroke 1999;30:867–872.
79 McGirt MJ, Parra A, Sheng H, Higuchi Y, Oury TD, Laskowitz DT, Pearlstein RD, Warner DS:
Attenuation of cerebral vasospasm following subarachnoid hemorrhage in mice overexpressing
extracellular superoxide dismutase. Stroke 2002;33:2317–2323.
80 Nakane H, Chu Y, Faraci FM, Oberley LW, Heistad DD: Gene transfer of extracellular super-
oxide dismutase increases superoxide dismutase activity in cerebrospinal fluid. Stroke 2001;32:
184–189.
81 Watanabe Y, Chu Y, Andresen JJ, Nakane H, Faraci FM, Heistad DD: Gene transfer of extracellu-
lar superoxide dismutase reduces cerebral vasospasm following subarachnoid hemorrhage. Stroke
2003;34:434–440.
82 Zimmerman M, Seifert V: Endothelin and subarachnoid hemorrhage: An overview. Neurosurgery
1998;43:863–876.
83 Chow M, Dumont AS, Kassell NF: Endothelin receptor antagonists and cerebral vasospasm: An
update. Neurosurgery 2002;51:1333–1342.
84 Ohkuma H, Parney I, Megyesi J, Ghahary A, Findlay JM: Antisense preproendothelin-oligoDNA
therapy for vasospasm in a canine model of subarachnoid hemorrhage. J Neurosurg 1999;90:
1105–1114.
85 Ogut O, Brozovich FV: Regulation of force in vascular smooth muscle. J Mol Cell Cardiol
2003;35:347–355.
86 Fujikawa H, Tani E, Yamaura I, Ozaki I, Miyaji K, Sato M, Takahashi K, Imajoh-Ohmi S:
Activation of protein kinases in canine basilar artery in vasospasm. J Cereb Blood Flow Metab
1999;19:44–52.
87 Laher I, Zhang JH: Protein kinase C and cerebral vasospasm. J Cereb Blood Flow Metab 2001;
21:887–906.
88 Sato M, Parent AD, Zhang JH: Inhibitory effect with antisense mitogen-activated protein kinase
oligodeoxynucleotide against cerebral vasospasm in rats. Stroke 2002;33:775–781.
89 de Martin R, Hoeth M, Hofer-Warbinek R, Schmid JA: The transcription factor NF-␬B and the
regulation of vascular cell function. Arterioscler Thromb Vasc Biol 2000;20:e83–e88.
90 Aihara Y, Kasuya H, Onda H, Hori T, Takeda J: Quantitative analysis of gene expressions related to
inflammation in canine spastic artery after subarachnoid hemorrhage. Stroke 2001;32:212–217.
91 Fassbender K, Hodapp B, Rossol S, Bertsch T, Schmeck J, Fritzinger M, Horn P, Vajkoczy P,
Kreisel S, Brunner J, Schmiedek P, Hennerici M: Inflammatory cytokines in subarachnoid haem-
orrhage: Association with abnormal blood flow velocities in basal cerebral arteries. J Neurol
Neurosurg Psychiatry 2001;70:534–537.
92 Mocco J, Mack WJ, Kim GH, Lozier AP, Laufer I, Kreiter KT, Sciacca RR, Solomon RA, Mayer SA,
Connolly ES: Rise in serum soluble intercellular adhesion molecule-1 levels with vasospasm
following aneurysmal subarachnoid hemorrhage. J Neurosurg 2002;97:537–541.
93 Kibbe MR, Billiar TR, Tzeng E: Gene therapy for restenosis. Circ Res 2000;86:829–833.
94 Beebe HG, Kritpracha B: Carotid endoarterectomy versus carotid angioplasty: Comparison of
current results. Semin Vasc Surg 2000;13:109–116.
95 Strandness DE Jr: Screening for carotid disease and surveillance for carotid restenosis. Semin
Vasc Surg 2001;14:200–205.
96 Chakhtoura EY, Hobson RW II, Goldstein J, Simonian GT, Lal BK, Haser PB, Silva MB Jr,
Padberg FT Jr, Pappas PJ, Jamil Z: In-stent restenosis after carotid angioplasty-stenting: Incidence
and management. J Vasc Surg 2001;33:220–226.
97 Simons M, Edelman ER, Dekeyser J-L, Langer R, Rosenberg RD: Antisense c-myb oligonucleotides
inhibit intimal arterial smooth muscle cell accumulation in vivo. Nature 1992;359:67–70.
98 Edelman ER, Simons M, Sirois MG, Rosenberg RD: c-myc in vasculoproliferative disease. Circ Res
1995;76:176–182.
99 Villa AE, Guzman LA, Poptic EJ, Labhasetwar V, D’Souza S, Farrell CL, Plow EF, Levy RJ,
DiCorleto PE, Topol EJ: Effects of antisense c-myb oligonucleotides on vascular smooth muscle
cell proliferation and response to vessel wall injury. Circ Res 1995;76:505–513.
100 Bennet MR, Anglin S, McEwan JR, Jagoe R, Newby AC, Evan GI: Inhibition of vascular smooth
muscle cell proliferation in vitro and in vivo by c-myc antisense oligodeoxynucleotides. J Clin
Invest 1994;93:820–828.
101 Kutryk MJB, Foley DP, van den Brand M, Hamburger JN, van der Giessen WJ, deFeyter PJ,
Bruining N, Sabate M, Serruys PW: Local intracoronary administration of antisense oligonucleotide
against c-myc for the prevention of in-stent restenosis. J Am Coll Cardiol 2002;39:281–287.
102 Morishita R, Gibbons GH, Ellison KE, Nakajima M, Zhang L, Kaneda Y, Ogihara T, Dzau VJ: Single
intraluminal delivery of antisense cdc2 kinase and proliferating-cell nuclear antigen oligonucleotides
results in chronic inhibition of neointimal hyperplasia. Proc Natl Acad Sci USA 1993;90:8474–8478.
103 Morishita R, Gibbons GH, Ellison KE, Nakajima M, von der Leyen H, Zhang L, Kaneda Y,
Ogihara T, Dzau VJ: Intimal hyperplasia after vascular injury is inhibited by antisense cdk 2
kinase oligonucleotides. J Clin Invest 1994;93:1458–1464.
104 Abe J, Zhou W, Taguchi J, Takuwa N, Miki K, Okazaki H, Kurokawa K, Kumada M, Takuwa Y:
Suppression of neointimal smooth muscle cell accumulation in vivo by antisense cdc2 and cdk2
oligonucleotides in rat carotid artery. Biochem Biophys Res Commun 1994;198:16–24.
medwedi.ru
105 Morishita R, Gibbons GH, Kaneda Y, Ogihara T, Dzau VJ: Pharmacokinetics of antisense
oligodeoxyribonucleotides (cyclin B1 and CDC 2 kinase) in the vessel wall in vivo: Enhanced
therapeutic utility for restenosis by HVJ-liposome delivery. Gene 1994;149:13–19.
106 Simons M, Edelman ER, Rosenberg RD: Antisense proliferating cell nuclear antigen oligonu-
cleotides inhibit intimal hyperplasia in a rat carotid artery injury model. J Clin Invest 1994;93:
2351–2356.
107 Zhu NL, Wu L, Liu PX, Gordon EM, Anderson WF, Starnes VA, Hall FL: Downregulation of
cyclin G1 expression by retrovirus-mediated antisense gene transfer inhibits vascular smooth
muscle cell proliferation and neointima formation. Circulation 1997;96:628–635.
108 Chang MW, Barr E, Lu MM, Barton K, Leiden MJ: Adenovirus-mediated over-expression of the
cyclin/cyclin-dependent kinase inhibitor, p21 inhibits vascular smooth muscle cell proliferation
and neointima formation in the rat carotid artery model of balloon angioplasty. J Clin Invest
1995;96:2260–2268.
109 Ueno H, Masuda S, Nishio S, Li J-J, Yamamoto H, Takeshita A: Adenovirus-mediated transfer of
cyclin-dependent kinase inhibitor-p21 suppresses neointimal formation in the balloon-injured rat
carotid arteries in vivo. Ann NY Acad Sci 1997;811:401–411.
110 Luo Z, Sata M, Nguyen T, Kaplan JM, Akita GY, Walsh K: Adenovirus-mediated delivery of Fas
ligand inhibits intimal hyperplasia after balloon injury in immunologically primed animals.
Circulation 1999;99:1776–1779.
111 Chen D, Krasinski K, Chen D, Sylvester A, Chen J, Nisen PD, Andrés V: Down regulation of cyclin-
dependent kinase 2 activity and cyclin a promoter activity in vascular smooth muscle cells by p27KIP,
and inhibitor of neointima formation in the rat carotid artery. J Clin Invest 1997;99:2334–2341.
112 Tsui LV, Camrud A, Mondesire J, Carlson P, Zayek N, Camrud L, Donahue B, Bauer S, Lin A,
Frey D, Rivkin M, Subramanian A, Falotico R, Gyuris J, Schwartz R, McArthur JG: p27-p16
fusion gene inhibits angioplasty-induced neointimal hyperplasia and coronary artery occlusion.
Circ Res 2001;89:323–328.
113 Yonemitsu Y, Kaneda Y, Tanaka S, Nakashima Y, Komori K, Sugimachi K, Sueishi K: Transfer of
wild-type p53 gene effectively inhibits vascular smooth muscle cell proliferation in vitro and
in vivo. Circ Res 1998;82:147–156.
114 Scheinman M, Ascher E, Levi GS, Hingorani A, Shirazian D, Seth P: p53 gene transfer to the
injured rat carotid artery decreases neointimal formation. J Vasc Surg 1999;29:360–369.
115 Smith RC, Branellec D, Gorski DH, Guo K, Perlman H, Dedieu JF, Pastore C, Mahfoudi A,
Denefle P, Isner JM, Walsh K: p21CIP1-mediated inhibition of cell proliferation by overexpres-
sion of the gax homeodomain gene. Gene Dev 1997;11:1674–1689.
116 Mano T, Luo Z, Malendowicz SL, Evans T, Walsh K: Reversal of GATA-6 downregulation
promotes smooth muscle differentiation and inhibits intimal hyperplasia in balloon-injured rat
carotid artery. Circ Res 1999;84:647–654.
117 Chang MW, Barr E, Seltzer J, Jiang Y-Q, Nabel GJ, Nabel EG, Parmacek MS, Leiden JM:
Cytostatic gene therapy for vascular proliferative disorders with a constitutively active form of the
retinoblastoma gene product. Science 1995;267:518–522.
118 Smith RC, Wills KN, Antelman D, Perlman H, Truong LN, Krasinski K, Walsh K: Adenoviral con-
structs encoding phosphorylation-competent full-length and truncated forms of the human retinoblas-
toma protein inhibit myocyte proliferation and neointima formation. Circulation 1997; 96:1899–1905.
119 Claudio PP, Fratta L, Farina F, Howard CM, Stassi G, Numata S, Pacilio C, Davis A, Lavitrano M,
Volpe M, Wilson JM, Trimarco B, Giordano A, Condorelli G: Adenoviral RB2/p130 gene transfer
inhibits smooth muscle cell proliferation and prevents restenosis after angioplasty. Circ Res
1999;85:1032–1039.
120 Wills KN, Mano T, Avanzini JB, Nguyen T, Antelman D, Gregory RJ, Smith RC, Walsh K: Tissue-
specific expression of an anti-proliferative hybrid transgene from the human smooth muscle ␣-actin
promoter suppresses smooth muscle cell proliferation and neointima formation. Gene Ther 2001;8:
1847–1854.
121 Morishita R, Gibbons GH, Horiuchi M, Ellison KE, Nakajima M, Zhang L, Kaneda Y,
Ogihara T, Dzau VJ: A gene therapy strategy using a transcription factor decoy of the E2F binding
site inhibits smooth muscle proliferation in vivo. Proc Natl Acad Sci USA 1995;92:
5855–5859.
122 Dzau VJ, Braun-Dullaeus RC, Sedding DG: Vascular proliferation and atherosclerosis: New
perspectives and therapeutic strategies. Nat Med 2002;8:1249–1256.
123 Guzman RJ, Hirschowitz EA, Brody SL, Crystal RG, Epstein SE, Finkel T: In vivo suppression
of injury-induced vascular smooth muscle cell accumulation using adenovirus-mediated transfer
of the herpes simplex virus thymidine kinase gene. Proc Natl Acad Sci USA 1994;91:
10732–10736.
124 Sata M, Perlman H, Muruve DA, Silver M, Ikebe M, Libermann TA, Oettgen P, Walsh K: Fas ligand
gene transfer to the vessel wall inhibits neointima formation and overrides the adenovirus-mediated
T cell response. Proc Natl Acad Sci USA 1998;95:1213–1217.
125 Pollman MJ, Hall JL, Mann MJ, Zhang L, Gibbons GH: Inhibition of neointimal cell bcl-x expression
induces apoptosis and regression of vascular disease. Nat Med 1998;4:222–227.
126 Tulis DA, Durante W, Liu X, Evans AJ, Peyton KJ, Schafer AI: Adenovirus-mediated heme
oxygenase-1 gene delivery inhibits injury-induced vascular neointima formation. Circulation 2001;
104:2710–2715.
127 Hanna AK, Fox JC, Neschis DG, Safford SD, Swain JL, Golden MA: Antisense basic fibroblast
growth factor gene transfer reduces neointimal thickening after arterial injury. J Vasc Surg 1997;
25:320–325.
128 Sirois MG, Simons M, Edelman ER: Antisense oligonucleotide inhibition of PDGFR-␤ receptor
subunit expression directs suppression of intimal thickening. Circulation 1997;95:669–676.
129 Deguchi J, Namba T, Hamada H, Nakaoka T, Abe J, Sato O, Miyata T, Makuuchi M, Kurokawa K,
Takuwa Y: Targeting endogenous platelet-derived growth factor B-chain by adenovirus-mediated
gene transfer potently inhibits in vivo smooth muscle proliferation after arterial injury. Gene Ther
1999;6:956–965.
130 Iaccarino G, Smithwick LA, Lefkowitz RJ, Koch WJ: Targeting G␤␥ signaling in arterial vascular
smooth muscle proliferation: A novel strategy to limit restenosis. Proc Natl Acad Sci USA 1999;
96:3945–3950.
131 Indolfi C, Avvedimento EV, Rapacciuolo A, Di Lorenzo E, Esposito G, Stabile E, Feliciello A,
Mele E, Giuliano P, Condorelli G: Inhibition of cellular ras prevents smooth muscle cell prolifer-
ation after vascular injury in vivo. Nat Med 1995;1:541–545.
132 Ueno H, Yamamoto H, Ito S, Li J-J, Takeshita A: Adenovirus-mediated transfer of a dominant-neg-
ative H-ras suppresses neointimal formation in balloon-injured arteries in vivo. Arterioscler
Thromb Vasc Biol 1997;17:898–904.
133 Indolfi C, Avvedimento EV, Rapacciuolo A, Esposito G, Di Lorenzo E, Leccia A, Pisani A,
Chieffo A, Coppola A, Chiariello M: In vivo gene transfer: Prevention of neointima formation by
inhibition of mitogen-activated protein kinase kinase. Basic Res Cardiol 1997;92:378–384.
134 Kim S, Izumi Y, Yano M, Hamaguchi A, Miura K, Yamanaka S, Miyazaki H, Iwao H: Angiotensin
blockade inhibits activation of mitogen-activated protein kinases in rat balloon-injured artery.
Circulation 1998;97:1731–1737.
135 Kume M, Komori K, Matsumoto T, Onohara T, Takeuchi K, Yonemitsu Y, Sugimachi K:
Administration of a decoy against the activator protein-1 binding site suppresses neointimal thick-
ening in rabbit balloon-injured arteries. Circulation 2002;105:1226–1232.
136 Nakaoka T, Gonda K, Ogita T, Otawara-Hamamoto Y, Okabe F, Kira Y, Harii K, Miyazono K,
Takuwa Y, Fujita T: Inhibition of rat vascular smooth muscle proliferation in vitro and in vivo by
bone morphogenetic protein-2. J Clin Invest 1997;100:2824–2832.
137 Tulis DA, Mnjoyan ZH, Schiesser RL, Shelat HS, Evans AJ, Zoldhelyi P, Fujise K: Adenoviral
gene transfer of fortilin attenuates neointima formation through suppression of vascular smooth
muscle cell proliferation and migration. Circulation 2003;107:98–105.
138 Rade JJ, Schulick AH, Virmani R, Dichek DA: Local adenoviral-mediated expression of recom-
binant hirudin reduces neointima formation after arterial injury. Nat Med 1996;2:293–298.
139 Atsuchi N, Nishida T, Marutsuka K, Asada Y, Kamikubo Y, Takeshita A, Ueno H: Combination of
a brief irrigation with tissue factor pathway inhibitor (TFPI) and adenovirus-mediated local TFPI
gene transfer additively reduces neointima formation in balloon-injured rabbit carotid arteries.
Circulation 2001;103:570–575.
140 Bennet MR, O’Sullivan M: Mechanisms of angioplasty and stent restenosis: Implications for
design of rational therapy. Pharmacol Ther 2001;91:149–166.
medwedi.ru
141 Dollery CM, Humphries SE, McClelland A, Latchman DS, McEwan JR: Expression of tissue
inhibitor of matrix metalloproteinases 1 by use of an adenoviral vector inhibits smooth muscle cell
migration and reduces neointimal hyperplasia in the rat model of vascular balloon injury.
Circulation 1999;99:3199–3205.
142 Furman C, Luo Z, Walsh K, Duverger N, Copin C, Fruchart JC, Rouis M: Systemic tissue
inhibitor of metalloproteinase-1 gene delivery reduces neointimal hyperplasia in balloon-injured
rat carotid artery. FEBS Lett 2002;531:122–126.
143 Cheng L, Mantile G, Pauly R, Nater C, Felici A, Monticone R, Bilato C, Gluzband YA, Crow MT,
Stetler-Stevenson W, Capogrossi MC: Adenovirus-mediated gene transfer of the human tissue
inhibitor of metalloproteinase-2 blocks vascular smooth muscle cell invasiveness in vitro and
modulates neointimal development in vivo. Circulation 1998;98:2195–2201.
144 Merrilees M, Beaumont B, Scott L, Hermanutz V, Fennessy P: Effect of TGF-␤1 antisense
S-oligonucleotide on synthesis and accumulation of matrix proteoglycans in balloon catheter-
injured neointima of rabbit carotid arteries. J Vasc Res 2000;37:50–60.
145 Welt FGP, Rogers C: Inflammation and restenosis in the stent era. Arterioscler Thromb Vasc Biol
2002;22:1769–1776.
146 Szöcs K, Lassègue B, Sorescu D, Hilenski LL, Valppu K, Couse TL, Wilcox JN, Quinn MT,
Lambeth JD, Griendling KK: Upregulation of nox-based NAD(P)H oxidases in restenosis after
carotid injury. Arterioscler Thromb Vasc Biol 2002;22:21–27.
147 Usui M, Egashira K, Ohtani K, Kataoka C, Ishibashi M, Hiasa K, Katoh M, Zhao Q, Kitamoto S,
Takeshita A: Anti-monocyte chemoattractant protein-1 gene therapy inhibits restenotic changes
(neointimal hyperplasia) after balloon injury in rats and monkeys. FASEB J 2002;16: 1838–1840.
148 Autieri MV, Yue T-L, Ferstein GZ, Ohlstein E: Antisense oligonucleotides to the p65 subunit of
NF-␬B inhibit human vascular smooth muscle cell adherence and proliferation and prevent neoin-
tima formation in rat carotid arteries. Biochem Biophys Res Commun 1995;213: 827–836.
149 Laukkanen MO, Kivelä A, Rissanen T, Rutanen J, Karkkainen MK, Leppanen O, Bräsen JH, Yla-
Herttuala S: Adenovirus-mediated extracellular superoxide dismutase gene therapy reduces neoin-
tima formation in balloon-denuded rabbit aorta. Circulation 2002;106:1999–2003.
150 von der Leyen HE, Dzau VJ: Therapeutic potential of nitric oxide synthase gene manipulation.
Circulation 2001;103:2760–2765.
151 von der Leyen HE, Gibbons GH, Morishita R, Lewis NP, Zhang L, Nakajima M, Kaneda Y,
Cooke JP, Dzau VJ: Gene therapy inhibiting neointimal vascular lesion: In vivo transfer of
endothelial cell nitric oxide synthase gene. Proc Natl Acad Sci USA 1995;92:1137–1141.
152 Janssens S, Flaherty D, Nong Z, Varenne O, van Pelt N, Haustermans C, Zoldhelyi P, Gerard R, Collen
D: Human endothelial nitric oxide synthase gene transfer inhibits vascular smooth muscle cell prolif-
eration and neointima formation after balloon injury in rats. Circulation 1998;97: 1274–1281.
153 Qian H, Neplioueva V, Shetty GA, Channon KM, George SE: Nitric oxide synthase gene therapy
rapidly reduces adhesion molecule expression and inflammatory cell infiltration in carotid arter-
ies of cholesterol-fed rabbits. Circulation 1999;99:2979–2982.
154 Shears LL II, Kibbe MR, Murdock AD, Billiar TR, Lizonova A, Kovesdi I, Watkins SC, Tzeng E:
Efficient inhibition of intima hyperplasia by adenovirus-mediated inducible nitric oxide synthase
gene transfer to rats and pigs in vivo. J Am Coll Surg 1998;187:295–306.
155 Sinnaeve P, Chiche J-D, Gillijns H, Pelt NV, Wirthlin D, van de Werf F, Collen D, Bloch KD,
Janssens S: Overexpression of a constitutively active protein kinase G mutant reduces neointima
formation and in-stent restenosis. Circulation 2002;105:2911–2916.
156 Ueno H, Haruno A, Morisaki N, Furuya M, Kangawa K, Takeshita A, Saito Y: Local expression
of C-type natriuretic peptide markedly suppresses neointimal formation in rat injured arteries
through an autocrine/paracrine loop. Circulation 1997;96:2272–2279.
157 Qian J-Y, Haruno A, Asada Y, Nishida T, Saito Y, Matsuda T, Ueno H: Local expression of C-type
natriuretic peptide suppresses inflammation, eliminates shear stress-induced thrombosis, and pre-
vents neointima formation through enhanced nitric oxide production in rabbit injured carotid
arteries. Circ Res 2002;91:1063–1069.
158 Murakami H, Yayama K, Miao RQ, Wang C, Chao L, Chao J: Kallikrein gene delivery inhibits
vascular smooth muscle cell growth and neointima formation in the rat artery after balloon angio-
plasty. Hypertension 1999;34:164–170.
159 Murakami H, Miao RQ, Chao L, Chao J: Adenovirus-mediated kallikrein gene transfer inhibits
neointima formation via production of nitric oxide in rat artery. Immunopharmacology 1999;44:
137–143.
160 Emanueli C, Salis MB, Chao J, Chao L, Agata J, Lin K-F, Munaò A, Straino S, Minasi A,
Capogrossi MC, Madeddu P: Adenovirus-mediated human tissue kallikrein gene delivery inhibits
neointima formation induced by interruption of blood flow in mice. Arterioscler Thromb Vasc
Biol 2000;20:1459–1466.
161 Numaguchi Y, Naruse K, Harada M, Osanai H, Mokuno S, Murase K, Matsui H, Toki Y, Ito T,
Okumura K, Hayakawa T: Prostacyclin synthase gene transfer accelerates reendothelialization and
inhibits neointimal formation in rat carotid arteries after balloon injury. Arterioscler Thromb Vasc
Biol 1999;19:727–733.
162 Todaka T, Yokoyama C, Yanamoto H, Hashimoto N, Nagata I, Tsukahara T, Hara S, Hatae T,
Morishita R, Aoki M, Ogihara T, Kaneda Y, Tanabe T: Gene transfer of human prostacyclin
synthase prevents neointimal formation after carotid balloon injury in rats. Stroke 1999;30:
419–426.
163 Yamada M, Numaguchi Y, Okumura K, Harada M, Naruse K, Matsui H, Ito T, Hayakawa T:
Prostacyclin synthase gene transfer modulates cyclooxygenase-2-derived prostanoid synthesis and
inhibits neointimal formation in rat balloon-injured arteries. Arterioscler Thromb Vasc Biol 2002;
22:256–262.
164 Asahara T, Chen D, Tsurumi Y, Kearney M, Rossow S, Passeri J, Symes JF, Isner JM: Accelerated
restitution of endothelial integrity and endothelium-dependent function after phVEGF165 gene
transfer. Circulation 1996;94:3291–3302.
165 Yoshimura S, Morishita R, Hayashi K, Kokuzawa J, Aoki M, Matsumoto K, Nakamura T, Ogihara T,
Sakai N, Kaneda Y: Gene transfer of hepatocyte growth factor to subarachnoid space in cerebral
hypoperfusion model. Hypertension 2002;39:1028–1034.
166 Yukawa H, Takahashi JC, Miyatake S-I, Saiki M, Matsuoka N, Akimoto M, Yanamoto H, Nagata I,
Kikuchi H, Hashimoto N: Adenoviral gene transfer of basic fibroblast growth factor promotes
angiogenesis in rat brain. Gene Ther 2000;7:942–949.
167 Lüders JC, Weihl CC, Lin G, Ghadge G, Stoodley M, Roos RP, Macdonald RL: Adenoviral gene
transfer of nitric oxide synthase increases cerebral blood flow in rats. Neurosurgery 2000;47:
1206–1215.
168 Peterson JW, Kwun BD, Hackett JD, Zervas NT: The role of inflammation in experimental cere-
bral vasospasm. J Neurosurg 1990;72:767–774.
169 Handa Y, Kabuto M, Kobayashi H, Kawano H, Takeuchi H, Hayashi M: The correlation between
immunological reaction in the arterial wall and the time course of the development of cerebral
vasospasm in a primate model. Neurosurgery 1991;28:542–549.
170 Laitinen M, Zachary I, Breier G, Pakkanen T, Hakkinen T, Luoma J, Abedi H, Risau W, Soma M,
Laakso M, Martin JF, Yla-Herttuala S: VEGF gene transfer reduces intimal thickening via
increased production of nitric oxide in carotid arteries. Hum Gene Ther 1997;8:1737–1744.
171 Varenne O, Pislaru S, Gillijns H, Van Pelt N, Gerard RD, Zoldhelyi P, Van de Werf F, Collen D,
Janssens P: Local adenovirus-mediated transfer of human endothelial nitric oxide synthase
reduces luminal narrowing after coronary angioplasty in pigs. Circulation 1998;98:919–926.
172 Marshall DJ, Palasis M, Lepore JJ, Leiden JM: Biocompatibility of cardiovascular gene delivery
catheters with adenovirus vectors: An important determinant of the efficiency of cardiovascular
gene transfer. Mol Ther 2000;1:423–429.
173 Nakamura T, Morishita R, Asai T, Tsuboniwa N, Aoki M, Sakonjo H, Yamasaki K, Hashiya N,
Kaneda Y, Ogihara T: Molecular strategy using cis-element ‘decoy’ of E2F binding site inhibits
neointimal formation in porcine balloon-injured coronary artery model. Gene Ther 2002;9:
488–494.
174 Ye Y-W, Landau C, Willard JE, Rajasubramanian G, Moskowitz A, Aziz S, Meidell RS, Eberhart RC:
Bioresorbable microporous stents deliver recombinant adenovirus gene transfer vectors to the arter-
ial wall. Ann Biomed Eng 1998;26:398–408.
175 Klugherz BD, Song C, Defelice S, Cui X, Lu Z, Connolly J, Hinson JT, Wilensky RL, Levy RJ:
Gene delivery to pig coronary arteries from stents carrying antibody-tethered adenovirus. Hum
Gene Ther 2002;13:443–454.
medwedi.ru
176 Porter TR, Xie F: Therapeutic ultrasound for gene delivery. Echocardiography 2001;18:349–353.
177 Porter TR, Hiser WL, Kricsfeld D, Deligonul U, Xie F, Iversen P, Radio S: Inhibition of carotid
artery neointimal formation with intravenous microbubbles. Ultrasound Med Biol 2001;27:
259–265.
178 Taniyama Y, Tachibana K, Hiraoka K, Namba T, Yamasaki K, Hashiya N, Aoki M, Ogihara T,
Yasufumi K, Morishita R: Local delivery of plasmid DNA into carotid artery using ultrasound.
Circulation 2002;105:1233–1239.
179 Waugh JM, Kattash M, Li J, Yuksel E, Kuo MD, Lussier KM, Weinfeld AB, Saxena R,
Rabinovsky ED, Thung S, Woo SLC, Shenaq SM: Gene therapy to promote thromboresistance:
Local overexpression of tissue plasminogen activator to prevent arterial thrombosis in an in vivo
rabbit model. Proc Natl Acad Sci USA 1999;96:1065–1070.
180 Iwaguro H, Yamaguchi J, Kalka C, Murasawa S, Masuda H, Hayashi S, Silver M, Li T, Isner JM,
Asahara T: Endothelial progenitor cell vascular endothelial growth factor gene transfer for vascular
regeneration. Circulation 2002;105:732–738.
181 Hess DC, Hill WD, Martin-Studdard A, Carroll J, Brailer J, Carothers J: Bone marrow as a source
of endothelial cells and NeuN-expressing cells after stroke. Stroke 2002;33:1362–1368.
182 Peng K-W, Russell SJ: Viral vector targeting. Curr Opin Biotechnol 1999;10:454–457.
183 Wickham TJ: Targeting adenovirus. Gene Ther 2000;7:110–114.
184 Pislaru S, Janssens SP, Gersh BJ, Simari RD: Defining gene transfer before expecting gene therapy.
Putting the horse before the cart. Circulation 2002;106:631–636.
Donald D. Heistad, MD
Department of Internal Medicine, University of Iowa College of Medicine
200 Hawkins Dr., Iowa City, IA 52242 (USA)
Tel. ⫹1 319 356 2706, Fax ⫹1 319 353 6343, E-Mail donald-heistad@uiowa.edu
Ex vivo Gene Therapy and Cell

Therapy for Stroke
Douglas Kondziolka a, Jason Sheehanb, Ajay Niranjana
a
Department of Neurological Surgery, University of Pittsburgh, Pittsburgh, Pa.,
b
Department of Neurological Surgery, University of Virginia,
Charlottesville, Va., USA
Introduction
The field of neural transplantation for treatment of neurological diseases

began decades ago when Bjorklund and Stenevi [1] demonstrated that transplan-
tation of dopamine-secreting neurons into the rat striatum improved functionality
in previously damaged nigrostriatal pathways. These results were later expanded
on by other groups [2]. This initial work in cell transplantation spawned addi-
tional research to understand the pathophysiology of neurodegenerative diseases
such as Parkinson’s (PD) and Huntington’s (HD) diseases as well as injury-related
neurological disorders such as ischemia or trauma. Preclinical and clinical stud-
ies have demonstrated proof-of-principle for restorative neurosurgical procedures
that replace missing neurotransmitters and rebuild damaged neuronal circuitry.
Recent advances in molecular biology, cell engineering, and stem cell technology
may afford even more novel approaches for neural transplantation.
Cell and Tissue Transplantation in the Brain
Schmidt et al. [3] first proposed the concept of using dissociated tissue
from the central nervous system for intraparenchymal implantation. In a series
of reports, they demonstrated that cellular suspensions of mesencephalic,
dopaminergic cells could be effectively implanted into rat parenchyma, sur-
vive, and promote regeneration [4–8]. PD, which is a complex neurological
disorder characterized in part by degeneration of dopaminergic neurons in
the substantia nigra par compacta, became the first widely applied model for
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neural transplantation. The successes and failures of neural transplantation with
regard to PD have yielded valuable therapeutic strategies for other neurological
diseases. For instance, neurotransplantation research has been extended to other
neurodegenerative diseases such as HD and to spinal cord regeneration and
posthypoxic injury. All of these neurological conditions share common chal-
lenges related to neurotransplantation: (1) selection of an appropriate and read-
ily available source of tissue; (2) refinement of tissue preparation protocols;
(3) development of safe and effective techniques for surgical implantation of
cells and tissue, and (4) creation of a suitable environment in vivo, which
fosters survival of transplanted neuronal tissue and restoration of damaged neu-
ronal connections.
Tissue Sources and Preparation for Neuronal Implantation
Finding a suitable source for neuronal implants poses both scientific and eth-
ical challenges. Prior to 1988, researchers were free to conduct federally financed
research using fetal tissue sources. However, research on human fetal tissue trans-
plants using material derived from elective abortions was banned from National
Institutes of Health (NIH) funding during the administration of President Ronald
Reagan due to the pressure from conservative religious groups, which influenced
scientific policy. Despite the ban on federal funding in human subjects, research
continued in animal models, and private funding also ensured that transplanta-
tions continued. In 1993, with groups from around the world reporting promising
results with fetal cell implantation and a change to a Democratic administration,
which lifted the NIH funding ban, fetal neurotransplantation research in human
subjects was allowed to proceed in the USA. Much has been written about ethi-
cal issues surrounding fetal tissue research; the facts remain that fetal tissue
research has a long track record of success and is legal [9–13].
One major problem of donated tissue is that it is often not suitable for in
vivo use. In a recent examination of approximately 1,500 embryonic donors in
five NIH-funded tissue banks, only seven samples were believed to be suitable
for transplantation in PD patients [14]. Other studies have indicated that multi-
ple donors or first-trimester embryonic tissues are not absolute criteria for suc-
cessful human grafting [15–17]. The cryopreservation of tissue is believed to be
less favorable for long-term neuronal survival and maturation [18]. Fetal cell
aggregates in long-term suspension cultures are another viable tissue culturing
protocol that has been explored. Logistically and economically, cryopreserved
cell lines are the most favorable approach and have been shown to be effective
in a stroke transplantation trial at the University of Pittsburgh [19, 20]. Despite
the limited number of suitable embryonic donor tissue in NIH-funded banks,
Kondziolka/Sheehan/Niranjan 440
a degree of optimism is warranted with the stem cell and neuronal precursor
line research that may soon replace primary human brain tissue as the major
sources of donor cells, unless further legislation impairs access to that material.
Prior to implantation of cells, in vitro processing is beneficial. Cell cultur-
ing techniques permit the selection of a viable and well characterized subset of
neuronal cells for implantation. Glial-neuronal interactions and neurotrophin
exposure in vitro can help to protect grafted cells from detrimental host response
and promote long-term survival in vivo [21, 22]. Some grafted cells have been
shown to undergo apoptosis or programmed cell death mediated by caspase
activity within the first 15 days postimplantation [23, 24]. Therefore, in vitro
inhibition of caspase activity prior to implantation or other ‘conditioning’ could
lead to decreased apoptosis and, in turn, improved overall viability of grafts [25].
Immunosuppression and Overall Success of the Graft
The need for either short- or long-term immunosuppression has been the
subject of much debate in the neural transplantation field. The necessity and
duration of immunosuppression are generally a function of the degree of graft-
versus-host response induced by the implanted tissue. Most agree that xenografts
require long-term immunosuppression. However, in 1991, Henderson et al. [18]
demonstrated that long-term clinical benefits were afforded in 12 advanced PD
patients who underwent graft implantation despite the absence of immunosup-
pression. Although human and primate studies of allogeneic transplants do not
induce immunity, definitive rejection has not been demonstrated [26–30]. Others
argue that immunosuppression may affect symptoms associated with PD and
other neurological conditions [30]. The annual cost (approximately USD 10,000)
and 1–10% per year complication risk associated with immunosuppression
must be factored into the equation [31]. Despite the cost, risks, and equivocal
research results, most researchers still favor a short-term course of immuno-
suppression with agents such as cyclosporine (5–10 mg/kg) and corticosteroids
[32]. Little is known about the utility of other immunosuppression agents such
as tacrolimus for brain transplantation [33].
The reaction of the host’s cells to the graft is critical in terms of the graft’s
survival, integration, and functionality. Research in murine transplant models
has suggested that a portion of the functional benefit of fetal transplantation may
be due to the host’s reaction alone or the surgical procedure itself. Release of
neurotrophic factors by the host parenchyma may enhance the surrounding neu-
ropil. Host sprouting adjacent to a dopaminergic graft has been demonstrated to
be quite substantial [34, 35]. Bankiewicz et al. [36] have demonstrated clinical
improvement in hemiparkinsonian monkeys after a surgical procedure that
Neural Transplantation for Stroke 441
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resulted in cavitation alone. As such, the regenerative capacity of the host
parenchyma may play a considerable role in the degree of functional recovery
achieved following neuronal grafting.
The microglial response also plays a role in the overall success or failure
of the graft. Microglia contribute to the immune-mediated response of the host
by functioning as antigen-presenting cells [37]. The role of microglia is impor-
tant not just following the brain injury or development of intracranial pathology
but also for the functioning of the brain under normal conditions [38]. Microglia
have been shown to produce growth factors and trigger a regenerative cascade
following central nervous system injury [39–43]. In fact, through the secre-
tion of neurotrophins, microglia can promote the survival and development of
TH-positive neurons in vitro and nigrostriatal dopaminergic neurons in vivo
[44–46]. Thus, the host’s neuroglial response plays a pivotal role in the graft’s
survival, differentiation, and function.
Cerebral Infarction as a Model for Cell Transplantation
Stroke is the third leading cause of death and the most common cause of
lasting disability in the USA [47]. The incidence of strokes in the USA is
approximately 750,000 cases per year [47]. One third of the strokes are fatal and
the majority of survivors demonstrate significant residual impairments [48, 49].
As the population shifts to an older median age and people live longer as a
result of improved therapies for other diseases, the prevalence of stroke-related
morbidity and mortality will increase. Recent research and clinical efforts have
focused on developing and refining treatments for acute stroke with a typically
narrow peri-ischemic window for the administration of therapeutic agents. At
present, no effective treatment for chronic stroke patients with fixed neurolog-
ical deficits is available.
Transplantation of neural cells into the stroke penumbra has been evalu-
ated by a number of investigators as an approach for ameliorating functional
deficits [50]. The two most common models of brain ischemia are the murine
hippocampal stroke model, which produces well-defined lesions particularly in
the CA1 region, and the middle cerebral artery occlusion model in the rat
[51–55]. A number of reports using both models have demonstrated survival
and integration of cortical transplants within infarcted regions [52, 56–59].
Moreover, stroke models have shown improved function following neuronal
transplantation [60–66].
Treatment of human cortical stroke by transplant has been discouraging
because of the heterogeneous make-up and the large volume of neuronal tissue
affected [67]. However, as a result of the successes with transplantation for
neurodegenerative diseases (e.g., PD and HD), some consensus exists that
transplantation has potential for treatment of striatal stroke [67].
Use of a Human Neuronal Cell Line for Neurotransplantation
The logistical difficulties inherent in obtaining large quantities of human

fetal neurons stimulated a search for alternative sources of tissue for transplan-
tation. One such source of tissue is the human teratocarcinoma-initiated neuronal
cell line (NT2). Andrews et al. [68] initially established this nontumorigenic,
embryonic cell line from a human teratocarcinoma. Treatment of NT2 cells with
retinoic acid causes differentiation into a mature neuronal phenotype, similar
in morphology and behavior to terminally differentiated, postmitotic neurons
[69, 70]. Following transplantation, NT2 cells demonstrate survival, formation of
synaptic processes, expression of neurotransmitters, and integration into the host’s
parenchyma [70–72]. In particular, the cells express three neurofilament proteins
(NFL, NFM, and NFH); microtubule-associated protein 2; glial-derived neuro-
trophic factor (GDNF); and possibly the axonal protein tau [Borlongan, pers.
commun.]. As a result of the neuronal phenotype and virtually unlimited supply,
NT2 cells are a useful source for neurotransplantation in animal models. NT2
cells have the following advantages: (1) they do not harbor known pathogens or
infectious agents potentially present in allografts or xenografts; (2) they have been
extensively characterized in vitro; (3) they are amenable to genetic engineering,
and (4) they have been utilized in animal models for PD and trauma [73–76].
The notion of restoring function postischemic injury by transplanting
human neuronal cells has attracted a number of proponents [67]. There is evi-
dence for the feasibility of this approach; for example, fetal tissue transplantation
in a rat model of transient focal cerebral ischemia demonstrated a restoration of
behavioral and motor functions [77]. Borlongan et al. [50, 67] reported that
transplantation of neuronally differentiated NT2 cells could ameliorate the
deficits following stroke. These preclinical studies were conducted in a transient,
focal ischemic model rather than a global one, so as to maximize the chances
of functional recovery. Animals treated with NT2 neurons and cyclosporine
demonstrated improvement in behavioral deficits during a 6-month window of
observation and evaluation. In particular, animals treated with NT2 neurons
exhibited recovery in the passive avoidance test (i.e., behavioral assessment)
and elevated body swing test (i.e., motor assessment) [78, 79]. Control groups
receiving fetal rat cerebellar cells, cell culture medium alone, or cyclosporine,
failed to demonstrate significant behavioral improvement. A similarly favorable
behavioral result following NT2 neuronal transplantation was seen in a middle
cerebral artery occlusion (MCAO) rat model [80].
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In view of these results, the initial objectives in a 12-patient Phase I clini-
cal trial performed at the University of Pittsburgh were to demonstrate the safety
of neuronal-cell transplantation in immunosuppressed striatal stroke patients
[81, 82]. In a 24-month follow-up period, no adverse events related to the
implantation occurred. A postmortem examination of one patient from this study
who died of unrelated causes clearly demonstrated NT2 neuron survival 27
months after surgery and the absence of any adverse brain parenchymal effects
[20]. The Phase I clinical stroke study also provided some evidence of possible
functional recovery from stroke-related neurological deficits following neuronal
cell transplantation. The mean National Institutes of Health Stroke Scale score
decreased and the mean European Stroke Scale total scores increased, suggest-
ing improvement in transplanted groups. Furthermore, total European Stroke
Scale scores and composite motor subscale scores of the European Stroke Scale
increased to a larger extent in the 4 patients who received 6 million cell implan-
tation as compared to those receiving only 2 million cells. The Barthel Index and
SF-36 scores decreased in the group receiving 2 million cells and increased in
the group receiving 6 million cells. Overall, in the group receiving 6 million
cells, outcome measurements demonstrated some improvement in stroke-related
deficits [82].
More recently, serial [18F] fluorodeoxyglucose positron emission tomogra-
phy after human neuronal implantation for stroke has been conducted. Fluoro-
deoxyglucose positron emission tomography can be utilized to map metabolic
brain response at the site of cell implantation. In that same group of 12 patients
who underwent implantation of human neuronal cells, fluorodeoxyglucose
positron emission tomography imaging was performed 1 week, 6 months, and
12 months postoperatively [19, 82]. Increased glucose metabolic activity in the
stereotactic target and surrounding tissue was noted at 6 and 12 month time-
points; this increase correlated with improved motor performance scores [19].
Thus, it would appear that enhanced cellular function at the implantation site
occurred in some patients and correlated with improved neurological function.
Although the Phase I clinical stroke trial at the University of Pittsburgh
demonstrated encouraging preliminary results, many questions remain unan-
swered and new questions have arisen. Underlying neuronal mechanisms that led
to improved neurological function have yet to be established, and could be non-
specifically related to cell grafting. For example, instrumentation of the striatum
or use of immunosuppressive drugs may have contributed to the observed
effects. Some possible cellular mechanisms for neurological improvement
include the following: neurotrophin secretion by grafted cells; increased neuro-
transmitter production by resident cells; re-establishment of local interneuronal
connections in response to grafting; improvement in regional oxygen tension
in response to grafting; or reduction in glial reactivity in response to grafting.
A two-center, Phase 2 (dose response) trial is underway to evaluate the utility of
neurotransplantation for patients who have suffered basal ganglia infarcts.
Eighteen patients have received either 5 or 10 million cells delivered into twenty-
five implants along five trajectories. Four additional patients were assigned to an
observational control group. All patients received a focused stroke rehabilitation
program. Further studies are planned to examine the role of immunosuppression,
patient age, severity of neurological deficit, number of implants and implanted
cells, choice of outcome measures, and randomization schemes.
Cellular Support for Graft Survival and Axonal Reconnection
One of the most important factors in neuronal survival and integration

of the graft is the creation of a receptive milieu in the host. In 1951, Levi-
Montalcini [83] first presented her work on growth factors at the New York
Academy of Sciences. While working for Viktor Hamburger, Levi-Montalcini
had grafted a malignant mouse tumor into a developing embryo. She noted that
nerve fibers had emerged not only from an adjacent sensory ganglia but even
more from the sympathetic ones. These nerve fibers had extended chaotically
and thereby completely invaded both the neoplastic tumor tissue and practically
all of the embryo’s organs [84]. The humoral factor secreted by the tumor was
eventually characterized as nerve growth factor (NGF) [85]. NGF was the first
member of a family of endogenous growth factors. Many of these growth fac-
tors or neurotrophins play a role in the central and peripheral nervous system
under normal and pathological conditions. Neurotrophins interact with cells
through high affinity, tyrosine kinase receptors (trk A, trk B) or lower affinity
p75 receptor [86]. The broad extent of neurotrophin functional roles and their
complex cell signaling pathways make them a continued focus of research
efforts. Depending upon the cell type and conditions, a particular neurotrophin
may have either pro- or anti-apoptotic cellular responses [87].
Neurotrophins have been the subject of studies to evaluate their therapeu-
tic role in neurodegenerative diseases [88–90]. Growth factors are crucial for
the development and survival of neural progenitor cells in both the central and
peripheral nervous system [91]. Even factors that were once thought to be neu-
rotoxic (e.g., tumor necrosis factor) have been shown to have neuroprotective
effects under certain circumstances [92]. Neurotrophins have been used as
neuroprotectants to slow, halt, or reverse the progression of neuronal degenera-
tion [90]. They also have been utilized to enhance survival of neuronal and
non-neuronal grafts [90]. Unfortunately, neurotrophins themselves do not pass
the blood-brain barrier in significant amounts. Several novel techniques have
been utilized to circumvent this problem and include grafting of cells which
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secrete selective neurotrophins; stereotactic intraparenchymal or intraventricu-
lar delivery of neurotrophins; conjugation of neurotrophins to antibodies and
delivery via receptor-mediated transport; peripheral administration of bioactive
neurotrophin subunits [93–95].
In brain transplantation, neurotrophins have favorably influenced the
survival, integration, and neurotransmitter expression of grafted and host
cells. Treatment of nigral dopaminergic neurons or fetal cerebral cortex with
brain-derived neurotrophic factor (BDNF) was found to improve the functional
performance and neuronal graft survival in a rat Parkinsonian model [96].
Interestingly, BDNF produced by dorsal interneurons was found to stimulate pro-
liferation and differentiation of motor neuron precursors after anterograde trans-
port [97]. Neurotrophin-3 enhanced survival of dopaminergic neurons in striatal
grafting of fetal mesencephalic cells [98]. Glial cell line-derived neurotrophic fac-
tor (GDNF) not only promoted survival, but also the sprouting of fetal transplants
in PD models [98–101]. Unfortunately, results of the intraventricular delivery of
GDNF in the treatment of PD has been discouraging [102]. NGF-supplemented
cellular grafts can prevent degeneration of basal forebrain cholinergic neurons,
increase the number of neurons, and stimulate the neuronal maturation [103–105].
The trophic effects of NGF-secreting stem cells prevented degeneration of vul-
nerable cholinergic striatal neurons in a rodent model of HD [106].
Neurotrophins could also be delivered following a stroke in order to com-
pensate for the death or impairment of ischemic cells. Using a transient middle
cerebral artery rat stroke model, Li et al. [107] demonstrated that neurological
improvement resulting from human marrow stromal intravenous therapy was
most likely derived from the increased production of NGF and BDNF. In a sim-
ilar animal model of stroke, overexpression of NGF, BDNF, GDNF, and ciliary
neurotrophic factor through recombinant adeno-associated viral gene transfer
was found to reduce neuronal death and lead to functional sparing [108, 109].
As noted previously, the neuron-like cells utilized in the stroke trial at the
University of Pittsburgh secrete GDNF, which may have contributed to the
favorable results observed thus far in terms of both graft survival and improved
functional outcome. Delivery of neurotrophins themselves appears to be neuro-
protective following infarction in other animal models [110–113]. Fibroblast
growth factor gene expression was observed to be upregulated within 3 days of
a focal cerebral infarction in rats [114]. Moreover, exogenous administration of
fibroblast growth factor after infarction reduced infarct size and enhanced func-
tional recovery [114].
Apoptosis or programmed cell death, which plays a major role in neuronal
development, may also occur in neurodegenerative diseases such as Alzheimer’s
and PD [115, 116]. In animal models of stroke, apoptosis has been implicated
in neuronal loss following ischemic injury [117, 118]. Neurotrophins appear to
attenuate the apoptotic signaling pathways, and may have value in this context.
Anti-apoptotic signaling activates transcription factors (e.g., nuclear factor-␬B)
and induces expression of stress proteins, antioxidant enzymes, calcium regu-
latory agents, ion channel modulators, and mitochondrial stabilizing proteins
(e.g., Bcl-2) [119]. Neurotrophins may have value in the treatment of neuronal
injury or loss from ischemic or degenerative conditions.
Gene Therapy for Ischemia with Genes for Neurotrophic Factors
Gene delivery systems using nonviral and viral vectors have been reported
for the treatment of genetic diseases, cancers, and other conditions [120, 129,
130]. Replication-defective herpes simplex virus (HSV) type 1 was among the
first vectors to be used in experimental gene transfer into the brain [121–123].
Retroviral vectors also were developed and tested for this purpose [124, 125].
However, many retroviral (nonlentiviral) vectors do not infect nondividing cells
such as neurons, and may be associated with risks of mutagenesis and tumoro-
genesis. [126, 127]. Replication-defective lentivirus can transduce a gene to
nondividing cells and affords long-term expression, but retains the potential to
cause insertional mutagenesis [128].
To deliver neurotrophic factor protein in ischemic brain, Kitagawa et al. [131]
chose an adenovirus vector containing the GDNF gene (Ad-GDNF). The protec-
tive effect of the Ad-GDNF transfer was examined after transient MCAO in the rat.
That study demonstrated reduction in infarct volume in the animals that were
pretreated with Ad-GDNF 24 h before a subsequent 90 min of transient MCAO.
The reduction in the infarct size was not associated with changes in regional
cerebral blood flow, as compared with the vehicle or Ad-LacZ animal groups.
Immunohistological analyses showed that treatment with Ad-GDNF greatly
reduced the number of TUNEL, caspase-3, and cytochrome c-positive cells. No
leukocyte infiltration was detected in Ad-GDNF-treated rats. Inhibition of the
cytosolic release of cytochrome c by Ad-GDNF suggests a target of protection in
an apoptotic pathway through cytochrome c and caspase-3 in the penumbra [132].
In another study Casper et al. [133] performed ex vivo transduction of
neural cell aggregates with replication defective HSV containing cDNA of
human vascular endothelial growth factor (HSVhvegf) and transplanted the
cells into the rat brain. They demonstrated a dose-dependent increase in blood
vessel density within transplants transduced with HSVvegf. The transplants
were vascularized at a faster rate for up to 4 weeks and the size of HSVvegf
transplant was twice that of control at 8 weeks. The rate of vascularization may
be a major factor in survival of transplanted cells into the brain and can be
improved by ex vivo transduction with HSVvegf.
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Several other gene transfer studies for cerebral ischemia have been per-
formed in animal models; for example, studies of the protective effects of
adenovirus-mediated neuronal apoptosis inhibitory protein and an interleukin-1
receptor antagonist against ischemic brain injury [134, 135]. HSV-mediated
HSP72 or Bcl-2 gene transfer also showed an improvement in focal ischemia-
induced brain damage [135–139]. These viral vectors have certain disadvan-
tages such as toxicity and low efficacy of gene expression; development of
other vectors is necessary for in vivo gene therapy approaches in patients.
Potential of Neural Stem Cell Transplantation

to the Ischemic Brain
Several studies have been performed to transplant neural cells into the
ischemic brain. For example, fetal rat hippocampal neurons were stereotaxi-
cally transplanted into 5-day-old ischemic hippocampal CA1 lesions of the
adult rat [52]. In the recipient brain, the transplanted cells survived well and
formed clusters in the host CA1 subfield at 14 or 100 days after the transplan-
tation. Fetal CA1 grafts also promoted recovery from cognitive deficits in mar-
mosets induced by excitotoxic damage to the CA1 field [140]. Li et al. [141]
used hematopoietic myeloid progenitor cells (1 ⫻ 105 per ␮l) cultured from
adult mouse bone marrow to implant into the penumbra of the ischemic stria-
tum at 4 or 24 h after 3 h of transient MCAO in mice. In another experiment,
fresh bone marrow cells (1 ⫻ 106 per 10 ␮l), including the supportive stromal
tissue obtained directly from adult rats, was grafted into the penumbra of the
ischemic striatum at 4 or 24 h after 2 h of transient MCAO in rats. The adult
hematopoietic bone marrow cells survived in the adult mouse and rat brains
after ischemia.
In other experiments, neuroepithelial stem cell lines (MHP36) were genet-
ically engineered with a temperature-sensitive gene so that the cells would repli-
cate at 33⬚C, but cease replication and differentiate into neurons or glia at the
body temperature of 37⬚C [142]. With this cell line (MHP36), Hodges et al.
[143], Virley et al. [144] showed functional recovery in adult rats after 15 min of
global ischemia, and also found an extensive repopulation of the grafted cells
into neuronal and glial phenotypes in the hippocampus. In further studies
MHP36 stem cell restored cognitive function in marmosets after NMDA-
induced excitotoxic hippocampal CA1 lesions as effectively as fetal homografts.
Marrow stromal cells (MSCs) have also been used in experimental stroke
models. MSCs also have the capacity to pass through the blood-brain barrier
and migrate throughout the forebrain and cerebellum, and they differentiate
into astrocytes and neurons after injection into neonatal mouse brain [145].
Transplantation of adult MSCs directly into the adult rat brain and spinal cord
has resulted in the reduction of functional deficits associated with stroke [146],
traumatic brain injury, and spinal cord injury [147]. MSCs can form neural phe-
notypes and migrate when placed in damaged brain [146, 147].
The route of administration is an important aspect of genetic and cellular
therapies for stroke. Direct injection of vectors into the brain parenchyma or
ventricle is one option that has been tested extensively, but less invasive proce-
dures would be preferable if possible. To test whether the intravenous route
could be used to send MSCs into the brain to reduce neurological functional
deficits after stroke in rats, Chen et al. [148] performed an intravenous infusion
of bone marrow derived-MSCs. These investigators subjected rats to 2 h of
MCAO and treated the animals with infusion of MSCs. They demonstrated sig-
nificant recovery of somatosensory behavior and Neurological Severity Score
(p ⬍ 0.05) in animals infused with 3 ⫻ 106 MSCs at day 1 or 7 days after MCAO
compared with control animals. Morphological analysis of the tissue indicated
that BrdU-labeled MSCs were more likely to enter into damaged brain than into
contralateral nonischemic brain. Many MSCs survived, and a few cells expressed
protein markers for parenchymal cells.
Neural stem cells have wide appeal as vectors for gene transfer, and may
represent the next logical step in clinical trials for stroke treatment. The stem
cell possesses the potential of self-renewal as well as multidirectional differen-
tiation. Stem cells are influenced by neurotrophic factors to differentiate into a
given neural lineage. It is expected that neural stem cells have the potential to
compensate and recover neural functions that were lost because of ischemic
stroke. Neural stem cells are believed to be present even in mature adult mam-
malian brain [149, 150], and could be directed toward expansion and self-repair
if the proper stimuli could be applied.
Immortalized multipotent neural stem and progenitor cells are another pre-
ferred source of tissue for gene manipulation and ex vivo gene transfer to the
brain [151]. These cells can be genetically transduced ex vivo (e.g., with growth
factors) and maintained as cell lines in culture. Targeted intracerebral delivery
of neurotrophic factors has emerged as an interesting therapeutic strategy to
repopulate an ischemic area of the brain. The concept behind immortalization
is to halt the program of development by inducing the cell in a mode of contin-
uous cell cycle. The most common method of immortalizing cells is to introduce
an oncogene such as large T antigen. tsA58 is a temperature-sensitive mutated
allele of the large T antigen that is stable at 33⬚C in cultured cells but degrades
at body temperature (37–39⬚C). The resultant conditionally immortalized
neural progenitors have the ability to differentiate and integrate after implanta-
tion into adult brain. Martinez-Serrano and Bjorklund [151] studied protection
of ischemia-induced striatal cell death using transplants of conditionally
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immortalized NGF-secreting H1B5 cells. They demonstrated that NGF-secret-
ing H1B5 cell implantation resulted in the reduction of the effect of subsequent
MCAO. The results obtained with the NGF-secreting H1B5 cell line have stim-
ulated the development of procedures for ex vivo gene transfer of other CNS-
active neurotrophic factors, such as BDNF, neurotrophin-3, NT-4/5, ciliary
neurotropic factor and GDNF.
Conclusions
Tremendous accomplishments in neuroscience, molecular biology, and

genetics provide a solid foundation for neurotransplantation. Restorative neuro-
surgical procedures will continue to evolve as advances are made in these fields.
Selection of a suitable graft material, safe and effective implantation techniques,
and establishment of a hospitable milieu for graft survival and integration will be
necessary to achieve optimal patient outcomes. Cerebral infarction and neurode-
generative disorders are appropriate initial candidates for neurotransplantation
research.
In future, it may be possible to employ multiple gene therapies strategy
targeted to specific areas in the ischemic brain. Neurons may be protected by
virus-mediated gene expression of neurotrophic factors, and angiogenic genes
such as VEGF or bFGF may be used to generate new vessels and improve col-
lateral circulation. Ex vivo-manipulated stem cells could be transplanted into
the ischemic lesion for integration and differentiation into neurons, to assist
with restoring neuronal density to the ischemic or infarcted brain.
References
1 Bjorklund A, Stenevi U: Reformation of the severed septohippocampal cholinergic pathway in the

adult rat by transplanted septal neurons. Cell Tissue Res 1977;185:289–302.
2 Perlow MJ, Kumakura K, Guidotti A: Prolonged survival of bovine adrenal chromaffin cells in rat
cerebral ventricles. Proc Natl Acad Sci USA 1980;77:5278–5281.
3 Schmidt RH, Bjorklund A, Stenevi U: Intracerebral grafting of dissociated CNS tissue suspensions:
A new approach for neuronal transplantation to deep brain sites. Brain Res 1981;218:347–356.
4 Gage FH, Bjorklund A, Stenevi U, Dunnett SB: Intracerebral grafting of neuronal cell suspen-
sions. VIII. Survival and growth of implants of nigral and septal cell suspensions in intact brains
of aged rats. Acta Physiol Scand Suppl 1983;522:67–75.
5 Bjorklund A, Stenevi U, Schmidt RH, Dunnett SB, Gage FH: Intracerebral grafting of neuronal
cell suspensions. II. Survival and growth of nigral cell suspensions implanted in different brain
sites. Acta Physiol Scand Suppl 1983;522:9–18.
6 Bjorklund A, Gage FH, Schmidt RH, Stenevi U, Dunnett SB: Intracerebral grafting of neuronal
cell suspensions. VII. Recovery of choline acetyltransferase activity and acetylcholine synthesis
in the denervated hippocampus reinnervated by septal suspension implants. Acta Physiol Scand
Suppl 1983;522:59–66.
7 Bjorklund A, Gage FH, Stenevi U, Dunnett SB: Intracerebral grafting of neuronal cell suspensions.
VI. Survival and growth of intrahippocampal implants of septal cell suspensions. Acta Physiol
Scand Suppl 1983;522:49–58.
8 Bjorklund A, Stenevi U, Schmidt RH, Dunnett SB, Gage FH: Intracerebral grafting of neuronal
cell suspensions. I. Introduction and general methods of preparation. Acta Physiol Scand Suppl
1983;522:1–7.
9 Grisolia JS: CNS stem cell transplantation: Clinical and ethical perspectives. Brain Res Bull 2002;
57:823–826.
10 De WG, Berghmans RL, Boer GJ, Andersen S, Brambati B, Carvalho AS, Dierickx K, Elliston S,
Nunez P, Osswald W, Vicari M: Ethical guidance on human embryonic and fetal tissue transplan-
tation: A European overview. Med Health Care Philos 2002;5:79–90.
11 Sparks RC: Ethical issues of fetal tissue transplantation: Research, procurement, and complicity
with abortion. Annu Soc Christ Ethics 1990:199–221.
12 McGee G, Caplan A: The ethics and politics of small sacrifices in stem cell research. Kennedy
Inst Ethics J 1999;9:151–158.
13 Robertson JA: Ethics and policy in embryonic stem cell research. Kennedy Inst Ethics J 1999;9:
109–136.
14 Branch DW, Ducat L, Fantel A, Low WC, Zhou FC, Dayton DH, Gill TJ 3rd: Suitability of fetal
tissues from spontaneous abortions and from ectopic pregnancies for transplantation. Human Fetal
Tissue Working Group. JAMA 1995;273:66–68.
15 Sladek JR Jr, Collier TJ, Elsworth JD, Roth RH, Taylor JR, Redmond DE Jr: Intrastriatal grafts
from multiple donors do not result in a proportional increase in survival of dopamine neurons in
nonhuman primates. Cell Transplant 1998;7:87–96.
16 Achim CL, Miners DK, Burrola PG, Martin FC, Wiley CA: In vivo model of HIV infection of the
human brain. Dev Neurosci 1993;15:423–432.
17 Sanders VJ, Mehta AP, White MG, Achim CL: A murine model of HIV encephalitis:
Xenotransplantation of HIV-infected human neuroglia into SCID mouse brain. Neuropathol Appl
Neurobiol 1998;24:461–467.
18 Henderson BT, Clough CG, Hughes RC, Hitchcock ER, Kenny BG: Implantation of human fetal
ventral mesencephalon to the right caudate nucleus in advanced Parkinson’s disease. Arch Neurol
1991;48:822–827.
19 Meltzer CC, Kondziolka D, Villemagne VL, Wechsler L, Goldstein S, Thulborn KR, Gebel J,
Elder EM, DeCesare S, Jacobs A: Serial [18F] fluorodeoxyglucose positron emission tomography
after human neuronal implantation for stroke. Neurosurgery 2001;49:586–592.
20 Nelson PT, Kondziolka D, Wechsler L, Goldstein S, Gebel J, DeCesare S, Elder EM, Zhang PJ,
Jacobs A, McGrogan M, Lee VM, Trojanowski JQ: Clonal human (hNT) neuron grafts for stroke
therapy: Neuropathology in a patient 27 months after implantation. Am J Pathol 2002;160:
1201–1206.
21 Bronstein DM, Perez-Otano I, Sun V, Mullis Sawin SB, Chan J, Wu GC, Hudson PM, Kong LY,
Hong JS, McMillian MK: Glia-dependent neurotoxicity and neuroprotection in mesencephalic
cultures. Brain Res 1995;704:112–116.
22 White MG, Hammond RR, Sanders VJ, Bonaroti EA, Mehta AP, Wang G, Wiley CA, Achim CL:
Neuron-enriched second trimester human cultures: Growth factor response and in vivo graft sur-
vival. Cell Transplant 1999;8:59–73.
23 Mahalik TJ, Hahn WE, Clayton GH, Owens GP: Programmed cell death in developing grafts of
fetal substantia nigra. Exp Neurol 1994;129:27–36.
24 D’Sa-Eipper C, Roth KA: Caspase regulation of neuronal progenitor cell apoptosis. Dev Neurosci
2000;22:116–124.
25 Schierle GS, Hansson O, Leist M, Nicotera P, Widner H, Brundin P: Caspase inhibition reduces
apoptosis and increases survival of nigral transplants. Nat Med 1999;5:97–100.
26 Ansari AA, Mayne A, Freed CR, Breeze RE, Schneck SA, O’Brien CF, Kriek EH, Zhang YB,
Mazziotta JC, Hutchinson M, Schroter G, Bakay RAE, Boyer K, Sundstrom JB: Lack of detectable
systemic humoral/cellular allogeneic response in human and nonhuman primate recipients of
embryonic mesencephalic allografts for the therapy of Parkinson’s disease. Transplant Proc 1995;
27:1401–1405.
medwedi.ru
27 Bakay RAE, Boyer KL, Freed CR, Ansari AA: Immunological responses to injury and grafting in
the central nervous system of nonhuman primates. Cell Transplant 1998;7:109–120.
28 Freed CR, Breeze RE, Schneck SA, Bakay RAE, Ansari AA: Fetal neural transplantation for
Parkinson’s disease; in Rich RR (ed): Clinical Immunology: Principles and Practice. St. Louis,
Mosby-Year Book, 1995, pp 1677–1687.
29 Lindvall O, Sawle G, Widner H, Rothwell JC, Bjorklund A, Brooks D, Brundin P, Frackowiak R,
Marsden CD, Odin P: Evidence for long-term survival and function of dopaminergic grafts in pro-
gressive Parkinson’s disease. Ann Neurol 1994;35:172–180.
30 Widner H, Brundin P: Immunological aspects of grafting in the mammalian central nervous sys-
tem: A review and speculative synthesis. Brain Res 1988;472:287–324.
31 Freed WJ, Poltorak M: Comments on brain tissue transplantation without immunosuppression.
Arch Neurol 1991;48:259–262.
32 Bakay RAE: Is transplantation to treat Parkinson’s disease dead? Neurosurgery 2001;49:
576–580.
33 Thompson TP, Lunsford LD, Kondziolka D: Restorative neurosurgery: Opportunities for restora-
tion of function in acquired, degenerative, and idiopathic neurological diseases. Neurosurgery
1999;45:741–752.
34 Dunn-Meynell AA, Levin BE: Selective sprouting of injured dopaminergic neurons induced by
embryonic brain grafts. Exp Neurol 1993;124:231–241.
35 Howells DW, Liberatore GT, Wong JY, Donnan GA: Dopaminergic responses to striatal damage.
J Neurol Sci 1996;139:125–130.
36 Bankiewicz KS, Palmatier M, Plunkett RJ, Cummins A, Oldfield EH: Reversal of hemiparkinson-
ian syndrome in nonhuman primates by amnion implantation into caudate nucleus. J Neurosurg
1994;81:869–876.
37 Poltorak M, Freed WJ: Immunological reactions induced by intracerebral transplantation:
Evidence that host microglia but not astroglia are the antigen-presenting cells. Exp Neurol 1989;
103:222–233.
38 Nakajima K, Kohsaka S: Functional roles of microglia in the brain. Neurosci Res 1993;17:
187–203.
39 Elkabes S, DiCicco-Bloom EM, Black IB: Brain microglia/macrophages express neurotrophins
that selectively regulate microglial proliferation and function. J Neurosci 1996;16:2508–2521.
40 Soontornniyomkij V, Wang G, Pittman CA, Wiley CA, Achim CL: Expression of brain-derived
neurotrophic factor protein in activated microglia of human immunodeficiency virus type 1
encephalitis. Neuropathol Appl Neurobiol 1998;24:453–460.
41 Achim CL, Wiley CA: Inflammation in AIDS and the role of the macrophage in brain pathology.
Curr Opin Neurol 1996;9:221–225.
42 Hamanoue M, Takemoto N, Matsumoto K, Nakamura T, Nakajima K, Kohsaka S: Neurotrophic
effect of hepatocyte growth factor on central nervous system neurons in vitro. J Neurosci Res 1996:
43:554–564.
43 Lazarov-Spiegler O, Solomon AS, Zeev-Brann AB, Hirschberg DL, Lavie V, Schwartz M:
Transplantation of activated macrophages overcomes central nervous system regrowth failure.
FASEB J 1996;10:1296–1302.
44 Nagata K, Takei N, Nakajima K, Saito H, Kohsaka S: Microglial conditioned medium promotes sur-
vival and development of cultured mesencephalic neurons from embryonic rat brain. J Neurosci Res
1993;34:357–363.
45 Nagata K, Nakajima K, Takemoto N, Saito H, Kohsaka S: Microglia-derived plasminogen
enhances neurite outgrowth from explant cultures of rat brain. Int J Dev Neurosci 1993;11:
227–237.
46 Batchelor PE, Liberatore GT, Wong JY, Porritt MJ, Frerichs F, Donnan GA, Howells DW:
Activated macrophages and microglia induce dopaminergic sprouting in the injured striatum and
express brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor. J Neurosci
1999;19:1708–1716.
47 Williams GR, Jiang JG, Matchar DB, Samsa GP: Incidence and occurrence of total (first-ever)
and recurrent stroke. Stroke 1999;30:2523–2528.
48 Wolf PA, Kannel WB, Verter J: Current status of risk factors for stroke. Neurol Clin 1983;1:317–343.
49 Kojima S, Omura T, Wakamatsu W, Kishi M, Yamazaki T, Lida M, Komachi Y: Prognosis and
disability of stroke patients after 4 years in Akita, Japan. Stroke 1990;21:72–77.
50 Borlongan CV, Tajima Y, Trojanowski JQ, Lee VM, Sanberg PR: Cerebral ischemia and CNS
transplantation: Differential effects of grafted fetal rat striatal cells and human neurons derived
from a clonal cell line. Neuroreport 1998;9:3703–3709.
51 Grabowski M, Johansson BB, Brundin P: Neocortical grafts placedin the infarcted brain of
adult rats: Few or no efferent fibers grow from transplant to host. Exp Neurol 1995;134:
273–276.
52 Aoki H, Onodera H, Yae T, Jian Z, Kogure K: Neural grafting to ischemic CA1 lesions in the rat
hippocampus: An autoradiographic study. Neuroscience 1993;56:345–354.
53 Tamura A, Graham D, McCulloch J, Teasdale G: Focal cerebral ischaemia in the rat. J Cereb Blood
Flow Metab 1981;1:53–60.
54 Grabowshi M, Nordborg C, Brundin P, Johansson BB: Middle cerebral artery occlusion in the
hypertensive and normotensive rat: A study of histopathology and behaviour. J Hypertens 1988;
6:405–411.
55 Koizumi J, Yoshida Y, Nakazawa T, Ooneda G: Experimental studies of ischemic brain edema:
A new experimental model of cerebral embolism in rats in which recirculation can be introduced
in the ischemic rats. Jpn J Stroke 1986;8:1–8.
56 Elsayed MH, Hogan T, Shaw P, Castro A: Use of fetal cortical grafts in hypoxic-ischemic brain
injury in neonatal rats. Exp Neurol 1996;137:127–141.
57 Grabowski M, Johansson BB, Brundin P: Survival of fetal neocortical grafts implanted in brain
infarcts of adult rats: The influence of postlesion time and age of donor tissue. Exp Neurol 1994;
127:126–136.
58 Hadani M, Freeman T, Munsiff A, Young W, Flamm E: Fetal cortical cells survive in focal cere-
bral infarct after permanent occlusion of the middle cerebral artery in adult rats. J Neurotrauma
1992;9:107–112.
59 Katayama Y, Tsubokawa T, Koshinaga M, Takahata T: Temporal pattern of survival and dendritic
growth of fetal hippocampal cells transplanted into ischemic lesions of the adult rat hippocampus.
Brain Res 1991;562:352–355.
60 Grabowski M, Brundin P, Hohansson BB: Functional integration of cortical grafts placed in brain
infarcts of rats. Ann Neurol 1993;34:362–368.
61 Johansson BB, Grabowski M: Functional recovery after brain infarction: Plasticity and neural
transplantation. Brain Pathol 1994;4:85–95.
62 Koide K, Hashitani T, Aihara N, Mabe H, Nishino H: Improvement of passive avoidance task after
grafting of fetal striatal cell suspensions in ischemic striatum in the rat. Restor Neurol Neurosci
1993;5:205–214.
63 Nishino H, Aihara N, Czurko A, Hashitani T, Isobe Y, Ichikawa O, Watari H: Reconstruction of
GABAergic transmission and behavior by striatal cell grafts in rats with ischemic infarcts in the
middle cerebral artery. J Neural Transplant Plast 1993;4:147–155.
64 Nunn J, Hodges H: Cognitive deficits induced by global cerebral ischaemia: Relationship to brain
damage and reversal by transplants. Behav Brain Res 1994;65:1–31.
65 Onifer SM, Low W: Spatial memory deficit resulting from ischemia-induced damage to the hip-
pocampus is ameliorated by intra-hippocampal transplants of fetal hippocampal neurons. Prog
Brain Res 1990;82:359–366.
66 Onizuka K, Fukuda A, Kunimatsu M, Kumazaki M, Sasaki M, Takaku A, Nishino H: Early cyto-
pathic features in rat ischemia model and reconstruction by neural graft. Exp Neurol 1996;137:
324–332.
67 Borlongan CV, Koutouzis T, Jorden J, Martinez R, Rodriguez A, Poulos S, Freeman T, Mckeown P,
Cahill D, Nishino H, Sanberg PR: Neural transplantation as an experimental treatment modality for
cerebral ischemia. Neurosci Biobehav Rev 1997;21:79–90.
68 Andrews PW, Damjanov I, Simon D, Banting G, Carlin C, Dracopoli N, Fogh J: Pluripotent
embryonal carcinoma clones derived from the human teratocarcinoma cell line Tera-2. Lab Invest
1984;50:147–162.
69 Pleasure SJ, Lee V: Ntera 2 cells: A human cell line which displays characteristics expected of a
human committed progenitor cell. J Neurosci Res 1993;35:585–602.
medwedi.ru
70 Trojanowski JQ, Kleppner SR, Hartley RS, Miyazono M, Fraser NW, Kesari S, Lee VMY:
Transfectable and transplantable postmitotic human neurons: A potential platform for gene ther-
apy of nervous system diseases. Exp Neurol 1997;144:92–97.
71 Andrews PW: Retinoic acid induces neuronal differentiation of a cloned human embryonal carci-
noma cell line in vitro. Dev Biol 1984;103:285–293.
72 Kleppner SR, Robinson KA, Trojanowski JQ, Lee VM: Transplanted human neurons derived from
a teratocarcinoma cell-line (ntera-2) mature, integrate, and survive for over 1 year in the nude-
mouse brain. J Comp Neurol 1995;357:618–632.
73 Pleasure SJ, Page C, Lee VM-Y: Pure, postmitotic, polarized human neurons derived from Ntera 2
cells provide a system for expressing exogenous proteins in terminally differentiated neurons.
J Neurosci 1992;12:1802–1815.
74 Lee VM-Y, Hartley RS, Trojanowski JQ: Neurobiology of human neurons (NT2N) grafted into
mouse spinal cord: Implications for improved therapy of spinal cord injury. Prog Brain Res 2000;
128:299–307.
75 Baker KA, Hong M, Sadi D, Mendez I: Intrastriatal and intranigral grafting of hNT neurons in the
6-OHDA rat model of Parkinson’s disease. Exp Neurol 2000;162:350–360.
76 Philips MG, Muir JK, Saatman KE, Raghupathi R, Lee VM, Trojanowski JQ, McIntosh TK:
Survival and integration of transplanted postmitotic human neurons following experimental brain
injury in immunocompetent rats. J Neurosurg 1999;90:116–124.
77 Nishino H, Koide K, Aihara N, Kumazaki M, Sakurai T, Nagai H: Striatal grafts in the ischemic
striatum improve pallidal GABA release and passive avoidance. Brain Res Bull 1993;32:517–520.
78 Borlongan CV, Saporta S, Poulos SG, Othberg A, Sanberg PR: Viability and survival of hNT
neurons determine degree of functional recovery in grafted ischemic rats. Neuroreport 1998;9:
2837–2842.
79 Borlongan CV, Tajima Y, Trojanowski JQ, Lee VM-Y, Sandberg PR: Transplantation of cryopre-
served human embryonal carcinoma-derived neurons (NT2N cells) promotes functional recovery
in ischemic rats. Exp Neurol 1998;149:310–321.
80 Saporta S, Borlongan CV, Sanberg PR: Neural transplantation of human neuroteratocarcinoma
(hNT) neurons into ischemic rats. A quantitative dose-response analysis of cell survival and
behavioral recovery. Neuroscience 1999;91:519–525.
81 Thompson T, Lunsford LD, Kondziolka D: Restorative neurosurgery: Opportunities for restora-
tion of function in acquired, degenerative, and idiopathic neurological diseases. Neurosurgery
1999;45:741–752.
82 Kondziolka D, Wechsler L, Goldstein S, Meltzer C, Thulborn K, Gebel J, Jannetta P, DeCesare S,
Elder E, McGrogan M, Reitman M, Bynum L: Transplantation of cultured human neuronal cells
for patients with stroke. Neurology 2000;55:565–569.
83 Levi-Montalcini R: From Turin to Stockholm via St. Louis and Rio de Janeiro. Science 2000;
287:809.
84 Levi-Montalcini R, Dal Toso R, della Valle F, Skaper SD, Leon A: Update of the NGF saga.
J Neurol Sci 1995;130:119–127.
85 Levi-Montalcini R: Morphological and metabolic effects of the nerve growth factor. Arch Biol
(Liege) 1965;76:387–417.
86 Dechant G: Molecular interactions between neurotrophin receptors. Cell Tissue Res 2001;305:
229–238.
87 Chao MV, Bothwell M: Neurotrophins: To cleave or not to cleave. Neuron 2002;33:9–12.
88 Elkabes S, Diciccobloom EM, Black IB: Brain microglia macrophages express neurotrophins that
selectively regulate microglial proliferation and function. J Neurosci 1996;16:2508–2521.
89 Hefti F: Neurotrophic factor therapy for nervous system degenerative diseases. J Neurobiol 1994;
25:1418–1435.
90 Lindsay RM, Altar CA, Cedarbaum JM, Hyman C, Wiegand SJ: The therapeutic potential of neu-
rotrophic factors in the treatment of Parkinson’s disease. Exp Neurol 1993;124:103–118.
91 Olson L: Toward trophic treatment in parkinsonism – A primate step. Nat Med 1996;2:400–401.
92 Fontaine V, Mohand-Said S, Hanoteau N, Fuchs C, Pfizenmaier K, Eisel U: Neurodegenerative
and neuroprotective effects of tumor necrosis factor (TNF) in retinal ischemia: Opposite roles of
TNF receptor 1 and TNF receptor 2. J Neurosci 2002;22:RC216.
93 Granholm AC, Albeck D, Backman C, Curtis M, Ebendal T, Frieden P, Henry M, Hoffer B,
Kordower J, Rose GM, Soderstrom S, Bartus RT: A non-invasive system for delivering neural
growth factors across the blood-brain barrier: A review. Rev Neurosci 1998;9:31–55.
94 Pan W, Banks WA, Kastin AJ: Permeability of the blood-brain barrier to neurotrophins. Brain Res
1998;788:87–94.
95 Friden PM, Walus LR, Watson P, Doctrow SR, Kozarich JW, Backman C, Bergman H, Hoffer B,
Bloom F, Granholm AC: Blood-brain barrier penetration and in vivo activity of an NGF conjugate.
Science 1993;259:373–377.
96 Zhou J, Bradford HF, Stern GM: Influence of BDNF on the expression of the dopaminergic phe-
notype of tissue used for brain transplants. Brain Res Dev Brain Res 1997;100:43–51.
97 Kawaja MD, Rosenberg MB, Yoshida K, Gage FH: Somatic gene transfer of nerve growth factor
promotes the survival of axotomized septal neurons and the regeneration of their axons in adult
rats. J Neurosci 1992;12:2849–2864.
98 Espejo M, Cutillas B, Arena TE, Ambrosio S: Increased survival of dopaminergic neurons in stri-
atal grafts of fetal ventral mesencephalic cells exposed to neurotrophin-3 or glial cell line-derived
neurotrophic factor. Cell Transplant 2000;9:45–53.
99 Kordower JH, Emborg ME, Bloch J, Ma SY, Chu Y, Leventhal L, McBride J, Chen EY, Palfi S,
Roitberg BZ, Brown WD, Holden JE, Pyzalski R, Taylor MD, Carvey P, Ling Z, Trono D, Hantraye P,
Deglon N, Aebischer P: Neurodegeneration prevented by lentiviral vector delivery of GDNF in
primate models of Parkinson’s disease. Science 2000;290:767–773.
100 Granholm AC, Mott JL, Bowenkamp K, Eken S, Henry S, Hoffer BJ, Lapchak PA, Palmer MR,
van Horne C, Gerhardt GA: Glial cell line-derived neurotrophic factor improves survival of ven-
tral mesencephalic grafts to the 6-hydroxydopamine lesioned striatum. Exp Brain Res 1997;116:
29–38.
101 Stromberg I, Bjorklund L, Johansson M, Tomac A, Collins F, Olson L, Hoffer B, Humpel C: Glial
cell line-derived neurotrophic factor is expressed in the developing but not adult striatum and
stimulates developing dopamine neurons in vivo. Exp Neurol 1993;124:401–412.
102 Kordower JH, Palfi S, Chen EY, Ma SY, Sendera T, Cochran EJ, Mufson EJ, Penn R, Goetz CG,
Comella CD: Clinicopathological findings following intraventricular glial-derived neurotrophic
factor treatment in a patient with Parkinson’s disease. Ann Neurol 1999;46:419–424.
103 Tuszynski MH, Roberts J, Senut MC, U HS Gage FH: Gene therapy in the adult primate brain:
Intraparechymal grafts of cells genetically modified to produce nerve growth factor prevent
cholinergic neuronal degeneration. Gene Ther 1996;3:305–314.
104 Diaz-Cintra S, Rivas P, Cintra L, Aguilar A, Guiterrez G, Perez E, Excobar M, Bermudez-Rattoni F:
Morphometric study of fetal brain transplants in the insular cortex and NGF effects on neuronal
and glial development. Cell Transplant 1995;4:505–513.
105 Westlund KN, Lu Y, Kadekaro M, Harmann P, Terrell ML, Pizzo DP, Hulsebosch CE, Eisenberg HM,
Perez-Polo JR: NGF-producing transfected 3T3 cells: Behavioral and histological assessment of
transplants in nigral lesioned rats. J Neurosci Res 1995;41:367–373.
106 Kordower JH, Chen EY, Winkler C, Fricker R, Charles V, Messing A, Mufson EJ, Wong SC,
Rosenstein JM, Bjorklund A, Emerich DF, Hammang J, Carpenter MK: Grafts of EGF-responsive
neural stem cells derived from GFAP-hNGF transgenic mice: Trophic and tropic effects in a
rodent model of Huntington’s disease. J Comp Neurol 1997;387:96–113.
107 Li Y, Chen J, Chen XG, Wang L, Gautam SC, Xu YX, Katakowski M, Zhang LJ, Lu M,
Janakiraman N, Chopp M: Human marrow stromal cell therapy for stroke in rat: Neurotrophins
and functional recovery. Neurology 2002;59:514–523.
108 Andsberg G, Kokaia Z, Klein RL, Muzyczka N, Lindvall O, Mandel RJ: Neuropathological and
behavioral consequences of adeno-associated viral vector-mediated continuous intrastriatal neu-
rotrophin delivery in a focal ischemia model in rats. Neurobiol Dis 2002;9:187–204.
109 Hermann DM, Kilic E, Kugler S, Isenmann S, Bahr M: Adenovirus-mediated GDNF and CNTF
pretreatment protects against striatal injury following transient middle cerebral artery occlusion in
mice. Neurobiol Dis 2001;8:655–666.
110 Zhao LR, Risedal A, Wojcik A, Hejzlar J, Johansson BB, Kokaia Z: Enriched environment influ-
ences brain-derived neurotrophic factor levels in rat forebrain after focal stroke. Neurosci Lett
2001;305:169–172.
medwedi.ru
111 Zhang Y, Pardridge WM: Neuroprotection in transient focal brain ischemia after delayed intra-
venous administration of brain-derived neurotrophic factor conjugated to a blood-brain barrier
drug targeting system. Stroke 2001;32:1378–1384.
112 Ferrer I, Krupinski J, Goutan E, Marti E, Ambrosio S, Arenas E: Brain-derived neurotrophic fac-
tor reduces cortical cell death by ischemia after middle cerebral artery occlusion in the rat. Acta
Neuropathol (Berl) 2001;101:229–238.
113 Hortobagyi T, Harkany T, Reisch R, Urbanics R, Kalman M, Nyakas C, Nagy Z: Neurotrophin-
mediated neuroprotection by solid fetal telencephalic graft in middle cerebral artery occlusion:
A preventive approach. Brain Res Bull 1998;47:185–191.
114 Kawamata T, Speliotes EK, Finklestein SP: The role of polypeptide growth factors in recovery
from stroke. Adv Neurol 1997;73:377–382.
115 Kanazawa I: How do neurons die in neurodegenerative diseases? Trends Mol Med 2001;7:339–344.
116 Jellinger KA: Cell death mechanisms in Parkinson’s disease. J Neural Transm 2000;107:1–29.
117 Morita-Fujimura Y, Fujimura M, Yoshimoto T, Chan PH: Superoxide during reperfusion contributes
to caspase-8 expression and apoptosis after transient focal stroke. Stroke 2001;32:2356–2361.
118 Benchoua A, Guegan C, Couriaud C, Hosseini H, Sampaio N, Morin D, Onteniente B: Specific
caspase pathways are activated in the two stages of cerebral infarction. J Neurosci 2001;21:
7127–7134.
119 Mattson MP, Culmsee C, Yu ZF: Apoptotic and antiapoptotic mechanisms in stroke. Cell Tissue
Res 2000;301:173–187.
120 Breakfield XO: Gene delivery into the brain using virus vectors. Nat Genet 1993;3:187–189.
121 Pallela TD, Hidaka Y, Silverman LJ, Levine M, Glorioso J, Kelley WN: Expression of human
HPRT mRNA in brains of mice infected with a recombinant herpes simplex virus-1 vector. Gene
1989;80:137–144.
122 Fink DJ, Sternberg LR, Weber PC, Mata M, Goins WF, Glorioso JC: In vivo expression of [beta]-
galactosidase in hippocampal neurons by HSV-mediated gene transfer. Hum Gene Ther 1992;3:
11–19.
123 Wolfe JH, Deshmann SL, Fraser NW: Herpes virus vector gene transfer and expression of [beta]-
glucuronidase in the central nervous system of MPS VII mice. Nat Genet 1992;1:379–384.
124 Price J, Turner D, Cepko C: Lineage analysis in the vertebrate nervous system by retrovirus-
mediated gene transfer. Proc Natl Acad Sci USA 1987;84:156–160.
125 Walsh C, Cepko CL: Clonally related cortical cells show several migration patterns. Science 1988;
241:1342–1345.
126 Mulligan RC: The basic science of gene therapy. Science1993;260:926–932.
127 Karpati G, Lochmuller H, Nalbantoglu J, Durham H: The principles of gene therapy for the ner-
vous system. Trends Neurosci 1996;19:49–54.
128 Kafri T, Blomer U, Peterson DA, Gage FH, Verma IM: Sustained expression of genes delivered
directly into liver and muscle by lentiviral vectors. Nat Genet 1997;17:314–317.
129 Ono T, Fujino Y, Tsuchiya T, Tsuda M: Plasmid DNAs directly injected into mouse brain with lipo-
fectin can be incorporated and expressed by brain cells. Neurosci Lett 1990;117:259–263.
130 Danko I, Fritz JD, Latendresse JS, Herweijer H, Schltz E, Wolf JA: Dystrophin expression
improves myofiber survival in mdx muscle following intramuscular plasmid DNA injection. Hum
Mol Genet 1993;2:2055–2061.
131 Kitagawa H, Sasaki C, Sakai K, Mori A, Mitsumoto Y, Mori T, Fukuchi Y, Setoguchi Y, Abe K:
Adenovirus-mediated gene transfer of GDNF prevents ischemic brain injury after transient MCA
occulusion in rats. J Cereb Blood Flow Metab 1999;19:1336–1344.
132 Fujimura M, Morita-Fujimura Y, Murakami K, Kawase M, Chan PH: Cytosolic redistribution of
cytochrome c after transient focal cerebral ischemia in rats. J Cereb Blood Flow Metab 1998;18:
1239–1247.
133 Casper D, Engstrom SJ, Mirchandani GR, Pidel A, Palencia D, Cho PH, Brownlee M, Edelstein D,
Federoff HJ, Sonstein WJ: Enhanced vascularization and survival of neural transplants with ex vivo
angiogenic gene transfer. Cell Transplantat 2002;11:331–349.
134 Betz AL, Yang GY, Davidson BL: Attenuation of stroke size in rats using an adenoviral vector to
induce overexpression of interleukin-1 receptor antagonist in brain. J Cereb Blood Flow Metab
1995;15:547–551.
135 Xu DG, Crocker SJ, Doucet J-P, St-Jean M, Tamai K, Hakim AM, Ikeda J-E, Liston P, Thompson CS,
Korneluk RG, MacKenzie A, Robertson GS: Elevation of neuronal expression of NAIP reduces
ischemic damage in the rat hippocampus. Nat Med 1997;3:997–1004.
136 Linnik MD, Zahos P, Geschwind MD, Federoff HJ: Expression of bcl-2 from a defective herpes sim-
plex virus-1 vector limits neuronal death in focal cerebral ischemia. Stroke 1995;26:1670–1674.
137 Lawrence MS, McLaughlin JR, Sun G-H, Ho DY, McIntosh L, Kunis DM, Sapolsky RM,
Steinberg GK: Herpes simplex viral vectors expressing bcl-2 are neuroprotective when delivered
after a stroke. J Cereb Blood Flow Metab 1997;17:740–744.
138 Yang GY, Zhao YJ, Davidson BL, Betz AL: Overexpression of interleukin-1 receptor antagonist
in the mouse brain reduces ischemic brain injury. Brain Res 1997;751:181–188.
139 Yenari MA, Fink SL, Sun GH, Chang LK, Patel MK, Kunis DM, Onley D, Ho DY, Sapolsky RM,
Steinberg GK: Gene therapy with HSP72 is neuroprotective in rat models of stroke and epilepsy.
Ann Neurol 1998;44:584–591.
140 Hodges H, Virley D, Williams C, Ridley RM, Sinden JD, Kershaw TR, Harland S, Gray JA,
Lantos PL: Fetal CA1 grafts promote recovery from cognitive deficits induced by excitotoxic
damage to the CA1 field in marmosets. J Cereb Blood Flow Metab 1997;17(suppl 1):S451.
141 Li Y, Chopp M, Chen J, Gautam SC, Dou D, Zhang L, Wang L, Xu Y, Powers C, Zhang X, Jiang F:
Intracerebral grafting of adult bone marrow cells after stroke in adult mice and rats. J Cereb Blood
Flow Metab 1999;19(suppl 1):S615.
142 Sinden JD, Rashid-Doubell F, Kershaw TR, Nelson A, Chadwick A, Jat PS, Noble MD, Hodges H,
Gray JA: Recovery of spatial learning by grafts of a conditionally immortalized hippocampal neu-
roepithelial cell line into the ischemia-lesioned hippocampus. Neuroscience 1997;81:599–608.
143 Hodges H, Virley D, Ridley RM, Sinden JD, Kershaw TR, French S, Harland S, Gray JA, Lantos PL:
Conditionally immoral MHP36 cells restore cognitive function in marmosets after excitotoxic
hippocampal CA1. J Cereb Blood Flow Metab 1999;19(suppl 1):S617.
144 Virley D, Ridley RM, Sinden JD, Kershaw TR, Harland S, Rashid T, French S, Sowinski P, Gray JA,
Lantos PL, Hodges H: Primary CA1 and conditionally immortal MHP36 cell grafts restore condi-
tional discrimination learning and recall in marmosets after excitotoxic lesions of the hippocampal
CA1 field. Brain 1999;22:2321–2335.
145 Kopen GC, Prockop DJ, Phinney DG: Marrow stromal cells migrate throughout forebrain and
cerebellum, and they differentiate into astrocytes after injection into neonatal mouse brains. Proc
Natl Acad Sci USA 1999;96:10711–10716.
146 Li Y, Chopp M, Chen J, Wang L, Gautam SC, Xu Y, Zhang ZG: Intrastriatal transplantation of
bone marrow stromal cells (MSCs) improves functional recovery after stroke in adult mice.
147 Chopp M, Zhang XH, Li Y, Wang L, Chen J, Lu D, Lu M, Rosenblum M: Spinal cord injury in
rat: Treatment with bone marrow stromal cell transplantation. Neuroreport 2000;11:3001–3005.
148 Chen J, Li Yi, Wang L, Zhang Z, Lu D, Lu M, Chopp M: Therapeutic benefit of intravenous
administration of bone marrow stromal cells after cerebral ischemia in rats. J Neurol Sci 2001;
189:49–57.
149 Eriksson PS, Perfilieva E, Bjork-Eriksson T, Alborn AM, Nordborg C, Peterson DA, Gage FH:
Neurogenesis in the adult human hippocampus. Nat Med 1998;4:1313–1317.
150 Gould E, Reeves AJ, Graziano MS, Gross CG: Neurogenesis in the neocortex of adult primates.
Science 1999;286:548–552.
151 Martinez-Serrano A, Bjorklund A: Ex vivo nerve growth factor gene transfer to the basal forebrain
in presymptomatic middle-aged rats prevents the development of cholinergic neuron atrophy and
cognitive impairment during aging. Proc Natl Acad Sci USA 1998;17;95:1858–1863.
Douglas Kondziolka, MD
Department of Neurological Surgery
Suite B-400, UPMC, 200 Lothrop Street, Pittsburgh, PA 15213 (USA)
Tel. ⫹1 412 647 6782, Fax ⫹1 412 647 0989, E-Mail Kondziol@neuronet.pitt.edu
medwedi.ru
Neuro-Oncology

Neurosurgical Applications for

Polymeric Drug Delivery Systems
Paul P. Wang a, Henry Brema,b
Departments of aNeurological Surgery and bOncology,
The Johns Hopkins Hospital, Baltimore, Md., USA
Introduction
Malignant brain tumors represent one of the most devastating forms of

human illness. Each year, approximately 16,800 Americans are diagnosed with
primary brain tumors and 13,100 die from the disease [1]. Half of all primary
brain tumors originate from glial cells and are thus classified as gliomas.
Astrocytomas, which represent 3 out of 4 gliomas, include a heterogeneous
group of tumors ranging from low-grade lesions and high-grade anaplastic
astrocytomas, to its most common and aggressive variant, the glioblastoma
multiforme (GBM).
Historically, the standard of care for high-grade malignant gliomas includes
surgical biopsy for pathological diagnosis [2], surgical debulking of accessible
tumor, and adjuvant radiation therapy and chemotherapy [3–7]. Unfortunately,
despite recent advances in neuroradiology and neurosurgical techniques, long-
term patient survival remains dismal. Median survival after surgical resection
alone is 6 months, and only 7.5% of patients survive 2 years. Adjuvant radiation
therapy extends median survival to 9 months, and adjuvant systemic chemother-
apy has been minimally effective [8, 9].
Due to the highly invasive nature of malignant gliomas, a gross total resection
of the tumor almost always leaves behind remnants at the microscopic level.
Broader surgical resection results in an increased risk of damaging functional brain
tissue, resulting in immediate morbidity and neurological deficit. Similarly,
increasing the dose or field of a radiation protocol introduces unacceptable side
effects such as tissue edema or necrosis. Therefore, efforts have been made to
improve adjuvant chemotherapy modalities for treating malignant brain tumors,
both in the development of more effective agents and in the advancement of drug
delivery methods. The introduction of biodegradable controlled-release local-
delivery polymers to treat malignant brain tumors has been an important advance-
ment in improving chemotherapy delivery methods. While the benefits of
local-delivery polymers are still debatable, local administration has a strong ratio-
nale, which stands to improve patient outcomes as the currently available
chemotherapeutic drugs improve. This chapter chronicles the history of local
delivery techniques for treating gliomas, their clinical applications, and future
technologies that may advance the use of local-delivery systems in neurosurgery.
Unique Issues of Drug Delivery into the Central Nervous System
The unique environment of the central nervous system (CNS) presents

significant challenges for delivering chemotherapy to brain tumors. The CNS is
physiologically isolated by the blood-brain barrier (BBB), composed of tight
junctions between endothelial cells of capillaries supplying the CNS [10], as
well as anchoring astrocytic foot processes. The BBB forms a pharmacological
barrier that under most conditions can prevent the influx of large molecules into
the brain. Anti-neoplastic agents that are large, ionically charged, or hydrophilic
do not readily penetrate the BBB [11] (fig. 1), and intolerably high systemic
drug levels may be necessary to achieve therapeutic doses within the CNS [12].
The approaches to improving drug delivery to brain tumor patients fall into
three categories: (1) tailoring existing agents to maximally utilize the natural
permeability properties of the BBB; (2) disrupting the BBB before systemic
delivery of agents, and (3) circumventing the BBB with direct delivery of
agents to the CNS. Using the first approach, existing drugs can be manipulated
pharmacologically to create smaller, more lipophilic, or ligand-adapted agents,
which traverse the BBB more readily. For example, both lumustine (CCNU)
and semustine (methyl-CCNU) are lipophilic variants of carmustine (BCNU)
[13], a previously existing chemotherapeutic agent with modest efficacy in the
treatment of malignant brain tumors. Clinical trials have not shown any signifi-
cant efficacy of either agent when given systemically [5]. Anti-neoplastic agents
also can be linked with a carrier or ligand capable of penetrating the BBB. For
example, dihydropyridine is a lipophilic carrier that readily crosses the BBB, and
has been shown to deliver a variety of drugs intracranially, including chemother-
apy agents, antibiotics, and neurotransmitters [14]. Receptor-specific monoclonal
antibodies also have been used to deliver drugs across the BBB [15].
The second approach to delivering drugs across the BBB involves two
steps, disruption of the BBB and systemic delivery of drugs across the com-
promised BBB. For example, the diuretic mannitol can cause acute dehydration
of endothelial cells, which in turn shrinks the cells and widens the tight junctions
Polymeric Drug Delivery Systems 459
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10⫺2
10⫺3
Cerebrovascular permeability (cm s⫺1)
CCNU
10⫺4 Caffeine BCNU
Antipyrine
Procarbazine PCNU
Propylene glycol Metronidazole Spirohydantoin

10⫺5 Misonidazole mustard
Ethylene Ftorafur
Glycerol glycol Acetamide
5-FU
Curare Dianhydrogalacticol
10⫺6 Thiourea
Vinblastine
N-methyl Urea Formamide
nicotinamide Vincristine
Methotrexate
10⫺7 Mannitol
Arabinose
Epipodophyllotoxin
Sucrose
10⫺8
10⫺4 10⫺3 10⫺2 10⫺1 1 10 100 1,000
Octanol–water partition coefficient
Fig. 1. Cerebrovascular permeability versus octanol-water partition coefficient of

selected chemicals and drugs. (Reprinted with permission from Elsevier Science [12].)
of the BBB, when infused intra-arterially in hyperosmolar concentrations. In a

study investigating the efficacy of premedicating brain tumor patients with
mannitol before the administration of carboplatin and etoposide, Williams et al.
[16] demonstrated that 4 out of 4 patients with primitive neuroectodermal
tumors and 2 out of 4 patients with CNS lymphomas had a clinical response fol-
lowing mannitol and drug administration. However, mannitol as an adjuvant to
chemotherapy has showed no benefit to patients with GBM, oligodendrogliomas,
or metastatic carcinomas, probably because it does not improve the delivery of
drug to the tumor site despite its ability to increase penetrance of agents across
the BBB [17]. Another drug that directly disrupts the BBB is the bradykinin
agonist RMP-7, which has been shown in experimental brain tumor models to
increase the uptake of carboplatin [18]. However, in vivo efficacy is limited
because like mannitol, it does not necessarily improve the delivery of the anti-
neoplastic agents to the tumor site after it has crossed the BBB [19].
The third approach to overcoming the BBB is to completely bypass it
by local delivery of the drug. The three main strategies within this approach
Wang/Brem 460
are: (1) delivery via cerebrospinal fluid (CSF) exposure; (2) drug administration
via catheters, and (3) delivery via sustained-release local-delivery polymers.
The local-delivery approach theoretically provides for the maintenance of
sustained significant local levels of the drug at the tumor site while avoiding dan-
gerously high systemic exposure. Such a strategy seems particularly appropriate
for the management of malignant gliomas, since 80–90% recur after conventional
treatment within 2 cm of the original resection bed [20].
CSF infusion requires the introduction of the drug via either an intraven-
tricular catheter or lumbar puncture. The BBB does not extend to the ependy-
mal lining of the ventricular system and delivery via CSF infusion bypasses the
BBB. Initial results have been disappointing mainly due to poor penetration
of the drug into the brain parenchyma [21]. Delivery of anti-neoplastic agents
through catheter systems requires accurately placing a catheter’s tip within the
tumor site and infusing from a reservoir of drug, through the catheter, and directly
to the tumor. One of the earliest such systems is the Ommaya reservoir [22],
which can deliver intermittent bolus injections of chemotherapy to the tumor. The
introduction of implantable pumps such as Infusaid (Norwood, Mass., USA)
[23], MiniMed PIMS (Sylmar, Calif., USA) [24], and Medtronic SynchroMed
(Minneapolis, Minn., USA) [25] allows for the constant infusion of drug over
extended periods of time. These devices may be limited by mechanical failure,
obstruction by clot or tissue debris, and infection.
Biocompatible sustained-release polymers are impregnated with chemother-
apeutic agents and surgically implanted in a tumor cavity after surgical resec-
tion and debulking of malignant gliomas. The polymers are designed to slowly
release the drug over time. They fall into two categories, biodegradable and
nonbiodegradable. The former release drug as the polymer matrix breaks down
over time, while the latter leaves behind an intact polymer matrix after all of the
therapeutic agents have been released [26].
Development of Biocompatible Polymers
Historical Perspective
In 1976, Langer and Folkman [27] reported the sustained and predictable
release of macromolecules from the ethylene vinyl acetate (EVAc) copolymer,
a nonbiodegradable polymer initially developed for possible applications within
the clothing industry. Drug incorporated into an EVAc polymer matrix diffuses
through the micropores of the matrix at a rate dependent on multiple chemical
properties of the drug, including molecular weight, charge, and water solubil-
ity. The polymer was deemed safe after rabbit cornea studies demonstrated its
biocompatibility [28] and rat brain studies demonstrated its inertness [29–33].
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Bulk erosion Surface erosion
Time
a b
Fig. 2. a Bulk erosion of biodegradable controlled-release polymer implants leads to

unpredictable release profiles. b Polymers exhibiting surface erosion release drug at nearly
constant rate (zero-order kinetics) as they dissolve in water. (Reprinted with permission [56].)
Since its introduction, EVAc polymers have been incorporated into numerous
clinical applications, including glaucoma treatment, dental care, contraception,
insulin therapy, asthma treatment, and chemotherapy. Recently, EVAc polymers
have been successfully used to deliver neurotrophic growth factors to treat
peripheral nerve injuries in a rat brachial plexus injury model [34]. However,
EVAc polymers have not been approved by the Food and Drug Administration
(FDA) for intracranial use [35]. In addition to the disadvantage of a permanent
foreign body left behind after drug delivery, EVAc polymers demonstrate first-
order release kinetics, with the release rate decreasing over time [36].
To avoid the disadvantages of a residual indwelling matrix, a series of
biodegradable polymer systems were developed. By releasing drugs with a com-
bination of diffusion and polymer degradation, sustained release of drugs could
be accomplished without leaving behind a permanent foreign body (fig. 2, 3).
The first such biodegradable polymer included the family of lactide/glycolide
polyesters, where the lactic acid and glycolic acid monomers are polymerized
with ester bonds. The polylacticcoglycolic acid (PLGA) polymers exhibited sev-
eral advantages over the nonbiodegradable EVAc polymers. In addition to
biodegradability, PLGA polymers could be tailored to provide a range of drug
delivery rates by varying the ratio of lactic acid to glycolic acid monomers [37].
Biocompatibility was demonstrated in the rat brain [38–40], as well as by poly-
mers fashioned into sutures, which are now in wide clinical use [41, 42]. Often
shaped into injectable microspheres [43], PLGA polymers have been success-
fully used to deliver a variety of drugs, including steroids, anti-inflammatory
agents, antibiotics, anesthetics, narcotics, and chemotherapeutic agents
[37, 44–53]. A microsphere variant also has been designed specifically for
stereotactic implantation into the brain [54]. Another variant, manufactured by
covalently linking the polymer matrix to a polyethylene glycol coating, has been
Wang/Brem 462
Diffusion
Degradation
Fig. 3. Polymer implants releasing drug by degradation provide more predictable

release profiles compared to diffusion. (Reprinted with permission [56].)
shown to reduce opsonization and elimination in the immune system [55]. A dis-
advantage to PLGA polymers is the drug release characteristic of bulk erosion,
similar to the dissolution of a sugar cube in water (fig. 2a), which can lead to
sporadic dumping of the drug, suboptimal tissue exposure, and unexpected acute
toxicity [56].
In 1985, Leong et al. [62] formulated the polyanhydride poly[bis(p-
carboxyphenoxyl)] propane-sebacic acid (pCPP:SA) matrix. Like the PLGA poly-
mer, pCPP:SA is biodegradable by spontaneously breaking down to dicarboxylic
acids in water. Unlike PLGA polymer, the pCPP:SA matrix offers several impor-
tant properties. First, because of its extreme hydrophobicity, the matrix shields the
incorporated drug from aqueous media, an important feature for compounds with
short biological half-lives. Second, in contrast to the PLGA breakdown by bulk
erosion (i.e., like a sugar cube), pCPP:SA breakdown is limited to its surface, a
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property called surface erosion (i.e., like peeling an onion) (fig. 2b). This property
confers a theoretical advantage of constant-rate or zero-order drug delivery. Like
the previously introduced polymer systems, the pCPP:SA matrix can also be man-
ufactured into an almost endless variety of shapes, including microspheres, sheets,
rods, and wafers [57–63]. Also like the PLGA polymer, altering the ratio osf the
two monomers CPP and SA can vary the rate of breakdown of pCPP:SA, and thus
the rate of drug delivery. For example, a 1 mm thick polymer composed of pure
CPP would require 3 years to degrade, but a mixture of 20% CPP and 80% SA or
pCPP:SA (20:80) would only require 3 weeks [60].
Multiple studies have demonstrated the biocompatibility of pCPP:SA. The
dicarboxylic acid breakdown products are neither mutagenic, teratogenic, nor
cytotoxic in standard assays [64]. In vitro proliferation assays with endothelial
cells and smooth muscle cells showed no inhibition properties in the pCPP:SA
matrix [64]. There was no identifiable inflammation in the rabbit cornea assay
[28, 62]. Biocompatibility with neural tissue was demonstrated in the brains of
rats [65], rabbits [66], and monkeys [67]. Due to its favorable drug release char-
acteristics and biocompatibility, pCPP:SA has been used extensively in the clin-
ical setting, and its current applications in neuro-oncology are discussed in the
next section.
A variety of other local-delivery sustained-release systems have been
developed for intracranial use. The fatty acid dimmer-sebacic acid (FAD:SA)
polymers [68–70] are well suited for releasing hydrophobic chemotherapeutic
agents [71–73]. Polyethylene glycol-coated liposomes and gelatin chondroitin
sulfate-coated microspheres have been developed to successfully release
anthracyclines and cytokines, respectively [74, 75]. Commonly used surgical
materials, such as fibrin glue [76], gelatin sponges [77], Surgicel (oxidized regen-
erated cellulose) [77], polymethylmethacrylate [78], and silastic tubing [79], have
all been used for intracranial local delivery of drugs.
Clinical Applications of Polyanhydride Polymers

for Drug Delivery BCNU (Gliadel)
Development and Clinical Use

The initial drug chosen for the development of sustained local delivery of
intracranial chemotherapy was carmustine (BCNU) due to its widespread use as
a systemic agent for treating malignant brain tumors and its established mecha-
nism of action against glioma cells. Belonging to the family of nitrosureas, BCNU
is a DNA alkylating agent (i.e., chloroethylation of guanine at the O6 position)
and causes DNA chain termination, impairing the mitotic activity of cancer cells.
Its relatively low molecular weight and lipid solubility allows it to cross the BBB
Wang/Brem 464
and accumulate in the CNS [13]. However, its effectiveness as a systemic agent
for brain tumors is limited by its relatively short half-life (⬍15 min) and numer-
ous serious side effects, including bone marrow suppression and pulmonary
fibrosis. Clinical trials investigating its efficacy with systemic delivery demon-
strated only modest improvements in survival [4, 6]. However, by incorporating
BCNU into local-delivery polymers, it was hoped that the efficacy would be
significantly increased and dose-limiting side effects could be avoided.
Preclinical Trials
Preclinical studies investigating BCNU-loaded polymer preparations first
studied the in vivo release kinetics of BCNU-loaded polymers. The initial study
used an EVAc copolymer loaded with BCNU and implanted into the rat brain
[33]. BCNU concentrations were measured with the Bratton-Marshall assay in
both cerebral hemispheres, as well as in serum samples collected at various time-
points after polymer placement. In the hemisphere ipsilateral to the polymer,
BCNU levels peaked at 4 h and remained elevated through day 7. Levels in the
contralateral hemisphere and in the serum were lower by at least an order of mag-
nitude throughout the time course of the experiment. This study demonstrated
the proof of principle of the ability of polymer technology to provide both local
delivery and sustained release of chemotherapy within the CNS. A second exper-
iment, again using a rat intracranial model, compared kinetics and biodistribution
of a BCNU and pCPP:SA (80:20) copolymer with direct stereotactic injection of
BCNU [80]. Using tritiated BCNU, the distribution of drug was assessed by
quantitative autoradiography in brain sections collected at various timepoints
following implantation/injection. Animals with BCNU polymers had approxi-
mately 50% of the ipsilateral hemisphere exposed to BCNU at day 3, and 10%
exposed at day 14. A polymer containing only 600 ␮g of BCNU provided tissue
concentrations of up to 6 mM at 10 mm from the implantation site on both days 3
and 7. In contrast, animals directly injected with BCNU showed an initial broadly
distributed BCNU spike from 1 to 3 h postinjection, which then rapidly disap-
peared. A third experiment implanted 20% (w/w) BCNU-loaded pCPP:SA
polymers in a primate intracranial model; tumoricidal concentrations of BCNU
was demonstrated up to 4 cm from the polymer site at 24 h after surgery [81]. At
30 days, active drug was still present up to 1 cm from the polymer. This set of
experiments confirmed that polymer technology can provide local, sustained, and
clinically significant in vivo delivery of chemotherapy agents within the CNS.
A second phase of preclinical experiments investigated the efficacy of
BCNU-loaded polymers in animal models of brain cancer. In 1993, Tamargo
et al. [29] compared the efficacy of polymer-delivered BCNU with systemic
BCNU using both the rat flank and intracranial 9L gliosarcoma models. In
the flank model using EVAc polymers, tumor growth delay was significantly
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longer in polymer-delivered BCNU compared to systemic delivery (16.3 vs.
11.2 days, p ⬍ 0.05). In the intracranial model, a 10 mg polymer with 20% (w/w)
BCNU polymers improved survival in animals with established 9L gliosarcoma.
Compared to controls, the EVAc and pCPP:SA polymers increased median sur-
vival times 7.3-fold and 5.4-fold, respectively. In contrast, systemic BCNU
increased survival only 2.4-fold. A second study using the same established rat
intracranial 9L gliosarcoma model compared the efficacy of 20% (w/w)
BCNU-loaded pCPP:SA polymers with direct stereotactic injection of an
equivalent dose of BCNU [82]. Median survival improved by 271% compared
to that of control animals in the polymer group. In contrast, stereotactic injec-
tion improved median survival only by 36%. There were also twice as many
long-term survivors in the polymer group.
A third set of studies established the optimal pCPP:SA monomer ratio and
dosing percentage of BCNU [83]. First, in vitro release kinetics were compared
between 50:50 and 20:80 pCPP:SA formulations with ranges of drug con-
centration between 4% and 32% BCNU-loaded polymers. In theory, lowering
the proportion of CPP should slow polymer degradation, and thus increase
sustained release. In fact, the release kinetics revealed minimal differences
between the two formulations loaded at 4% BCNU, but comparisons between
the 32% polymers revealed a 150% increase in release duration (18 vs. 7 days)
for the 50:50 formulation. Next, the efficacy of polymer loads of 0, 4, 8, 12, 20,
and 32% (w/w) were compared in both 50:50 and 20:80 pCPP:SA polymer for-
mulation. When tested against the established rat intracranial 9L gliosarcoma
model, survival was maximized in the 20% loaded pCPP:SA (20:80) formula-
tion, which provided a 63% survival rate at 200 days compared to a median
survival of ⬍20 days in controls. An additional toxicity study using the
cynomolgus monkey intracranial model demonstrated that 20% BCNU-loaded
pCPP:SA (20:80) polymers had no systemic or local morbidities, and MRI
images obtained 150 days after implantation showed no evidence of edema or
mass effect [83].
A final set of preclinical experiments investigated the efficacy of local
polymer delivery of various anti-neoplastic agents, including BCNU, to combat
brain metastases [84]. First, maximum nontoxic doses of each agent in pCPP:SA
polymer were established in the mouse intracranial model. Next, efficacy was
tested with and without concurrent radiation therapy in several metastatic tumor
lines, including the B16 melanoma, RENCA renal cell carcinoma, CT26 colon
cancer, and Lewis lung carcinoma. While both BCNU-loaded polymers and
radiation treatments were effective alone, they were much more effective in
combination against B16 melanoma (median survival 35 vs. 21.5 days for con-
trols; p ⫽ 0.0005), RENCA renal cell carcinoma (38.5 vs. 12 days; p ⬍ 0.007),
and Lewis lung carcinoma (23 vs. 21 days; p ⫽ 0.001). A later study showed
Wang/Brem 466
intracranial BCNU polymers to be effective with (41 vs. 17 days; p ⫽ 0.02) and
without irradiation (⬎200 vs. 17 days; p ⬍ 0.0001) against the EMT-6 breast
cancer in mice [85]. These encouraging findings have led to clinical trials of
BCNU-loaded pCPP:SA treatment for metastatic brain tumors.
Clinical Experience with Recurrent Gliomas

Based on the preclinical findings described above, approval was obtained
for a multicenter phase I-II human trial using pCPP:SA for the treatment of
malignant gliomas [86]. Enrollment criteria limited patients to those with
recurrent malignant gliomas who had previously undergone a craniotomy for
debulking and in whom standard therapy had failed. Other eligibility require-
ments included an indication for reoperation, a single tumor focus with ⱖ1 cm3
of enhancing volume on radiographic imaging, completion of external beam
radiotherapy, a Karnofsky Performance Scale (KPS) score of ⱖ60, and no expo-
sure to nitrosureas during the 6 weeks prior to polymer implantation. Twenty-one
patients were enrolled, and three different polymer loads were tested: 1.93, 3.85,
and 6.35% (w/w). Each polymer weighed 200 mg, and most patients received a
maximum eight wafers within the tumor cavity following debulking (fig. 4).
Tumor volumes were similar in all groups.
Following treatment with polymeric BCNU as described above, there was
no evidence of systemic toxicity, measured by frequent blood chemistry, hema-
tological, and urinalysis exams. Although KPS scores fell slightly during the
immediate postoperative period, they returned to baseline and remained stable
for at least 7 weeks, indicating quality of life preservation during the chemother-
apeutic period. The implanted polymers were detectable on postoperative CT
and MRI. They appeared as areas of decreased signals on T1-weighted MRIs,
and some were visible on CT as long as 49 days after surgery. In 13 of 21
patients, routine protocol scans revealed some areas of enhancement around the
implant sites, which generally resolved spontaneously and did not correlate with
any neurological decline or other toxic sequelae [87]. Over the course of study,
10 patients required reoperation for declining neurological status with increas-
ing enhancement on MRI or CT. The most notable intraoperative finding was a
rim of necrotic tissue up to 1 cm thick around the wafer(s), similar to that
described in interstitial brachytherapy. The KPS scores were generally improved
following the removal of this tissue. The overall median survival times were
46 weeks after implant and 87 weeks after initial diagnosis, with 86% of patients
alive after more than one year of diagnosis. On the basis of this work, the 3.85%
BCNU-loaded polymer was chosen for further study.
The next clinical trial was a rigorous multicenter, prospective, random-
ized, double-blinded, and placebo controlled phase III study [88] investigating
the efficacy of 3.85% BCNU (w/w) pCPP:SA polymers in treating 222 patients
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Fig. 4. After surgical debulking, the tumor resection cavity is lined with up to eight
200 mg 3.8% (w/w) BCNU pCPP:SA (20:80) (Gliadel®) polymer, where the loaded drug is
gradually released as they dissolve.
with recurrent malignant gliomas at 27 medical centers in the North America.

Patients were randomized to receive either a BCNU treatment polymer or a
blank placebo. Selection criteria was the same as for the preceding study, except
that no chemotherapy was allowed for 4 weeks preceding surgery, and systemic
chemotherapy was allowed as early as 2 weeks after surgery. Individual patients
and their surgeons decided upon additional operations for tumor recurrence
during the study period. Mean age was 47.8 years, and randomization rendered
the treatment and placebo groups well matched for age, tumor type, and preop-
erative KPS scores. All patients previously underwent external beam radiother-
apy, and 52.7% of the treatment group and 48.2% of the control group had
previously undergone chemotherapy.
Wang/Brem 468
1.00
0.9
0.8
0.7
Probability of survival
0.6
0.5
Carmustine polymer
0.4
0.3
0.2
0.1
Placebo polymer
0
0 20 40 60 80 100 120 140 160 180 200
Time (weeks)
Fig. 5. The overall survival for patients receiving BCNU-loaded polymers versus
controls at the time of the operation for recurrent malignant gliomas. (Reprinted with
permission from Elsevier Science [88].)
The median postoperative survival of the patients implanted with treatment

polymer was 34 weeks compared to 23 weeks in the placebo group (hazard ratio
0.67, p ⫽ 0.006) (fig. 5). The 6-month survival rate was 60% in the treatment
group and 47% in the placebo group. Remarkably, among GBM patients
(n ⫽ 145), there was a 50% increase in 6-month survival within the treatment
population compared to control population (p ⫽ 0.02). The BCNU-loaded
polymer was again shown to be safe and well tolerated, with no evidence of
bone marrow suppression or systemic toxicity. Within 6 months of polymer
placement, 11.8% of treatment and 11.6% of control patients underwent reop-
eration. Intracranial infection was more common in the treatment group than
the placebo group (4/110 or 3.6 vs. 1/112 or 0.9%), but this adverse event
did not reach statistical significance. Of the eleven brains from the treatment
group that were examined postmortem, there were mild inflammatory reactions
without marked necrosis.
These studies established that BCNU-polymers are safe and relatively effec-
tive in the treatment of recurrent malignant gliomas. Based on the mean survival
rates and safety data, the FDA in 1996 approved 3.85% BCNU-loaded PCPP:SA
polymer (Gliadel®) for the treatment of recurrent GBM. This was the first time in
23 years that the FDA approved a new treatment for malignant gliomas.
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Clinical Experience with Gliadel Used as Initial Therapy
In general, any oncology treatment found effective at recurrence has
been subsequently shown to be even more effective as initial therapy. After
the establishment of Gliadel’s role in treating recurrent malignant gliomas,
attention naturally turned to examining its role during initial disease pre-
sentation. A phase I-II trial with 22 enrolled patients in three medical cen-
ters was designed to determine the safety of Gliadel polymers at the time
of initial surgery [89]. Like the previous studies, the polymer wafers weighed
200 mg, and most patients received a maximum of eight wafers. Inclusion
criteria required a single enhancing tumor focus ⱖ1 cm3, age ⬎18 years,
and a KPS score of ⱖ60. The mean age was 60, and all the patients received
postoperative external beam radiation therapy averaging 5000 rad. No
patients received additional chemotherapy during the first 6 months after
surgery.
There was no perioperative mortality, nor was there evidence of systemic
or local toxicity attributable to the polymer. Twenty-one of 22 patients received
a pathological diagnosis of GBM. Median survival was 44 weeks from implan-
tation with 4 patients surviving ⬎18 months. This phase I-II study demon-
strated that Gliadel was a safe treatment option for the patients newly diagnosed
with malignant gliomas.
Based on these encouraging findings, a phase III, multicenter, randomized,
double-blinded, placebo-controlled study of the efficacy of locally implanted
Gliadel against newly diagnosed malignant gliomas was begun [90]. Admission
criteria included the presence of a single tumor focus with ⱖ1 cm3 of enhance-
ment, age between 18 and 65 years, a KPS score ⱖ60, and a histopathological
diagnosis of malignant glioma on intraoperative frozen section. Again, 200 mg
wafers were used. Originally intended for 100 patients, the study was termi-
nated prematurely at 32 patients due to the unavailability of the drug. There
were 16 patients in each arm of the study. The median age was 55.5 years for
the BCNU group and 53 years for placebo. The median KPS score was 75 for
the treatment group and 90 in the control group. There was a discrepancy in
tumor pathology; all placebo patients harbored GBMs, but only 11 of 16 BCNU
patients had the same diagnosis.
With all 32 patients included in the analysis, the median survival of the
treatment group was 58.1 weeks compared to 39.9 weeks for the control group
(p ⫽ 0.012) (fig. 6). When limited to the subgroup of GBM patients, the treat-
ment group had a median survival of 53.3 weeks versus 39.9 weeks for the
placebo group (p ⫽ 0.008). Perhaps more striking was the fact that 3 out of 4
enrolled patients with GBM alive 3 years after the termination of the study
were in the BCNU-loaded polymer group. Age and preoperative KPS score
significantly impacted survival, while tumor type impacted survival but not in a
Wang/Brem 470
1.00
0.75
0.50 Gliadel
0.25
Placebo N ⫽ 32
0.00
0 20 40 60 80 100 120 140 160
Time from surgery to death in weeks
Follow-up time⫽ 156 weeks
Fig. 6. Kaplan-Meier survival curve for patients with Grade III or IV gliomas treated
with BCNU-loaded polymer implants versus placebo. (Reprinted with permission [90].)
statistically significant way. Again, no local or systemic toxicity attributable to

the polymers was noted.
A larger phase IV, multicenter, randomized, double-blinded, placebo-
controlled study was recently carried out involving 240 patients to definitively
assess the role of Gliadel in initial therapy [91]. This study demonstrated
that treatment with Gliadel increased median survival (13.9 vs. 11.6 months,
p ⫽ 0.003) compared to controls treated with blank polymers. Based partially
on these results as well as the results of previous studies, in 2003 the FDA
approved Gliadel for the treatment of initially diagnosed malignant glioma. This
step represents another major milestone in the evolution of local-delivery poly-
mer chemotherapy for the treatment of brain tumors.
Clinical studies investigating the efficacy of BCNU-polymer treatment
has by no means concluded. A phase I-II dose-escalation study was recently
completed to establish the maximal nontoxic BCNU loading in 20:80
PCPP:SA polymers. Similar to the results in animal models [83], the maximal
tolerated dose was determined to be 20% BCNU (w/w) [92]. At this time,
Gliadel is still loaded only at 3.85%. Future clinical trials include a phase III
trial to evaluate a 20% BCNU-loaded wafer, as well as trials investigating the
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use of Gliadel for metastatic tumors and in combination with other drugs, and
the use of chemotherapy resistance modification in combination with Gliadel.
Other Neuro-Oncology Applications

The successful development of BCNU-loaded sustained-release polymers
from the laboratory to standard clinical use has served as a model for future
investigation. With local delivery, agents that have never been seriously con-
sidered for neuro-oncology due to systemic toxicity can now be examined. The
following agents have been either successfully incorporated into polymers or
successfully used as adjuvants for polymer-based treatment.
O6-Benzylguanine
A limitation of Gliadel is the acquired resistance of many brain tumors to
BCNU through the expression of O6-alkylguanine-DNA alkyltransferase
(AGT), a DNA-repair protein found in the majority of brain tumors treated with
BCNU [93]. BCNU exerts its anti-neoplastic effects by chloroethylation of DNA
at the O6 position of guanine. By removing adducts at this position, AGT is able
to protect tumor cells from BCNU. O6-Benzylguanine (O6-BG) is a substrate
analog that can irreversibly inactivate AGT by transferring a benzyl group to a
cysteine residue at the active site of AGT [94].
The O6-BG-mediated AGT inhibition increases tumor sensitivity to
BCNU, but when given in combination with systemic BCNU, maximal toler-
ated doses of BCNU were reduced up to 6-fold in animal models due to bone
marrow toxicity [95]. Presumably, the same increased BCNU sensitivity O6-BG
imparted to tumor cells also affected cells vulnerable to BCNU-related toxicity.
Rhines et al. [96] postulated that by combining systemic O6-BG with intracra-
nial local-delivered BCNU polymers instead of systemic BCNU, the side
effects of combined treatment could be significantly reduced. Using the estab-
lished rat intracranial F98 glioma model (a tumor line with high AGT activity),
they showed that by giving systemic O6-BG with BCNU-loaded pCPP:SA
polymers, median survival was improved in the combination therapy over
animals receiving O6-BG alone (34 vs. 22 days, p ⫽ 0.0002) or BCNU polymer
alone (34 vs. 25 days, p ⫽ 0.0001). They also found that it was not necessary
to reduce BCNU polymer load when systemic O6-BG was introduced. The
animals did not exhibit bone marrow or gastrointestinal toxicity.
These results suggest that the concurrent use of O6-BG and BCNU poly-
mers may be an important addition to the treatment of malignant brain tumors.
In fact, preliminary phase I clinical trials investigating O6-BG as an adjuvant to
BCNU treatment have been completed [97]. Current ongoing efforts include
incorporating O6-BG within polymers for local delivery, and dose-escalation
studies for systemic and local-delivered O6-BG.
Wang/Brem 472
Paclitaxel
Paclitaxel (Taxol) is a microtubule-binding agent, which has been shown
to have tumoricidal activity against non-small cell lung cancer, breast cancer,
and ovarian cancer. In vitro studies have shown paclitaxel to be potent against
rat and human glioma cell lines [98], but not permeable across the BBB [99],
thus making it an excellent candidate for local polymeric delivery.
In vitro kinetics studies demonstrated that 20–40% (w/w) paclitaxel-
loaded pCPP:SA (20:80) polymers released for up to 1000 h. Biodistribution
studies revealed tumoricidal concentrations of paclitaxel in the rat brain for
⬎30 days after implantation. In the established rat intracranial 9L gliosarcoma
model, median survival was improved from 19.5 days in rats treated with blank
polymers to 61.5 days with 20% paclitaxel-loaded polymers (p ⬍ 0.001) [100].
Clinical trials utilizing a paclitaxel-loaded PACLIMER [101] microsphere
delivery system are currently in progress for ovarian cancer. Toxicity trials on
dogs for the treatment of intracranial tumors are ongoing; the initial results
show no early mortality or significant morbidity that can be attributed to the
paclitaxel polymer [102]. Phase I-II clinical trials are being planned for imple-
mentation as soon as these canine toxicity studies are completed and can
demonstrate safety.
Camptothecin
The camptothecins are a family of inhibitors of DNA-replicating enzyme
topoisomerase I [103]. While in vitro and in vivo studies showed great promise,
clinical trials demonstrated unexpected toxicities with systemic administration
[104]. This prevented its use as a systemic agent for gliomas, but made it an
excellent candidate for local polymer delivery [32]. Among the family of camp-
tothecins, sodium camptothecin was selected due to its chemical properties
which make it easy to load onto polymers. Initially incorporated onto the EVAc
polymers, where in vitro kinetics experiments demonstrated its sustained
release, local-delivery sodium camptothecin demonstrated dramatically
increased efficacy in the established rat intracranial 9L gliosarcoma model,
where 50% (w/w) polymers extended median survival from 19 days in control
animals to ⬎120 days (p ⬍ 0.001). In addition, while none of the control rats
survived beyond 32 days, 59% of treatment animals survived ⬎120 days.
In contrast, systematic camptothecin had no impact on survival. No local or
systemic toxicity was observed in the polymer-implanted animals.
Sodium camptothecin loaded onto pCPP:SA polymers was also tested,
both in the previously described mouse metastatic tumor study [84], and,
most recently, in the established rat intracranial 9L gliosarcoma model [105].
The metastatic study showed that the camptothecin-loaded pCPP:SA polymer
was effective only in combination with radiation therapy and only against the
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B16 melanoma (median survival 27.5 vs. 19 days; p ⫽ 0.043). In the 9L
gliosarcoma model, median survival was extended with 50% (w/w) camp-
tothecin-loaded polymers from 17 days in control animals to 69 days
(p ⬍ 0.001). No local or systemic toxicity was noted. The polymers were also
shown to release active camptothecin for up to 1000 h. Current ongoing
efforts include preclinical studies examining the efficacy of various camp-
tothecin analogs [106].
Quinacrine
Quinacrine was an early anti-malarial drug and is rarely used anymore in
the clinical setting. While its exact mechanism of action is unknown, it demon-
strates a wide variety of intracellular actions, including the ability to reduce
mutagenicity in leukemia and glial cell lines [107, 108]. In the rat flank C6
glioma model, quinacrine has been shown to reduce tumor growth when deliv-
ered orally at its maximally tolerated dose (20 mg/kg) in conjunction with
systemic BCNU therapy, compared to systemic BCNU therapy alone [108].
However, preliminary work by us has shown that in the established rat intracra-
nial 9L gliosarcoma model, oral quinacrine exhibits no effect when given with
intracranial BCNU-loaded 3.85% (w/w) pCPP:SA polymers (fig. 7a). It is
likely that the CNS delivery of quinacrine to the tumor site is inadequate, since
quinacrine exhibits a 10% penetrance into the CNS. However, the coimplanta-
tion of 15% (w/w) quinacrine-loaded pCPP:SA polymers with BCNU-loaded
polymers does result in a significant increase in survival (fig. 7b). To exhibit
this synergistic effect, it is necessary that the quinacrine polymer be implanted
2 days before the BCNU polymer; placing both polymers in the tumor bed con-
currently results in no benefit whatsoever (fig. 7c). Current ongoing work
includes investigations into the molecular basis behind this synergistic effect.
Possible mechanisms include quinacrine’s documented anti-mutagenicity, inter-
ference with the outward transport of BCNU out of the glial cell, enhancement
of the BCNU-induced apoptosis, and anti-AGT activity.
Mitoxantrone
Mitoxantrone is a dihydroxyanthracenedione derivative used in the treatment
of advanced breast cancer, non-Hodgkin’s lymphoma, acute nonlymphoblastic
leukemia, and chronic myelogenous leukemia in blast crisis, and has been
FDA approved for the treatment of hepatic and ovarian cancer. Its use for CNS
malignancies has been limited by its poor CNS penetrance and dose-limiting
myelosuppression. Recently, DiMeco et al. [109] demonstrated that with up to
10% (w/w) mitoxantrone-loaded pCPP:SA wafers median survival was signifi-
cantly improved compared to controls (50 vs. 19 days, p ⬍ 0.0001). Further
animal studies in combination with other chemotherapy agents are ongoing.
Wang/Brem 474
Immunotherapy
The role of local delivery of immunotherapy to combat malignant gliomas
has mainly involved the use of cytokines, such as interleukins (IL), interferons,
and colony-stimulating factors, which are produced by cells of the immune
system to generate and maintain an immune response. Because of genetic muta-
tions, tumor cells express proteins foreign to the host, thereby rendering them
vulnerable to an immune response. Effort has been made in using local delivery
to release cytokines into brain tumors because cytokines are impermeable to the
BBB and carry with them systemic toxicity.
Two general strategies have been made in providing for the local deliv-
ery of cytokines. The first is the sustained release of cytokine at the tumor
site using irradiated tumor cells transduced to secrete the cytokine in a
paracrine fashion. Thompson et al. [110] demonstrated with the C57BL/6
mouse intracranial melanoma model that IL-2-transduced tumor cells could
generate an immune response to wild-type tumor via direct injection to
the tumor site, but not when injected in the flank. In contrast, GM-CSF-
transduced cells generated an immune response via flank injection, but not
when injected intracranially. Synergy was noted when intracranial IL-2-
transduced cells and subcutaneously flank GM-CSF-transduced cells were
injected simultaneously.
A study using the same animal model demonstrated several interesting
findings [111]. First, rats with intracranially implanted IL-2-transduced cells
demonstrated increased survival compared to controls when challenged with
tumor both intracranially and in sites distal to the brain. Second, after success-
ful rejection of an initial challenge, these animals also exhibited immunological
memory by mounting an immune response and increasing survival in the face
of a second tumor challenge, both intracranial and in sites distal to the brain. In
contrast, identical or 10-fold increased doses of subcutaneously injected IL-2-
transduced cells failed to elicit such memory responses. Finally, the study
demonstrated with gene knockout mice that natural killer cells but not CD4⫹
T cells were most responsible for the anti-tumor immune response. Because
flank-injected IL-2 paracrine cells did not elicit a similar response, the study
postulated that immune cells within the CNS had different cytokine require-
ments than their counterparts in the periphery.
A third study identified IL-12 as a potential candidate for local paracrine
delivery [112]. In addition to its immune regulatory effects, IL-12 also exhibits
anti-angiogenesis properties. By challenging the established rat intracranial
9L gliosarcoma model, tumor cells transduced with IL-12 were implanted
intracranially. Expression of IL-12 was confirmed by reverse transcriptase-
polymerase chain reaction. Furthermore, in addition to prolonging survival,
local paracrine delivery of IL-12 also induced immunological memory to the
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IC BCNU and po quinacrine vs. IC 9L gliosarcoma
Control (n⫽ 7) Quinacrine (n⫽ 8)

Day 3 IC quinacrine and day 5 IC BCNU vs. 9L
BCNU/Quinacrine (n⫽ 8) BCNU (n ⫽ 8) gliosarcoma
100
Control (n ⫽10) BCNU (n⫽8)
Quinacrine (n⫽8) Quinacrine/BCNU (n⫽8)
80 100
80
Survival (%)
60
Survival (%)
60
40
40
20
20
0 0
0 20 40 60 80 100 120 0 20 40 60
a Time (days) b Time (days)
Day 5 IC BCNU and quinacrine vs. 9L

gliosarcoma
Control (n⫽ 10) BCNU (n⫽ 8)

Quinacrine (n⫽ 8) Quinacrine/BCNU (n⫽8)
100
80
Survival (%)
60
40
20
0
0 20 40 60
c Time (days)
Fig. 7. a Kaplan-Meier survival curve for animals treated with intracranial 10 mg 3.8%
(w/w) BCNU pCPP:SA (20:80) polymers placed day 5 after 9L gliosarcoma implantation
with or without daily oral quinacrine gavages (20 mg/kg) starting day 5 and lasting 14 days.
b Kaplan-Meier survival curve for animals treated with intracranial BCNU polymers placed
day 5 after 9L gliosarcoma implantation with or without intracranial 10 mg 15% (w/w)
Wang/Brem 476
animals. A second injection of wild-type 9L gliosarcoma tumor cells also
elicited an immune response.
Several experiments have recently examined the interaction between local
paracrine immunotherapy and local polymer-delivered chemotherapy. Using the
mouse intracranial F16-B10 melanoma model, an experiment demonstrated syn-
ergy between either 10% (w/w) BCNU-loaded or 1% (w/w) carboplatin pCPP:SA
polymers with local paracrine IL-2-transduced cells [113]. When combining
BCNU-loaded polymers with immunotherapy, 70% of the animals receiving com-
bination therapy survived ⬎72 days, compared to none (with a median survival of
15.8 days) in controls (p ⫽ 0.0023). When combining carboplatin-loaded poly-
mers with immunotherapy, 80% of the animals survived ⬎72 days, compared to
none (with a median survival of 20.6 days) in controls (p ⫽ 0.0001). Histological
examination of animals receiving combination therapy revealed rare degenerating
tumors cells with a marked mixed inflammatory reaction on postimplantation day
14, and no tumor cells and resolution of the inflammatory reaction on day 72.
The second general strategy for sustained delivery of cytokines involves
loading them directly into polymers. In 1998, Wiranowska et al. [114] demon-
strated proof of principle that polymers could deliver sustained and biologically
active cytokines. By loading murine interferon-␣/␤ onto EVAc polymers, they
demonstrated with both in vitro and in vivo experiments that the released inter-
ferous were biologically active. In vitro assays determined most of the activity
was released within the first 4 days. In vivo trials demonstrated most of the activ-
ity was released within the first 24 h and gradually decreased over the next 3 days.
In 1993, Golumbek et al. [75] introduced the gelatin chondroitin sulfate
microsphere system for local delivery of drugs in an injectable mixture. In 2001,
Hanes et al. [115] encapsulated IL-2 into gelatin chondroitin sulfate and con-
firmed that the mixture maintained a sustained release of activity over 2 weeks
in vitro and up to 3 weeks in vivo. Using the rat intracranial 9L gliosarcoma
model, the mouse intracranial B16-F10 melanoma model, and the mouse liver
CT26 carcinoma model, they then demonstrated statistically increased effective-
ness in generating a protective immune response when injecting the gelatin chon-
droitin sulfate-IL-2 mixture into tumor compared to controls or local paracrine
delivery. In B16-F10 model, 42% of animals exhibited protection on a second
tumor challenge. Recently, Rhines et al. [116] demonstrated that the combination
of IL-2-loaded microspheres and BCNU-loaded pCPP:SA polymers showed a
statistically significant increase of median survival when compared to treatment
quinacrine pCPP:SA (20:80) polymers placed day 3 after tumor implantation. c Kaplan-
Meier survival curve for animals treated with intracranial BCNU polymers placed day 5 after
9L gliosarcoma implantation with or without intracranial 10 mg 15% (w/w) quinacrine
pCPP:SA (20:80) polymers concurrently day 3 after tumor implantation.
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with either modality alone. Therefore, brain tumor vaccines using polymer deliv-
ery of cytokines is a promising avenue of investigation.
Angiogenesis Inhibitors
Angiogenesis is the process where new blood vessels form, and is essen-
tial for the tumor growth [117]. Without angiogenesis, the source of nutrients is
limited to a few millimeters by diffusion from the tumor periphery, and tumor
size is arrested in equilibrium between peripheral cell proliferation and central
cell death. With angiogenesis, nutrient delivery can reach the central cells, and
tumor growth becomes exponential with the potential for metastatic spread
[118]. Since GBM is one of the most angiogenic of all neoplasms, the use of
angiogenesis inhibitors for treating brain tumors has generated much interest.
One of the earliest attempts at local polymer-delivered anti-angiogenesis
agents used heparin and cortisone [119], which exhibits angiogenesis inhibition
properties [120] when used in combination. Loading both drugs onto an EVAc
copolymer and testing against the rabbit cornea VX2 carcinoma model, angio-
genesis activity was reduced by 60% at 21 days after implantation (p ⬍ 0.05).
In the same study, the drug combination loaded in pCPP:SA polymers inhibited
growth in the rat flank 9L gliosarcoma model by 78% (p ⬍ 0.05).
Squalamine, an aminosterol isolated from the dogfish shark, has been shown
to exhibit anti-angiogenesis properties by inhibiting tumor mitogen-induced
endothelial cell proliferation [121]. In the rabbit cornea VX2 carcinoma model,
EVAc copolymers loaded with 20% (w/w) squalamine have been shown to inhibit
vascular ingrowth. Currently, squalamine is being evaluated in clinical trials for
a variety of advanced cancers [122]. Further work examining its efficacy in local
polymer delivery is ongoing.
Another anti-angiogenesis agent is minocycline, a broad-spectrum antibiotic
with anti-collagenase properties [123]. When loaded onto the EVAc polymer and
tested against the rabbit cornea VX2 carcinoma model, it inhibited neovascu-
larization by a factor of 4.5, 4.4, and 2.9 on days 7, 14, and 21, respectively
(p ⬍ 0.05 at all timepoints) [124]. In a follow-up study, the polymers were found
to deliver minocycline in a sustained fashion with 55% of the drug released at 90
days [31]. With 50% (w/w) minocycline-loaded EVAc polymers challenging the
rat intracranial 9L gliosarcoma model, median survival increased from 13 to 69
days (p ⬍ 0.001) when polymers were implanted simultaneously with tumor
injection. When treated in the established rat intracranial 9L gliosarcoma model,
minocycline-loaded polymers alone did not impact survival, but they did extend
median survival by 43% when used in conjunction with surgical resection
(p ⬍ 0.002), and by 90% with surgical resection and systemic BCNU. Recent
work with 40–50% (w/w) minocycline-loaded PCPP:SA polymers suggest that
their efficacy is significantly better than that of the EVAc polymers [125].
Wang/Brem 478
Cyclophosphamide and 4-Hydroxyperoxycyclophosphamide
Cyclophosphamide (Cytoxan) is an alkylating agent, widely used for the
treatment of a variety of malignancies. Its active metabolite, hydroxycy-
clophosphamide, poorly crosses the BBB. Therefore, the drug has not been
widely used for the treatment of malignant gliomas. Because cyclophos-
phamide requires enzymatic activation by the hepatic p450 cytochrome oxidase
system, its potential for effective utilization of local-delivery polymer systems
is low [126].
4-hydroxyperoxycyclophosphamide (4-HC) is a derivative of cyclophos-
phamide, which spontaneously converts in vivo into the active metabolite [127],
thus making it a candidate for polymeric local delivery. Because of 4-HC’s
hydrophilic properties, the FAD:SA system was chosen for delivery.
Pharmacokinetic and biodistribution studies demonstrated favorable release
kinetics and intracranial distribution [128]. In the rat model, cerebral drug lev-
els peaked between 5 and 20 days after implantation, in contrast to a rapid fall
in levels 48 h after systemic dosing. After toxicity studies demonstrated the
maximum tolerable polymer dose of 20% (w/w), efficacy was measured using
the established rat intracranial 9L gliosarcoma and F98 glioma model [72].
Animals treated with the blank polymer had a median survival of 14 days with
no long-term survivors, while animals treated with 4-HC loaded polymers had
a median survival of 77 days with 40% surviving beyond 80 days (p ⫽ 0.004).
Recently, it was shown that L-buthionine sulfoximine (BSO) potentiates
the anti-tumor effects of 4-HC in the rat 9L gliosarcoma model [129] by inhibit-
ing the glutathione S-transferase enzyme pathway, which appears to play an
important role in the inactivation of alkylating agents [130]. In the established
intracranial 9L gliosarcoma model, corelease of both 4-HC and BSO from the
FAD:SA polymer boosted median survival 4.6 times greater than rats treated
with empty polymer (61.5 vs. 13 days, p ⬍ 0.001), while release of 4-HC alone
improved median survival only 2.3 times (p ⬍ 0.001). Systemic delivery of
BSO in conjunction with polymer-delivered 4-HC did not impact survival. In
addition, a separate experiment in this study showed that local delivery of BSO
may be safer than systemic administration. BSO-loaded EVAc polymer
implanted in a rat brain depleted intracranial glutathione levels without impact-
ing hepatic levels, while systemic BSO delivery was shown to reduce hepatic
glutathione levels without impacting intracranial levels.
Adriamycin
Adriamycin, an anthracycline antibiotic that intercalates with DNA, caus-
ing strand scission and double stranded cross breaks, exhibits tumoricidal activ-
ity on breast cancer, acute leukemia, lymphoma, and other cancers [131]. In
1983, following surgical debulking for malignant glioma, Nakazawa et al. [132]
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treated 20 patients with adriamycin injections to the tumor bed via an Ommaya
reservoir. Total doses of 5.0–10.0 mg in daily 0.5-mg aliquots were injected
into the reservoir. In addition, all patients received cobalt-60 irradiation and
immunotherapy. This regimen achieved a one-year survival rate of 55%.
Compared to systemic delivery, local infusion with Ommaya reservoirs
increased the local concentration of adriamycin up to 38 times, and the drug
was noted to penetrate up to 3 cm into the brain parenchyma.
Subsequently, adriamycin was successfully incorporated into EVAc
polymers in the shape of needles [133]. Pharmacokinetic studies demonstrated
zero-order kinetics release, and further studies demonstrated that the needles
significantly inhibited the growth of brain tumor xenografts in nude mice.
Adriamycin has also been successfully incorporated into biodegradable pCPP:SA
polymers [134]. In vitro assays demonstrated sustained release, and in vivo
studies demonstrate improved median survival (33 vs. 13 days in controls,
p ⬍ 0.0006) in the established rat intracranial 9L glioma model. Further animal
studies of the adriamycin-loaded pCPP:SA polymers, including investigations
with concurrent immunotherapy, are ongoing.
5-Fluorouracil
5-Fluorouracil (5-FU) is a thymidine analog that blocks the conversion of
deoxyuridylic acid to thymidylic acid, thus depriving the cell of an essential
precursor for DNA synthesis. While effective in treating various nonintracranial
malignancies, its efficacy for brain tumors is limited by its systemic toxicities,
which include myelosuppression and gastrointestinal mucosal injury [131]. In
fact, early efficacy trials testing 5-FU’s role in treating brain tumors were dis-
appointing [135–137].
There is a long history in the investigation of 5-FU local delivery. The
earliest attempt was a 1968 study in which 5-FU was loaded into gelatin
sponges and Surgicel [77]. While no evidence of toxicity was noted, no thera-
peutic effect was demonstrated. In 1979, pharmacokinetic studies of silastic
tubes loaded with 5-FU and urokinase demonstrated continual release of both
drugs for over 5 weeks [79]. When tested against the rat flank ethylnitrosourea
induced gliomas, this drug-loaded silastic tube was found to be capable of
inhibiting tumor growth. This study led to a clinical trial of 14 patients with
malignant gliomas or metastatic disease. Thirteen patients lived for more than
8 months following implantation. Additional clinical trials using silastic tubes
loaded with a ‘chemotherapy cocktail’ of 250 mg 5-FU, 6,000 IU urokinase,
1.5 mg mitomycin C, and 250 mg bromodeoxyuridine showed a median sur-
vival of 18 months for patients with malignant gliomas, with a 3-year survival
rate of 16% [138, 139]. Clinically significant levels of 5-FU were measured as
long as 2 years after implantation.
Wang/Brem 480
In 1986, the first attempt to deliver 5-FU with polymers was made when
several anti-neoplastic agents, including 5-FU, adriamycin, and mitomycin C,
were loaded onto a matrix consisting of ‘glassified monomers’ with 10% poly-
metacrylic methyl acid and tested on 55 patients [140]. The one-year survival
rate for malignant gliomas was 47%. 5-FU was then successfully loaded onto
PLGA microspheres [141]. When tested against the established rat intracranial C6
glioma model, treatment with 5-FU-loaded microspheres significantly decreased
mortality (p ⫽ 0.017), while treatment with placebo and bolus 5-FU injections
had no effect [142]. There were no observed toxicities, and histological exami-
nation showed only mild tissue reaction.
Menei et al. [143] reported treating 8 newly diagnosed GBM patients with
surgical debulking and implantation of 5-FU-loaded PGLA microspheres. The
patients also underwent postoperative adjuvant external beam radiation therapy.
Clinically significant concentrations of 5-FU were measured in the CSF up to
one month after surgery, while 5-FU levels in the blood were low and transitory.
Median survival time was 98 weeks for the 8 patients, with 2 patients exhibit-
ing disease remission at 139 and 153 weeks.
Fluorodeoxyuridine, a compound related to 5-FU, has been successfully
delivered from FAD-SA polymers in vitro and in vivo [144]. This study was
based on the earlier work in which fluorodeoxyuridine was continuously infused
with a Medtronic SynchroMed pump to treat a single patient with intracranial
metastatic renal cell carcinoma [145]. A complete response was achieved in
3 months and maintained for 22 months.
Methotrexate
Methotrexate (MTX) is a folate antagonist widely used against a number of
malignancies. Its effectiveness against brain tumors is limited, again due to imper-
meability across the BBB and systemic side effects, which include myelosuppres-
sion and gastrointestinal necrosis [131]. Once introduced to the brain parenchyma
via direct injection, MTX disseminates widely in the brain [146]; its dissemination
is comparatively poor with cisternal or intraventricular infusion [21, 147].
The first large-scale clinical trial of intracranial MTX was reported in
1987, where 269 patients were divided into five treatment groups, each receiv-
ing a different postoperative strategy following surgical resection [148]. One of
the groups received ‘local chemotherapy,’ which was implantation of a Spongistan
matrix soaked with 50 mg MTX. No side effects or complications were observed.
While the local MTX had no statistically significant effect on overall survival,
there were more long-term survivors in the group receiving local MTX than in
the other groups.
In an effort to improve stability and biodistribution, MTX has also been
modified by covalent linking to dextran [149]. In vitro studies against the
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human H80 glioma line demonstrated that the presence of the conjugated
dextran did not adversely affect tumoricidal properties. On a three-dimensional
collagen lattice designed to simulate extracellular matrix, the MTX-dextran
conjugate showed superior penetrance when compared to unmodified MTX. In
the established rat intracranial 9L gliosarcoma model, FAD:SA polymers
loaded with the MTX-dextran conjugate offered modest, but significant,
improvement over controls.
Other attempts to produce local delivery include loading MTX onto poly-
methylmethacrolate pellets [78] and PLGA copolymer matrices [150]. Despite
release kinetics demonstrating release of 96–99% of the drug within only
2 days, the MTX-loaded pellets significantly improved median survival by 69%
compared to controls in the rat intracranial ethylnitrosourea-induced tumor
model. The MTX-loaded PLGA polymer inhibited glioma growth in the rat
flank. A fibrin glue-based MTX system has also been introduced that inhibited
glioma growth in the rat flank [76].
Although early clinical trials [148, 151] showed minimal toxicity of intratu-
moral MTX, other case reports have documented neurological side effects. For
example, one patient with meningeal carcinoma developed an abulic-hypokinetic
syndrome and left hemiparesis after receiving intraventricular MTX [152]. Two
patients with MTX administered via an Ommaya reservoir developed large cysts
at the catheter tip [153].
Platinum Drugs
Carboplatin is a second generation platinum analog that causes myelosup-
pression when administered systemically, but is less neurotoxic than its parent
compound, cisplatin [154]. Because of its solubility, carboplatin is optimally
released by the FAD:SA polymer. Using the rat intracranial F98 glioma model,
Olivi et al. [71] determined a maximum nontoxic dose of 5% (w/w) and then
tested for efficacy. Carboplatin-loaded polymers increased median survival
from 16 days in control animals (with all controls being dead by day 19) to
52 days. In a separate study, carboplatin polymers were assessed against vari-
ous mouse metastatic brain tumors [84]. In combination with radiation therapy,
carboplatin-loaded polymers prolonged survival against the CT26 colon carci-
noma (median survival 33 vs. 20.5 days for controls, p ⫽ 0.013) and RENCA
renal cell carcinoma (15 vs. 12 days, p ⬍ 0.01). The carboplatin-loaded poly-
mers alone demonstrated efficacy against the B16 melanoma (27 vs. 16.5 days,
p ⫽ 0.043), while combination with radiation was not effective.
In an attempt similar to combining 4-HC and BSO, carboplatin has also
been coupled with ␣-cyclodextrin to delay the decomposition and increase
bioavailability [155]. Both agents were incorporated into ethylcellulose micro-
capsules at a 2.2% (w/w) loading, and pharmacokinetic assays demonstrated
Wang/Brem 482
56% release of carboplatin over 110 days. When tested against the established
rat intracranial F98 glioma, animals implanted with microcapsules loaded with
only ␣-cyclodextrin exhibited a median survival of 20 days, compared to
34 days for microcapsules loaded with only carboplatin (p ⬍ 0.001), and 51
days for microcapsules loaded with both (p ⬍ 0.01 vs. carboplatin alone).
Despite its neurotoxicity, there has been significant effort investigating the
role of local delivery for carboplatin’s parent compound cisplatin. When loaded
onto biodegradable polylactic acid polymers and tested with the established rat
intracranial 9L gliosarcoma model, median survival was increased from
24 days in the control group to 32 days for animals treated with systemic cis-
platin, 39 days for animals treated with local bolus infusions of cisplatin, and
more than 60 days for the polymer delivery group (p ⬍ 0.00006 vs. systemic
group; p ⬍ 0.001 vs. bolus group). Furthermore, histopathological ‘cure’ was
seen in 8 of 12 cisplatin-loaded polymer animals versus 3 of 13 local bolus
infusion animals (p ⬍ 0.01). No cures were seen in the other groups.
Biocompatibility was confirmed in a follow-up study [38].
Dexamethasone
Vasogenic edema is a major source of morbidity in brain tumors and is
induced by malignant gliomas secondary to breakdown of the BBB [131]. High
dose corticosteroid therapy can significantly alleviate the edema [156], but
systemic sustained exposure leads to significant side effects, including diabetes
mellitus, skin atrophy, Cushing’s syndrome, weight gain, hemorrhagic gas-
trointestinal ulcers, myopathies, osteoporosis, and pathological fractures [157].
When 35% dexamethasone (w/w) was loaded onto the EVAc copolymer, clini-
cally significant delivery was demonstrated in the rat brain for up to 21 days
[158]. Concurrent plasma levels were noted to be low. To assess efficacy, both
systemic dexamethasone and drug-loaded EVAc polymers were tested in the rat
intracranial 9L gliosarcoma-induced model [30]. Measuring edema as percent-
age water weight, it was demonstrated that both intracranial dexamethasone-
loaded polymer (79.15%; p ⬍ 0.05) and intraperitoneal dexamethasone
injections (79.16%; p ⬍ 0.05) were more efficacious compared to controls
(79.45%) and intraperitoneal polymer implantation (79.39%).
Bleomycin
Bleomycin is a tumoricidal antibiotic used to treat testicular cancer, squa-
mous cell carcinoma, and other malignancies. Dose-limiting systemic side
effects include gastrointestinal and pulmonary toxicities. Intracranial polymeric
delivery of bleomycin has been limited to two reports, where the drug was loaded
into a compressed tablet form of lactose and encapsulated with ethylcellulose
[159, 160]. In the canine intracranial model, the system demonstrated a release
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half life of 11 days, with CSF bleomycin levels detected for up to 20 days. In a
Wistar rat intracranial glioma model, the tablet demonstrated statistically signif-
icant decreased tumor growth compared to systemically delivered bleomycin.
The preparation was then placed into 6 patients undergoing craniopharyngioma
resection and recurrence was prolonged in one patient.
Other Neuro-Oncology-Related Agents

Radiosensitizers, such as 5-iodo-2⬘-deoxyuridine (IudR), which act by
replacing thymidine in replicating DNA, have been incorporated into pCPP:SA
polymers and tested in animal models as adjuvants to radiation therapy [161,
162]. Anticonvulsants, such as phenytoin, have been successfully incorporated
into the EVAc copolymer, and decreased cobalt-induced seizures in Sprague-
Dawley rats [163]. Neither class of drug has seen clinical use in conjunction
with local polymer drug delivery.
Other Neurosurgery Applications of Polymeric Drug Delivery
The neurosurgical applications for polymer-based drug delivery systems

are not limited to treating the malignancies. A growing number of neurosurgi-
cal research endeavors to utilize this technology, including clinical applications
for vasospasm, peripheral nerve injuries, and spinal fusion.
Vasospasm
Delayed vasospasm occurs in approximately 30% of patients 4–14 days
following aneurysmal subarachnoid hemorrhage. Despite early surgical inter-
vention and advances in microsurgical techniques and neuroimaging, it remains
the leading cause of delayed morbidity and mortality in subarachnoid hemor-
rhage. The etiology of delayed vasospasm is unclear. However, there has been
an increasing recent evidence suggesting that both inflammation and depleted
levels of nitric oxide (NO) play a important role in its presentation [164, 165].
Ibuprofen, an anti-inflammatory agent, and (Z)-1-[2-(2-aminoethyl)-
N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA/NO), an NO donor
molecule, have both been studied as potential drugs that can be loaded onto
polymers to treat vasospasm.
The anti-inflammatory properties of ibuprofen include its ability to
inhibit the expression of certain cell adhesion molecules and therefore disrupt
leukocyte endothelial cell interactions. However, dose-limiting side effects
prevent its systemic administration in obtaining clinically significant levels in
the CNS. In 1999, Thai et al. [166] loaded 50% ibuprofen (w/w) onto EVAc
Wang/Brem 484
polymers and demonstrated the complete release of all drugs within a 12-day
period. In the rat femoral artery model, ibuprofen-loaded polymers demon-
strated statistically significant reduction in vasospasm compared to blank
polymers, when the treatment was initiated within 6 h (98.0% vs. 69.2%
vessel lumen patency, p ⬍ 0.002). No adverse reactions were observed in the
animals.
DETA/NO is a water soluble NO donor whose application in vasospasm
treatment is under active investigation. A recent study performed by Aihara
et al. [167] using a monkey intracranial subarachnoid hemorrhage model
treated animals with continuous intercisternal infusion of DETA/NO via
pumps for up to 7 days. No statistically significant reduction in vasospasm was
encountered. However, it should be noted that in their treatment regimen, the
reservoirs of the pumps were filled every 24 h with freshly prepared DETA in
sterile water. Without a buffered solution, it is possible that the DETA/NO
decomposed immediately on contact with aqueous carrier. On the other hand,
by loading DETA/NO in EVAc polymers, this problem is avoided since the
loaded drug is sequestered away from the aqueous environment due to the
anhydrous nature of the EVAc lattice and will not decompose until it is
released. In fact, Tamargo and colleagues [168] have shown that local 20%
(w/w) DETA/NO-loaded polymers significantly reduces vasospasm in multi-
ple animal models, including the rat femoral artery model, rabbit basilar artery
model [169], and monkey intracranial model [170]. The 20% DETA/NO-
loaded polymers exhibited no apparent toxic side effects in any of the animal
models, and further efficacy and toxicity studies are ongoing.
Peripheral Nerve Injuries

The incidence of obstetric brachial plexus injuries in the USA has been
estimated to be as high as 2.5 per 1,000 live births [171]. Recently, using EVAc
polymers loaded with neurotrophic factors glial cell-line-derived neurotrophic
factor and brain-derived neurotrophic factor, Aszmann et al. [34] demonstrated
with a rat neonate brachial plexus crush injury model that the combination of
glial cell-line-derived neurotrophic factor/brain derived neurotrophic factor
polymers resulted in dramatic motoneuron rescue effect as well as greater
post-treatment strength and voluntary functionality. This finding suggests that
the exogenous trophic support of motoneurons using polymer delivery systems
may have a significant role in the treatment of all types of severe neonatal
plexopathies.
Spinal Fusion
Bone morphogenic proteins (BMPs) represent a family of bone growth fac-
tors whose existence was first postulated as early as 1965 by Urist [172]. There
medwedi.ru
has been much excitement over its possible clinical applications following its iso-
lation, cloning, and sequencing. Active areas of research include its use in ortho-
pedic surgery, plastic surgery and reconstruction, dentistry, and maxillofacial
surgery.
The most obvious clinical neurosurgical application of BMP is its role in
spinal fusion (with or without instrumentation). In fact, investigations have
recently culminated in the presentation of two clinical trials. Boden et al. [173]
demonstrated significantly higher fusion rates in Grade I spondylolisthesis
when recombinant BMP with biphasic calcium phosphate granules were intro-
duced in posterolateral fusion procedures with or without fixation. (Also see
chapter by Kang et al. in this volume.) However, Johnsson et al. [174] discov-
ered no significant effect with an implant consisting of recombinant BMP
reconstituted in a Type I bone collagen carrier for noninstrumented posterolat-
eral fusion of Grade I-II L5-S1 spondylolisthesis. With differences in results
between these studies, it is clear that the clinical role of BMP in spinal fusion
will remain an active area of interest in the near future.
There have been multiple trials investigating the use of polymer-based sys-
tems to deliver BMP in a spinal fusion setting (e.g., [175, 176]). Most use some
form of PLGA polymer system; animal models include sheep, canine, and rab-
bit. To date, the research has not reached the level of human clinical trials.
Future Directions
Polymer-based delivery systems represent a proof of principle that con-

trolled drug delivery can play a significant role in the treatment of various neu-
rosurgical pathologies, including malignancies. Active investigation is ongoing
in several other exciting approaches to sustained drug delivery in the brain.
Convection-Enhanced Delivery Systems

In tissue, compounds travel by diffusion, which is dependent on both the
free concentration gradient and the diffusivity of the compound in the tissue.
Convection, which can be used to supplement diffusion, relies on a simple pres-
sure gradient, and is independent of molecular weight. When a drug is infused
into the cerebral white matter, a pressure gradient is created, and can be used to
introduce high concentrations of drug throughout the brain without structural or
functional side effects [177–179]. Primate trials investigating the treatment of
Parkinson’s disease symptoms have been conducted using convection-enhanced
drug delivery (CEDD) [180]. (Also see chapter by Bankiewicz in this volume.)
A recent study using CEDD to deliver the monoclonal antibody trastuzumab to
treat metastatic breast cancer in a rat model demonstrated an increase of median
Wang/Brem 486
survival of 52 days as compared to 26.5 days for intraperitoneal injection of
trastuzumab (p ⫽ 0.009) and 16 days for controls [181]. Recently, CEDD was
used in a study of taxol to treat 3 brain tumor patients [182]. The investigation
focused on the use of diffusion-weighted MRI to monitor the effects of drug
delivery.
Microchip Drug Delivery

A novel method of drug delivery with clinical potential is the use of newly
developed microchips to control drug delivery (fig. 8a) [183]. Using solid-state
silicon technology, microchip systems provide controlled release of multiple
microreservoirs, thus providing for single or multiple agent delivery. Drugs, in
the form of solids, liquids, or gels are released by electrochemical dissolution
of a thin anode membrane covering each microreservoir. The delivery time of
each reservoir can be programmed independently, thus providing for a seemingly
endless permutation of release profiles and therapy combinations. The device is
an integrated circuit, capable of providing its own microbattery, memory, and
processing. It can be implanted surgically, mounted on the tip of a small probe, or
even swallowed. Current research in this technology includes efficacy and bio-
compatibility studies.
Alternative biodegradable ‘passive chips’ (fig. 8b) are also being devel-
oped based solely on biodegradable polymer technology [184]. Instead of an
integrated silicon based circuit, the release mechanism of each microreservoir
is controlled by slow degradation of a thin polymeric membrane covering each
reservoir. Like the active microchip described above, current research includes
efficacy and biocompatibility studies. Both ‘pharmacy-on-a-chip’ systems may
potentially be used to deliver up to 1,000 different drugs on demand.
Conclusions and Future Directions in Solid Phase Drug Delivery
Tumors of the CNS represent a significant pharmacological and clinical

challenge. The physiological barriers that isolate the CNS make difficult the
delivery of high tumoricidal CNS concentrations of anti-neoplastic agents with-
out causing unacceptably toxic systemic levels of drug. Biodegradable polymer
technology has allowed a new approach to treating brain tumors. Gliadel, the
3.85% (w/w) BCNU-loaded pCPP:SA (20:80) polymer, represents the first suc-
cessful delivery system developed from this technology, and the first new treat-
ment for malignant gliomas approved by the FDA in two decades. Many other
drug-polymer combinations are now undergoing active investigation. These
studies are focusing on combinations of local-delivery approaches, such as the
promising mix of immunotherapy and chemotherapy. Still newer technologies,
medwedi.ru
Silicon nitride Anode
or dioxide
Silicon
Cathode
Active
substance
Small reservoir opening

(usually covered by gold membrane)
Silicon side wall
Large reservoir
a b opening (for reservoir filling)
Fig. 8. a Microchip. Front (left) and back views of a new microchip for controlled
local release of chemicals. Dots between the three large bars (cathodes) on the front are the
caps (anodes) covering the reservoirs holding the chemicals. Electrical voltage applied
between the cap and cathode causes a reaction that dissolves the cap, releasing the reservoir’s
contents. The back view shows larger openings through which the contents of the reservoirs
are deposited (these openings are sealed after filling.) Photo by Paul Horwitz, Atlantic Photo
Service, Inc. b Schematic of passive microchip. Initial models use PLGA and other existing
polymer matrices for the substrate and the entire chip will be biodegradable.
such as the microchip and CEDD, may hopefully enhance the contributions that
polymer drug delivery systems have made.
The future of neurosurgical oncology is an exciting one. With the introduc-
tion of new technology, perhaps when a patient undergoes an operative resection/
debulking of a malignant tumor, his treatment may soon include a microchip
programmed and loaded with a combination of chemotherapy agents tailored to
intraoperative pathology and molecular biological diagnosis of the tumor. Drugs
such as dexamethasone and phenytoin could be loaded onto several microre-
sevoirs to treat cerebral edema and prevent postoperative seizures. Cytokine-
loaded microspheres and irradiated tumor cells from resected specimens could
be placed directly onto the tumor site, or loaded onto other wells of the chip. The
microchip(s) could then be implanted into the tumor cavity intraoperatively.
Should there be a recurrence, stereotactic biopsy for diagnosis and biolog-
ical properties could be followed by the implantation of microspheres loaded
Wang/Brem 488
with angiogenesis inhibitors and additional chemotherapy agents. Such exciting
possibilities for both the patient and neurosurgeon have been made possible by
the development of local and controlled drug delivery to the CNS.
Acknowledgements
Research presented in this work has been supported by the National Cooperative Drug
Discovery Group (UO1-CA52857 and AI 47739) of the National Cancer Institute (NCI-NIH),
Bethesda, MD. Dr. Wang is a neurosurgery resident and neuro-oncology surgery fellow whose
research is supported by a grant (T32 CA-09574) from the National Institute of Health/The
Johns Hopkins University, Bethesda/Baltimore, Md., USA.
Under a licensing agreement between Guilford Pharmaceuticals and the Johns Hopkins
University (JHU), Dr. Brem is entitled to a share of royalty received by the University on
sales of products described in this work. Dr. Brem and JHU own Guilford Pharmaceuticals
stock, which is subject to certain restrictions under University policy. Dr. Brem is also a paid
consultant to Guilford Pharmaceuticals. The terms of this arrangement are being managed
by JHU in accordance with conflict of interest policies.
References
1 Legler LM, Gloeckler Ries LA, Smith MA, Warren LL, Heineman EF, Kaplan RS, Linet MS:
Brain and other central nervous system cancers: Recent trends in incidence and mortality. J Natl
Cancer Inst 1999;91:1382–1390.
2 Rostomily R, Keles GE, Berger MS: Radical surgery in the management of low-grade and high-
grade gliomas. Baillieres Clin Neurol 1996;5:345–369.
3 Chang CH, Horton J, Schoenfeld D, Salazer O, Perez-Tamayo R, Kramer S, Weinstein A, Nelson JS,
Tsukada Y: Comparison of postoperative radiotherapy and combined postoperative radiotherapy and
chemotherapy in the multidisciplinary management of malignant gliomas. A joint Radiation Therapy
Oncology Group and Eastern Cooperative Oncology Group study. Cancer Res 1983;52:997–1007.
4 Green SB, Byar DP, Walker MD, Pistenmaa DA, Alexander EJ, Batzdorf U, Brooks WH, Hunt WE,
Mealey JJ, Odom GL, Paoletti P, Ransohoff JI, Robertson JT, Selker RG, Shapiro WR, Smith KR Jr,
Wilson CB, Strike TA: Comparisons of carmustine, procarbazine, and high-dose methylprednisolone
as additions to surgery and radiotherapy for the treatment of malignant glioma. Cancer Treat Rep
1983;67:121–132.
5 Kornblith PL, Walker M: Chemotherapy for malignant gliomas. J Neurosurg 1988;68:1–17.
6 Walker MD, Green SB, Byar DP, Alexander EJ, Batzdorf U, Brooks WH, Hunt WE, MacCarty CS,
Mahaley MSJ, Mealey JJ, Owens G, Ransohoff JI, Robertson JT, Shapiro WR, Smith KRJ,
Wilson CB, Strike TA: Randomized comparisons of radiotherapy and nitrosoureas for the treatment
of malignant glioma after surgery. N Engl J Med 1980;303:1323–1329.
7 Walker MD, Alexander EJ, Hunt WE, MacCarty CS, Mahaley MSJ, Mealey JJ, Norrell HA,
Owens G, Ransohoff J, Wilson CB, Gehan EA, Strike TA: Evaluation of BCNU and/or radiotherapy
in the treatment of anaplastic gliomas. A cooperative clinical trial. J Neurosurg 1978;49: 333–343.
8 Barker FG, Chang SM, Gutin PH, Malek MK, McDermott MW, Prados MD, Wilson CB: Survival
and functional status after resection of recurrent glioblastoma multiforme. Neurosurgery 1998;42:
709–720.
9 Mohan DS, Suh JH, Phan JL, Kupelian PA, Cohen BH, Barnett GH: Outcome in elderly patients
undergoing definitive surgery and radiation therapy for supratentorial glioblastoma multiforme at
a tertiary institution. Int J Radiat Oncol Biol Phys 1998;42:981–987.
medwedi.ru
10 Reese TS, Karnovsky MJ: Fine structural localization of a blood-brain barrier to exogenous per-
oxidase. J Cell Biol 1967;34:207–217.
11 Grieg NH: Opitimizing drug delivery to brain tumors. Cancer Treat Rev 1987;14:1–28.
12 Abbott NJ, Romero IA: Transporting therapeutics across the blood-brain barrier. Mol Med Today
1996;2:106–113.
13 Loo TL, Dion RL, Dixon RL, Rall DP: The antitumor agent, 1,3-bis(2-chloroethyl)-1-nitrosourea.
J Pharm Sci 1966;55:492–497.
14 Prokai L, Prokai-Tatrai K, Bodor N: Targeting drugs to the brain by redox chemical delivery sys-
tems. Med Res Rev 2000;20:367–416.
15 Pardridge WM: Vector-mediated drug delivery to the brain. Adv Drug Deliv Rev 1999;36:299–321.
16 Williams PC, Henner WD, Roman-Goldstein S, Dahlborg SA, Brummett RE, Tableman M,
Dana BW, Neuwelt EA: Toxicity and efficacy of carboplatin and etoposide in conjunction with
disruption of the blood-brain barrier in the treatment of intracranial neoplasms. Neurosurgery
1995;37:17–28.
17 Warnke PC, Blasberg RG, Groothuis DR: The effect of hyperosmotic blood-brain barrier disrup-
tion on blood-to-tissue transport in ENU-induced gliomas. Ann Neurol 1987;22:300–305.
18 Elliott PJ, Hayward NJ, Dean RL, Blunt DG, Bartus RT: Intravenous RMP-7 selectively increases
uptake of carboplatin in experimental brain tumors. Cancer Res 1996;56:3998–4005.
19 Prados MD, Schold SJS, Fine HA, Jaeckle K, Hochberg F, Mechtler L, Fetell MR, Phuphanich S,
Feun L, Janus TJ, Ford K, Graney W: A randomized, double-blind, placebo-controlled, phase 2
study of RMP-7 in combination with carboplatin administered intravenously for the treatment of
recurrent malignant glioma. Neuro-oncol 2003;5:96–103.
20 Hochberg FH, Pruitt A: Assumptions in the radiotherapy of glioblastoma. Neurology 1980;30:
907–911.
21 Blasberg RG: Methotrexate, cytosine arabinoside, and BCNU concentration in the brain after
ventriculocisternal perfusion. Cancer Treat Rep 1977;61:625–631.
22 Ratcheson RA, Ommaya AK: Experience with the subcutaneous cerebrospinal-fluid reservoir.
Preliminary report of 60 cases. N Engl J Med 1968;279:1025–1031.
23 Chandler WF, Greenberg HS, Ensminger WD, Diaz RF, Junck LR, Hood TW, Gebarski SS, Page MA:
Use of implantable pump systems for intraarterial, intraventricular, and intratumoral treatment of
malignant brain tumors. Ann NY Acad Sci 1988;531:206–212.
24 Lord P, Allami H, Davis M, Diaz R, Heck P, Fischell R: MiniMed Technologies programmable
implantable infusion system. Ann NY Acad Sci 1988;531:66–71.
25 Heruth KT: Medtronic SynchroMed drug administration system. Ann NY Acad Sci 1988;531:
72–75.
26 Langer R: New methods of drug delivery. Science 1990;249:1527–1533.
27 Langer R, Folkman J: Polymers for the sustained release of proteins and other macromolecules.
Nature 1976;263:797–800.
28 Langer R, Brem H, Tapper D: Biocompatibility of polymeric delivery systems for macromole-
cules. J Biomed Mater Res 1981;15:267–277.
29 Tamargo RJ, Myseros JS, Epstein JI, Yang MB, Chasin M, Brem H: Interstitial chemotherapy of
the 9L gliosarcoma: Controlled release polymers for drug delivery in the brain. Cancer Res 1993;
53:329–333.
30 Tamargo RJ, Sills AKJ, Reinhard CS, Pinn ML, Long DM, Brem H: Interstitial delivery of dexa-
methasone in the brain for the reduction of peritumoral edema. J Neurosurg 1991;74:956–961.
31 Weingart JD, Sipos EP, Brem H: The role of minocycline in the treatment of intracranial 9L glioma.
J Neurosurg 1995;82:635–640.
32 Weingart JD, Thompson RC, Tyler B, Colvin OM, Brem H: Local delivery of the topoisomerase I
inhibitor camptothecin sodium prolongs survival in the rat intracranial 9L gliosarcoma model. Int
J Cancer 1995;62:605–609.
33 Yang MB, Tamargo RJ, Brem H: Controlled delivery of 1,3-bis(2-chloroethyl)-1-nitrosourea from
ethylene-vinyl acetate copolymer. Cancer Res 1989;49:5103–5107.
34 Aszmann OC, Korak KJ, Kropf N, Fine E, Aebischer P, Frey M: Simultaneous GDNF and BDNF
application leads to increased motoneuron survival and improved functional outcome in an exper-
imental model for obstetric brachial plexus lesions. Plast Reconstr Surg 2002;110: 1066–1072.
Wang/Brem 490
35 Langer RS, Wise DL: Medical applications of controlled release. Boca Raton, CRC Press, 1984.
36 Brem H, Walter K, Langer R: Polymers as controlled drug delivery devices for the treatment of
malignant brain tumors. Eur J Pharm Biopharm 1993;27:2–7.
37 Lewis DH: Controlled release of bioactive agents from lactide/glycolide polymers; in Chasin M,
Langer R (eds): Biodegradable Polymers as Drug Delivery Systems. New York, Marcel Dekker,
1990, pp 1–41.
38 Kong Q, Kleinschmidt-Demasters BK, Lillehei KO: Intralesionally implanted cisplatin cures pri-
mary brain tumor in rats. J Surg Oncol 1997;64:268–273.
39 Lillehei KO, Kong Q, Withrow SJ, Kleinschmidt-Demasters BK: Efficacy of intralesionally
administered cisplatin-impregnated biodegradable polymer for the treatment of 9L gliosarcoma in
the rat. Neurosurgery 1996;39:1191–1199.
40 Menei P, Daniel V, Montero-Menei C, Brouillardm M, Pouplard-Barthelaix A, Benoit JP:
Biodegradation and brain tissue reaction to poly(D,L-lactide-co-glycolide) microspheres. Bio-
materials 1993;14:470–478.
41 Frazza EJ, Schmitt EE: A new absorbable suture. J Biomed Mater Res 1971;5:43–58.
42 Brady JM, Cutright DE, Miller RA, Barristone GC: Resorption rate, route, route of elimination,
and ultrastructure of the implant site of polylactic acid in the abdominal wall of the rat. J Biomed
Mater Res 1973;7:155–166.
43 Spenlehauer G, Vert M, Benoit JP, Boddaert A: In vitro and in vivo degradation of poly
(D,L-lactide/glycolide) type microspheres made by solvent evaporation method. Biomaterials
1989; 10:557–563.
44 Burt HM, Jackson JK, Bains SK, Liggins RT, Oktaba AM, Arsenault AL, Hunter WL: Controlled
delivery of taxol from microspheres composed of a blend of ethylene-vinyl acetate copolymer and
poly (d,l-lactic acid). Cancer Lett 1995;88:73–79.
45 Sato H, Wang YM, Adachi I, Horikoshi I: Pharmacokinetic study of taxol-loaded poly(lactic-
co-glycolic acid) microspheres containing isopropyl myristate after targeted delivery to the lung
in mice. Biol Pharm Bull 1996;19:1596–1601.
46 Peyman GA, Conway M, Khoobehi B, Soike K: Clearance of microsphere-entrapped 5-fluorouracil
and cytosine arabinoside from the vitreous of primates. Int Ophthalmol 1992;16: 109–113.
47 Torres AI, Boisdron-Celle M, Benoit JP: Formulation of BCNU-loaded microspheres: Influence
of drug stability and solubility on the design of the microencapsulation procedure. J Micro-
encapsul 1996;13:41–51.
48 Williams RC, Paquette DW, Offenbacher S, Adams DF, Armitage GC, Bray K, Caton J, Cochran DL,
Drisko CH, Fiorellini JP, Giannobile WV, Grossi S, Guerrero DM, Johnson GK, Lamster IB,
Magnusson I, Oringer RJ, Persson GR, Van Dyke TE, Wolff LF, Santucci EA, Rodda BE, Lessem J:
Treatment of periodontitis by local administration of minocycline microspheres: A controlled trial.
J Periodontol 2001;72:1535–1544.
49 Lamprecht A, Ubrich N, Yamamoto H, Schafer U, Takeuchi H, Maincent P, Kawashima Y, Lehr CM:
Biodegradable nanoparticles for targeted drug delivery in treatment of inflammatory bowel disease.
J Pharmacol Exp Ther 2001;299:775–781.
50 Lee M, Browneller R, Wu Z, Jung A, Ratanawong C, Sharifi R: Therapeutic effects of leuprorelin
microspheres in prostate cancer. Adv Drug Deliv Rev 1997;28:121–138.
51 Benoit JP, Faisant N, Venier-Julienne MC, Menei P: Development of microspheres for neurologi-
cal disorders: From basics to clinical applications. J Control Release 2000;65:285–296.
52 Sugiyama T, Kumagai S, Nishida T, Ushijima K, Matsuo T, Yakushiji M, Hyon SH, Ikada Y:
Experimental and clinical evaluation of cisplatin-containing microspheres as intraperitoneal
chemotherapy for ovarian cancer. Anticancer Res 1998;18:2837–2842.
53 Singh M, Saxena BB, Singh R, Kaplan J, Ledger WJ: Contraceptive efficacy of norethindrone
encapsulated in injectable biodegradable poly-dl-lactide-co-glycolide microspheres (NET-90):
Phase III clinical study. Adv Contracept 1997;13:1–11.
54 Menei P, Benoit JP, Boisdron-Celle M, Fournier D, Mercier P, Guy G: Drug targeting into the
central nervous system by stereotactic implantation of biodegradable microspheres. Neurosurgery
1994;34:1058–1064.
55 Gref R, Minamitake Y, Peracchia MT, Trubetskoy V, Torchilin V, Langer R: Biodegradable long-
circulating polymeric nanospheres. Science 1994;263:1600–1603.
medwedi.ru
56 Brem H, Langer R: Polymer-based drug delivery to the brain. Sci Med 1996;July/August:52–61.
57 Mathiowitz E, Saltzman M, Domb A: Polyanhydride microspheres as drug carriers. II. Micro-
encapsulation by solvent removal. J Appl Polym Sci 1988;35:755–774.
58 Mathiowitz E, Langer R: Polyanhydride microspheres as drug carriers. I. Hot-melt microencap-
sulation. J Control Release 1987;5:13–22.
59 Bindschaedler C, Leong K, Mathiowitz E, Langer R: Polyanhydride microsphere formulation by
solvent extraction. J Pharm Sci 1988;77:696–698.
60 Chasin M, Domb A, Ron E: Polyanhydrides as drug delivery systems; in Chasin M, Langer R
(eds): Biodegradable Polymers as Drug Delivery Systems. New York, Marcel Dekker, 1990,
pp 43–70.
61 Howard MA III, Gross A, Grady MS, Langer RS, Mathiowitz E, Winn HR, Mayberg MR:
Intracerebral drug delivery in rats with lesion-induced memory deficits. J Neurosurg 1989;71:
105–112.
62 Leong KW, Kost J, Mathiowitz E, Langer R: Polyanhydrides for controlled release of bioactive
agents. Biomaterials 1986;7:364–371.
63 Mathiowitz E, Kline D, Langer R: Morphology of polyanhydride microsphere delivery systems.
Scanning Microsc 1990;4:329–340.
64 Leong KW, D’Amore PD, Marletta M, Langer R: Bioerodible polyanhydrides as drug-carrier
matrices. II. Biocompatibility and chemical reactivity. J Biomed Mater Res 1986;20:51–64.
65 Tamargo RJ, Epstein JI, Reinhard CS, Chasin M, Brem H: Brain biocompatibility of a biodegrad-
able, controlled-release polymer in rats. J Biomed Mater Res 1989;23:253–266.
66 Brem H, Kader A, Epstein JI, Tamargo RJ, Domb A, Langer R, Leong KW: Biocompatibility of a
biodegradable, controlled-release polymer in the rabbit brain. Sel Cancer Ther 1989;5:55–65.
67 Brem H, Tamargo RJ, Olivi A, Pinn M, Weingart JD, Wharam M, Epstein JI: Biodegradable poly-
mers for controlled delivery of chemotherapy with and without radiation therapy in the monkey
brain. J Neurosurg 1994;80:283–290.
68 Dang W, Saltzman WM: Dextran retention in the rat brain following release from a polymer
implant. Biotechnol Prog 1992;8:527–532.
69 Domb A, Bogdansky S, Olivi A, Judy K, Dureza C, Lenartz D, Pinn MI, Colvin M, Brem H:
Controlled delivery of water soluble and hydrolytically unstable anti-cancer drugs for polymeric
implants. Polymer Prepri 1991;32:219–220.
70 Shieh L, Tamada J, Chen I, Pang J, Domb A, Langer R: Erosion of a new family of biodegradable
polyanhydrides. J Biomed Mater Res 1994;28:1465–1475.
71 Olivi A, Ewend MG, Utsuki T, Tyler B, Domb AJ, Brat DJ, Brem H: Interstitial delivery of carbo-
platin via biodegradable polymers is effective against experimental glioma in the rat. Cancer
Chemother Pharmacol 1996;39:90–96.
72 Judy KD, Olivi A, Buahin KG, Domb A, Epstein JI, Colvin OM, Brem H: Effectiveness of con-
trolled release of a cyclophosphamide derivative with polymers against rat gliomas. J Neurosurg
1995;82:481–486.
73 Shikani AH, Eisele DW, Domb AJ: Polymer delivery of chemotherapy for squamous cell carci-
noma of the head and neck. Arch Otolaryngol Head Neck Surg 1994;120:1242–1247.
74 Gabizon AA: Liposomal anthracyclines. Hematol Oncol Clin North Am 1994;8:431–450.
75 Golumbek PT, Azhari R, Jaffee EM, Levitsky HI, Lazenby A, Leong K, Pardoll DM: Controlled
release, biodegradable cytokine depots: A new approach in cancer vaccine design. Cancer Res 1993;
53:5841–5844.
76 Hirakawa W, Kadota K, Asakura T, Niiro M, Yokoyama S, Hirano H, Yatsushiro K, Kubota Y,
Shimodozono Y: Local chemotherapy for malignant brain tumors using methotrexate-containing
fibrin glue. Gan To Kagaku Ryoho 1995;22:805–809.
77 Ringkjob R: Treatment of intracranial gliomas and metastatic carcinomas by local application of
cytostatic agents. Acta Neurol Scand 1968;44:318–322.
78 Rama B, Mandel T, Jansen J, Dingeldein E, Mennel HD: The intraneoplastic chemotherapy in a
rat brain tumour model utilizing methotrexate-polymethylmethacrylate-pellets. Acta Neurochir
1987;87:70–75.
79 Oda Y, Tokuriki Y, Tsuda E, Handa H, Kieler J: Trial of anticancer pellet in malignant brain
tumours: 5 FU and urokinase embedded in silastic. Acta Neurochir Suppl 1979;28:489–490.
Wang/Brem 492
80 Grossman SA, Reinhard C, Colvin OM, Chasin M, Brundrett R, Tamargo RJ, Brem H: The intra-
cerebral distribution of BCNU delivered by surgically implanted biodegradable polymers.
J Neurosurg 1992;76:640–647.
81 Fung LK, Ewend MG, Sills A, Sipos EP, Thompson R, Watts M, Colvin OM, Brem H, Saltzman WM:
Pharmacokinetics of interstitial delivery of carmustine, 4-hydroperoxycyclophosphamide, and pacli-
taxel from a biodegradable polymer implant in the monkey brain. Cancer Res 1998;58: 672–684.
82 Buahin KG, Brem H: Interstitial chemotherapy of experimental brain tumors: Comparison of
intratumoral injection versus polymeric controlled release. J Neurooncol 1995;26:103–110.
83 Sipos EP, Tyler B, Piantadosi S, Burger PC, Brem H: Optimizing interstitial delivery of BCNU
from controlled release polymers for the treatment of brain tumors. Cancer Chemother Pharmacol
1997;39:383–389.
84 Ewend MG, Williams JA, Tabassi K, Tyler BM, Babel KM, Anderson RC, Pinn ML, Brat DJ,
Brem H: Local delivery of chemotherapy and concurrent external beam radiotherapy prolongs
survival in metastatic brain tumor models. Cancer Res 1996;56:5217–5223.
85 Ewend MG, Sampath P, Williams JA, Tyler BM, Brem H: Local delivery of chemotherapy prolongs
survival in experimental brain metastases from breast carcinoma. Neurosurgery 1998; 43:1185–1193.
86 Brem H, Mahaley MS Jr, Vick NA, Black KL, Schold SC Jr, Burger PC, Friedman AH, Ciric IS,
Eller TW, Cozzens JW, Kenealy JN: Interstitial chemotherapy with drug polymer implants for the
treatment of recurrent gliomas. J Neurosurg 1991;74:441–446.
87 Hammoud DA, Belden CJ, Ho AC, Dal Pan GJ, Herskovits EH, Hilt DC, Brem H, Pomper MG:
The surgical bed after BCNU polymer wafer placement for recurrent glioma: Serial assessment
on CT and MR imaging. AJR Am J Roentgenol 2003;180:1469–1475.
88 Brem H, Piantadosi S, Burger PC, Walker M, Selker R, Vick NA, Black K, Sisti M, Brem S,
Mohr G, Muller P, Morawetz R, Schold SC: Placebo-controlled trial of safety and efficacy of
intraoperative controlled delivery by biodegradable polymers of chemotherapy for recurrent
gliomas. The Polymer-brain Tumor Treatment Group. Lancet 1995;345:1008–1012.
89 Brem H, Ewend MG, Piantadosi S, Greenhoot J, Burger PC, Sisti M: The safety of interstitial
chemotherapy with BCNU-loaded polymer followed by radiation therapy in the treatment of
newly diagnosed malignant gliomas: Phase I trial. J Neurooncol 1995;26:111–123.
90 Valtonen S, Timonen U, Toivanen P, Kalimo H, Kivipelto L, Heiskanen O, Unsgaard G, Kuurne T:
Interstitial chemotherapy with carmustine-loaded polymers for high-grade gliomas: A randomized
double-blind study. Neurosurgery 1997;41:44–48.
91 Westphal M, Hilt DC, Bortey E, Delavault P, Olivares R, Warnke PC, Whittle IR, Jääskeläinen J,
Ram Z: A phase 3 trial of local chemotherapy with biodegradable carmustine (BCNU) wafers
(Gliadel wafers) in patients with primary malignant glioma. J Neurooncol 2003;5.
92 Olivi A, Grossman SA, Tatter S, Barker FG 2nd, Judy K, Olson J, Hilt D, Fisher JD, Piantadosi S:
Results of a phase I dose escalation study using BCNU impregnated polymers in patients with
recurrent malignant gliomas. J Clin Oncol 2003;in press.
93 Silber JR, Bobola MS, Ghatan S, Blank A, Kolstoe DD, Berger MS: O6-methylguanine-DNA
methyltransferase activity in adult gliomas: Relation to patient and tumor characteristics. Cancer
Res 1998;58:1068–1073.
94 Pegg AE, Boosalis M, Samson L, Moschel RC, Byers TL, Swenn K, Dolan ME: Mechanism of
inactivation of human O6-alkylguanine-DNA alkyltransferase by O6-benzylguanine. Biochemistry
1993;32:11998–12006.
95 Page JG, Giles HD, Phillips W, Gerson SL, Smith AC, Tomaszewski JE: Preclinical toxicology
study of O6-benzylguanine (NSC-637037) and BCNU (carmustine, NSC-409962) in male and
female beagle dogs. Presented at Proc Am Assoc Cancer Res, 1993.
96 Rhines LD, Sampath P, Dolan ME, Tyler BM, Brem H, Weingart J: O6-benzylguanine potentiates
the antitumor effect of locally delivered carmustine against an intracranial rat glioma. Cancer Res
2000;60:6307–6310.
97 Rosenblum M, Weingart J, Dolan E, Tatter S, Judy K, Olson J, Bohan E, Fisher J: Phase I study
of Gliadel combined with a continuous intravenous infusion of O6-benzylguanine in patients with
recurrent malignant glioma. Presented at 38th ASCO Annual Meeting, Orlando, FL, 2002.
98 Cahan MA, Walter KA, Colvin OM, Brem H: Cytotoxicity of taxol in vitro against human and rat
malignant brain tumors. Cancer Chemother Pharmacol 1994;33:441–444.
medwedi.ru
99 Klecker R, Jamis-Dow C, Egorin M: Distribution and metabolism of 3-H Taxol in the rat (abstract).
Proc Am Assoc Cancer Res 1992;34:381.
100 Walter KA, Cahan MA, Gur A, Tyler B, Hilton J, Colvin OM, Burger PC, Domb A, Brem H:
Interstitial taxol delivered from a biodegradable polymer implant against experimental malignant
glioma. Cancer Res 1994;54:2207–2212.
101 Harper EWD, Lapidus RG, Garver RI Jr: Enhanced efficacy of a novel controlled release pacli-
taxel formulation (PACLIMER delivery system) for local-regional therapy of lung cancer tumor
nodules in mice. Clin Cancer Res 1999;5:4242–4248.
102 Gabikian P, Li KW, Magee C, Morrell C, Tyler BM, Foster F, Dang W, Brem H, Walter KA: Safety
of intracranial paclitaxel: Polilactofate microspheres in dogs (poster). Presented at American
Association of Neurological Surgeons Annual Meeting, Chicago, IL, 2002.
103 Hsiang YH, Liu LF: Identification of mammalian DNA topoisomerase I as an intracellular target
of the anticancer drug camptothecin. Cancer Res 1988;48:1722–1726.
104 Slichenmyer WJ, Rowinsky EK, Donehower RC, Kaufmann SH: The current status of camp-
tothecin analogues as antitumor agents. J Natl Cancer Inst 1993;85:271–291.
105 Storm PB, Moriarity JL, Tyler B, Burger PC, Brem H, Weingart JD: Polymer delivery of camp-
tothecin against 9L gliosarcoma: Release, distribution, and efficacy. J Neurooncol 2002;in
press.
106 Sampath P, Amundson E, Wall ME, Tyler BM, Wani MC, Alderson LM, Colvin M, Brem H,
Weingart JD: Camptothecin analogs in malignant gliomas: Comparative analysis and characteri-
zation. J Neurosurg 2003;98:570–577.
107 Giampietri A, Fioretti MC, Goldin A, Bonmassar E: Drug-mediated antigenic changes in murine
leukemia cells: Antagonistic effects of quinacrine, an antimutagenic compound. J Natl Cancer Inst
1980;64:279–301.
108 Reyes S, Herrera LA, Ostrosky P, Sotelo J: Quinacrine enhances carmustine therapy of experi-
mental rat glioma. Neurosurgery 2001;49.
109 DiMeco F, Li KW, Tyler BM, Wolf AS, Brem H, Olivi A: Local delivery of mitoxantrone for the
treatment of malignant brain tumors in rats. J Neurosurg 2002;97:1173–1178.
110 Thompson RC, Pardoll DM, Jaffee EM, Ewend MG, Thomas MC, Tyler BM, Brem H: Systemic
and local paracrine cytokine therapies using transduced tumor cells are synergistic in treating
intracranial tumors. J Immunother Emphasis Tumor Immunol 1996;19:405–413.
111 Ewend MG, Thompson RC, Anderson R, Sills AK, Staveley-O’Carroll K, Tyler BM, Hanes J,
Brat D, Thomas M, Jaffee EM, Pardoll DM, Brem H: Intracranial paracrine interleukin-2 therapy
stimulates prolonged antitumor immunity that extends outside the central nervous system.
J Immunother 2000; 23:438–448.
112 DiMeco F, Rhines LD, Hanes J, Tyler BM, Brat D, Torchiana E, Guarnieri M, Colombo MP,
Pardoll DM, Finocchiaro G, Brem H, Olivi A: Paracrine delivery of IL-12 against intracranial 9L
gliosarcoma in rats. J Neurosurg 2000;92:419–427.
113 Sampath P, Hanes J, DiMeco F, Tyler BM, Brat D, Pardoll DM, Brem H: Paracrine immunother-
apy with interleukin-2 and local chemotherapy is synergistic in the treatment of experimental
brain tumors. Cancer Res 1999;59:2107–2114.
114 Wiranowska M, Ransohoff J, Weingart JD, Phelps C, Phuphanich S, Brem H: Interferon-containing
controlled-release polymers for localized cerebral immunotherapy. J Interferon Cytokine Res
1998;18:377–385.
115 Hanes J, Sills A, Zhao Z, Suh KW, Tyler B, DiMeco F, Brat DJ, Choti MA, Leong KW, Pardoll DM,
Brem H: Controlled local delivery of interleukin-2 by biodegradable polymers protects animals
from experimental brain tumors and liver tumors. Pharm Res 2001;18:899–906.
116 Rhines LD, DiMeco F, Lawson HC, Tyler BM, Hanes J, Olivi A, Brem H: Local immunotherapy
with interleukin-2 delivered from biodegradable polymer microspheres combined with interstitial
chemotherapy: A novel treatment for experimental malignant glioma. Neurosurgery 2003; 52:1–8.
117 Folkman J: Tumor angiogenesis: Therapeutic implications. N Engl J Med 1971;285:1182–1186.
118 Gimbrone MA Jr, Leapman SB, Cotran RS, Folkman J: Tumor dormancy in vivo by prevention of
neovascularization. J Exp Med 1972;136:261–276.
119 Tamargo RJ, Leong KW, Brem H: Growth inhibition of the 9L glioma using polymers to release
heparin and cortisone acetate. J Neurooncol 1990;9:131–138.
Wang/Brem 494
120 Folkman J, Langer R, Linhardt RJ, Haudenschild C, Taylor S: Angiogenesis inhibition and tumor
regression caused by heparin or a heparin fragment in the presence of cortisone. Science 1983;
221:719–725.
121 Sills AK Jr, Williams JI, Tyler BM, Epstein DS, Sipos EP, Davis JD, McLane MP, Pitchford S,
Cheshire K, Gannon FH, Kinney WA, Chao TL, Donowitz M, Laterra J, Zasloff M, Brem H:
Squalamine inhibits angiogenesis and solid tumor growth in vivo and perturbs embryonic vascu-
lature. Cancer Res 1998;58:2784–2792.
122 Bhargava P, Marshall JL, Dahut W, Rizvi N, Trocky N, Williams JI, Hait H, Song S, Holroyd KJ,
Hawkins MJ: A phase I and pharmacokinetic study of squalamine, a novel antiangiogenic agent,
in patients with advanced cancers. Clin Cancer Res 2001;7:3912–3919.
123 Golub LM, Wolff M, Lee HM, McNamara TF, Ramamurthy NS, Zambon J, Ciancio S: Further
evidence that tetracyclines inhibit collagenase activity in human cervicular fluid and from other
mammalian sources. J Periodontal Res 1985;20:12–23.
124 Tamargo RJ, Bok RA, Brem H: Angiogenesis inhibition by minocycline. Cancer Res 1991;51:
672–675.
125 Frazier JL, Wang PP, Case D, Tyler BM, Pradilla G, Weingart JD, Brem H: Local delivery of
minocycline and systemic BCNU have synergistic activity in the treatment of intracranial glioma.
J Neurooncol 2003;in press.
126 Chabner BA, Collins JM (eds): Cancer Chemotherapy: Principles and Practice. Philadelphia,
Lippencott, 1990.
127 Colvin M, Hilton J: Pharmacology of cyclophosphamide and metabolites. Cancer Treat Rep 1981;
65(suppl 3):89–95.
128 Buahin KG, Judy KD, Hartke C, Domb A, Maniar M, Colvin O, Brem H: Controlled release of
4-hydroperoxycyclophosphamide from the fatty acid dimer-sebacic acid copolymer. Polym Adv
Technol 1992;3:311.
129 Sipos EP, Witham TF, Ratan R, Burger PC, Baraban J, Li KW, Piantadosi S, Brem H: L-buthionine
sulfoximine potentiates the antitumor effect of 4-hydroperoxycyclophosphamide when adminis-
tered locally in a rat glioma model. Neurosurgery 2001;48:392–400.
130 Colvin OM, Friedman HS, Gamcsik MP, Fenselau C, Hilton J: Role of glutathione in cellular
resistance to alkylating agents. Adv Enzyme Regul 1993;33:19–26.
131 Goodman LS, Hardman JG, Limbird LE, Gilman AG: Goodman and Gilman’s: The pharmaco-
logical basis of therapeutics. New York, McGraw-Hill, 2001.
132 Nakazawa S, Itoh Y, Shimura T, Matsumoto M, Yajima K: New management of brain neoplasms.
II. Local injection of adriamycin. No Shinkei Geka 1983;11:821–827.
133 Lin SY, L.F. C, Lui WY, Chen CF, Han SH: Tumoricidal effect of controlled-release polymeric
needle devices containing adriamycin HCl in tumor-bearing mice. Biomater Artif Cells Artif
Organs 1989;17:189–203.
134 Watts MC, Lesniak MS, Burke M, Samdani AF, Tyler BM, Brem H: Controlled release of
adriamycin in the treatment of malignant glioma (poster). Presented at American Association of
Neurological Surgeons Annual Meeting, Denver, CO, 1997.
135 Levin VA, Edwards MS, Wara WM, Allen J, Ortega J, Vestnys P: 5-Fluorouracil and
1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) followed by hydroxyurea, misonidazole,
and irradiation for brain stem gliomas: A pilot study of the Brain Tumor Research Center and the
Childrens Cancer Group. Neurosurgery 1984;14:679–681.
136 Shapiro WR: Studies on the chemotherapy of experimental brain tumors: Evaluation of
1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea, vincristine, and 5-fluorouracil. J Natl Cancer Inst
1971;46:359–368.
137 Shapiro WR, Green SB, Burger PC, Selker RG, VanGilder JC, Robertson JT, Mealey J Jr,
Ransohff J, Mahaley MS Jr: A randomized comparison of intra-arterial versus intravenous BCNU,
with or without intravenous 5-fluorouracil, for newly diagnosed patients with malignant glioma.
J Neurosurg 1992;76:772–781.
138 Oda Y, Uchida Y, Murata T, Mori K, Tokuriki Y, Handa H, Kobayashi A, Hashi K, Kieler J:
Treatment of brain tumors with anticancer pellet – Experimental and clinical study. No Shinkei
Geka 1982;10: 375–381.
medwedi.ru
139 Oda Y, Kamijyo Y, Okumura T, Tokuriki Y, Yamashita J, Handa H, Aoyama I, Hashi K, Mori K:
Clinical application of a sustained release anticancer pellet. No Shinkei Geka 1985;13:
1305–1311.
140 Kubo O, Himuro H, Inoue N, Tajika Y, Tajika T, Tohyama T, Sakairi M, Yoshida M, Kaetsu I,
Kitamura K: Treatment of malignant brain tumors with slowly releasing anticancer drug-polymer
composites. No Shinkei Geka 1986;14:1189–1195.
141 Boisdron-Celle M, Menei P, Benoit JP: Preparation and characterization of 5-fluorouracil-
loaded microparticles as biodegradable anticancer drug carriers. J Pharm Pharmacol 1995;47:
108–114.
142 Menei P, Boisdron-Celle M, Croue A, Guy G, Benoit JP: Effect of stereotactic implantation of
biodegradable 5-fluorouracil-loaded microspheres in healthy and C6 glioma-bearing rats. Neuro-
surgery 1996;39:117–124.
143 Menei P, Venier MC, Gamelin E, Saint-Andre JP, Hayek G, Jadaud E, Fournier D, Mercier P,
Guy G, Benoit JP: Local and sustained delivery of 5-fluorouracil from biodegradable micro-
spheres for the radiosensitization of glioblastoma: A pilot study. Cancer 1999;86:325–330.
144 Choti MA, Saenz J, Yang X, Brem H: Intrahepatic FUdR delivered from biodegradable
polymer in experimental liver metastases from colorectal carcinoma. Presented at Proc of
AACR, 1995.
145 Damascelli B, Marchiano A, Frigerio LF, Salvetti M, Spreafico C, Garbagnati F, Zanoni F, Radice F:
Flexibility and efficacy of automatic continuous fluorodeoxyuridine infusion in metastases from
a renal cell carcinoma. Cancer 1991;68:995–998.
146 Sendelbeck SL, Urquhart J: Spatial distribution of dopamine, methotrexate and antipyrine during
continuous intracerebral microperfusion. Brain Res 1985;328:251–258.
147 Blasberg RG, Patlak CS, Shapiro WR: Distribution of methotrexate in the cerebrospinal fluid and
brain after intraventricular administration. Cancer Treat Rep 1977;61:633–641.
148 Diemath HE: Local use of cytostatic drugs following removal of glioblastomas. Wien Klin
Wochenschr 1987;99:674–676.
149 Dang W, Colvin OM, Brem H, Saltzman WM: Covalent coupling of methotrexate to dextran
enhances the penetration of cytotoxicity into a tissue-like matrix. Cancer Res 1994;54:1729–1735.
150 Zeller WJ, Bauer S, Remmele T, Wowra B, Sturm V, Stricker H: Interstitial chemotherapy of exper-
imental gliomas. Cancer Treat Rep 1990;7:183–189.
151 Weiss SR, Raskind R: Treatment of malignant brain tumors by local methotrexate. A preliminary
report. Int Surg 1969;51:149–155.
152 Uldry PA, Teta D, Regli L: Focal cerebral necrosis caused by intraventricular chemotherapy with
methotrexate. Neurochirurgia 1991;37:72–74.
153 Shimura T, Nakazawa S, Ikeda Y, Node Y: Cyst formation following local chemotherapy of
malignant brain tumor: A clinicopathological study of two cases. No Shinkei Geka 1992;20:
1179–1183.
154 Olivi A, Gilbert M, Duncan KL, Corden B, Lenartz D, Brem H: Direct delivery of platinum-based
antineoplastics to the central nervous system: A toxicity and ultrastructural study. Cancer
Chemother Pharmacol 1993;31:449–454.
155 Utsuki T, Brem H, Pitha J, Loftsson T, Kristmundsdottir T, Tyler BT, Olivi A: Potentiation of anti-
cancer effects of microencapsulated carboplatin by hydroxypropyl ␣-cyclodextrin. J Control
Release 1996;40:251–260.
156 Maxell RE, Long DM, French LA: The clinical effects of a synthetic gluco-corticoid used for
brain edema in the practice of neurosurgery; in Reulen HJ, Schurmann K (eds): Steroids and Brain
Edema. Berlin, Springer-Verlag, 1972, pp 219–232.
157 Melby JC: Drug spotlight program: Systemic corticosteroid therapy: Pharmacology and
endocrinologic considerations. Ann Intern Med 1974;81:505–512.
158 Reinhard CS, Radomsky ML, Saltzman WM, Brem H: Polymeric controlled release of dexa-
methasone in normal rat brain. J Control Rel 1991;16:331–340.
159 Katakura R, Mori T, Mineura K, Suzuki J: A device for prolonged releasing of anticancer
drug–bleomycin. No Shinkei Geka 1980;8:1057–1062.
160 Katakura R, Mori T, Suzuki J: A device for prolonged releasing of anticancer drug–bleomycin:
Second report. No Shinkei Geka 1982;10:941–944.
Wang/Brem 496
161 Williams JA, Dillehay LE, Tabassi K, Sipos E, Fahlman C, Brem H: Implantable biodegradable
polymers for IUdR radiosensitization of experimental human malignant glioma. J Neurooncol
1997;32:181–192.
162 Williams JA, Yuan X, Dillehay LE, Shastri VR, Brem H, Williams JR: Synthetic, implantable poly-
mers for local delivery of IUdR to experimental human malignant glioma. Int J Radiat Oncol Biol
Phys 1998;42:631–639.
163 Tamargo RJ, Rossell LA, Tyler BM, Aryanpur JJ: Interstitial delivery of diphenylhydantoin in the
brain for the treatment of seizures in the rat model (poster). Presented at American Association of
Neurological Surgeons Annual Meeting, San Diego, CA, 1994.
164 Polin RS, Bavbek M, Shaffrey ME, Billups K, Bogaev CA, Kassell NF, Lee KS: Detection of
soluble E-selectin, ICAM-1, VCAM-1, and L-selectin in the cerebrospinal fluid of patients after
subarachnoid hemorrhage. J Neurosurg 1998;89:559–567.
165 Pluta RM, Thompson BG, Dawson TM, Snyder SH, Boock RJ, Oldfield EH: Loss of nitric oxide
synthase immunoreactivity in cerebral vasospasm. J Neurosurg 1996;84:648–654.
166 Thai QA, Oshiro EM, Tamargo RJ: Inhibition of experimental vasospasm in rats with the periadven-
titial administration of ibuprofen using controlled-release polymers. Stroke 1999;30: 140–147.
167 Aihara Y, Jahromi BS, Yassari R, Sayama T, Macdonald RL: Effects of a nitric oxide donor on and
correlation of changes in cyclic nucleotide levels with experimental vasospasm. Neurosurgery
2003;52:661–667.
168 Tierney TS, Clatterbuck RE, Lawson C, Thai QA, Rhines LD, Tamargo RJ: Prevention and rever-
sal of experimental posthemorrhagic vasospasm by the periadventitial administration of nitric
oxide from a controlled-release polymer. Neurosurgery 2001;49:945–951; discussion 943–951.
169 Gabikian P, Clatterbuck RE, Eberhart CG, Tyler BM, Tierney TS, Tamargo RJ: Prevention of
experimental cerebral vasospasm by intracranial delivery of a nitric oxide donor from a
controlled-release polymer: Toxicity and efficacy studies in rabbits and rats. Stroke 2002;33:
2681–2686.
170 Clatterbuck RE, Gailloud P, Tierney TS, Murphy K, Tamargo RJ: Controlled release of nitric oxide
donor DETA-NO prevents cerebral vasospasm following experimental subarachnoid hemorrhage
in monkeys. Neurosurgery 2002;51:545–546.
171 Jackson ST, Hoffer MM, Parrish N: Brachial-plexus palsy in the newborn. J Bone Joint Surg Am
1988;70:1217–1220.
172 Urist MR: Bone: Formation by autoinduction. Science 1965;150:893–899.
173 Boden SD, Kang J, Sandhu H, Heller JG: Use of recombinant human bone morphogenetic protein-2
to achieve posterolateral lumbar spine fusion in humans: A prospective, randomized clinical pilot trial:
2002 Volvo Award in clinical studies. Spine 2002;27:2662–2673.
174 Johnsson R, Stromqvist B, Aspenberg P: Randomized radiostereometric study comparing
osteogenic protein-1 (BMP-7) and autograft bone in human noninstrumented posterolateral
lumbar fusion: 2002 Volvo Award in clinical studies. Spine 2002;27:2654–2661.
175 Toth JM, Estes BT, Wang M, Seim HB 3rd, Scifert JL, Turner AS, Cornwall GB: Evaluation of
70/30 poly(L-lactide-co-D,L-lactide) for use as a resorbable interbody fusion cage. J Neurosurg
2002;97:423–432.
176 Kandziora F, Bail H, Schmidmaier G, Schollmeier G, Scholz M, Knispel C, Hiller T, Pflugmacher R,
Mittlmeier T, Raschke M, Haas NP: Bone morphogenetic protein-2 application by a poly(D,L-lac-
tide)-coated interbody cage: In vivo results of a new carrier for growth factors. J Neurosurg
2002;97:40–48.
177 Bobo RH, Laske DW, Akbasak A, Morrison PF, Dedrick RL, Oldfield EH: Convection-enhanced
delivery of macromolecules in the brain. Proc Natl Acad Sci USA 1994;91:2076–2080.
178 Laske DW, Morrison PF, Lieberman DM, Corthesy ME, Reynolds JC, Stewart-Henney PA,
Koong SS, Cummins A, Paik CH, Oldfield EH: Chronic interstitial infusion of protein to primate
brain: Determination of drug distribution and clearance with single-photon emission computer-
ized tomography imaging. J Neurosurg 1997;87:586–594.
179 Lieberman DM, Corthesy ME, Cummins A, Oldfield EH: Reversal of experimental parkinsonism
by using selective chemical ablation of the medial globus pallidus. J Neurosurg 1999;90:928–934.
180 Lonser RR, Gogate N, Morrison PF, Wood JD, Oldfield EH: Direct convective delivery of macro-
molecules to the spinal cord. J Neurosurg 1998;89:616–622.
medwedi.ru
181 Grossi PM, Ochiai H, Archer GE, McLendon RE, Zalutsky MR, Friedman AH, Friedman HS,
Bigner DD, Sampson JH: Efficacy of intracerebral microinfusion of trastuzumab in an athymic rat
model of intracerebral metastatic breast cancer. Clin Cancer Res 2003;9:5514–5520.
182 Mardor Y, Roth Y, Lidar Z, Jonas T, Pfeffer R, Maier SE, Faibel M, Nass D, Hadani M, Orenstein A,
Cohen JS, Ram Z: Monitoring response to convection-enhanced taxol delivery in brain tumor
patients using diffusion-weighted magnetic resonance imaging. Cancer Res 2001;61:4971–4973.
183 Santini JT Jr, Richards AC, Scheidt RA, Cima MJ, Langer RS: Microchip technology in drug
delivery. Ann Med 2000;32:377–379.
184 Richards Grayson AC, Choi IS, Tyler BM, Wang PP, Brem H, Cima MJ, Langer R: Multi-pulse
drug delivery from a resorbable polymeric microchip device. Nat Mater 2003;2:767–772.
185 Li Y, Shawgo RS, Tyler B, Henderson PT, Vogel JS, Rosenberg A, Storm PB, Langer R, Brem H,
Cima MJ: In vivo release from a drug delivery MEMS device. J Control Release, in press.
Henry Brem, MD
Departments of Neurological Surgery and Oncology
The Johns Hopkins Hospital, Hunterian 817, 725 North Wolfe Street
Baltimore, MD 21205 (USA)
Tel. ⫹1 410 614 0477, Fax ⫹1 410 614 0478, E-Mail hbrem@jhmi.edu
Wang/Brem 498
Immunotherapy Strategies for

Treatment of Malignant Gliomas
Larry Harshynea, Phyllis Flomenbergb, David W. Andrewsa
a
Department of Neurosurgery and bDepartment of Internal Medicine,
Thomas Jefferson University, Philadelphia, Pa., USA
Malignant astrocytomas are primary intracranial tumors that are resistant

to treatment with surgery, radiation, and chemotherapy. With rare exceptions,
all of these tumors recur after initial treatment, requiring repeat surgery and
adjuvant treatments. Irrespective of interventions, the median survival for
WHO Grade 4 tumors (glioblastomas) remains a dismal 47 weeks [1] and
despite fundamental advances in chemotherapy, little progress has been made
in the treatment of malignant astrocytoma. Several factors contribute to the lack
of progress in this area, including suboptimal drug delivery through the blood-
brain barrier and refractory hypoxic cell subpopulations. Drug delivery to the
tumor is impeded by the blood-brain barrier, limiting the choice of CNS agents
to certain lipophilic drugs. Glioma cell subpopulations are markedly heteroge-
neous and show phenotypic differences in their sensitivity to established drugs
such as lipophilic variants of carmustine (BCNU) and cisplatin. Hypoxic
glioma cell subpopulations are also refractory to therapy due to their radio- and
chemo-resistant properties. Over the past decade, these issues have been
addressed by intratumoral drug delivery trials (as discussed by Brem et al. in
another chapter in this volume), external beam radiation, or brachytherapy, all
of which have shown only marginal success.
Conventional treatment failure has prompted development and utilization
of novel strategies such as gene therapy, antisense therapy, and immunotherapy.
In the case of immunotherapy, a number of strategies have emerged and collec-
tively represent a promising new approach to the treatment of malignant
glioma. At the same time, information about native glioma immunomodulatory
capabilities may help explain why immunotherapies have failed in early human
trials. This chapter will serve to review what is currently known about glioma
immunobiology and review strategies currently being developed as anti-glioma
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immunotherapies. We provide a review of cell-mediated immunity and current
assays of immune response.
Antigen Presenting Cells and the Immune Response
The cell-mediated immune response plays a vital role in anti-tumor

immunity. An effective cellular immune response requires the coordination of
activated helper (CD4) and killer (CD8) T cells. T cells cannot act indis-
criminately and must first be exposed to foreign antigens by antigen presenting
cells (APCs). Dendritic cells (DCs) are the most potent APCs in the immune
system. APCs secrete many immune activators and modifiers into the local
environment, which not only augment adaptive immunity but also amplify the
innate immune response; for example, activation and recruitment of other
APCs, macrophages, natural killer (NK) cells, eosinophils and mast cells.
Furthermore, APCs assume many different roles throughout their life and must
remain extremely plastic, capable of quickly modifying their function to meet
the needs of the host. Their flexible nature and central role within the immune
system makes them attractive candidates as targets for therapy aimed at boost-
ing immunity.
Networks of APCs exist in nearly every organ. APCs are most prevalent in
epithelial barriers that serve as the first line of defense. Increased exposure to
pathogens in mucosal tissue and skin necessitate increased surveillance. While
APCs were described 30 years ago in the peripheral lymphoid tissue of mice [2],
APCs actually were first identified in the epidermis in 1868 by Paul Langerhans,
a German anatomist, who mistook them for nerve endings. Later, they were
found to be APCs and still bear his name, Langerhans cells (LC). A LC-like pop-
ulation of APCs also can be found in the epithelial lining of the gut [3], airways
[4, 5], and reproductive tract [6, 7]. A discrete subset of interstitial APCs (iAPCs)
resides in the dermis [8] and submucosa. The blood contains APCs [9–11] and
APC precursors [12] which are en route to tissues. APCs exist throughout the
periphery and act as sentinels for the immune system. They are extremely effi-
cient at capturing antigens from a variety of sources including soluble proteins
and peptides bound by heat shock proteins, as well as cell-associated antigens
from other live or dead cells (fig. 1).
Upon acquiring an antigen, APCs receive an activation signal, which occurs
when they sense foreign pathogens or cells. APCs are also extremely sensitive to
inflammatory cytokines. Following activation, they down-regulate the ability to
acquire antigen and shift their focus towards antigen presentation and T cell
stimulation. Activated APCs enter lymphatic ducts and migrate to the draining
lymph nodes where they encounter large numbers of T lymphocytes. During
Harshyne/Flomenberg/Andrews 500
Fig. 1. Immature DC efficiently capture apoptotic bodies. Equal number of DiD-
labeled immature DC (red) and DiO-labeled apoptotic T cells (green) were cocultured
together for 4 h and analyzed by confocal microscopy. Numerous DC-containing multiple
apoptotic bodies are observed in these low and high power fields.
migration, APCs process and present captured antigens with the MHC class II
complex or cross-present antigens with the MHC class I complex (fig. 2). In
addition, APCs, like all nucleated cells, are able to present endogenous antigen
in the context of MHC class I.
CD4 T cells recognize antigens bound to MHC class II molecules,
whereas CD8 T cells respond to antigens bound by MHC class I molecules.
T cells recognize antigen/MHC complexes via their T cell receptors. Following
stimulation through MHC/T cell receptor interactions, T cells begin to prolifer-
ate. In order to become fully functional effectors, however, T cells must receive
an additional, costimulatory signal from the APC. Activated APCs express high
levels of costimulatory molecules, including CD40, CD80, and CD86, all of
which are capable of providing this second signal to T cells. T cells which only
receive one signal, through the T cell receptor, become ‘tolerant’ and are unable
to respond to antigen.
The primary role of activated helper cells is to provide support to the
immune system. For the most part, this helper cell support involves secretion of
cytokines such as -interferon (IFN-) and interleukins (IL-2, IL-4, IL-5,
IL-12), which perpetuate and direct the immune response. Helper T cells are
divided into two main subsets, Th1 and Th2. IFN-, IL-2, and IL-12 are con-
sidered to be Th1 cytokines, which are proinflammatory and strengthen the
Immunotherapy for Treatment of Malignant Gliomas 501
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TGF-, PGE-2 and IL-10
Tumor cell
IL-2, IL-6, IFN-
TK TH1
IL-6, IL-12
CD28 TCR TCR CD40L
CD86 MHC I MHC II

CD40
APC
Tumor antigen
Fig. 2. Mechanism for tumor antigen loading and T cell cross-priming through
antigen-presenting cells.
cell-based immune response. IL-4, IL-5, IL-6, IL-10, and TGF- belong to the
Th2 family of cytokines. In contrast to Th1 cytokines, Th2 cytokines are anti-
inflammatory and result in the down-regulation of the cell-mediated immune
response. Activated killer cells, or cytotoxic T lymphocytes (CTL) search the
periphery for target cells, or cells which present the proper antigen in MHC
class I molecules on their surface. Upon identifying a target cell, CTL employ
one of two different mechanisms to rapidly destroy it. Target cells can be killed
by the release of lytic granules containing two classes of effector proteins. One
class consists of pore-forming proteins called perforins. Perforins polymerize in
the target cell membrane to form tiny holes throughout the lipid bilayer. The
second class of effector molecules consists of a family of serine proteases
called granzymes, which set the cell death cascade in motion. While nearly all
target lysis by CTL occurs via the perforin or granzyme pathway, perforin
knockout mice are still able to lyse target cells. An alternative lysis pathway
exists which involves Fas receptor/ligand interaction. Binding of Fas on the sur-
face of target cells by CTL initiates a proteolytic cascade, which results in the
apoptosis of the target cell. Several important questions remain unanswered in
tumor immunology which include how more potent effector T cells could be
generated, what conditions facilitate better tumor cell recognition, and what is
required to achieve memory T cells that results in life-long protection against
specific tumors and/or metastases.
Novel Methods for Monitoring the Immune Response in Glioma
Glioma patients may have immune dysregulation. Therefore, precise and

consistent assays to assess T cell numbers and function are vital for an accurate
estimation of immune competency in glioma. Quantitative and qualitative mea-
sures of immune function are therefore important criteria when designing clin-
ical trials for glioma therapy.
Antigen-specific T cells circulate through the blood and lymphatic sys-
tem. In order to become activated, T cells recognize peptide bound to MHC
molecules on APCs. In 1996, an innovative system for quantitating antigen-
specific T cells was developed [13]. MHC class I molecules were bound to
peptides and attached to a fluorescenated substrate, generating tetramers.
Tetramers have proven to be a powerful tool when quantifying T cell reper-
toires. Tetramers permit the estimation of circulating precursor frequencies of
antigen-specific T cells before therapy, and the extent of expansion following
immune boosting. Tetramer assays have been broadened to include the obser-
vation of antigen-specific T cells in situ [14]. Similarly, investigators have
developed CD1 tetramers to look at glycolipid reactive T cells [15] and MHC
class II tetramers to monitor peptide-specific CD4 T cell populations [16].
Carboxyfluorescein diacetate succinimidyl ester is a cell-permeable dye
that nonspecifically labels intracellular proteins within an intact, functioning
cell [17]. As a labeled cell divides, so too does its fluorescent profile, with each
daughter cell receiving approximately half of the fluorescently labeled proteins.
Serial dilution of the vital dye resulting from proliferation can be tracked for up
to eight to ten cycles, after which the dye becomes extinct. Carboxyfluorescein
diacetate succinimidyl ester enables quantitative kinetic studies of the immune
response, detailed analysis of proliferative profiles within minor subsets, and
the ability to address T cell differentiation.
When T cells proliferate, they also differentiate into fully armed effector
cells. For CD4 helper T cells, this means production and secretion of
immunomodulatory cytokines. CD8 killer T cells, on the other hand, produce
cytolytic molecules. Cytokine production is most often documented utilizing
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monoclonal antibodies. Enzyme-linked immunosorbent assay (ELISA) has
been the classical standard for measuring cytokine secretion [18]. An ELISA
utilizes two antibodies that recognize different epitopes. A capture antibody,
immobilized to one plate, binds to cytokine molecules present in a serum or
supernatant sample. A second antibody is used to detect the bound cytokine in
an enzyme catalyzed reaction. While ELISAs are very sensitive they are also
labor intensive and only detect a single cytokine. A novel flow cytometry-based
method has recently been developed by BD Biosciences (Cytokine Bead Array,
CBA). The CBA achieves sensitivity comparable to an ELISA, but can detect
multiple cytokines in a single sample [19]. The CBA assay is very efficient, as
it generates up to seven times the amount of data in less time than a conven-
tional ELISA with a sensitivity of CBA (⬃4 pg/ml) that rivals ELISA. The
enzyme-linked immunospot assay is similar to ELISA, except that cytokines
are captured onto a membrane [20] instead of a plastic plate. The ELISPOT is
also a very sensitive assay (as few as 100 molecules of specific protein are
detected) that capitalizes on the high concentration of cytokines in the environ-
ment immediately surrounding the producer cell. A spot forms when detector
antibodies undergo a colorimetric reaction in regions of high cytokine produc-
tion. The spot represents a trace of the cytokine-producing cell and vary in size
depending on the amount of cytokine produced by a given cell.
Flow cytometry achieves both rapid detection and semi-quantitative mea-
surements of rare cytokine-producing cells [21, 22]. Intracellular cytokine
staining with fluorescent antibodies can detect subsets of rare cells producing
multiple cytokines when analyzed by flow cytometry. Stimulation cultures are
inhibited by the addition of monensin or brefeldin A, which stop vesicular
transport [21]. Instead of secreting cytokines, substantial quantities of cytokines
accumulate within cells. Following overnight incubation, cultures are fixed,
permeabilized, and probed with cytokine-specific antibodies. Subsets of cells
are defined by labeling with lineage markers before fixation. A drawback is that
intracellular cytokine staining kills cells and prevents one from conducting
additional experiments. An alternative is cytokine capture assay, which retains
many advantages of intracellular cytokine staining and also maintains cell via-
bility. In this assay, a cytokine catch reagent is attached to the outside of the cell
via CD45, which is found on all hematopoietic cells. Secreted cytokine is cap-
tured on the cell surface and then detected using a fluorescent, phycoerythrin-
conjugated antibody specific for cytokines. MAgnetic Cell Sorting microbead
technology (Miltenyi Biotech) uses miniature magnetic beads and separation
columns that are gentle to cells to obtain a highly purified population of cells
in less than an hour [23]. By combining cytokine capture and magnetic selec-
tion, the authors of one study were able to purify IFN- producing, melan-
A-specific T cells using anti-PE microbeads [24]. This technology enables
subsequent experiments to be performed on highly purified and well-defined
populations of tumor-specific effector cells.
Tetramers and enzyme-linked immunospots provide innovative ways to
measure the frequency of antigen-specific CD8 T cells, but they do not assess
function of these responders. While many novel methods have emerged to
assess CD4 helper T cell function, antigen specific lysis remains the gold
standard. Traditionally, cytolytic activity was defined by 51Chromium (51Cr)
release assays [25]. Target cells were loaded with antigen, labeled with 51Cr, and
cocultured with effector T cells. Destruction of target cells was indirectly mea-
sured by 51Cr release into the supernatant within 4 h of adding killer T cells. The
hazards associated with radioactivity as well as high background levels due to
spontaneous release of 51Cr have prompted researchers to develop novel ways
to identify cell-mediated cytotoxic lysis. Several nonradioactive alternatives for
antigen-specific lysis have included detecting the release of intracellular
enzymes [26] or the release of fluorescenated proteins [27, 28]. Direct mea-
sures of cell death can be combined with the fluorescent protein release [29, 30]
to further pinpoint the mechanism of cell death.
Target cells whose membrane and intracellular protein have been labeled
with fluorescent dyes (e.g., DID or PKH-27, and carboxyfluorescein diacetate
succinimidyl ester, respectively) can provide a bright and narrow fluorescent
profile when analyzed by flow cytometry. These dyes form stable conjugates
and do not readily cross intact cell membranes, resulting in much lower back-
ground levels when compared to classical 51Cr release assays. Lysis by effector
cells, however, does result in an immediate loss of fluorescence due to the
extravasation of labeled proteins through the compromised membrane.
Moreover, APCs that endocytose released labeled protein may interfere with the
interpretation of results. To control for the latter, Sheely et al. [28] labeled the
cell membrane of target cells with an additional dye to be sure that they
observed a loss of fluorescence in target cells and not a gain of fluorescence in
bystander APCs. Labeling of intracellular and/or membrane proteins of target
cells can be combined with probes of cell death including propidium iodide
[29], 7-AAD [30], or TUNEL labeling. In our hands, the fluorescent-based
assays for cytolytic activity have proven to be more sensitive due to low levels
of spontaneous release and have enabled direct detection of lysed target cells
via high-throughput flow cytometric analysis.
Two molecular immunological techniques have recently emerged to effec-
tively monitor the immune response at the population level: gene expression
arrays [31] and reverse-transcriptase-polymerase chain reaction (RT-PCR)
[32–34]. With expression arrays, it is possible to look at thousands of genes and
determine which are differentially expressed in tumor-specific T cells when
compared to naïve T cells. Large amounts of data are generated by arrays, but
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software specifically designed to interpret and quantify an immune response is
currently unavailable. Protocols are under development to evaluate global anti-
tumor responses within individual patients. In addition to microarray profiling,
RT-PCR has advanced to a level where it has become a very valuable tool with
which to monitor immune responses within the circulating peripheral blood
mononuclear cells (PBMC). RT-PCR provides oncologists with a molecular
genetic signature of the immune response in cancer; for example, Kammula et al.
[33] monitored gene expression in anti-tumor lymphocytes. Copies of CD8,
IFN, GM-CSF, IL-2, and TNF- mRNA increased significantly in PBMC taken
from patients who were vaccinated with tumor-derived peptides. Furthermore,
copies of mRNA encoding markers of early T cell activation, including CD25
and CD69, increased shortly after immunization. There was a correlation
between increasing levels of gp100 mRNA and IFN- mRNA in patients receiv-
ing the vaccine that was not observed in the control group. RT-PCR results were
confirmed by tetramer analysis. This study provides a compelling evidence sup-
porting the efficacy of RT-PCR-based monitoring of immune responses.
Immune Dysfunction in Glioma and Constraints Imposed on

Proinflammatory Responses in the CNS
In the periphery, the body’s innate response to anything foreign includes a

massive infiltration of inflammatory cells and proteins that aid in defense and
tissue repair at the site of insult. Extensive cell death and tissue destruction
often accompany an inflammatory immune response. As a host organ, the CNS
poses an unique set of constraints for inflammatory responses. The brain is
composed of resting phase (postmitotic) and predominantly nonregenerating
cells and is enclosed within a fixed cranial vault. Recognizing the physiologi-
cal relationship between volume and pressure, brain compliance cannot tolerate
swelling beyond a certain point which would result from the influx of excess
immune cells, nor can the host afford tissue loss, particularly in eloquent
regions of the brain.
An example of an autoimmune T cell-mediated destruction in the brain is
acute disseminated encephalomyelitis [35]. The body has in place several phys-
iological barriers and mechanisms to safeguard the brain from such a destruc-
tive inflammatory response. The blood-brain barrier largely restricts the
trafficking of immune cells and mediators from the periphery to the brain.
Furthermore, brain tissue neither contains endogenous APCs, nor does it pos-
sess conventional lymphatics to carry cells and/or antigen to local nodes. By
restricting the communication between the two compartments, these measures
serve to reduce the risk of a full-fledged immunological attack within the brain.
a b
c d
Fig. 3. a–d Photomicrographs of malignant glioma permanent sections in patients

previously treated with an antisense paradigm, hematoxylin and eosin stain, 400. All four
photomicrographs represent examples of acquired T cell lymphocytic infiltration after
abdominal implantation of apoptotic autologous malignant glioma cells.
In the event that an activated immune cell gets past the blood-brain barrier,
many cells within the brain express FasL, and Fas-bearing lymphocytes would
undergo apoptosis before an immune response could be initiated.
Studies have demonstrated naturally occurring cellular immune responses
in glioma as well as a positive correlation between lymphocytic infiltration and
median survival prognosis [36–40]. Our experience in modifying the immune
response in glioma involves antisense directed against the insulin-type growth
factor type one receptor (IGF-IR/AS ODN) in glioma patients [41, 42]. In this
work, we measured an acquired intratumoral lymphocytic infiltration, which is
six-fold higher than previously reported (see fig. 3). Using Western blot, we
demonstrated IGF-I receptor down-regulation after IGF-IR/AS ODN treatment
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Table 1. Immune modulatory capabilities of gliomas
Glioma modulations Consequence
Systemic and regional depletion Lack of antigen-specific recognition

of Th cells of tumor cells
Lack of MHC Class I molecules on Loss of recognition by cytotoxic T cells
tumor cells
MHC class II expression on Regional depletion of Th 1 cells
tumor cells (T cell anergy)
TGF- production by tumor cells Profound suppression of T-, B- and
NK-cells and macrophages
Prostaglandin E2 and IL-10 Suppression of Th1 and professional
production by tumor cells APC functions
Production of colony-stimulating factors Activation of macrophages
Chemokine production by tumor cells Chemotaxis of T cells and
macrophages into tumor tissue
Presence of IL-1 autocrine loop in Partial activation of T cells and
tumor cells macrophages
Production of IL-1RA by tumor cells Regulation of IL-1 autocrine loop;
suppression of IL-1-mediated
immune cascade reaction
Lack of IFN- genes in tumor cells Suppression of MHC molecules
and induction of cytokines
Adapted from [128].
of patient tumor cells into vitro, and tumor cell apoptosis in vivo after implan-
tation of treated tumor cells into the host abdomen [41]. It was hypothesized
that acquired lymphocytic tumor infiltration may have resulted from an
immune cross-priming mechanism invoked by in vivo tumor cell apoptosis.
Some studies have suggested a relationship between lymphocyte compe-
tence and clinical outcome in breast cancer [43–45]. Effects of lymphocyte
competence are unknown in glioma patients, although some glioma patients are
known to have impaired cell-mediated immunity (e.g., decreased cellular immu-
nity when assessed by delayed-type cutaneous reactions) [46]. Among other
possibilities, this may be due to the impairment of mitogen- and antigen-induced
T cell reactivity, impaired production of IL-2, or decreased expression of the
IL-2 receptor p55 chain [46]. Whether host immune deficiencies are directly
related to immune modulation by gliomas remains unclear, but glioma cells are
known to produce a variety of cytokines that modulate host immune reactions
(table 1). Gliomas also were shown to produce FasL and confer resistance to
host immunity by inducing apoptosis in Fas receptor-bearing T cells [47]. Thus,
in addition to any immunological safeguards present in normal brain tissue,
gliomas possess additional mechanisms to subvert immunological detection and
attack. Their primary means of immune evasion elicits immune dysfunction.
By deploying anti-inflammatory Th2 cytokines (e.g., TGF-, IL-10,
PGE2) gliomas can circumvent immune responses in the brain. Although
tumor-infiltrating lymphocytes (TIL) can be observed in gliomas, often there
are multiple immunological defects among TIL consisting of decreased
responses to mitogens and reduced killing capacity, which can be attributed to
the local immunosuppressive environment.
While there are no resident APCs in the brain, dendritic cells and
macrophages also can be found circulating in the cerebrospinal fluid. Similarly,
blood dendritic cells can extravasate from vessels within the brain, enabling
transient migration through tissue. These cells are fully capable of acquiring
antigen and migrating to cervical lymph nodes. The immunosuppressive envi-
ronment in the brain, compounded by the presence of tumor, prevents the acti-
vation of APCs that are essential for generating effector T cells. Instead,
presentation to T cells by an inactivated APC results in tolerance and anergy. By
maintaining an immunosuppressive environment that affects both APC and
immune effector cells, gliomas induce a response that favors immunological
tolerance instead of a destructive inflammatory response. Recognizing this phe-
nomenon, one can devise ways to overcome tumor-induced immunosuppres-
sion; when designing an anti-glioma immunotherapy, great care must be taken
to generate an anti-glioma immune response, which preserves normal brain
tissues in order to avoid generalized inflammation and tissue necrosis.
Current Strategies under Consideration for

Immunotherapy of Gliomas
Anti-glioma immunotherapy efforts can be divided into two strategies:

those that target tumors directly and those that are designed to boost the exist-
ing immune response. The first strategy uses cytokines or antibodies to neu-
tralize molecules required for tumor survival or functioning. The second
strategy may boost the immune response by enhancing NK-mediated or T cell-
mediated killing; for example, by vaccinating the host with tumor antigens or
with autologous dendritic cells primed with tumor antigens.
Cytokines are intercellular mediators, which provide an important role in
coordinating and skewing the immune response [48]. Expression of many
cytokines is regulated by the presence of opposing cytokines. As an example,
Th1 cytokines will cause the down-regulation of Th2 cytokines. Cytokines can
be administered directly inside biodegradable polymers [49], with direct infusion
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into the brain [50, 51], or with intravenously injections [52, 53]. Alternatively,
cytokines can be administered with gene therapy expression vectors [54–57].
IL-2, IL-12, TNF-, and IFN- producing gene transfer vectors have exhibited
limited success in treating gliomas in animal models. In a recent review of
glioma clinical trials, however, cytokine administration yielded little benefit [58].
Antibodies are highly specific molecules of the immune system with long
half-lives that allow them to persist within the body for extended periods of
time. Tumor antigen biospecificity and stability make antibodies attractive can-
didates for glioma immunotherapy. Antibodies employ several modes of action
to target gliomas. The structure of antibodies enable them to bind target mole-
cules, thus preventing interaction with natural ligands such as growth factors
required for glioma survival. Epidermal growth factor receptor (EGFR) and a
major splice variant, EGFRvIII are often overexpressed in gliomas [59, 60] and
are targets for glioma therapies [61, 62]. Vascular endothelial growth factor is
another target for interference by antibody-based glioma therapy [63, 64].
In addition to blocking vital tumor growth factors, antibodies can uncloak
glioma immune evasion. NK cells play an important role in innate immune detec-
tion of tumors. NK cells are capable of recognizing cells coated with antibodies
and killing them, known as antibody-dependent cell-mediated cytotoxicity.
Stimulating antibody-dependent cell-mediated cytotoxicity with antibodies spe-
cific for EGFR (or EGFRvIII) [65–67], vimentin [68], or ganglioside GD2 [69]
has been attempted for the experimental treatment of glioma.
Additional glioma targets have included CD95 (Fas) and TNFR, often
referred to as ‘death receptors.’ When these receptors are bound by their respec-
tive ligands, cell death is initiated. Interestingly, death receptors play an impor-
tant role in maintaining the immune-privileged status of the brain. Gliomas
have been shown to down-regulate these receptors in order to gain a survival
advantage; there has been some limited success in targeting this pathway in
glioma therapy [70] by driving expression of Fas/TNFR [71] or transducing
gliomas [72, 73].
Antibodies also can be functionalized for tumor ablation by conjugating
them to toxins, drugs, or radionuclides. This kind of targeted delivery of anti-
tumor agents provides a major advantage. Antibodies linked to ricin [74–76] or
pseudomonas toxin [77, 78] have been used to treat gliomas in preclinical stud-
ies. In a similar manner, radioimmunotherapy with antibodies specific for
tenascin and conjugated to 131I [79–81] and 90Y [80, 82, 83] have exhibited lim-
ited success in a clinical setting. Similar data have been reported using radioac-
tive antibodies specific for EGFR [84–86]. The drawback is that continued
exposure to irradiation, due to the persistence of antibodies in the serum, deliv-
ers a substantial dose of irradiation to unaffected organs and limits the quantity
that can be safely administered.
SSC PBMC Gated on R1
R2 R3
R1
FSC CD8 CD4
CD8 T cells CD4 T cells

(R1 and R2) (R1 and R3)
0.00% 0.00%
Unloaded
DC
2.81% 0.10%
U118 lysate-
loaded DC
CD69
IFN-
Fig. 4. Glioma-specific T cells respond to U118 lysate-loaded DC. PBMC were cocul-
tured for 6 h with autologous U118 lysate-loaded or control DC (matured with IL-1, IL-6,
PGE2, and TNF-). Cocultures were harvested, stained with fluorescent antibodies, and ana-
lyzed by flow cytometry. U118 lysate-loaded DC activate both CD4 and CD8 T cells.
Additionally, CD8 T cells produced IFN- following antigen presentation by DC.
Strategies designed to boost the immune response include the use of tumor
vaccines and/or primed APCs. The use of allogeneic cell lines for glioma vac-
cines has been explored in orthotopic rodent models by Ashley et al. [87].
Priming of the anti-glioma immune response using cellular vaccines is not
dependent on the expression of syngeneic class I MHC molecules on the
immunizing cell [88, 89]. The immune priming mechanism is optimized by
generating antigens from apoptosis induced in the immunizing cells [90–92].
Our lab has explored the use of allogeneic glioma cells in cancer therapy by
loading human dendritic cells with antigen and cross-priming autologous
CTLs. As a preliminary finding, we detected CD8 and CD4 T cell responses to
allogeneic tumor lysate-loaded dendritic cells in the peripheral blood from
glioma patients (fig. 4). These T cells exhibited cytotoxicity against autologous
glioma cells in vitro.
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While preliminary data suggest the value of using an APC-based
immunotherapy, many basic questions remain unanswered. For example, is it
best to target APCs in vivo or ex vivo? Which type of APCs generate the best
anti-tumor response: immature APCs or mature APCS? If mature APCs, what
are the maturation stimuli?, What is the best source of antigen to ‘feed’ APCs
(apoptotic cells, tumor peptides, DNA-encoding tumor antigens)? How many
APCs should be utilized for each vaccination, and how frequently should vac-
cinations be given? Which injection route will generate the best immunity
(subcutaneous, intravenous, intramuscular, intranodal)? More clinical trials
and studies comparing immunization protocols are necessary to answer these
questions.
It is possible to enrich mature APCs populations directly from human tissue
[93, 94] or circulating PBMC [95, 96]. However, the low frequency and avail-
ability of tissue have limited these approaches. Instead, well-established methods
for generating APCs in vitro have been described [97–99]. These cells resemble
their in vivo counterparts, LC and iAPCs, and function similarly. Ex vivo
approaches begin with the purification or enrichment of CD14 and CD34
precursors, which can differentiate into APCs in the presence of cytokines. APCs
that have been loaded with antigen from various sources (e.g., glioma RNA [100],
glioma peptides [101, 102], apoptotic tumor cells [103], tumor lysate [104],
fusion of glioma cells and APCs [105]) all have been shown to induce anti-tumor
responses. When using ex vivo APCs for therapy, one can choose the maturation
stimuli given to APCs. In vitro maturation of APCs can be achieved by any num-
ber of stimuli: bacterial components (cell wall, DNA) [106], double-stranded
RNA (mimicking viral infection) [107], CpG DNA [108], monocyte-conditioned
medium [109], CD40L (mimicking CD4 T cell helper function) [110], and
inflammatory cytokines [111, 112]. CD40L and a cytokine cocktail consisting of
TNF, IL1, IL6, IFN, and PGE2 can be used to obtain clinical grade, mature
APCs [112, 113].
Given the complexity of generating ex vivo GMP-grade APC vaccine,
APCs also may be targeted in vivo. Some systems have utilized viral vectors to
target LC in skin [114, 115]. Alternatively, APCs possess many antigen-uptake
receptors, which represent feasible candidates for APC therapy. To date, most
efforts have focused on one receptor in particular called Dec-205. Dec-205 is a
C-type lectin (binding to oligosaccharides) and its expression is restricted
solely to APCs. An antibody specific for Dec-205 has been used to successfully
deliver antigen to APCs in vivo [116]. Antigen was presented in both MHC
class I and class II molecules [116–118]. APCs [119–121] and tumors [122] are
capable of producing exosomes, which are tiny, antigen-presenting vesicles that
can induce potent anti-tumor responses [119, 122] and are under investigation
in a phase I/II study involving non-small cell lung cancer patients.
A recent study detailed an innovative, two-step approach that targeted
APCs in situ. Vaccination resulted in protective, tumor-specific immunity
[123]. Macrophage inflammatory protein 3, a chemotactic protein that attracts
APCs, was encased in a polymer rod. Similar rods were constructed that con-
tained tumor lysate or peptide. Macrophage inflammatory protein 3 was
released in a controlled fashion and resulted in an accumulation of LC in the
vicinity of the rod following subcutaneous implantation. Coimplantation of
rods encasing macrophage inflammatory protein 3 and tumor lysate or pep-
tide into mice produced potent, tumor-specific CTL activity. As a comparison,
mice were vaccinated with either polymer rods or conventional ex vivo gener-
ated APCs. Similar anti-tumor responses were seen in both groups, supporting
a vaccination strategy.
Preclinical studies reflecting dendritic cell manipulation in glioma treat-
ments are summarized in a review by Parney et al. [58]. In addition to glioma
[102] other human trials using autologous APCs to treat tumors have been pub-
lished, including lymphoma [124], melanoma [125], renal cell carcinoma [126],
and prostate cancer [127]. In the case of glioma, Yu et al. [102] demonstrated
that APC vaccination utilizing autologous APCs pulsed with peptides acid-
eluted from the surface of autologous glioma cells elicited systemic cytotoxic-
ity in 4 of 7 patients treated. This study demonstrated the feasibility, safety, and
bioactivity of an autologous peptide-pulsed APC vaccine for patients with
malignant gliomas. Our laboratory is currently exploring the use of a novel
class of sulfones, which induce apoptosis in a variety of glioma and other cell
lines, suggesting another means to prime the immune system and induce anti-
tumor responses in glioma patients. The challenge remains not only in under-
standing and exploiting glioma immune modulation, but also in gaining a more
thorough understanding of human immunobiology and refining efficient, quan-
titative means of measuring immune response.
References
1 Galanis E, Buckner JC, Dinapoli RP, Scheithauer BW, Jenkins RB, Wang CH, O’Fallon JR, Farr G Jr:
Clinical outcome of gliosarcoma compared with glioblastoma multiforme: North Central Cancer
Treatment Group results. J Neurosurg 1998;89:425–430.
2 Steinman RM, Cohn ZA: Identification of a novel cell type in peripheral lymphoid organs of
mice. I. Morphology, quantitation, tissue distribution. J Exp Med 1973;137:1142–1162.
3 Wilders MM, Sminia T, Janse EM: Ontogeny of non-lymphoid and lymphoid cells in the rat gut with
special reference to large mononuclear Ia-positive dendritic cells. Immunology 1983;50:303–314.
4 Sertl K, Takemura T, Tschachler E, Ferrans VJ, Kaliner MA, Shevach EM: Dendritic cells with
antigen-presenting capability reside in airway epithelium, lung parenchyma, and visceral pleura.
J Exp Med 1986;163:436–451.
5 Mootz W, Mausle E, Schondorf J: Electronmicroscopic determination of Langerhans cells in the
crypt epithelium of human tonsils. Z Haut Geschlechtskr 1972;47:213–216.
medwedi.ru
6 Thiery M, Willighagen GJ: Langerhans cells in cervix. Am J Obstet Gynecol 1969;104:1223.
7 Edwards JN, Morris HB: Langerhans’ cells and lymphocyte subsets in the female genital tract.
Br J Obstet Gynaecol 1985;92:974–982.
8 Murphy GF, Bhan AK, Harrist TJ, Mihm MC Jr: In situ identification of T6-positive cells in nor-
mal human dermis by immunoelectron microscopy. Br J Dermatol 1983;108:423–431.
9 Siegal FP, Kadowaki N, Shodell M, Fitzgerald-Bocarsly PA, Shah K, Ho S, Antonenko S, Liu YJ:
The nature of the principal type 1 interferon-producing cells in human blood. Science 1999;284:
1835–1837.
10 Olweus J, BitMansour A, Warnke R, Thompson PA, Carballido J, Picker LJ, Lund-Johansen F:
Dendritic cell ontogeny: A human dendritic cell lineage of myeloid origin. Proc Natl Acad Sci
USA 1997;94:12551–12556.
11 O’Doherty U, Peng M, Gezelter S, Swiggard WJ, Betjes M, Bhardwaj N, Steinman RM: Human
blood contains two subsets of dendritic cells, one immunologically mature and the other imma-
ture. Immunology 1994;82:487–493.
12 Grouard G, Rissoan MC, Filgueira L, Durand I, Banchereau J, Liu YJ: The enigmatic plasmacy-
toid T cells develop into dendritic cells with interleukin (IL)-3 and CD40-ligand. J Exp Med
1997;185:1101–1111.
13 Altman JD, Moss PA, Goulder PJ, Barouch DH, McHeyzer-Williams MG, Bell JI, McMichael
AJ, Davis MM: Phenotypic analysis of antigen-specific T lymphocytes. Science 1996;274:
94–96.
14 Skinner PJ, Haase AT: In situ tetramer staining. J Immunol Methods 2002;268:29–34.
15 Sidobre S, Kronenberg M: CD1 tetramers: A powerful tool for the analysis of glycolipid-reactive
T cells. J Immunol Methods 2002;268:107–121.
16 Kwok WW, Ptacek NA, Liu AW, Buckner JH: Use of class II tetramers for identification of CD4
T cells. J Immunol Methods 2002;268:71–81.
17 Lyons AB, Parish CR: Determination of lymphocyte division by flow cytometry. J Immunol
Methods 1994;171:131–137.
18 Kilburn DG, Shematek G, Crane S: Quantitation of helper factor activity. I. Utilization of the
ELISA method to measure helper activity in the secondary antibody response. J Immunol Methods
1981;44:301–310.
19 Cook EB, Stahl JL, Lowe L, Chen R, Morgan E, Wilson J, Varro R, Chan A, Graziano FM,
Barney NP: Simultaneous measurement of six cytokines in a single sample of human tears using
microparticle-based flow cytometry: Allergics vs. non-allergics. J Immunol Methods 2001;254:
109–118.
20 Czerkinsky C, Andersson G, Ekre HP, Nilsson LA, Klareskog L, Ouchterlony O: Reverse
ELISPOT assay for clonal analysis of cytokine production. I. Enumeration of gamma-interferon-
secreting cells. J Immunol Methods 1988;110:29–36.
21 Jung T, Schauer U, Heusser C, Neumann C, Rieger C: Detection of intracellular cytokines by flow
cytometry. J Immunol Methods 1993;159:197–207.
22 Donnenberg VS, O’Connell PJ, Logar AJ, Zeevi A, Thomson AW, Donnenberg AD: Rare-event
analysis of circulating human dendritic cell subsets and their presumptive mouse counterparts.
Transplantation 2001;72:1946–1951.
23 Manz R, Assenmacher M, Pfluger E, Miltenyi S, Radbruch A: Analysis and sorting of live cells
according to secreted molecules, relocated to a cell-surface affinity matrix. Proc Natl Acad Sci
USA 1995;92:1921–1925.
24 Oelke M, Moehrle U, Chen JL, Behringer D, Cerundolo V, Lindemann A, Mackensen A:
Generation and purification of CD8 melan-A-specific cytotoxic T lymphocytes for adoptive
transfer in tumor immunotherapy. Clin Cancer Res 2000;6:1997–2005.
25 Brunner KT, Mauel J, Cerottini JC, Chapuis B: Quantitative assay of the lytic action of immune
lymphoid cells on 51-Cr-labelled allogeneic target cells in vitro: Inhibition by isoantibody and by
drugs. Immunology 1968;14:181–196.
26 Korzeniewski C, Callewaert DM: An enzyme-release assay for natural cytotoxicity. J Immunol
Methods 1983;64:313–320.
27 Karawajew L, Jung G, Wolf H, Micheel B, Ganzel K: A flow cytometric long-term cytotoxicity
assay. J Immunol Methods 1994;177:119–130.
28 Sheely ME, McDermott AB, Furlan SN, Klenerman P, Nixon DF: A novel technique for the
fluorometric assessment of T lymphocyte antigen specific lysis. J Immunol Methods 2001;249:
99–110.
29 Johann S, Blumel G, Lipp M, Forster R: A versatile flow cytometry-based assay for the determi-
nation of short- and long-term natural killer cell activity. J Immunol Methods 1995;185:209–216.
30 Lecoeur H, Fevrier M, Garcia S, Riviere Y, Gougeon ML: A novel flow cytometric assay for quan-
titation and multiparametric characterization of cell-mediated cytotoxicity. J Immunol Methods
2001;253:177–187.
31 Zhang X, Chen Z, Huang H, Gordon JR, Xiang J: DNA microarray analysis of the gene expres-
sion profiles of naive versus activated tumor-specific T cells. Life Sci 2002;71:3005–3017.
32 Kammula US, Lee KH, Riker AI, Wang E, Ohnmacht GA, Rosenberg SA, Marincola FM:
Functional analysis of antigen-specific T lymphocytes by serial measurement of gene expression
in peripheral blood mononuclear cells and tumor specimens. J Immunol 1999;163:6867–6875.
33 Kammula US, Marincola FM, Rosenberg SA: Real-time quantitative polymerase chain reaction
assessment of immune reactivity in melanoma patients after tumor peptide vaccination. J Natl
Cancer Inst 2000;92:1336–1344.
34 Hempel DM, Smith KA, Claussen KA, Perricone MA: Analysis of cellular immune responses in
the peripheral blood of mice using real-time RT-PCR. J Immunol Methods 2002;259:129–138.
35 Garg RK: Acute disseminated encephalomyelitis. Postgrad Med J 2003;79:11–17.
36 Kuppner MC, Hamou MF, de Tribolet N: Immunohistological and functional analyses of lymphoid
infiltrates in human glioblastomas. Cancer Res 1988;48:6926–6932.
37 Brooks WH, Markesbery WR, Gupta GD, Roszman TL: Relationship of lymphocyte invasion and
survival of brain tumor patients. Ann Neurol 1978;4:219–224.
38 Rainbird S, Allwood G, Ridley A: Lymphocyte-mediated cytotoxicity against gliomas. Brain
1981;104:451–464.
39 Safdari H, Hochberg FH, Richardson EP Jr: Prognostic value of round cell (lymphocyte) infiltra-
tion in malignant gliomas. Surg Neurol 1985;23:221–226.
40 Ridley A, Cavanagh JB: Lymphocytic infiltration in gliomas: Evidence of possible host resistance.
Brain 1971;94:117–124.
41 Andrews DW, Resnicoff M, Flanders AE, Kenyon L, Curtis M, Merli G, Baserga R, Iliakis G,
Aiken RD: Results of a pilot study involving the use of an antisense oligodeoxynucleotide directed
against the insulin-like growth factor type I receptor in malignant astrocytomas. J Clin Oncol
2001;19:2189–2200.
42 Palma L, Di Lorenzo N, Guidetti B: Lymphocytic infiltrates in primary glioblastomas and recidi-
vous gliomas: Incidence, fate, and relevance to prognosis in 228 operated cases. J Neurosurg
1978;49:854–861.
43 Humphrey L, Singla O: Immune responsiveness of patients with malignant melanoma and carci-
noma of the breast. Semin Surg Oncol 1991;7:230–238.
44 Dean JH, McCoy JL, Cannon GB, Leonard CM, Perlin E, Kreutner A, Oldham RK, Herberman RB:
Cell-mediated immune responses of breast cancer patients to autologous tumor-associated antigens.
J Natl Cancer Inst 1977;58:549–555.
45 McCoy JL, Rucker R, Petros JA: Cell-mediated immunity to tumor-associated antigens is a better
predictor of survival in early stage breast cancer than stage, grade or lymph node status. Breast
Cancer Res Treat 2000;60:227–234.
46 Roszman T, Elliott L, Brooks W: Modulation of T-cell function by gliomas. Immunol Today 1991;
12:370–374.
47 Didenko VV, Ngo HN, Minchew C, Baskin DS: Apoptosis of T lymphocytes invading glioblas-
tomas multiforme: A possible tumor defense mechanism. J Neurosurg 2002;96:580–584.
48 Kurt-Jones EA, Hamberg S, Ohara J, Paul WE, Abbas AK: Heterogeneity of helper/inducer T lym-
phocytes. I. Lymphokine production and lymphokine responsiveness. J Exp Med 1987;166:
1774–1787.
49 Rhines LD, Sampath P, DiMeco F, Lawson HC, Tyler BM, Hanes J, Olivi A, Brem H: Local
immunotherapy with interleukin-2 delivered from biodegradable polymer microspheres combined
with interstitial chemotherapy: A novel treatment for experimental malignant glioma.
medwedi.ru
50 Brandes AA, Scelzi E, Zampieri P, Rigon A, Rotilio A, Amista P, Berti F, Fiorentino MV: Phase II
trial with BCNU plus alpha-interferon in patients with recurrent high-grade gliomas. Am J Clin
Oncol 1997;20:364–367.
51 Giussani C, Carrabba G, Pluderi M, Lucini V, Pannacci M, Caronzolo D, Costa F, Minotti M,
Tomei G, Villani R, Carroll RS, Bikfalvi A, Bello L: Local intracerebral delivery of endogenous
inhibitors by osmotic minipumps effectively suppresses glioma growth in vivo. Cancer Res
2003;63:2499–2505.
52 Allen J, Packer R, Bleyer A, Zeltzer P, Prados M, Nirenberg A: Recombinant interferon beta:
A phase I-II trial in children with recurrent brain tumors. J Clin Oncol 1991;9:783–788.
53 Johansson M, Henriksson R, Bergenheim AT, Koskinen LO: Interleukin-2 and histamine in com-
bination inhibit tumour growth and angiogenesis in malignant glioma. Br J Cancer 2000;83:
826–832.
54 Ehtesham M, Samoto K, Kabos P, Acosta FL, Gutierrez MA, Black KL, Yu JS: Treatment of
intracranial glioma with in situ interferon-gamma and tumor necrosis factor-alpha gene transfer.
Cancer Gene Ther 2002;9:925–934.
55 Liu Y, Ehtesham M, Samoto K, Wheeler CJ, Thompson RC, Villarreal LP, Black KL, Yu JS: In situ
adenoviral interleukin 12 gene transfer confers potent and long-lasting cytotoxic immunity in
glioma. Cancer Gene Ther 2002;9:9–15.
56 Chen B, Timiryasova TM, Gridley DS, Andres ML, Dutta-Roy R, Fodor I: Evaluation of cytokine
toxicity induced by vaccinia virus-mediated IL-2 and IL-12 antitumour immunotherapy. Cytokine
2001;15:305–314.
57 van Herpen CM, De Mulder PH: Locoregional immunotherapy in cancer patients: Review of clin-
ical studies. Ann Oncol 2000;11:1229–1239.
58 Parney IF, Hao C, Petruk KC: Glioma immunology and immunotherapy. Neurosurgery 2000;46:
778–791; discussion 791–792.
59 Wong AJ, Bigner SH, Bigner DD, Kinzler KW, Hamilton SR, Vogelstein B: Increased expression
of the epidermal growth factor receptor gene in malignant gliomas is invariably associated with
gene amplification. Proc Natl Acad Sci USA 1987;84:6899–6903.
60 Wong AJ, Ruppert JM, Bigner SH, Grzeschik CH, Humphrey PA, Bigner DS, Vogelstein B:
Structural alterations of the epidermal growth factor receptor gene in human gliomas. Proc Natl
Acad Sci USA 1992;89:2965–2969.
61 Stragliotto G, Vega F, Stasiecki P, Gropp P, Poisson M, Delattre JY: Multiple infusions of anti-
epidermal growth factor receptor (EGFR) monoclonal antibody (EMD 55,900) in patients with
recurrent malignant gliomas. Eur J Cancer 1996;32A:636–640.
62 Wersall P, Ohlsson I, Biberfeld P, Collins VP, von Krusenstjerna S, Larsson S, Mellstedt H,
Boethius J: Intratumoral infusion of the monoclonal antibody, mAb 425, against the epidermal-
growth-factor receptor in patients with advanced malignant glioma. Cancer Immunol Immunother
1997;44:157–164.
63 Rubenstein JL, Kim J, Ozawa T, Zhang M, Westphal M, Deen DF, Shuman MA: Anti-VEGF anti-
body treatment of glioblastoma prolongs survival but results in increased vascular cooption.
Neoplasia 2000;2:306–314.
64 Stefanik DF, Fellows WK, Rizkalla LR, Rizkalla WM, Stefanik PP, Deleo AB, Welch WC:
Monoclonal antibodies to vascular endothelial growth factor (VEGF) and the VEGF receptor,
FLT-1, inhibit the growth of C6 glioma in a mouse xenograft. J Neurooncol 2001;55:91–100.
65 Eller JL, Longo SL, Hicklin DJ, Canute GW: Activity of anti-epidermal growth factor receptor
monoclonal antibody C225 against glioblastoma multiforme. Neurosurgery 2002;51:1005–1013;
discussion 1013–1014.
66 Mishima K, Johns TG, Luwor RB, Scott AM, Stockert E, Jungbluth AA, Ji XD, Suvarna P,
Voland JR, Old LJ, Huang HJ, Cavenee WK: Growth suppression of intracranial xenografted
glioblastomas overexpressing mutant epidermal growth factor receptors by systemic administra-
tion of monoclonal antibody (mAb) 806, a novel monoclonal antibody directed to the receptor.
Cancer Res 2001;61:5349–5354.
67 Faillot T, Magdelenat H, Mady E, Stasiecki P, Fohanno D, Gropp P, Poisson M, Delattre JY:
A phase I study of an anti-epidermal growth factor receptor monoclonal antibody for the treatment
of malignant gliomas. Neurosurgery 1996;39:478–483.
68 Kokunai T, Tamaki N, Matsumoto S: Antigen related to cell proliferation in malignant gliomas rec-
ognized by a human monoclonal antibody. J Neurosurg 1990;73:901–908.
69 Shitara K, Kuwana Y, Nakamura K, Tokutake Y, Ohta S, Miyaji H, Hasegawa M, Hanai N:
A mouse/human chimeric anti-(ganglioside GD3) antibody with enhanced antitumor activities.
Cancer Immunol Immunother 1993;36:373–380.
70 Kawaguchi S, Mineta T, Ichinose M, Masuoka J, Shiraishi T, Tabuchi K: Induction of apoptosis in
glioma cells by recombinant human Fas ligand. Neurosurgery 2000;46:431–438; discussion
438–439.
71 Weller M, Frei K, Groscurth P, Krammer PH, Yonekawa Y, Fontana A: Anti-Fas/APO-1 antibody-
mediated apoptosis of cultured human glioma cells: Induction and modulation of sensitivity by
cytokines. J Clin Invest 1994;94:954–964.
72 Mizuno M, Yoshida J: Tumor necrosis factor-alpha gene transfer augments anti-Fas antibody-
mediated apoptosis in human glioma cells. Jpn J Cancer Res 1996;87:543–547.
73 Shinoura N, Yoshida Y, Sadata A, Hanada KI, Yamamoto S, Kirino T, Asai A, Hamada H:
Apoptosis by retrovirus- and adenovirus-mediated gene transfer of Fas ligand to glioma cells:
Implications for gene therapy. Hum Gene Ther 1998;9:1983–1993.
74 Ohtomo T, Kawata H, Sekimori Y, Shimizu K, Kishima H, Moriuchi S, Miyao Y, Akamatsu K,
Tsuchiya M: A humanized single-chain Fv fragment with high targeting potential against human
malignant gliomas. Anticancer Res 1998;18:4311–4315.
75 Laske DW, Ilercil O, Akbasak A, Youle RJ, Oldfield EH: Efficacy of direct intratumoral therapy
with targeted protein toxins for solid human gliomas in nude mice. J Neurosurg 1994;80:520–526.
76 Martell LA, Agrawal A, Ross DA, Muraszko KM: Efficacy of transferrin receptor-targeted
immunotoxins in brain tumor cell lines and pediatric brain tumors. Cancer Res 1993;53:
1348–1353.
77 Rand RW, Kreitman RJ, Patronas N, Varricchio F, Pastan I, Puri RK: Intratumoral administration
of recombinant circularly permuted interleukin-4-Pseudomonas exotoxin in patients with high-
grade glioma. Clin Cancer Res 2000;6:2157–2165.
78 Husain SR, Behari N, Kreitman RJ, Pastan I, Puri RK: Complete regression of established human
glioblastoma tumor xenograft by interleukin-4 toxin therapy. Cancer Res 1998;58:3649–3653.
79 Cokgor I, Akabani G, Kuan CT, Friedman HS, Friedman AH, Coleman RE, McLendon RE,
Bigner SH, Zhao XG, Garcia-Turner AM, Pegram CN, Wikstrand CJ, Shafman TD, Herndon JE
2nd, Provenzale JM, Zalutsky MR, Bigner DD: Phase I trial results of iodine-131-labeled anti-
tenascin monoclonal antibody 81C6 treatment of patients with newly diagnosed malignant
gliomas. J Clin Oncol 2000;18:3862–3872.
80 Goetz C, Riva P, Poepperl G, Gildehaus FJ, Hischa A, Tatsch K, Reulen HJ: Locoregional radioim-
munotherapy in selected patients with malignant glioma: Experiences, side effects and survival
times. J Neurooncol 2003;62:321–328.
81 Reardon DA, Akabani G, Coleman RE, Friedman AH, Friedman HS, Herndon JE 2nd, Cokgor I,
McLendon RE, Pegram CN, Provenzale JM, Quinn JA, Rich JN, Regalado LV, Sampson JH,
Shafman TD, Wikstrand CJ, Wong TZ, Zhao XG, Zalutsky MR, Bigner DD: Phase II trial of
murine (131)I-labeled antitenascin monoclonal antibody 81C6 administered into surgically cre-
ated resection cavities of patients with newly diagnosed malignant gliomas. J Clin Oncol 2002;20:
1389–1397.
82 Grana C, Chinol M, Robertson C, Mazzetta C, Bartolomei M, De Cicco C, Fiorenza M, Gatti M,
Caliceti P, Paganelli G: Pretargeted adjuvant radioimmunotherapy with yttrium-90-biotin in
malignant glioma patients: A pilot study. Br J Cancer 2002;86:207–212.
83 Riva P, Franceschi G, Frattarelli M, Lazzari S, Riva N, Giuliani G, Casi M, Sarti G, Guiducci G,
Giorgetti G, Gentile R, Santimaria M, Jermann E, Maeke HR: Loco-regional radioimmunother-
apy of high-grade malignant gliomas using specific monoclonal antibodies labeled with 90Y:
A phase I study. Clin Cancer Res 1999;5:s3275-s3280.
84 Emrich JG, Brady LW, Quang TS, Class R, Miyamoto C, Black P, Rodeck U: Radioiodinated (I-125)
monoclonal antibody 425 in the treatment of high grade glioma patients: Ten-year synopsis of a
novel treatment. Am J Clin Oncol 2002;25:541–546.
85 Miyamoto CT, Brady LW, Rackover MA, Emrich J, Class R, Bender H, Micaily B, Steplewski Z:
The use of epidermal growth factor receptor-425 monoclonal antibodies radiolabeled with
medwedi.ru
iodine-125 in the adjuvant treatment of patients with high grade gliomas of the brain. Recent
Results Cancer Res 1996;141:183–192.
86 Snelling L, Miyamoto CT, Bender H, Brady LW, Steplewski Z, Class R, Emrich J, Rackover MA:
Epidermal growth factor receptor 425 monoclonal antibodies radiolabeled with iodine-125 in the
adjuvant treatment of high-grade astrocytomas. Hybridoma 1995;14:111–114.
87 Ashley DM, Sampson JH, Archer GE, Batra SK, Bigner DD, Hale LP: A genetically mod-
ified allogeneic cellular vaccine generates MHC class I-restricted cytotoxic responses against
tumor-associated antigens and protects against CNS tumors in vivo. J Neuroimmunol 1997;78:
34–46.
88 Huang AY, Golumbek P, Ahmadzadeh M, Jaffee E, Pardoll D, Levitsky H: Bone marrow-derived
cells present MHC class I-restricted tumour antigens in priming of antitumour immune responses.
Ciba Found Symp 1994;187:229–240; discussion 240–244.
89 Huang AY, Golumbek P, Ahmadzadeh M, Jaffee E, Pardoll D, Levitsky H: Role of bone marrow-
derived cells in presenting MHC class I-restricted tumor antigens. Science 1994;264: 961–965.
90 Henry F, Boisteau O, Bretaudeau L, Lieubeau B, Meflah K, Gregoire M: Antigen-presenting cells
that phagocytose apoptotic tumor-derived cells are potent tumor vaccines. Cancer Res
1999;59:3329–3332.
91 Hoffmann TK, Meidenbauer N, Dworacki G, Kanaya H, Whiteside TL: Generation of tumor-
specific T-lymphocytes by cross-priming with human dendritic cells ingesting apoptotic tumor
cells. Cancer Res 2000;60:3542–3549.
92 Schnurr M, Scholz C, Rothenfusser S, Galambos P, Dauer M, Robe J, Endres S, Eigler A:
Apoptotic pancreatic tumor cells are superior to cell lysates in promoting cross-priming of cyto-
toxic T cells and activate NK and gammadelta T cells. Cancer Res 2002;62:2347–2352.
93 Inaba K, Schuler G, Witmer MD, Valinksy J, Atassi B, Steinman RM: Immunologic properties of
purified epidermal Langerhans cells: Distinct requirements for stimulation of unprimed and sen-
sitized T lymphocytes. J Exp Med 1986;164:605–613.
94 Blauvelt A, Katz SI, Udey MC: Human Langerhans cells express E-cadherin. J Invest Dermatol
1995;104:293–296.
95 Coates PT, Barratt-Boyes SM, Donnenberg AD, Morelli AE, Murphey-Corb M, Thomson AW:
Strategies for preclinical evaluation of dendritic cell subsets for promotion of transplant tolerance
in the nonhuman primate. Hum Immunol 2002;63:955–965.
96 MacDonald KP, Munster DJ, Clark GJ, Dzionek A, Schmitz J, Hart DN: Characterization of
human blood dendritic cell subsets. Blood 2002;100:4512–4520.
97 Caux C, Vanbervliet B, Massacrier C, Dezutter-Dambuyant C, de Saint-Vis B, Jacquet C, Yoneda K,
Imamura S, Schmitt D, Banchereau J: CD34 hematopoietic progenitors from human cord blood
differentiate along two independent dendritic cell pathways in response to GM-CSFTNF alpha.
J Exp Med 1996;184:695–706.
98 O’Doherty U, Steinman RM, Peng M, Cameron PU, Gezelter S, Kopeloff I, Swiggard WJ, Pope M,
Bhardwaj N: Dendritic cells freshly isolated from human blood express CD4 and mature into typ-
ical immunostimulatory dendritic cells after culture in monocyte-conditioned medium. J Exp Med
1993;178:1067–1076.
99 Zhou LJ, Tedder TF: CD14 blood monocytes can differentiate into functionally mature CD83
dendritic cells. Proc Natl Acad Sci USA 1996;93:2588–2592.
100 Insug O, Ku G, Ertl HC, Blaszczyk-Thurin M: A dendritic cell vaccine induces protective immu-
nity to intracranial growth of glioma. Anticancer Res 2002;22:613–621.
101 Liau LM, Black KL, Prins RM, Sykes SN, DiPatre PL, Cloughesy TF, Becker DP, Bronstein JM:
Treatment of intracranial gliomas with bone marrow-derived dendritic cells pulsed with tumor
antigens. J Neurosurg 1999;90:1115–1124.
102 Yu JS, Wheeler CJ, Zeltzer PM, Ying H, Finger DN, Lee PK, Yong WH, Incardona F, Thompson RC,
Riedinger MS, Zhang W, Prins RM, Black KL: Vaccination of malignant glioma patients with
peptide-pulsed dendritic cells elicits systemic cytotoxicity and intracranial T-cell infiltration. Cancer
Res 2001;61:842–847.
103 Nakahara N, Pollack IF, Storkus WJ, Wakabayashi T, Yoshida J, Okada H: Effective induction of
antiglioma cytotoxic T cells by coadministration of interferon-beta gene vector and dendritic cells.
104 Yoshida S, Morii K, Watanabe M, Saito T, Yamamoto K, Tanaka R: The generation of anti-tumoral
cells using dentritic cells from the peripheral blood of patients with malignant brain tumors.
Cancer Immunol Immunother 2001;50:321–327.
105 Kikuchi T, Akasaki Y, Irie M, Homma S, Abe T, Ohno T: Results of a phase I clinical trial of vacci-
nation of glioma patients with fusions of dendritic and glioma cells. Cancer Immunol Immunother
2001;50:337–344.
106 Kopp E, Medzhitov R: Recognition of microbial infection by Toll-like receptors. Curr Opin
Immunol 2003;15:396–401.
107 Cella M, Salio M, Sakakibara Y, Langen H, Julkunen I, Lanzavecchia A: Maturation, activation,
and protection of dendritic cells induced by double-stranded RNA. J Exp Med 1999;189:
821–829.
108 Hartmann G, Weiner GJ, Krieg AM: CpG DNA: A potent signal for growth, activation, and mat-
uration of human dendritic cells. Proc Natl Acad Sci USA 1999;96:9305–9310.
109 Reddy A, Sapp M, Feldman M, Subklewe M, Bhardwaj N: A monocyte conditioned medium is
more effective than defined cytokines in mediating the terminal maturation of human dendritic
cells. Blood 1997;90:3640–3646.
110 Ridge JP, Di Rosa F, Matzinger P: A conditioned dendritic cell can be a temporal bridge between
a CD4 T-helper and a T-killer cell. Nature 1998;393:474–478.
111 Morse MA, Zhou LJ, Tedder TF, Lyerly HK, Smith C: Generation of dendritic cells in vitro from
peripheral blood mononuclear cells with granulocyte-macrophage-colony-stimulating factor,
interleukin-4, and tumor necrosis factor-alpha for use in cancer immunotherapy. Ann Surg
1997;226:6–16.
112 Lee AW, Truong T, Bickham K, Fonteneau JF, Larsson M, Da Silva I, Somersan S, Thomas EK,
Bhardwaj N: A clinical grade cocktail of cytokines and PGE2 results in uniform maturation of
human monocyte-derived dendritic cells: Implications for immunotherapy. Vaccine 2002;20
(suppl 4):A8–A22.
113 Feuerstein B, Berger TG, Maczek C, Roder C, Schreiner D, Hirsch U, Haendle I, Leisgang W,
Glaser A, Kuss O, Diepgen TL, Schuler G, Schuler-Thurner B: A method for the production of
cryopreserved aliquots of antigen-preloaded, mature dendritic cells ready for clinical use.
J Immunol Methods 2000;245:15–29.
114 de Gruijl TD, Luykx-de Bakker SA, Tillman BW, van den Eertwegh AJ, Buter J, Lougheed SM,
van der Bij GJ, Safer AM, Haisma HJ, Curiel DT, Scheper RJ, Pinedo HM, Gerritsen WR:
Prolonged maturation and enhanced transduction of dendritic cells migrated from human skin
explants after in situ delivery of CD40-targeted adenoviral vectors. J Immunol 2002;169:
5322–5331.
115 Esslinger C, Chapatte L, Finke D, Miconnet I, Guillaume P, Levy F, MacDonald HR: In vivo
administration of a lentiviral vaccine targets DCs and induces efficient CD8() T cell responses.
J Clin Invest 2003;111:1673–1681.
116 Hawiger D, Inaba K, Dorsett Y, Guo M, Mahnke K, Rivera M, Ravetch JV, Steinman RM,
Nussenzweig MC: Dendritic cells induce peripheral T cell unresponsiveness under steady state
conditions in vivo. J Exp Med 2001;194:769–779.
117 Mahnke K, Guo M, Lee S, Sepulveda H, Swain SL, Nussenzweig M, Steinman RM: The dendritic
cell receptor for endocytosis, DEC-205, can recycle and enhance antigen presentation via major
histocompatibility complex class II-positive lysosomal compartments. J Cell Biol 2000;151:
673–684.
118 Bonifaz L, Bonnyay D, Mahnke K, Rivera M, Nussenzweig MC, Steinman RM: Efficient target-
ing of protein antigen to the dendritic cell receptor DEC-205 in the steady state leads to antigen
presentation on major histocompatibility complex class I products and peripheral CD8 T cell
tolerance. J Exp Med 2002;196:1627–1638.
119 Zitvogel L, Regnault A, Lozier A, Wolfers J, Flament C, Tenza D, Ricciardi-Castagnoli P, Raposo G,
Amigorena S: Eradication of established murine tumors using a novel cell-free vaccine: Dendritic
cell-derived exosomes. Nat Med 1998;4:594–600.
120 Clayton A, Court J, Navabi H, Adams M, Mason MD, Hobot JA, Newman GR, Jasani B: Analysis
of antigen presenting cell derived exosomes, based on immuno-magnetic isolation and flow
cytometry. J Immunol Methods 2001;247:163–174.
medwedi.ru
121 Raposo G, Nijman HW, Stoorvogel W, Liejendekker R, Harding CV, Melief CJ, Geuze HJ: B lym-
phocytes secrete antigen-presenting vesicles. J Exp Med 1996;183:1161–1172.
122 Wolfers J, Lozier A, Raposo G, Regnault A, Thery C, Masurier C, Flament C, Pouzieux S, Faure F,
Tursz T, Angevin E, Amigorena S, Zitvogel L: Tumor-derived exosomes are a source of shared
tumor rejection antigens for CTL cross-priming. Nat Med 2001;7:297–303.
123 Kumamoto T, Huang EK, Paek HJ, Morita A, Matsue H, Valentini RF, Takashima A: Induction of
tumor-specific protective immunity by in situ Langerhans cell vaccine. Nat Biotechnol 2002;20:
64–69.
124 Hsu FJ, Benike C, Fagnoni F, Liles TM, Czerwinski D, Taidi B, Engleman EG, Levy R:
Vaccination of patients with B-cell lymphoma using autologous antigen-pulsed dendritic cells.
Nat Med 1996;2:52–58.
125 Nestle FO, Alijagic S, Gilliet M, Sun Y, Grabbe S, Dummer R, Burg G, Schadendorf D:
Vaccination of melanoma patients with peptide- or tumor lysate-pulsed dendritic cells. Nat Med
1998;4:328–332.
126 Kugler A, Stuhler G, Walden P, Zoller G, Zobywalski A, Brossart P, Trefzer U, Ullrich S, Muller CA,
Becker V, Gross AJ, Hemmerlein B, Kanz L, Muller GA, Ringert RH: Regression of human metasta-
tic renal cell carcinoma after vaccination with tumor cell-dendritic cell hybrids. Nat Med 2000;6:
332–336.
127 Tjoa BA, Simmons SJ, Bowes VA, Ragde H, Rogers M, Elgamal A, Kenny GM, Cobb OE,
Ireton RC, Troychak MJ, Salgaller ML, Boynton AL, Murphy GP: Evaluation of phase I/II clin-
ical trials in prostate cancer with dendritic cells and PSMA peptides. Prostate 1998;36:39–44.
128 Tada M, DeTribolet ND: Tumor immunology; in Berger MS, Wilson CB (eds): The Gliomas.
Philadelphia, WB Saunders, 1999, pp 579–587.
David W. Andrews, MD
Professor of Neurosurgery
909 Walnut Street, 2nd floor, Philadelphia, PA 19107 (USA)
Tel. 1 215 503 7005, Fax 1 215 503 7007, E-Mail david.andrews@jefferson.edu
Glioma-Genesis
Signaling Pathways for the Development of
Molecular Oncotherapy
Gurpreet S. Kapoor a, Donald M. O’Rourkea,b

Departments of aNeurosurgery and bPathology and Laboratory Medicine,
University of Pennsylvania School of Medicine, Philadelphia, Pa., USA
Introduction to Gliomas
Gliomas are the most common primary central nervous system (CNS) tumors
that arise from astrocytes, oligodendrocytes, or their precursors. Gliomas are clas-
sified according to whether they exhibit features of astrocytic, oligodendroglial, or
ependymal cells. They are graded on a scale of I, II, III, or IV according to their
degree of malignancy as judged by histological features [1]. Grade IV or glioblas-
toma multiforme (GBM) is the most aggressive glioma [2]. GBM either arise de
novo (primary) or progress to GBM from low-grade gliomas (secondary). The
malignant progression of gliomas involves a stepwise accumulation of genetic
alterations that generally affect either signal transduction pathways activated by
receptor tyrosine kinases (RTK) such as platelet-derived growth factor/receptor
(PDGF/PDGFR), fibroblast growth factor 2, insulin-like growth factor receptor,
and epidermal growth factor receptor (EGF-R) [3–9], or cell cycle arrest pathways
involving regulators such as CDK4, CDK6, cyclin D1, MDM2, P16INK4a, P14ARF,
RB, and p53 (fig. 1) [10–17].
RTK, which play a critical role in normal cell proliferation and differentia-
tion, have been extensively studied by various laboratories for their possible role
in ‘glioma-genesis’, or the process of glial transformation. RTK constitute a fam-
ily of at least twenty members containing an extracellular ligand-binding domain,
a single transmembrane domain, and an intracellular cytoplasmic domain with
intrinsic tyrosine kinase activity [18]. Many studies have shown aberrant signal-
ing by RTK in a variety of cancers, including human brain tumors. Constitutive
activation of RTK is one of the important features of aberrant signaling leading
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Grade I and II Grade III Grade IV
Low-grade Anaplastic Glioblastoma

astrocytoma astrocytoma
p53 mutations RB mutation EGFR amplification

PDGF/R overexpression CDK4 amplification EGFR mutation
INK4a/ARF loss INK4a/ARF loss
PTEN loss PTEN loss
DMBT1/mxi loss RB mutation
19q loss
11p loss
Fig. 1. Stepwise accumulation of genetic alterations in gliomagenesis. PDGF/R,

platelet-derived growth factor/receptor; RB, retinoblastoma; CDK4, cyclin-dependent kinase
4; INK4a, inhibitor of cyclin-dependent kinase on chromosome 4; ARF, alternative reading
frame on INK4a locus; PTEN, phosphatase/tensin homolog on chromosome 10; DMBT1,
deleted in malignant brain tumors 1; EGFR, epidermal growth factor receptor.
to malignant transformation and tumor proliferation, and can occur by several

mechanisms [19]. Deregulated RTK signaling can occur via gene amplification,
overexpression, and activating mutations, including deletions in the extracellular
domain or alterations in the RTK cytoplasmic domain. Another mechanism of
aberrant RTK signaling involves the activation of autocrine growth factor and
receptor loops [20]. In CNS tumors, particularly astrocytomas, the classical
examples of autocrine growth factor/receptor loops involve production of PDGF,
epidermal growth factor (EGF), transforming growth factor-␣ (TGF-␣), and
their respective receptors [21]. Therefore, it has become imperative to focus on
mitogenic and transforming signaling cascades generated by PDGF/PDGFR,
EGF/EGF-R and TGF-␣/TGF␣-R autocrine loops in order to understand the
molecular mechanisms underlying glioma formation, and to use these signaling
modules as novel therapeutic targets.
PDGF/PDGF-R Signaling in Human and Mouse Cell Tumors
Accumulated evidence suggests that PDGF and PDGFR play a significant

role in glial development and lineage commitment [22]. In cell culture, PDGF
functions to block differentiation and promote proliferation of O2A glial prog-
enitors that give rise to either oligodendrocytes or type-2 astrocytes [23, 24].
Although coexpression of PDGF and PDGFR has been shown in all brain
Kapoor/O’Rourke 522
tumor stages, including low-grade astrocytomas, anaplastic astrocytomas, and
GBM [21, 25–27], PDGF/PDGFR overexpression has been most commonly
observed in low-grade astrocytomas in association with loss-of-function of the
p53 tumor suppressor [28, 29]. These observations suggest a cooperative rela-
tionship between the PDGF/PDGF-R and p53 signaling pathways. A recent
study using cell culture and a transgenic mouse model showed that overexpres-
sion of PDGF in neural progenitors induced the formation of oligoden-
drogliomas, whereas PDGF transfer into differentiated astrocytes induced
either formation of oligodendrogliomas or mixed oligoastrocytomas [30]. The
observed histologies of these glial tumors were consistent with low-grade neo-
plasms. Collectively, these reports suggest that the PDGF mitogenic signaling
loop may be an early or initiating event in driving neural precursors or differ-
entiated glial cells to low-grade astrocytomas and/or oligodendrogliomas, prior
to malignant transformation of low-grade clones. It, therefore, appears that
PDGF/R alterations are most commonly observed in ‘secondary GBM’ or those
malignant gliomas that arise from lower-grade tumors [26].
The PDGF family consists of four members (PDGF-A, -B, -C, and -D) which
transduce signals through the PDGF-␣ and PDGF-␤ receptors. Biosynthesis
and processing of PDGFs involve the formation of dimers PDGF-AA, -BB, -CC,
and -DD and the heterodimer PDGF-AB [31]. Numerous studies have reported
the expression of PDGF-A and -B ligands in glioblastomas, and indicate that
autocrine signaling by these isoforms is required for cell survival [3, 32, 33].
Moreover, expression or alteration of PDGF-A, -B, -C and PDGFR also have
been implicated in medulloblastomas and ependymomas [34, 35]. Recently it
was reported that PDGF-B enhances glioma angiogenesis by stimulating vas-
cular endothelial growth factor (VEGF) expression in tumor endothelia and
promoting pericyte recruitment to neovessels [36]. A recent study in glioma cell
lines and primary glioblastoma tissues using quantitative reverse transcriptase-
PCR also implicated PDGF-C and -D ligands in the formation of brain tumors
and confirmed the existence of autocrine signaling by PDGF-A and -B in brain
tumors [4].
PDGF-AA and -CC selectively bind to PDGFR␣, whereas PDGF-DD pref-
erentially binds to PDGF␤, with PDGF-BB displaying affinity for both recep-
tors [31, 37–39]. Binding of PDGF stabilizes PDGFR dimerization, which is
followed by autophosphorylation of tyrosine residues, leading to an increased
tyrosine kinase activity [40, 41] and formation of docking sites for signal relay
molecules containing src homology 2 (SH2) domains. A large number of SH2
domain-containing enzymes, such as phosphatidylinositol 3-kinase, phospholi-
pase C-␥, src tyrosine kinases, protein tyrosine phosphatase SHP-2, and GTPase
activating proteins for Ras have been shown to bind to particular SH2 sites
on PDGF␣- and ␤-receptors and to modulate different signaling pathways.
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PDGFR binds to other molecules such as Grb2, Grb7, Nck, and Shc, which lack
enzymatic activities and have adapter functions, linking the activated receptor to
downstream signaling molecules and distinct pathways. PDGFR also binds to
STAT family transcription factors, which translocate to the nucleus to directly
activate the transcription of genes [42].
EGF-R/ErbB Signaling Cascades in Human

and Experimental Astrocytomas
EGF-R belongs to the Erb-B family of type I RTK, based on structural

homology to the v-erbB oncogene carried by the avian erythroblastosis virus
[43]. The family includes four members: EGF-R (also termed erbB1/HER1),
neu (erbB2 or HER2), erbB3 (HER3), and erbB4 (HER4). In high-grade astro-
cytomas or GBM, the majority of gene amplification events involve EGF-R
[8, 44, 45]. Approximately 50% of GBM, but only a small percentage of
anaplastic astrocytomas, express high levels of EGF-R [8]. These observations
suggest that EGF-R overexpression and/or gene alteration is a late event in
glioma-genesis and is frequently observed in ‘primary’ or ‘de novo’ glioblas-
tomas occurring in older patients [46–48]. Sequencing of the amplified EGF-R
genes revealed that frequent gene rearrangements result in essentially seven
classes of variant EGF-R transcripts [49]. The most common rearrangement is
a genomic deletion of exons 2–7, resulting in an in-frame deletion of 801 bp
of the coding sequence to generate a mutant receptor called de2–7 EGF-R,
⌬EGF-R, or EGF-RvIII which cannot bind ligand due to a truncated extracel-
lular domain but is constitutively active [9, 49–54]. This mutant receptor also
has been detected in cancers of the lung, breast, and prostate [55, 56], but not
in normal tissues [54]. Amplification of EGF-R genes has been implicated in
poor prognosis of patients with GBM [57], and it has been demonstrated that
patients with EGFRvIII-positive GBMs have shorter life expectancies [58].
Unlike wtEGF-R, EGF-RvIII transforms NIH3T3 cells [59] and strongly
enhances the tumorigenicity of human gliomas in nude mice [53, 60].
Overexpression of mutant EGF-R in astrocytes or their precursors in transgenic
mice has been shown to promote the development of glioblastoma [61].
However, the novel glycine residue resulting from gene rearrangement creates
a new epitope at the splice site and the tumor-specific expression of EGF-RvIII
makes this mutant a potential tumor-specific target for therapy in gliomas and
in other cancers [62–64]. Moreover, expression of EGF-RvIII in gliomas pro-
vides resistance to cisplatin, a commonly used chemotherapeutic agent [65],
which suggests a need for EGF-RvIII-targeted inhibition in combination with
chemotherapy.
Enhanced EGF-R signaling has also been reported to cooperate with other
alterations in the development of GBM. The most common alterations are those
that disrupt cell cycle arrest and include the deletion of p16INK4a/p19ARF [13, 16],
deletion of RB [14, 15], loss of function of p53 [14], amplification of CDK4
[17, 66], CDK6 [11], cyclin D1 [10] and MDM2 [16]. However, the associated
genetic lesions that cooperate with enhanced EGF-R signaling in the develop-
ment of GBM have not been completely characterized. In a recent study, it was
demonstrated that expression of EGF-RvIII, but not EGF-RWT in mouse astro-
cytes harboring activated oncogenic Ras resulted in the formation of oligoden-
droglioma and mixed oligoastrocytoma tumors [67]. Many reports evaluating
human tumor tissues have shown that combined loss of p16INK4a and p19ARF, but
not of either p53, p16INK4a or p19ARF alone, is associated with EGF-R activation
in GBM [29, 68, 69]. On the other hand, a study using a mouse model system
showed that human telomerase catalytic component overexpressed in normal
human astrocytes cooperates with p53/pRb inactivation and Ras pathway activa-
tion, but not PI3-kinase/Akt pathway or EGFR activation, to allow the formation
of intracranial tumors strongly resembling p53/pRb pathway-deficient,
telomerase-positive, Ras-activated human grade III anaplastic astrocytomas
[70]. The loss of p53 in combination with an NF1 (Neurofibromatosis type I)
loss leads to astrocytomas and glioblastoma formation in mice [71].
Furthermore, a study in mice showed that the combined activation of Ras and
Akt in neural progenitors induces the formation of glioblastoma [72]. A more
recent study showed that p16INK4a/p19ARF loss cooperates with Ras and Akt acti-
vation in astrocyte precursors and neural progenitors to generate glioblastomas
of various morphologies [73], indicating that glioma genesis occurs through an
intricate cooperativity between genetic alterations often involving RTK signal-
ing pathways and cell cycle regulatory molecules.
Elevated levels of activated Akt have also been associated with the loss of
the tumor suppressor phosphatase/tensin (PTEN) homolog, located on chromo-
some 10 in many glioblastomas [74, 75, 76]. Collectively, these studies suggest
that robust signaling by overexpressed EGF-R and/or loss of functional PTEN
and other genes leads to the increased activation of Akt in GBM and thus
increased transformation. However, it is unclear whether the specific state of
glial cell differentiation plays a restrictive role in glioma progression. Recent
work in mice has demonstrated that deregulation of specific genetic pathways
(i.e., Ink4a/Arf inactivation and EGF-R activation) may be more important than
the neural cell of origin in dictating the emergence and phenotype of malignant
gliomas [77].
Other ErbB proteins have been implicated in other CNS tumors. Both
ErbB2 and ErbB4 expression levels have been shown to predict the prognosis
of childhood medulloblastoma and ependymoma [78, 79]. In addition to their
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pathological role in brain tumors, ErbB proteins have been implicated in many
systemic human cancers such as cancer of the colon, head and neck, pancreas,
lung, breast, kidney, ovary, and bladder [80]. Studies in these cancers have
linked the prognosis to excessive receptor kinase activity, receptor overexpres-
sion, ligand-independent constitutive activation of receptor mutants, and/or
autocrine stimulation [81–84]. Increased receptor activity leads to increased
downstream signaling and enhanced cell transformation. Studies on ErbB fam-
ily members have demonstrated that homodimerization and heterodimerization
are the initial events in variety of cellular signals required for cell growth and
differentiation of many cell types under physiological conditions [85]. Thus,
upon EGF stimulation, EGF-R forms a homodimer and/or heterodimer with
other family members, which leads to auto- or transphosphorylation and acti-
vation of RTK activity, recruitment of various signaling relay molecules, and
initiation of variety of a intracellular signaling cascades including MAPK,
PI3-kinase/Akt, PLC-␥, and STAT (signal transducers and activators of tran-
scription). Among these, MAPK and PI3-kinase/Akt pathways have been more
extensively studied in glial tumors.
TGF-␣/EGF-R Autocrine Loop in Brain Tumors
TGF-␣ is a member of the EGF family and is a potent mitogen for a num-
ber of cell types in culture. Mature TGF-␣ is a 5.5-kDa peptide [86] sharing
30% structural homology with EGF. TGF-␣ binds to EGF-R and activates RTK
activity [87–89]. Binding of TGF-␣ to EGF-R initiates receptor dimerization
and autophosphorylation, followed by the recruitment of src-homology 2 (SH2)
domain-containing molecules, which link EGF-R to similar intracellular path-
ways initiated by the EGF/EGF-R loop [18, 90]. Several tumors and tumor cell
lines have been shown to coexpress EGF-R and TGF-␣ [9], indicating the exis-
tence of autocrine activation loop driving tumor growth. High levels of TGF-␣
have also been reported in human glioma [68, 92, 93]. Increased TGF-␣ levels
have been observed in many primary human glioblastomas and anaplastic
astrocytomas [93]. The highest level of TGF-␣ expression was found in recur-
rent tumors, which apparently had undergone transformation from low-grade to
high-grade malignant anaplastic tumors. The elevated expression of TGF-␣l has
also been reported in primitive neuroectodermal brain tumors including medul-
loblastomas [94].
It was reported that the induction of TGF-␣ via a tetracycline inducible
system (tet on/off) in the glioma cell line U1242MG resulted in an increased
motility at the single cell level, suggesting that coexpression of EGF-R and
TGF-␣ was capable of forming an independent autocrine locomotory loop and
that TGF-␣ may form an important component of the glioma cell invasion
machinery [95]. TGF-␣ also has been shown to decrease GFAP (marker for
astrocytic differentiation) mRNA levels in U373MG glioblastoma cells. On the
other hand, TGF-␣ up-regulates gene expression for nestin, a marker for undif-
ferentiated astrocytic precursors, without affecting vimentin gene transcription.
These changes in the gene expression of intermediate filament proteins were
correlated with an increased motility and less stellate morphology, suggesting
that TGF-␣ may be required to induce dedifferentiation in glial tumors in order
to promote motility [96]. In a recent study, U1242MG cells containing a TGF-␣
inducible transgene were used to determine the effect of autocrine TGF-␣ on
cell proliferation in vitro and on subcutaneous tumor growth in nude mice [97].
In this study, induction of TGF expression in the absence of tetracycline
resulted in increased cell proliferation in vitro, which was inhibited by block-
ing EGF-R with monoclonal antibody C225 and a tyrosine kinase inhibitor
(RTKI) specific to EGF-R. Similarly, U1242MG clones expressing TGF-␣
(in mice which were not fed with tetracycline) developed into tumors of varied
sizes. Moreover, mice injected with the TGF-␣ expressing clones developed
larger tumors than those injected with control clones. These studies clearly
indicate that the TGF-␣/EGF-R autocrine loop plays an important role in glial
tumor progression.
Mitogen-Activated Protein Kinase (MAPK) Signaling in Gliomas
Mitogen-activated protein kinases (MAPK) are proline-directed serine-

threonine kinases, which are activated by a variety of cellular stimuli and regu-
late a variety of cellular processes such as proliferation, differentiation,
development, and tumorigenesis. The MAPK superfamily has been clearly
divided into three subgroups: extracellularly responsive kinases (p42/44MAPK or
Erk1/2); c-jun N-terminal kinases (p46/54JNK) which are also known as the
stress-activated protein kinases (SAPKs); and p38MAPK (also known as RK,
Mxi-2, CSBP1/2, or HOG-1-related). Although the MAPK families are struc-
turally related, they are generally activated by distinct extracellular stimuli, thus
comprising a series of separate MAPK cascades. Genetic and biochemical
analyses have identified the universally conserved Ras/Raf/MEK/p42/44MAPK
pathway [98, 99] as one of the most ubiquitous MAPK pathways stimulated by
various growth factor receptors including PDGF-R, EGF-R and other RTK. In
general, receptor-ligand interaction leads to recruitment of adapter proteins
such as Grb2, which brings the guanine nucleotide exchange factor Sos to the
receptor to form a stable complex, which is required for the activation of
monomeric membrane-bound G-protein Ras by the exchange of GDP for GTP
Glioma-Genesis 527
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Ligand
Membrane
Sos Ras GTP
Grb2 Grb1
RTKs
SHP-2
Sos
Shc
Ras
GDP
Farnesyl transferase
Cytoplasm
Raf1
PI3-k Ral Rac
MEK 1/2
Akt
Rho
MAPK
Apoptosis
Nucleus
Elk-1, c-Myc, Ets, STAT 1/3, PPAR␥ Cytoskeletal
AFX forkhead
organization
transcription factor
Cell growth and

Transcription proliferation
Fig. 2. Receptor tyrosine kinase-induced activation of Ras/Raf/MEK/MAPK pathway.

RTK, receptor tyrosine kinase; Grb2, growth factor receptor 3-binding protein; Gab1, Grb2
associated binder1; Sos, son-of-sevenless (Drosophila homolog of ras); Ras, the protein
product of c-ras; Raf1, a kinase, the protein product of c-raf; MEK, MAP kinase kinase;
MAPK, mitogen-activated protein ser/threo kinases; PPAR, peroxisome proliferator-
activated receptor.
(fig. 2) [100]. In addition, EGF-induced activation of Ras may be transduced

via another adapter protein, Shc, which binds to activated EGF-R and becomes
phosphorylated, creating an additional binding site for Grb2 [101]. Recent work
indicates that the protein tyrosine phosphatase SHP-2 gets recruited to the
EGF-R bound adapter Gab1 and facilitates Sos relocation to GTP-bound Ras
[102, 103]. Studies in U87MG glioblastoma cells showed that activation of
Ras/Raf/MEK/p42/44MAPK pathway by EGF-RvIII was blocked by PI3-kinase
inhibitors, wortmannin and LY294002, whereas wild-type EGF-R induced Erk
activation was largely unaffected, suggesting that EGF-RvIII and EGF-R wild-
type preferentially use different signaling pathways to activate Ras/Raf/MEK/
p42/44MAPK pathways [104].
One of the best characterized effectors of Ras are the Raf serine/threonine
kinases, which are required for the activation of the MEK/p42/44 MAPK pathway
and are felt to be critical for Ras-mediated cell transformation (fig. 2) [100, 105,
106]. The other three effector pathways that have demonstrated roles in Ras
transformation are those mediated by Ras activation of PI3-kinase, by guanine
nucleotide exchange factors (GEFs) for the Ras-related small GTPase Ral
(RalGDS), and by Rac (Tiam 1). In the classical pathway, Ras-activated Raf
stimulates downstream MAPK kinase, which in turn phosphorylates MAP
kinases (also named ERKs) [107–110]. MAPKs or ERKs can translocate to the
nucleus to phosphorylate and activate several transcription factors for activation
of growth-inducing genes (fig. 2). A recent study showed that normal human
astrocytes undergo a p16INK4a-associated senescence-like growth arrest in
response to sustained activation of the Ras/Raf/MEK/p42/44MAPK pathway.
However, high-grade glioma cells that have dismantled p16INK4a-associated
senescence-like growth arrest pathways are potentially regulated by a second
p21cip1-dependent growth arrest pathway in response to sustained Ras/Raf/MEK/
p42/44MAPK pathway activation [111].
Mutated and constitutively activated forms of Ras are found in ⬃50% of
all human metastatic tumors [112]. In gliomas, specific mutations affecting Ras
have not been observed. However, high levels of Ras-GTP have been docu-
mented in high-grade astrocytomas [113, 114]. It was recently demonstrated
that a region on chromosome 10 (10p13), which carries a Ras suppressor, was
deleted in 30% of the high-grade gliomas tested and the majority of oligoden-
drogliomas, but not in other CNS tumors, bladder or colon tumors, or normal
tissue [115]. Ectopic expression of Ras Suppressor-1 inhibited tumorigenesis of
a glioblastoma cell line. Similarly, a novel Ras-related protein, Rig (i.e., Ras-
related inhibitor of cell growth) has been shown to be expressed at high levels
in normal cardiac and neural tissue. However, an expression of Rig protein was
frequently lost or down regulated in neural tumor-derived cell lines and in pri-
mary human astrocytomas. Moreover, ectopic Rig expression in human astro-
cytomas suppressed cell growth [116]. These studies suggest that high levels of
Ras-GTP in high-grade astrocytomas may be due in part to a gradual accumu-
lation of Ras suppressor protein mutations in primary glial tumors, as well as
from activated RTK signaling.
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Ras transformation of mouse NIH3T3 cells results in elevated levels of
cyclin D1 and accelerated G1 progression [117]. Thus, increased Ras activation
leads to an increased levels of the G1-specific protein complex CDK4/cyclin D
in gliomas, which drives cells into S phase and mitosis. In neurofibromatosis
type I (NF1), Ras is constitutively active due to a mutation in the NF1 gene,
which encodes the Ras GTPase activating protein-related protein neurofi-
bromin required to convert active GTP-bound Ras to inactive GDP-bound Ras
[118, 119]. Activation of other small G-proteins such as Rap1, which also acti-
vates the Raf1/MEK/MAPK pathway, may result in increased cell proliferation
in astrocytomas [120].
Tuberin, a tuberous sclerosis complex2 gene product, regulates the activ-
ity of Rap1, via a GTPase activating protein-related domain at its C-terminus
[121]. Analysis of sporadic astrocytomas and ependymomas demonstrated
either increased Rap1 or reduced tuberin protein expression in 50–60% of
gliomas, compared to a small percentage of schwannomas and meningiomas
and none of the oligodendrogliomas studied [120], suggesting that alterations
in Rap1 signaling may play an important role in the development of certain spo-
radic human gliomas.
Ral guanine nucleotide exchange factors have been associated with Ras-
induced transformation in very few cell lines [122, 123]. Their role in the devel-
opment of brain tumors and other human cancers is still unclear, but may be more
significant than originally thought [120]. Activated forms of Rac have been
reported to induce survival in Rat1 fibroblasts and M14 melanoma cells [124,
125], but their role in glial cell survival has not been studied extensively. A recent
study with primary glioma and astrocyte cell cultures showed that Rac1 regulates
a major survival pathway in most glioma cells, and that suppression of Rac1
activity stimulates death in virtually all glioma cells, regardless of the mutational
status [126]. However, normal astrocytes were not affected, suggesting that Rac
survival pathway is specific to transformed glial cells and could be used as a
potential therapeutic target for the treatment of malignant gliomas.
The stress-activated protein kinases, in particular c-jun N-terminal kinases
(JNKs), have been implicated in human tumorigenesis [127, 128]. It was
reported that the overexpression of EGF-RvIII in mouse NIH3T3 cells leads to
constitutive activation of the JNK pathway, which correlates with enhanced
transformation by EGF-RvIII [129]. Moreover, a recent study showed constitu-
tively active forms of JNK isoforms in primary glial tumors, indicating a pos-
sible association between EGF-RvIII and JNKs in the development of GBMs
[130]. Previously, we showed that overexpression of transforming ErbB recep-
tor complexes leads to constitutive activation of Erk MAPK and particularly
JNK MAPKs, including JNK2, enhanced transformation, and resistance to
apoptosis in primary human glioblastoma cells [131]. A single study in T98G
glioblastoma cells showed that JNK2 is required for growth of T98G cells in
non-stress conditions, and that p21cip1/waf1 may contribute to the sustained
growth arrest of JNK2-depleted T98G cultures [132]. Recently, we observed
that JNK pathway activation might play an important and specific role in cell
migration and motility in GBMs [O’Rourke, pers. commun.]. These observa-
tions clearly indicate that JNK pathway events may play a critical role in glial
tumor development and progression.
PI3-Kinase/Akt Pathway and Gliomas
The PI3-kinase/Akt pathway is one of the major survival pathways in

epithelial cells. In general, activation of the PI3-kinase/Akt pathway begins
with receptor activation, rapid stimulation of phosphoinositol metabolism [133,
134], and coupling of PI3-kinase to phosphorylated docking proteins such as
Gab1 [135]. PI3-kinase is a phospholipid kinase composed of a regulatory sub-
unit, p85, that contains two SH2 and one SH3 domain, and a catalytic subunit
designated p110. We have recently documented that coupling of the SHP-2
protein tyrosine phosphatase to Gab1 is essential for EGF-R mediated PI3-
kinase activation in glioblastoma cells [136–138]. Activated PI3-kinase phos-
phorylates phosphatidylinositol 4,5-biphosphate (PIP2) or PtdIns(4,5)P2 via
its p110 subunit to generate second messengers PtdIns(3,4)P2 (PIP2) and
PtdIns(3,4,5)P3 (PIP3). PIP3 mediates membrane translocation of several sig-
naling-proteins, such as the serine-threonine (Ser-Thr) kinases PDK1 and Akt,
the docking protein Gab1, and PLC-␥ [133, 134]. PDK1 phosphorylates Akt at
Thr308 [139]. It has been proposed that an unidentified protein kinase (PKC)
(a hypothetical PDK2) is responsible for the phosphorylation of Akt at Ser473
leading to its complete activation. Activated Akt phosphorylates and inhibits
several proapoptotic proteins such as Bad [140], and the Forkhead transcription
factor [141], and GSK-3 [142] to promote cell survival. It also promotes pro-
tein synthesis by activating p70S6 kinase via mammalian target of rapamycin
(mTOR) (fig. 3).
Approximately 80% of all human GBM express activated Akt, which might
account for increased cell survival and a worse prognosis for the patients with
these tumors [76]. Overexpression of constitutively activated Akt has been shown
to convert anaplastic astrocytomas to GBM in a human astrocyte glioma model
[70]. A previous study demonstrated an increased association of PI3-kinase with
focal adhesion kinase with sustained PI3-kinase activity, PIP3 levels, and Akt
phosphorylation in glioblastoma and breast cancer cells expressing a PTEN phos-
phatase-inactivate mutant, suggesting a possible role for PI3-kinase in cell migra-
tion, invasion, spreading, and focal adhesions [143] (cf., ref. 144). Another study
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Ligand
Membrane
PIP2 PIP3
P110
PTEN
p85
EGFR
Gabl
Deleted in GBMs
SHP-2 LY294002,
Wortmannin PDK 1/2
Grb2
Sos
Shc
Amino acids
Cytoplasm Upregulated in GBMs

Akt mTOR
Gsk-3 4E-BP1
p70S6K
Apoptosis
elF4E
Bad
Protein synthesis
Nucleus
Akt FKHR
Transcription
Transcription
Cell growth and
proliferation
Apoptosis
Fig. 3. EGFR-mediated activation and regulation of PI3-kinase/Akt pathway. PDK1/2,

phosphoinositide-dependent kinase 1 and 2; GSK-3, glycogen synthase kinase-3; mTOR,
mammalian target of rapamycin; FKHR, forkhead/winged helix protein; 4E-BP1, eIF4E-
binding protein 1; eIF4E, eukaryotic initiation factor 4E; p70S6K, ribosomal protein S6 kinase.
in PTEN mutant C6 glioma cells showed a positive association between increased

PI3-kinase/Akt signaling and increased invasiveness and gelatinase activity,
again suggesting that unchecked PI3-kinase/Akt activation in gliomas not only
serves as a survival signal but might also participate in tumor motility and infil-
tration [145]. Previously, we reported that EGF-R transcriptionally upregulates
VEGF in human glioblastoma cells via PI3-kinase-dependent pathway [146]. We
recently observed that PTEN mutation in human glioblastoma cells cooperates
with EGF-R activation to upregulate VEGF expression in a PI3-kinase/Akt-
dependent manner, suggesting that PI3-kinase/Akt axis constitutes an important
component of the angiogenic cascade required for the development of glioblas-
toma multiforme [147].
Activation of PI3-kinase/Akt axis is regulated by the lipid phosphatase activ-
ity of the PTEN tumor suppressor, which blocks Akt activation by converting PIP3
to PIP2, thereby mediating cell cycle arrest and/or apoptosis [74, 148–150].
Various groups have reported that a region including the PTEN locus on the long
arm of chromosome 10 is deleted in many tumors, including glioblastomas,
breast, prostrate, endometrial carcinoma, and melanoma [151–153], suggesting a
pathogenic role for constitutively active Akt survival pathway in these cancers.
Interestingly, it has been shown that PTEN suppresses growth of U87MG
glioblastoma cells by blocking cell cycle progression through G1, and this was cor-
related to a significant accumulation of the cell cycle kinase inhibitor p27kip1 [149],
suggesting that loss of function of PTEN is one of the prerequisites for GBM
development. It has also been observed that PTEN mutations are more frequent in
‘primary’ or de novo GBM, but not in secondary GBMs, which arise from low-
grade (grade II) or anaplastic astrocytomas (grade III) [154]. The observation that
secondary glioblastomas contain p53 mutations as a genetic hallmark but rare
PTEN mutations suggests that primary and secondary GBMs develop through dis-
tinct genetic pathways involving RTK signaling [154, 155]. Approximately 40% of
GBMs are associated with deletions of the PTEN locus on chromosome 10
[156–158]. Earlier studies using mini-chromosome-transfer experiments showed
that introduction of human chromosome 10q into glioblastoma cells suppressed
the growth in soft agar or formation of tumors in nude mice [159]. Overexpression
of PTEN in glioblastoma cells via adenovirus-mediated gene delivery generated
similar results in soft agar and in nude mice [160]. Furthermore, we found that
overexpression of signal regulatory protein SIRP␣1 in glioblastoma cell lines neg-
atively regulates EGF-R mediated PI3-kinase/Akt signaling and led to reduced
transformation, reduced cell migration and cell spreading, and enhanced apopto-
sis following DNA damage [136]. Studies are underway to establish the mecha-
nism by which SIRP␣1 proteins mediate their effects on PI3-K/Akt activation and
also to determine the factors regulating the expression of these proteins in normal
astrocytes and GBMs under normal physiological conditions.
Phospholipase C-␥ Pathway and Glioma
Phospholipase C-␥ (PLC-␥) is a phosphoinositide-specific phospholipase,

which plays an important role in tumor cell migration occurring via PDGF and
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Membrane
PLC-␥
PIP2
p110
Gab1
EGFR
p85
SHP-2
Grb2
IP3 DAG
Sos
Shc
Ca 2⫹
Cytoplasm
CaMK Endoplasmic reticulum PKC
⫹/⫺ ⫹/⫺
Ras/Raf-1/MEK/MAPK
Nucleus
Transcription
Cell growth and

proliferation
Fig. 4. EGFR-induced PLC-E pathway participates in activation of different pathways.

PLC-␥, phosphoinositide-specific phospholipase C-␥; DAG, diacylglycerol; IP3, inositol
1,4,5-triphosphate; CaMK, Ca2⫹/calmodulin-dependent protein kinases.
EGFR by an undefined mechanism [161]. One study showed that inhibition of

PLC-␥ activation by a pharmacological inhibitor or a dominant negative PLC-␥
(PLCz) blocked glioma cell motility and invasion of fetal rat brain aggregates,
suggesting PLC-␥ as a potential target for anti-invasive therapy for GBM [105].
Upon receptor activation, PLC-␥ is rapidly recruited to the receptor through the
binding of its SH2 domains to pTyr sites in adapter proteins such as Gab1 [97].
Coupling to the receptor activates PLC-␥, which hydrolyzes its substrate PIP2 to
generate two secondary messengers, diacylglycerol and inositol 1,4,5-triphos-
phate (IP3) [74]. IP3 binds to specific intracellular receptors and stimulates the
release of intracellular Ca2⫹. Free calcium then binds to calmodulin, which in
turn activates a family of Ca2⫹/calmodulin-dependent protein kinases (CaMK).
Diacylglycerol and Ca2⫹ also activate members of the PKC family [3, 114].
CaMKs and PKCs in turn exert both stimulatory and inhibitory effects on down-
stream Ras/Raf/MEK/MAPK signaling [121] (fig. 4).
Several in vitro studies have supported the involvement of PKCs in glioma
cell proliferation and invasion [10, 81, 150, 162, 163]. A recent paper suggests
that PKC-␣ and PKC-␧ cooperate with EGF-R in the induction of ornithine decar-
boxylase (ODC) to increase glioma cell proliferation [25]. Also it was shown that
a scaffolding protein RACK1 mediates interaction between integrin ␤ chain and
PMA-activated PKC-␧ resulting in increased focal adhesion and lamellipodia for-
mation, indicating that PKC-␧ positively regulate integrin-dependent adhesion,
spreading, and motility of human glioma cells. Another study showed that PKC-␧
differentially activated Erks at focal adhesions and was required for PMA-induced
adhesion and migration of human glioma cell [164]. Use of PKC inhibitors sug-
gest that PKCs also play fundamental role in regulating cell cycle and participat-
ing in cell survival mechanism. A recent study in human glioma cells reported that
PKC-␫ and PKC-␤ II phosphoylates cyclin-dependent kinase activating kinase,
suggesting their role in cell cycle regulation. Overexpression of PKC-␩ has been
shown to increase proliferative capacity of glioblastoma cells and block UV- and
gamma-irradiation induced apoptosis by inhibiting caspase-9 activation. These
studies suggest that broad spectrum targeting of PKC isoforms may form a basis
for the future glioma-therapy.
Signal Transducers and Activators of Transcription (STATs)
EGF-R activates three forms of STAT (STAT-1, -3, -5) [165], whereas
PDGFR binds and activates only STAT-5 [166]. Upon receptor stimulation,
STATs can be activated by phosphorylation via Janus kinase-dependent or Janus
kinase-independent pathways [113, 165]. Activated STATs bind to homotypic
or heterotypic STATs through their SH2 domains to form homodimers or
heterodimers and translocate into the nucleus to bind to sequence-specific STAT-
responsive elements on DNA, and activate the transcription of specific target
genes such as p21CIP1, cyclin D1, myc, Bcl-2, Bcl-xL, and caspase 1. Collectively,
these target genes can regulate cell cycle progression and apoptosis (fig. 5).
Of the three STATs, STAT-1 and -3 have been shown to be involved in
EGFR-mediated cell cycle regulation [165]. STAT-1 has been implicated as a
negative regulator of cell cycle progression and a promoter of apoptosis [37],
whereas STAT-3 acts as a positive regulator of cell cycle progression with anti-
apoptotic activities [128, 167]. A constitutively activated STAT-3␣ has been
reported in large percentage of gliomas and medulloblastomas, indicating that
STAT-3 may play an important role in EGF-R mediated oncogenesis [117].
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Membrane
PLC-␥
Receptor
p110
Gab1
Jak p85
Jak SHP-2
Grb2
STAT
Sos
STAT
Shc
Cytoplasm
STAT
STAT
Nucleus
STAT
STAT
STAT-responsive elements
p21CIP1, cyclin D1, c-myc, Bcl-2, Bcl-xL, and caspase 1
Cell cycle progression and apoptosis
Fig. 5. Activation of STAT pathway by EGFR or PDGFR. STAT, Signal Transducers

and Activators of Transcription; Janus kinase.
Targeted Molecular Therapy for Gliomas
Increased knowledge of the structure and activating mechanisms of RTKs

and distinct downstream signaling modules has substantially improved our under-
standing of the cellular machinery that mediates glioma-genesis and maintains
the malignant phenotype of transformed glia. There has been a search for new
approaches to target specific steps in the pathogenesis of high grade gliomas
because treatment with conventional cytotoxic agents has shown very little
progress [168]. The heterogeneous nature of malignant gliomas due to different
genetic lesions makes a compelling argument for more rational targeted therapies.
The molecular and pharmacotherapeutic approaches to gliomas can be
broadly divided into immunotherapy (monoclonal antibodies and tumor vacci-
nation), antisense oligonucleotides, gene therapy, and small molecules such as
Ligand
(a) Inhibiting receptor-ligand interaction (b) Inhibiting receptor tyrosine kinase activity
- Herceptin (Trastuzumab), C225 (Cetuximab) - ZD-1839 (IRESSA), OSI-774 (Tarceva),
PKI166, CI1033, EKB-569, STI571
EGFR, PDGFR
p110
(d) Inhibiting farnesyl transferase
Gab1
Gab1
p85
(c) Inhibiting P13-k/Akt pathway - SCH66336, R115777
PTP SH2
- Wortmannin and LY294002 PIP2 Grb2 Sos
Grb2
PTEN Sos
Shc Shc
PIP3 PDK 1/2 Ras
Pro-Ras Farnesyl
Akt transferase
(e) Inhibiting mTOR
activation mTOR
-CCl-779 GSK-3 Bad
PI3-K Raf Rac
Nucleus Akt MEK1/2 Rho
(f) Use of antisense

oligonucleotide strategy to P42/44MAPK
inhibit mRNA transcription
and translation mRNA (g) Inhibiting MAPK pathway
Fig. 6. Novel strategies to inhibit aberrant RTK signaling in glial tumor cells by inhibit-
ing receptor-ligand interactions, inhibiting the TK domain of RTK, inhibiting PI3-kinase/Akt
pathway, inhibiting farnesyl transferase, inhibiting MAPK pathway, or using antisense
oligonucleotides to inhibit the translation of key proteins. PDGFR, platelet-derived growth
factor receptor; mRNA, messenger ribonucleic acid.
tyrosine kinase inhibitors and farnesyl transferase inhibitors. In this section, we

will focus mainly on small molecule and immunotherapeutic approaches that
target RTK signaling with a brief overview on anti-angiogenesis therapy (fig. 6).
Immunotherapy: Monoclonal Antibodies in Glioma Therapy

Because ⬃50% of GBMs coexpress high levels of EGF-R and a mutant
EGF-R receptor EGF-RvIII [54, 8], generation of monoclonal antibodies
(mAbs) directed against the EGF-R and/or EGF-RvIII mutant may be useful in
blocking tumor progression mediated by aberrant EGF-R signaling. MAbs
directed against the extracellular domain of ErbB family RTKs have proven to
be an effective strategy to kill tumor cells derived from systemic epithelial can-
cers [169]. Well-known examples include Herceptin (Trastuzumab) against
HER2 or ErbB2 and IMC-C225 (Cetuximab or Erbitux, ImClone) against
ErbB1 receptor. The U.S. Food and Drug Administration (FDA) has approved
Herceptin for the breast cancer treatment [170–172]. C225 is in Phase III
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clinical trials and has been shown to promote growth inhibitory effects in a vari-
ety of tumors including pancreatic, colorectal, renal and breast carcinomas
[173, 174]. According to data presented at the 2003 American Association of
Clinical Oncology (ASCO), 329 patients with metastatic colorectal cancer were
enrolled in a randomized Phase II trial to receive either Cetuximab or a combi-
nation of cetuximab and irinotecan (CPT-11), the standard chemotherapeutic
agent. The study showed that tumors shrank in 22.9% of the patients receiving
two-drug regimen as compared to only 10.8% of those receiving cetuximab
alone [175]. In addition, two-drug regimen patients had no signs of tumor pro-
gression for a median of 4 months, whereas patients on cetuximab alone had no
tumor progression for a median of 1.5 months. These results suggest that the
anti-tumor efficacy can be enhanced in most cases, when Mabs are used in
combination with cytotoxic agents.
A number of monoclonal antibodies have also been generated against EGF-
RvIII [54, 176]. The most effective MAb was the murine IgG2a Mab Y10, which
recognizes both human EGF-RvIII and a murine homologue of this mutation
[177]. It was also shown that incubation of EGF-RvIII-expressing cells with
Y10 inhibited DNA synthesis, and cell proliferation leading to cell death. In
addition, Y10 was able to mediate cell death of EGF-RvIII-positive cells in the
presence of complement, as well as with both murine and human cells bearing
Fc receptors [177]. Intraperitoneal injection of Y10 led to increased survival in
the mice bearing subcutaneous EGF-RvIII-expressing tumors. However, it
failed to increase survival in animals with intracerebral-tumors because of its
inability to cross blood-brain barrier. Similarly, another Mab directed against
EGF-RvIII, Mab 806, showed reduced tumor volume and increased survival of
mice bearing xenografts of U87MG.EGFRvIII, LN-Z308.EGFRvIII, or A1207
EGF-RvIII gliomas, but is ineffective with mice bearing U87MG tumors, sug-
gesting the specificity of this antibody [178, 179]. In principle, Mab have been
shown to promote receptor internalization resulting in the attenuation of recep-
tor phosphorylation and downstream signaling [51, 179].
Alternatively, monoclonal antibodies can be ‘armed’ with toxins or
radionuclides [180]. An anti-EGFRvIII antibody fused to pseudomonas-exo-
toxin-A generates cytotoxicity in mouse fibroblasts and human-glioblastoma
cells expressing EGFRvIII without affecting parental cells, suggesting that
‘armed’ monoclonal antibodies can be more specific than ‘unarmed’ or ‘naked’
antibodies [64]. Monoclonal antibodies against EGF-R and EGF-RvIII have
also been used to deliver 125I to GBM cells in animal xenografts and in patients
[181–184]. However, iodinated antibodies may pose a risk of killing non-neo-
plastic cells.
A relatively new approach involves the use of immunoliposomes, in which
liposomes are attached to antibody fragments, to deliver variety of cytotoxic
agents, toxins, or even genes for therapy [180–187]. A practical limitation for
clinical use of full-length MAbs in the treatment of brain tumors is the require-
ment for these large molecules to permeate the blood-brain barrier. This limita-
tion is the rationale behind generation of smaller mAb fragments to treat
cancers [51, 177].
Immunotherapy: Tumor Vaccination for Gliomas

Recent advances in research on antigen presentation, use of specific
cytokines, and T-cell-mediated cytotoxicity have given new directions for glioma
immunotherapy. One of the recent approaches is the use of adoptive
immunotherapy, which can broadly grouped into two categories. The first
method of immunotherapy involves vaccination with dendritic cells pulsed with
tumor antigens. Antigen presentation by antigen presenting cells (APCs) is a
pivotal step in inducing antigen-specific immunity and is essential for tumor
vaccine design. Dendritic cells have always been considered as the most potent
APCs for initiating an immune response. Dendritic cells are bone-marrow-
derived cells, capable of presenting antigens in HLA-restricted manner. The abil-
ity to efficiently prime CD4 T-helper cells and to generate CD8 cytotoxic T-cells
make dendritic cells attractive in vaccine strategies for glioma therapy. One
study showed that immunizing tumor cells mixed with syngeneic spleen-derived
dendritc cells significantly prolonged mean survival for rats harboring pre-
established intracranial tumors [188]. Another group also reported a prolonged
survival in rats harboring pre-established intracranial 9L gliomas after vaccina-
tion with bone marrow-derived dendritic cells pulsed with acid-eluted protein
from 9L glioma cells [189]. Similar results were demonstrated by vaccinating
mice with dendritic cells pulsed with Semliki Forest virus-mediated glioma
complementary DNA [190] and dendritic cells fused to glioma cells [191].
These observations involving dendritic cell antigen presentation in animal
glioma models have prompted a number of vaccine clinical trials in patients
with glioma. In the first Phase I clinical trial, patients receiving an autologous-
peripheral blood-dendritic cells pulsed with peptides from autologous glioma
cell surface showed a prolonged survival time with an increased intratumoral
cytotoxicity and memory T-cell infilteration [192]. However, another Phase I
clinical trial involving patients vaccinated with a novel fusion product of autol-
ogous dendritic and glioma cells did not showed statistically significant treat-
ment-associated response to therapy [193]. However, there were no serious
adverse autoimmune-responses observed in this study, indicating that dendritic
cell-based immunotherapy can be used safely in humans as an adjunct to cur-
rently available glioma therapies [194].
Another immunotherapy technique uses gene-based vaccination. Genetic
manipulation of tumor cells to express certain cytokines like IFN-␥, GM-CSF, or
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IL-12 has been shown to stimulate a potent immunity against tumors within the
brain and provides a basis for gene-based immunotherapy [195, 196]. Vaccination
of allogeneic pre-B cells expressing EGF-RvIII has been shown to produce a
systemic immune response against autologous intracranial-tumor expressing
the same antigen [167]. Vaccination with genetically engineered glioma cells
expressing antisense molecules that block specific gene expression, such as
glioma-derived immunosuppressive factors TGF-␤2 and insulin-like growth fac-
tor-1, has also been shown to suppress intracranial tumor growth [197].
Two approaches have been used to introduce cytokines intracranially with
gene transfer techniques. The first approach involves direct intracranial implan-
tation of genetically engineered tumor cells, which secrete cytokines. Cytokines
such as IL-2, IL-4, GM-CSF, TNF-␣ and IFN-␥ all have been shown to demon-
strate a significant survival advantage in animal models [194, 198]. The second
approach involves direct cytokine gene transfer with viral vectors. Genetically
engineered adenovirus and herpes simplex virus expressing cytokines have
been tested and showed survival advantage, when used in experimental brain
tumor models [163, 199, 200]. Despite encouraging results, there have been
very few clinical trial results reported. A study involving single GBM patient
showed that repeated immunization of autologous tumor cells and a genetically
modified fibroblasts that secrete IL-2 promoted an antitumor response medi-
ated in part by cytotoxic T-cells [201]. Similarly, another Phase I clinical trial
involving 11 GBM patients immunized with autologous tumor cells modified
with Newcastle disease virus showed noticeable peripheral immune responses,
but no survival advantage over patients who received conventional combination
treatment of surgery, radiotherapy, and chemotherapy [202].
Small Molecule Therapy for RTK Signaling Pathways

One promising approach to inhibit aberrant RTK signaling is the genera-
tion of small-molecule drugs that selectively interfere with intrinsic tyrosine
kinase activity, and thereby inhibit receptor autophosphorylation and down-
stream signaling cascades [203]. RTK signaling can be targeted at three main
levels: the receptor itself or the PI3-kinase/Akt and/or Ras/MAPK signaling
modules (fig. 4).
Targeting at the Level of the Receptor: EGF-R, EGF-RvIII and PDGFR

One reasonable approach to inhibit RTK signaling is to generate small
molecule drugs that interfere with the function of the receptor. A number of
synthetic small molecules that are tyrosine kinase inhibitors have been gener-
ated, particularly for the EGFR/ErbB family. The most advanced of these are
ATP analogues of the quinazoline and pyridopyrimidine family that compete
with ATP for ATP-binding sites and thus block RTK activation [204].
Reversible inhibitors ZD-1839 (IRESSA, AstraZeneca) and OSI-774 (Tarceva,
Roche/OSI) are two potent EGF-R inhibitors currently being tested in clinical
trials against solid tumors [162, 205]. OSI-774 has been approved for the use in
Phase III clinical trials alone or in combination with conventional chemother-
apy, for non-small-cell lung and pancreatic cancer. Similarly, another reversible
ErbB inhibitor, PKI166 (Novartis) and the irreversible inhibitors CI1033
(Pfizer/Warner-Lambert) and EKB-569 (Wyeth-Ayerst) also inhibit EGF-R sig-
naling by competing with ATP for binding to the receptor [206, 207]. CI1033
has also been reported to block EGF-RvIII activation [207]. The FDA has
approved STI 571 (Gleevec, Novartis), another tyrosine kinase inhibitor, which
inhibits the non-RTK bcr-abl kinase, and two RTKs, PDGFR and c-Kit, for the
treatment of chronic-myeloid leukemia [208]. Gleevec also inhibits the growth
of GBM xenografts in vivo [209] and is currently in clinical trials for malignant
gliomas.
Targeting the PI3-Kinase/Akt Pathway

Bi-allelic PTEN loss with consequent deregulation of the PI3-knase/Akt
pathway is one of the major hallmarks of most of the GBMs [76, 158]. Since
this pathway is so crucial for glioblastoma cell proliferation and survival, the
PTEN/PI3-kinase/Akt pathway serves as a potential target for therapy. PI3-kinase
inhibitors such as wortmannin and LY294002 are effective but also quite toxic,
and it has been difficult to generate specific PI3-kinase/Akt inhibitors. An alter-
native approach is to inhibit FRAP/mTOR kinase, a downstream target of the
PI3-kinase/Akt pathway [210], with less toxicity [211]. The immunosuppres-
sant drug rapamycin forms a complex with the immunophilin FKB12 that binds
specifically to mTOR, and inhibits its kinase activity [211, 212]. An ester ana-
logue of Rapamycin, CCl-779 (Wyeth Ayerst) showed growth inhibition of
PTEN-deficient mouse as well as human cancer cells, indicating the possibility
of using mTOR inhibitors for treating PTEN-null human cancers, including
glioblastomas [211]. CCl-779 is currently being evaluated in Phase I testing in
human malignant gliomas. The main goal is to determine the efficacy of CCI-779
in these trials and to determine whether patient sensitivity correlates with
PTEN loss and PI3-kinase/Akt activation. It is also important to determine
whether EGF-R and/or EGF-RvIII expressions are associated with tumor
response to therapy.
Inhibition of the Ras/MAPK Pathway

As discussed earlier, specific mutations affecting Ras have not been
identified in human gliomas. Rather, constitutive Ras activation in GBMs
mainly results from overactivation of EGF-R, EGF-RvIII, and/or PDGFR sig-
naling. For signal-transduction, Ras must attach to the plasma membrane,
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which depends on the post-translational addition of a farnesyl group to its
C-terminal end by the enzyme farnesyl transferase. Thus, farnesyl transferase
inhibitors (FTIs) may be helpful in blocking Ras-mediated signaling and there
has been a great deal of effort into the development of FTIs as cancer thera-
peutics. FTIs may also target other pathways like Rho-mediated signaling and
the PI3-kinase/Akt pathway, which require farnesylation [213]. Two synthetic
FTIs, SCH66336 (Schering-Plough) [214] and R115777 (Zarnestra, Janssen
Research Foundation), have shown promising results in pre-clinical models,
including inhibition of growth of GBM cell lines [215–217]. Moreover, pre-
liminary findings suggest that EGFR overexpresssion in GBM cells can confer
increased sensitivity to SCH66336 [216]. R115777 is in Phase I/II clinical trial
evaluation involving patients with recurrent glioma [218].
We recently observed in our laboratory that overexpression of mutant
EGF-RvIII in human glioblastoma cells leads to constitutive activation of the
Erk MAPK kinase pathway in GBM cells, and pharmacological inhibition of
this pathway in turn downregulates EGFRvIII phosphorylation and transforma-
tion [O’Rourke, manuscript submitted], indicating that the use of inhibitors of
the MAPK pathway may be an alternative approach to treat EGF-RvIII-con-
taining malignant gliomas. There are several ongoing efforts to inhibit con-
stituents of the Ras/Raf/MEK/MAPK pathway, which may eventually achieve
enhanced efficacy with reduced toxicity in the management of malignant
gliomas that are often refractory to conventional therapy.
Anti-Angiogenesis Therapy
Malignant gliomas are among the most highly vascularized solid human
tumors [161]. The invasive nature of gliomas and accompanying neovasculature
results from the expression of stimulatory angiogenic factors, which induce
endothelial cell proliferation and promote complex interaction between cancer
cell and extracellular matrix (ECM). The most extensively studied factors
include VEGFs and VEGF receptors, integrins, and metalloproteinases. In
recent years, targeting these molecules to generate effective anti-angiogenic
drugs has become one of the major thrusts in the field of glioma therapy. VEGF
was shown to be differentially overexpressed in gliomas when compared to nor-
mal tissue [219, 220]. Furthermore, expression of VEGF has been strongly cor-
related to microvessel density in gliomas and meningiomas [221], indicating
their potential as a target for the drug development.
Antibodies against VEGF and VEGF receptors are currently being tested in
Phase II/III clinical trials. It was recently reported that a recombinant humanized
monoclonal antibody against VEGF, bevacizumab (AVASTIN, Genentech),
given with conventional chemotherapy significantly improved survival of
patients with metastatic colorectal cancers, suggesting that bevacizumab can be
effective in treatment of VEGF-overexpressing cancers [222]. Similarly, small
molecule inhibitors of VEGF-R tyrosine kinases such as SU5416, SU6668,
PTK787, and ZD4190 are already under clinical investigation [223].
The second category of target molecules for angiogenesis includes integrins
and metalloproteinases 2 and 9 (MMP-2 and MMP-9), which participate in
ECM attachment and degradation to promote neovascularization. Integrins inter-
act with ECM constituents through specific peptide motifs such as Arg-Gly-Asp
(RGD) present on ECM proteins to facilitate attachment. Synthetic analogs of
RGDs are now being considered as therapeutic tools to inhibit tumor growth
and invasion. It was recently demonstrated in an in vivo mouse model and by
vitro experiments that an ␣ v-integrin antogonist EMD 121974, a cyclic RGD-
penta-peptide was able to induce apoptosis in ␣ v-integrin-expressing U87MG
glioblastoma and DAOY medulloblastoma cell lines, suggesting a potential
of using RGDs for glioma therapy [224]. Also, conditionally replicative-
adenoviruses (CRAds) carrying RGD motifs are being used in pre-clinical stud-
ies to treat various cancers with antiangiogenesis approaches, including gliomas
[225, 226]. Furthermore, antibodies against variety of integrins have been shown
to inhibit matrigel invasion of glioma cell lines and primary cultures [227].
A recent study showed that intraperitoneal administration of IS20I, a specific
inhibitor of ␣(v)␤ 3 integrin, reduces tumor growth in nude mouse bearing
intracranial and subcutaneous glioma tumors [165]. Synthetic MMP inhibitors
such as Marimastat, Batimastat (BB-94), AG3340, and Bay12–9566 have been
used in Phase II and III clinical trials in several cancers including malignant
gliomas [223].
Only recently, a large number of naturally occurring endogenous inhibitors
of angiogenesis, such as platelet-derived factor 4 (PF 4), thrombospondin-1,
metallospondin, tissue inhibitors of metalloproteinases (TIMPs), plasminogen
activator/inhibitor, angiopoientin-2 (ANG-2), angiostatin (cleaved from plas-
minogen), and endostatin (cleaved from collagen XVIII) have been discovered,
which in theory might be exploited in anti-angiogenesis therapy. Among them,
angiostatin and endostatin are already in Phase I trials.
Previous studies showed that systemic administration of angiostatin suc-
cessfully treated subcutaneous and intracerebral gliomas of rat and human ori-
gin in nude mouse model [228, 229]. In addition, retroviral or adenoviral
transduction of the angiostatin gene into established brain tumors in mice inhib-
ited tumor growth effectively [166, 230, 231]. Similarly, endostatin has been
successfully used systemically or by gene transfer methods to treat a variety of
cancers, including fibrosarcoma, melanoma, hemangiothelioma, prostate, renal,
and mammary, lung, and colon carcinomas [232]. A recent study involving
immunological analysis of frozen tissues from 51 patients with astrocytic tumors
(Grade 2, 3 and 4) showed an increased levels of tissue endostatin, suggesting a
Glioma-Genesis 543
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definite role of the proteins in glioma formation [233]. Overexpression of endo-
statin has been shown to reduce tumor growth rate by 90% in rat C6 glioma
model [234]. Similarly, endostatin was shown to reduce vascularization, blood
flow, and tumor growth in rat gliosarcoma model [235]. Furthermore, combined
administration of endostatin and a PKC␣-inhibiting DNA enzyme improved sur-
vival rate in rats bearing intracranial malignant glioma BT(4)C, suggesting that
combined treatment may represent an attractive therapeutic strategy against
malignant gliomas [236]. A study in ovarian cancer cells showed that combined
application of angiostatin and endostatin produced synergistic effects on tumor
suppression and suggests that the molecular targets differs for the two inhibitory
molecules [237]. Taken together, these studies indicate that the angiogenic
machinery involves a plethora of interacting molecules, which could be
exploited as potential candidates for anti-angiogenesis drug development.
Conclusions and Future Directions
Over the past decade, progress has been made in understanding the mole-
cular pathogenesis of malignant gliomas. Empirical observations and experi-
mental studies have led to an emerging consensus that malignant progression of
gliomas occurs through either abnormal activation of signal transduction path-
ways down-stream of RTKs and/or disruption of cell cycle arrest pathways. It
has been clearly elucidated by genetic studies and animal models that there is
an intricate cooperativity between RTK signaling pathways and cell cycle reg-
ulatory molecules. Abnormal or deregulated RTK signaling can occur via gene
amplification or activating mutations of EGFR, overexpression of fibroblast
growth factor, FGF-R and PDGF and/or PDGFR, and overactivation of the
TGF-␣/EGF-R autocrine loop. This leads to constitutive activation of down-
stream signaling pathways, which in part involves the PI3-kinase/Akt pathway
and the Ras/Raf1/MEK/MAP kinase pathway.
The elucidation of the signaling pathways activated due to different genetic
alterations is now being translated into glial tumor treatment strategies. Initial
efforts have focused on the use of single agents directed at specific target mol-
ecules. However, the complexity and cross talk between different signaling cas-
cades may limit potential efficacy of targeting a single molecule. Therefore, it
has become imperative to consider combinatorial treatment regimens involving
either multiple inhibitors targeting different pathways or combination of these
inhibitors with traditional cytotoxic drugs. Furthermore, efforts should also be
made to design new strategies to determine the optimal dose and effectiveness
of these agents by evaluating their molecular effects as an endpoint rather than
assessing traditional maximally tolerated dose and/or overall tumor response.
Finally, the molecular heterogeneity of GBMs posses a great challenge for
any targeted molecular therapy. Therefore, it is essential to develop an expanded
molecular sub-classification of GBMs. This can be achieved by using genetic
approaches such as gene profiling coupled with careful biological validation
and development of new biochemical and clinical markers. Future treatment
strategies for malignant glial tumors will involve a fine integration between a
broad-spectrum drug regimen, advances in molecular profiling of tumors and
protocol design. The potential benefit of these strategies offers a hope for sig-
nificant improvement in the prognosis for patients with malignant glial brain
tumors.
Acknowledgments
Our research is supported by grants to D.M.O. from the National Institutes of Health,
the Department of Veterans Affairs (Merit Review Program), and The Brain Tumor Society.
References
1 Kleihues P, Louis DN, Scheithauer BW, Rorke LB, Reifenberger G, Burger PC, et al: The WHO
classification of tumors of the nervous system. J Neuropathol Exp Neurol 2002;61:215–225;
2 Kleihues P, Burger PC, Scheithauer BW: The new WHO classification of brain tumours. Brain
Pathol 1993;3:255–268.
3 Guha A, Dashner K, Black PM, Wagner JA, Stiles CD: Expression of PDGF and PDGF receptors
in human astrocytoma operation specimens supports the existence of an autocrine loop. Int
J Cancer 1995;60:168–173.
4 Lokker NA, Sullivan CM, Hollenbach SJ, Israel MA, Giese NA: Platelet-derived growth factor
(PDGF) autocrine signaling regulates survival and mitogenic pathways in glioblastoma cells:
Evidence that the novel PDGF-C and PDGF-D ligands may play a role in the development of brain
tumors. Cancer Res 2002;62:3729–3735.
5 Sara VR, Prisell P, Sjogren B, Persson L, Boethius J, Enberg G: Enhancement of insulin-like
growth factor 2 receptors in glioblastoma. Cancer Lett 1986;32:229–234.
6 Smith JS, Wang XY, Qian J, Hosek SM, Scheithauer BW, Jenkins RB, et al: Amplification of the
platelet-derived growth factor receptor-A (PDGFRA) gene occurs in oligodendrogliomas with
grade IV anaplastic features. J Neuropathol Exp Neurol 2000;59:495–503.
7 Trojan J, Blossey BK, Johnson TR, Rudin SD, Tykocinski M, Ilan J: Loss of tumorigenicity of rat
glioblastoma directed by episome-based antisense cDNA transcription of insulin-like growth
factor I. Proc Natl Acad Sci USA 1992;89:4874–4878.
8 Wong AJ, Bigner SH, Bigner DD, Kinzler KW, Hamilton SR, Vogelstein B: Increased expression
of the epidermal growth factor receptor gene in malignant gliomas is invariably associated with
gene amplification. Proc Natl Acad Sci USA 1987;84:6899–6903.
9 Wong AJ, Ruppert JM, Bigner SH, Grzeschik CH, Humphrey PA, Bigner DS, et al: Structural
alterations of the epidermal growth factor receptor gene in human gliomas. Proc Natl Acad Sci
USA 1992;89:2965–2969.
10 Buschges R, Weber RG, Actor B, Lichter P, Collins VP, Reifenberger G: Amplification and expres-
sion of cyclin D genes (CCND1, CCND2 and CCND3) in human malignant gliomas. Brain Pathol
1999;9:435–442; discussion 432–433.
Glioma-Genesis 545
medwedi.ru
11 Costello JF, Plass C, Arap W, Chapman VM, Held WA, Berger MS, et al: Cyclin-dependent kinase
6 (CDK6) amplification in human gliomas identified using two-dimensional separation of genomic
DNA. Cancer Res 1997;57:1250–1254.
12 Frankel RH, Bayona W, Koslow M, Newcomb EW: p53 mutations in human malignant gliomas:
Comparison of loss of heterozygosity with mutation frequency. Cancer Res 1992;52:1427–1433.
13 Giani C, Finocchiaro G: Mutation rate of the CDKN2 gene in malignant gliomas. Cancer Res
1994;54:6338–6339.
14 Henson JW, Schnitker BL, Correa KM, von Deimling A, Fassbender F, Xu HJ, et al: The
retinoblastoma gene is involved in malignant progression of astrocytomas. Ann Neurol 1994;36:
714–721.
15 Newcomb EW, Alonso M, Sung T, Miller DC: Incidence of p14ARF gene deletion in high-grade
adult and pediatric astrocytomas. Hum Pathol 2000;31:115–119.
16 Reifenberger G, Liu L, Ichimura K, Schmidt EE, Collins VP: Amplification and overexpression
of the MDM2 gene in a subset of human malignant gliomas without p53 mutations. Cancer Res
1993;53:2736–2739.
17 Rollbrocker B, Waha A, Louis DN, Wiestler OD, von Deimling A: Amplification of the cyclin-
dependent kinase 4 (CDK4) gene is associated with high cdk4 protein levels in glioblastoma mul-
tiforme. Acta Neuropathol (Berl) 1996;92:70–74.
18 Ullrich A, Schlessinger J: Signal transduction by receptors with tyrosine kinase activity. Cell
1990;61:203–212.
19 Kolibaba KS, Druker BJ: Protein tyrosine kinases and cancer. Biochim Biophys Acta 1997;
1333:F217–F248.
20 Hanahan D, Weinberg RA: The hallmarks of cancer. Cell 2000;100:57–70.
21 Maher EA, Furnari FB, Bachoo RM, Rowitch DH, Louis DN, Cavenee WK, et al: Malignant
glioma: Genetics and biology of a grave matter. Genes Dev 2001;15:1311–1333.
22 Yeh YL, Kang YM, Chaibi MS, Xie JF, Graves DT: IL-1 and transforming growth factor-beta
inhibit platelet-derived growth factor-AA binding to osteoblastic cells by reducing platelet-
derived growth factor-alpha receptor expression. J Immunol 1993;150:5625–5632.
23 Noble M, Murray K, Stroobant P, Waterfield MD, Riddle P: Platelet-derived growth factor pro-
motes division and motility and inhibits premature differentiation of the oligodendrocyte/type-2
astrocyte progenitor cell. Nature 1988;333:560–562.
24 Richardson WD, Pringle N, Mosley MJ, Westermark B, Dubois-Dalcq M: A role for platelet-
derived growth factor in normal gliogenesis in the central nervous system. Cell 1988;53:309–319.
25 Claesson-Welsh L: Signal transduction by the PDGF receptors. Prog Growth Factor Res 1994;5:
37–54.
26 Heldin CH, Westermark B: Platelet-derived growth factor: Mechanism of action and possible
in vivo function. Cell Regul 1990;1:555–566.
27 Westermark B, Heldin CH, Nister M: Platelet-derived growth factor in human glioma. Glia
1995;15:257–263.
28 Chung R, Whaley J, Kley N, Anderson K, Louis D, Menon A, et al: TP53 gene mutations and 17p
deletions in human astrocytomas. Genes Chromosomes Cancer 1991;3:323–331.
29 von Deimling A, Eibl RH, Ohgaki H, Louis DN, von Ammon K, Petersen I, et al: p53 mutations
are associated with 17p allelic loss in grade II and grade III astrocytoma. Cancer Res 1992;52:
2987–2990.
30 Dai C, Celestino JC, Okada Y, Louis DN, Fuller GN, Holland EC: PDGF autocrine stimulation
dedifferentiates cultured astrocytes and induces oligodendrogliomas and oligoastrocytomas from
neural progenitors and astrocytes in vivo. Genes Dev 2001;15:1913–1925.
31 Betsholtz C, Karlsson L, Lindahl P: Developmental roles of platelet-derived growth factors.
Bioessays 2001;23:494–507.
32 Hermanson M, Funa K, Hartman M, Claesson-Welsh L, Heldin CH, Westermark B, et al: Platelet-
derived growth factor and its receptors in human glioma tissue: Expression of messenger RNA and
protein suggests the presence of autocrine and paracrine loops. Cancer Res 1992;52:3213–3219.
33 Vassbotn FS, Ostman A, Langeland N, Holmsen H, Westermark B, Heldin CH, et al: Activated
platelet-derived growth factor autocrine pathway drives the transformed phenotype of a human
glioblastoma cell line. J Cell Physiol 1994;158:381–389.
34 Andrae J, Molander C, Smits A, Funa K, Nister M: Platelet-derived growth factor-B and -C and
active alpha-receptors in medulloblastoma cells. Biochem Biophys Res Commun 2002;296:
604–611.
35 Black P, Carroll R, Glowacka D: Expression of platelet-derived growth factor transcripts in medullo-
blastomas and ependymomas. Pediatr Neurosurg 1996;24:74–78.
36 Guo P, Hu B, Gu W, Xu L, Wang D, Huang HJ, et al: Platelet-derived growth factor-B enhances
glioma angiogenesis by stimulating vascular endothelial growth factor expression in tumor
endothelia and by promoting pericyte recruitment. Am J Pathol 2003;162:1083–1093.
37 Bergsten E, Uutela M, Li X, Pietras K, Ostman A, Heldin CH, et al: PDGF-D is a specific,
protease-activated ligand for the PDGF beta-receptor. Nat Cell Biol 2001;3:512–516.
38 Gilbertson DG, Duff ME, West JW, Kelly JD, Sheppard PO, Hofstrand PD, et al: Platelet-derived
growth factor C (PDGF-C), a novel growth factor that binds to PDGF alpha and beta receptor.
J Biol Chem 2001;276:27406–27414.
39 LaRochelle WJ, Jeffers M, McDonald WF, Chillakuru RA, Giese NA, Lokker NA, et al: PDGF-D,
a new protease-activated growth factor. Nat Cell Biol 2001;3:517–521.
40 Fantl WJ, Escobedo JA, Williams LT: Mutations of the platelet-derived growth factor receptor that
cause a loss of ligand-induced conformational change, subtle changes in kinase activity, and
impaired ability to stimulate DNA synthesis. Mol Cell Biol 1989;9:4473–4478.
41 Kazlauskas A, Cooper JA: Autophosphorylation of the PDGF receptor in the kinase insert region
regulates interactions with cell proteins. Cell 1989;58:1121–1133.
42 Heldin CH, Ostmanv A, Ronnstrand L: Signal transduction via platelet-derived growth factor
receptors. Biochim Biophys Acta 1998;1378:F79–F113.
43 Salomon DS, Brandt R, Ciardiello F, Normanno N: Epidermal growth factor-related peptides and
their receptors in human malignancies. Crit Rev Oncol-Hematol 1995;19:183–232.
44 Bigner SH, Humphrey PA, Wong AJ, Vogelstein B, Mark J, Friedman HS, et al: Characterization
of the epidermal growth factor receptor in human glioma cell lines and xenografts. Cancer Res
1990;50:8017–8022.
45 Libermann TA, Nusbaum HR, Razon N, Kris R, Lax I, Soreq H, et al: Amplification, enhanced
expression and possible rearrangement of EGF receptor gene in primary human brain tumours of
glial origin. Nature 1985;313:144–147.
46 Kleihues P, Lubbe J, Watanabe K, von Ammon K, Ohgaki H: Genetic alterations associated with
glioma progression. Verh Dtsch Ges Pathol 1994;78:43–47.
47 Kleihues P, Ohgaki H: Primary and secondary glioblastomas: From concept to clinical diagnosis.
Neuro oncology 1999;1:44–51.
48 Ohgaki H, Schauble B, zur Hausen A, von Ammon K, Kleihues P: Genetic alterations associated
with the evolution and progression of astrocytic brain tumours. Virchows Archiv 1995;427:
113–118.
49 Rasheed BK, Wiltshire RN, Bigner SH, Bigner DD: Molecular pathogenesis of malignant
gliomas. Curr Opin Oncol 1999;11:162–167.
50 Ekstrand AJ, Sugawa N, James CD, Collins VP: Amplified and rearranged epidermal growth fac-
tor receptor genes in human glioblastomas reveal deletions of sequences encoding portions of the
N- and/or C-terminal tails. Proc Natl Acad Sci USA 1992;89:4309–4313.
51 Kuan CT, Wikstrand CJ, Bigner DD: EGF mutant receptor vIII as a molecular target in cancer
therapy. Endocr Relat Cancer 2001;8:83–96.
52 Nagane M, Lin H, Cavenee WK, Huang HJ: Aberrant receptor signaling in human malignant
gliomas: Mechanisms and therapeutic implications. Cancer Lett 2001;(suppl 162):S17–S21.
53 O’Rourke DM, Nute EJ, Davis JG, Wu C, Lee A, Murali R, et al: Inhibition of a naturally occur-
ring EGFR oncoprotein by the p185neu ectodomain: Implications for subdomain contributions to
receptor assembly. Oncogene 1998;16:1197–1207.
54 Wikstrand CJ, Reist CJ, Archer GE, Zalutsky MR, Bigner DD: The class III variant of the epider-
mal growth factor receptor (EGFRvIII): Characterization and utilization as an immunotherapeu-
tic target. J Neurovirol 1998;4:148–158.
55 Moscatello DK, Holgado-Madruga M, Godwin AK, Ramirez G, Gunn G, Zoltick PW, et al:
Frequent expression of a mutant epidermal growth factor receptor in multiple human tumors.
Cancer Res 1995;55:5536–5539.
Glioma-Genesis 547
medwedi.ru
56 Olapade-Olaopa EO, Moscatello DK, MacKay EH, Horsburgh T, Sandhu DP, Terry TR, et al:
Evidence for the differential expression of a variant EGF receptor protein in human prostate
cancer. Br J Cancer 2000;82:186–194.
57 Schlegel J, Stumm G, Brandle K, Merdes A, Mechtersheimer G, Hynes NE, et al: Amplification
and differential expression of members of the erbB-gene family in human glioblastoma. Journal
of Neuro oncol 1994;22:201–207.
58 Feldkamp MM, Lala P, Lau N, Roncari L, Guha A: Expression of activated epidermal growth factor
receptors, Ras-guanosine triphosphate, and mitogen-activated protein kinase in human glioblastoma
multiforme specimens. Neurosurgery 1999;45:1442–1453.
59 Moscatello DK, Montgomery RB, Sundareshan P, McDanel H, Wong MY, Wong AJ:
Transformational and altered signal transduction by a naturally occurring mutant EGF receptor.
Oncogene 1996;13:85–96.
60 Nishikawa R, Ji XD, Harmon RC, Lazar CS, Gill GN, Cavenee WK, et al: A mutant epidermal
growth factor receptor common in human glioma confers enhanced tumorigenicity. Proc Natl
Acad Sci USA 1994;91:7727–7731.
61 Holland EC, Hively WP, DePinho RA, Varmus HE: A constitutively active epidermal growth fac-
tor receptor cooperates with disruption of G1 cell-cycle arrest pathways to induce glioma-like
lesions in mice. Genes Dev 1998;12:3675–3685.
62 Hills D, Rowlinson-Busza G, Gullick WJ: Specific targeting of a mutant, activated FGF receptor
found in glioblastoma using a monoclonal antibody. Intern J Cancer 1995;63:537–543.
63 Lorimer IA: Mutant epidermal growth factor receptors as targets for cancer therapy. Curr Cancer
Drug Targets 2002;2:91–102.
64 Lorimer IA, Keppler-Hafkemeyer A, Beers RA, Pegram CN, Bigner DD, Pastan I: Recombinant
immunotoxins specific for a mutant epidermal growth factor receptor: Targeting with a single chain
antibody variable domain isolated by phage display. Proc Natl Acad Sci USA 1996;93:
14815–14820.
65 Nagane M, Levitzki A, Gazit A, Cavenee WK, Huang HJ: Drug resistance of human glioblastoma
cells conferred by a tumor-specific mutant epidermal growth factor receptor through modulation
of Bcl-XL and caspase-3-like proteases. Proc Natl Acad Sci USA 1998;95:5724–5729.
66 Holland EC, Hively WP, Gallo V, Varmus HE: Modeling mutations in the G1 arrest pathway in
human gliomas: Overexpression of CDK4 but not loss of INK4a-ARF induces hyperploidy in
cultured mouse astrocytes. Genes Dev 1998;12:3644–3649.
67 Ding H, Shannon P, Lau N, Wu X, Roncari L, Baldwin RL, et al: Oligodendrogliomas result from
the expression of an activated mutant epidermal growth factor receptor in a RAS transgenic mouse
astrocytoma model. Cancer Res 2003;63:1106–1113.
68 Ekstrand AJ, James CD, Cavenee WK, Seliger B, Pettersson RF, Collins VP: Genes for epidermal
growth factor receptor, transforming growth factor alpha, and epidermal growth factor and their
expression in human gliomas in vivo. Cancer Res 1991;51:2164–2172.
69 Hayashi Y, Ueki K, Waha A, Wiestler OD, Louis DN, von Deimling A: Association of EGFR gene
amplification and CDKN2 (p16/MTS1) gene deletion in glioblastoma multiforme. Brain Pathol
1997;7:871–875.
70 Sonoda Y, Ozawa T, Aldape KD, Deen DF, Berger MS, Pieper RO: Akt pathway activation con-
verts anaplastic astrocytoma to glioblastoma multiforme in a human astrocyte model of glioma.
Cancer Res 2001;61:6674–6678.
71 Reilly KM, Loisel DA, Bronson RT, McLaughlin ME, Jacks T: Nf1;Trp53 mutant mice develop
glioblastoma with evidence of strain-specific effects. Nat Genet 2000;26:109–113.
72 Holland EC, Celestino J, Dai C, Schaefer L, Sawaya RE, Fuller GN: Combined activation of Ras
and Akt in neural progenitors induces glioblastoma formation in mice. Nat Genet 2000;25:
55–57.
73 Uhrbom L, Dai C, Celestino JC, Rosenblum MK, Fuller GN, Holland EC: Ink4a-Arf loss coop-
erates with KRas activation in astrocytes and neural progenitors to generate glioblastomas of
various morphologies depending on activated Akt. Cancer Res 2002;62:5551–5558.
74 Davies MA, Lu Y, Sano T, Fang X, Tang P, LaPushin R, et al: Adenoviral transgene expression of
MMAC/PTEN in human glioma cells inhibits Akt activation and induces anoikis. Cancer Res
1998;58:5285–5290.
75 Fujisawa H, Kurrer M, Reis RM, Yonekawa Y, Kleihues P, Ohgaki H: Acquisition of the glioblas-
toma phenotype during astrocytoma progression is associated with loss of heterozygosity on
10q25-qter. Am J Pathol 1999;155:387–394.
76 Haas-Kogan D, Shalev N, Wong M, Mills G, Yount G, Stokoe D: Protein kinase B (PKB/Akt)
activity is elevated in glioblastoma cells due to mutation of the tumor suppressor PTEN/MMAC.
Curr Biol 1998;8:1195–1198.
77 Bachoo RM, Maher EA, Ligon KL, Sharpless NE, Chan SS, You MJ, et al: Epidermal growth fac-
tor receptor and Ink4a/Arf: Convergent mechanisms governing terminal differentiation and trans-
formation along the neural stem cell to astrocyte axis. Cancer Cell 2002;1:269–277.
78 Gilbertson RJ, Bentley L, Hernan R, Junttila TT, Frank AJ, Haapasalo H, et al: ERBB receptor
signaling promotes ependymoma cell proliferation and represents a potential novel therapeutic
target for this disease. Clin cancer res 2002;8:3054–3064.
79 Gilbertson RJ, Perry RH, Kelly PJ, Pearson AD, Lunec J: Prognostic significance of HER2 and
HER4 coexpression in childhood medulloblastoma. Cancer Res 1997;57:3272–3280.
80 Lui VW, Grandis JR: EGFR-mediated cell cycle regulation. Anticancer Res 2002;22:1–11.
81 Chow NH, Liu HS, Lee EI, Chang CJ, Chan SH, Cheng HL, et al: Significance of urinary epi-
dermal growth factor and its receptor expression in human bladder cancer. Anticancer Res
1997;17:1293–1296.
82 Grandis JR, Melhem MF, Gooding WE, Day R, Holst VA, Wagener MM, et al: Levels of TGF-
alpha and EGFR protein in head and neck squamous cell carcinoma and patient survival. J Natl
Cancer Inst 1998;90:824–832.
83 Turkeri LN, Erton ML, Cevik I, Akdas A: Impact of the expression of epidermal growth factor,
transforming growth factor alpha, and epidermal growth factor receptor on the prognosis of super-
ficial bladder cancer. Urology 1998;51:645–649.
84 Zwick ME, Cutler DJ, Chakravarti A: Patterns of genetic variation in Mendelian and complex
traits. Annu Rev Genomics Hum Genet 2000;1:387–407.
85 Carraway KL 3rd, Cantley LC. A neu acquaintance for erbB3 and erbB4: A role for receptor
heterodimerization in growth signaling. Cell 1994;78:5–8.
86 Derynck R, Roberts AB, Winkler ME, Chen EY, Goeddel DV: Human transforming growth factor-
alpha: Precursor structure and expression in E. coli. Cell 1984;38:287–297.
87 Massague J: Epidermal growth factor-like transforming growth factor. II. Interaction with epidermal
growth factor receptors in human placenta membranes and A431 cells. J Biol Chem 1983;258:
13614–13620.
88 Shum L, Reeves SA, Kuo AC, Fromer ES, Derynck R: Association of the transmembrane TGF-alpha
precursor with a protein kinase complex. J Cell Biol 1994;125:903–916.
89 Yarden Y, Ullrich A: Growth factor receptor tyrosine kinases. Ann Rev Biochem 1988;57:443–478.
90 Tang P, Steck PA, Yung WK: The autocrine loop of TGF-alpha/EGFR and brain tumors. J Neuro
oncol 1997;35:303–314.
91 Derynck R, Goeddel DV, Ullrich A, Gutterman JU, Williams RD, Bringman TS, et al: Synthesis
of messenger RNAs for transforming growth factors alpha and beta and the epidermal growth
factor receptor by human tumors. Cancer Res 1987;47:707–712.
92 Schlegel U, Moots PL, Rosenblum MK, Thaler HT, Furneaux HM: Expression of transforming
growth factor alpha in human gliomas. Oncogene 1990;5:1839–1832.
93 Yung WK, Zhang X, Steck PA, Hung MC: Differential amplification of the TGF-alpha gene in
human gliomas. Cancer Commun 1990;2:201–205.
94 Huber H, Eggert A, Janss AJ, Wiewrodt R, Zhao H, Sutton LN, et al: Angiogenic profile of childhood
primitive neuroectodermal brain tumours/medulloblastomas. Eur J Cancer 2001;37:2064–2072.
95 El-Obeid A, Bongcam-Rudloff E, Sorby M, Ostman A, Nister M, Westermark B: Cell scattering
and migration induced by autocrine transforming growth factor alpha in human glioma cells
in vitro. Cancer Res 1997;57:5598–5604.
96 Zhou R, Skalli O: TGF-alpha differentially regulates GFAP, vimentin, and nestin gene expression
in U-373 MG glioblastoma cells: Correlation with cell shape and motility. Exp Cell Res 2000;254:
269–278.
97 El-Obeid A, Hesselager G, Westermark B, Nister M: TGF-alpha-driven tumor growth is inhibited
by an EGF receptor tyrosine kinase inhibitor. Biochem Biophy Res Commun 2002;290:349–358.
Glioma-Genesis 549
medwedi.ru
98 Garrington TP, Johnson GL: Organization and regulation of mitogen-activated protein kinase sig-
naling pathways. Curr Opin Cell Biol 1999;11:211–218.
99 Wilkinson MG, Millar JB: Control of the eukaryotic cell cycle by MAP kinase signaling path-
ways. FASEB J 2000;14:2147–2157.
100 Cox AD, Der CJ: Ras family signaling: Therapeutic targeting. Cancer Biol Ther 2002;1:599–606.
101 Schlessinger J: Cell signaling by receptor tyrosine kinases. Cell 2000;103:211–225.
102 Li W, Nishimura R, Kashishian A, Batzer AG, Kim WJ, Cooper JA, et al: A new function for a
phosphotyrosine phosphatase: Linking GRB2-Sos to a receptor tyrosine kinase. Mol Cell Biol
1994;14:509–517.
103 Yart A, Laffargue M, Mayeux P, Chretien S, Peres C, Tonks N, et al: A critical role for phospho-
inositide 3-kinase upstream of Gab1 and SHP2 in the activation of ras and mitogen-activated
protein kinases by epidermal growth factor. J Biol Chem 2001;276:8856–8864.
104 Lorimer IA, Lavictoire SJ: Activation of extracellular-regulated kinases by normal and mutant
EGF receptors. Biochimica et Biophysica Acta 2001;1538:1–9.
105 Hamad NM, Elconin JH, Karnoub AE, Bai W, Rich JN, Abraham RT, et al: Distinct requirements
for Ras oncogenesis in human versus mouse cells. Genes Dev 2002;16:2045–2057.
106 Katz ME, McCormick F: Signal transduction from multiple Ras effectors. Curr Opin Genet Dev
1997;7:75–79.
107 Boulton TG, Nye SH, Robbins DJ, Ip NY, Radziejewska E, Morgenbesser SD, et al: ERKs: A fam-
ily of protein-serine/threonine kinases that are activated and tyrosine phosphorylated in response
to insulin and NGF. Cell 1991;65:663–675.
108 Dent P, Haser W, Haystead TA, Vincent LA, Roberts TM, Sturgill TW: Activation of mitogen-
activated protein kinase kinase by v-Raf in NIH 3T3 cells and in vitro. Science 1992;257:1404–1407.
109 Howe LR, Leevers SJ, Gomez N, Nakielny S, Cohen P, Marshall CJ: Activation of the MAP kinase
pathway by the protein kinase raf. Cell 1992;71:335–342.
110 Kyriakis JM, Force TL, Rapp UR, Bonventre JV, Avruch J: Mitogen regulation of c-Raf-1 protein
kinase activity toward mitogen-activated protein kinase-kinase. J Biol Chem 1993;268:16009–16019.
111 Fanton CP, McMahon M, Pieper RO: Dual growth arrest pathways in astrocytes and astrocytic
tumors in response to Raf-1 activation. J Biol Chem 2001;276:18871–18877.
112 Chambers AF, Tuck AB: Ras-responsive genes and tumor metastasis. Crit Rev Oncog 1993;4:
95–114.
113 Guha A, Feldkamp MM, Lau N, Boss G, Pawson A: Proliferation of human malignant astrocy-
tomas is dependent on Ras activation. Oncogene 1997;15:2755–2765.
114 Gutmann DH, Giordano MJ, Mahadeo DK, Lau N, Silbergeld D, Guha A: Increased neurofibro-
matosis 1 gene expression in astrocytic tumors: Positive regulation by p21-ras. Oncogene 1996;12:
2121–2127.
115 Chunduru S, Kawami H, Gullick R, Monacci WJ, Dougherty G, Cutler ML: Identification of an
alternatively spliced RNA for the Ras suppressor RSU-1 in human gliomas. J Neuro Oncol 2002;
60:201–211.
116 Ellis CA, Vos MD, Howell H, Vallecorsa T, Fults DW, Clark GJ: Rig is a novel Ras-related protein
and potential neural tumor suppressor. Proc Natl Acad Sci USA 2002;99:9876–9881.
117 Liu JJ, Chao JR, Jiang MC, Ng SY, Yen JJ, Yang-Yen HF: Ras transformation results in an elevated
level of cyclin D1 and acceleration of G1 progression in NIH 3T3 cells. Mol Cell Biol 1995;15:
3654–3663.
118 Reed N, Gutmann DH: Tumorigenesis in neurofibromatosis: New insights and potential therapies.
Trends Mol Med 2001;7:157–162.
119 Zhu Y, Parada LF: A particular GAP in mind. Nat Genet 2001;27:354–355.
120 Gutmann DH, Saporito-Irwin S, DeClue JE, Wienecke R, Guha A: Alterations in the rap1 signal-
ing pathway are common in human gliomas. Oncogene 1997;15:1611–1616.
121 Jin F, Wienecke R, Xiao GH, Maize JC Jr, DeClue JE, Yeung RS: Suppression of tumorigenicity
by the wild-type tuberous sclerosis 2 (Tsc2) gene and its C-terminal region. Proc Natl Acad Sci
USA 1996;93:9154–9159.
122 Lu Z, Hornia A, Joseph T, Sukezane T, Frankel P, Zhong M, et al: Phospholipase D and RalA coop-
erate with the epidermal growth factor receptor to transform 3Y1 rat fibroblasts. Mol Cell Biol
2000;20:462–467.
123 Ward Y, Wang W, Woodhouse E, Linnoila I, Liotta L, Kelly K: Signal pathways which promote
invasion and metastasis: Critical and distinct contributions of extracellular signal-regulated kinase
and Ral-specific guanine exchange factor pathways. Mol Cell Biol 2001;21:5958–5969.
124 Nishida K, Kaziro Y, Satoh T: Anti-apoptotic function of Rac in hematopoietic cells. Oncogene
1999;18:407–415.
125 Ruggieri R, Chuang YY, Symons M: The small GTPase Rac suppresses apoptosis caused by serum
deprivation in fibroblasts. Mol Med 2001;7:293–300.
126 Senger DL, Tudan C, Guiot MC, Mazzoni IE, Molenkamp G, LeBlanc R, et al: Suppression of
Rac activity induces apoptosis of human glioma cells but not normal human astrocytes. Cancer
Res 2002;62:2131–2140.
127 Behrens A, Jochum W, Sibilia M, Wagner EF: Oncogenic transformation by ras and fos is medi-
ated by c-Jun N-terminal phosphorylation. Oncogene 2000;19:2657–2663.
128 Chen N, Nomura M, She QB, Ma WY, Bode AM, Wang L, et al: Suppression of skin tumorigene-
sis in c-Jun NH(2)-terminal kinase-2-deficient mice. Cancer Res 2001;61:3908–3912.
129 Antonyak MA, Moscatello DK, Wong AJ: Constitutive activation of c-Jun N-terminal kinase by a
mutant epidermal growth factor receptor. J Biol Chem 1998;273:2817–2822.
130 Tsuiki H, Tnani M, Okamoto I, Kenyon LC, Emlet DR, Holgado-Madruga M, et al: Constitutively
active forms of c-Jun NH2-terminal kinase are expressed in primary glial tumors. Cancer Res
2003;63:250–255.
131 Wu CJ, Qian X, O’Rourke DM: Sustained mitogen-activated protein kinase activation is induced
by transforming erbB receptor complexes. DNA Cell Biol 1999;18:731–741.
132 Potapova O, Gorospe M, Bost F, Dean NM, Gaarde WA, Mercola D, et al: c-Jun N-terminal
kinase is essential for growth of human T98G glioblastoma cells. J Biol Chem 2000;275:
24767–24775.
133 Czech MP: Lipid rafts and insulin action. Nature 2000;407:147–148.
134 Rameh LE, Cantley LC: The role of phosphoinositide 3-kinase lipid products in cell function.
J Biol Chem 1999;274:8347–8350.
135 Rodrigues GA, Falasca M, Zhang Z, Ong SH, Schlessinger J: A novel positive feedback loop
mediated by the docking protein Gab1 and phosphatidylinositol 3-kinase in epidermal growth fac-
tor receptor signaling. Mol Cell Biol 2000;20:1448–1459.
136 Wu CJ, Chen Z, Ullrich A, Greene MI, O’Rourke DM: Inhibition of EGFR-mediated phospho-
inositide-3-OH kinase (PI3-K) signaling and glioblastoma phenotype by signal-regulatory pro-
teins (SIRPs). Oncogene 2000;19:3999–4010.
137 Wu CJ, O’Rourke DM, Feng GS, Johnson GR, Wang Q, Greene MI: The tyrosine phosphatase
SHP-2 is required for mediating phosphatidylinositol 3-kinase/Akt activation by growth factors.
Oncogene 2001;20:6018–6025.
138 Kapoor GS, Zhan Y, Johnson GR, O’Rourke DM: Distinct domains in the SHP-2 phosphatase
differentially regulate epidermal growth factor receptor/NF-kappa B activation through Gablin
glioblastoma cells. Mol Cell Biol 2004;24:823–836.
139 Alessi DR, Andjelkovic M, Caudwell B, Cron P, Morrice N, Cohen P, et al: Mechanism of activation
of protein kinase B by insulin and IGF-1. Embo J 1996;15:6541–51.
140 Cardone MH, Roy N, Stennicke HR, Salvesen GS, Franke TF, Stanbridge E, et al: Regulation of
cell death protease caspase-9 by phosphorylation. Science 1998;282:1318–1321.
141 Brunet A, Bonni A, Zigmond MJ, Lin MZ, Juo P, Hu LS, et al: Akt promotes cell survival by phos-
phorylating and inhibiting a Forkhead transcription factor. Cell 1999;96:857–868.
142 Cross DA, Alessi DR, Cohen P, Andjelkovich M, Hemmings BA: Inhibition of glycogen synthase
kinase-3 by insulin mediated by protein kinase B. Nature 1995;378:785–789.
143 Tamura M, Gu J, Danen EH, Takino T, Miyamoto S, Yamada KM: PTEN interactions with focal
adhesion kinase and suppression of the extracellular matrix-dependent phosphatidylinositol
3-kinase/Akt cell survival pathway. J Biol Chem 1999;274:20693–20703.
144 Yamada KM, Araki M: Tumor suppressor PTEN: Modulator of cell signaling, growth, migration
and apoptosis. J Cell Sci 2001;114:2375–2382.
145 Kubiatowski T, Jang T, Lachyankar MB, Salmonsen R, Nabi RR, Quesenberry PJ, et al: Association
of increased phosphatidylinositol 3-kinase signaling with increased invasiveness and gelatinase
activity in malignant gliomas.[comment]. J Neurosurg 2001;95:480–488.
Glioma-Genesis 551
medwedi.ru
146 Maity A, Pore N, Lee J, Solomon D, O’Rourke DM: Epidermal growth factor receptor transcrip-
tionally up-regulates vascular endothelial growth factor expression in human glioblastoma cells
via a pathway involving phosphatidylinositol 3⬘-kinase and distinct from that induced by hypoxia.
Cancer Res 2000;60:5879–5886.
147 Pore N, Liu S, Haas-Kogan DA, O’Rourke DM, Maity A: PTEN mutation and epidermal growth
factor receptor activation regulate vascular endothelial growth factor (VEGF) mRNA expression
in human glioblastoma cells by transactivating the proximal VEGF promoter. Cancer Res 2003;
63:236–241.
148 Furnari FB, Lin H, Huang HS, Cavenee WK: Growth suppression of glioma cells by PTEN
requires a functional phosphatase catalytic domain. Proc Natl Acad Sci USA 1997;94:
12479–12484.
149 Li DM, Sun H: PTEN/MMAC1/TEP1 suppresses the tumorigenicity and induces G1 cell cycle
arrest in human glioblastoma cells. Proc Natl Acad Sci USA 1998;95:15406–15411.
150 Tian XX, Pang JC, To SS, Ng HK: Restoration of wild-type PTEN expression leads to apoptosis,
induces differentiation, and reduces telomerase activity in human glioma cells. J Neuropathol Exp
Neurol 1999;58:472–479.
151 Cantley LC, Neel BG: New insights into tumor suppression: PTEN suppresses tumor formation
by restraining the phosphoinositide 3-kinase/AKT pathway: Proc Natl Acad Sci USA 1999;96:
4240–4245.
152 Di Cristofano A, Pandolfi PP: The multiple roles of PTEN in tumor suppression. Cell 2000;100:
387–390.
153 Rasheed BK, Fuller GN, Friedman AH, Bigner DD, Bigner SH: Loss of heterozygosity for 10q
loci in human gliomas. Genes Chromosomes Cancer 1992;5:75–82.
154 Tohma Y, Gratas C, Biernat W, Peraud A, Fukuda M, Yonekawa Y, et al: PTEN (MMAC1) mutations
are frequent in primary glioblastomas (de novo) but not in secondary glioblastomas. J Neuropathol
Exp Neurol 1998;57:684–689.
155 Peraud A, Watanabe K, Plate KH, Yonekawa Y, Kleihues P, Ohgaki H: p53 mutations versus EGF
receptor expression in giant cell glioblastomas. J Neuropathol Expl Neurol 1997;56:1236–1241.
156 Liu W, James CD, Frederick L, Alderete BE, Jenkins RB: PTEN/MMAC1 mutations and EGFR
amplification in glioblastomas. Cancer Res 1997;57:5254–5257.
157 Schmidt EE, Ichimura K, Goike HM, Moshref A, Liu L, Collins VP: Mutational profile of the
PTEN gene in primary human astrocytic tumors and cultivated xenografts. J Neuropathol Exp
Neurol 1999;58:1170–1183.
158 Smith JS, Tachibana I, Passe SM, Huntley BK, Borell TJ, Iturria N, et al: PTEN mutation, EGFR
amplification, and outcome in patients with anaplastic astrocytoma and glioblastoma multiforme.
159 Pershouse MA, Stubblefield E, Hadi A, Killary AM, Yung WK, Steck PA: Analysis of the func-
tional role of chromosome 10 loss in human glioblastomas. Cancer Res 1993;53:5043–5050.
160 Cheney IW, Johnson DE, Vaillancourt MT, Avanzini J, Morimoto A, Demers GW, et al: Suppression
of tumorigenicity of glioblastoma cells by adenovirus-mediated MMAC1/PTEN gene transfer.
Cancer Res 1998;58:2331–2334.
161 Brem S, Cotran R, Folkman J: Tumor angiogenesis: A quantitative method for histologic grading.
162 Ciardiello F, Caputo R, Bianco R, Damiano V, Pomatico G, De Placido S, et al: Antitumor effect and
potentiation of cytotoxic drugs activity in human cancer cells by ZD-1839 (Iressa), an epidermal
growth factor receptor-selective tyrosine kinase inhibitor. Clin Cancer Res 2000;6:2053–2063.
163 Liu Y, Ehtesham M, Samoto K, Wheeler CJ, Thompson RC, Villarreal LP, et al: In situ adenoviral
interleukin 12 gene transfer confers potent and long-lasting cytotoxic immunity in glioma. Cancer
Gene Ther 2002;9:9–15.
164 Besson A, Davy A, Robbins SM, Yong VW: Differential activation of Erks to focal adhesions by
PKC epsilon is required for PMA-induced adhesion and migration of human glioma cells.
Oncogene 2001;20:7398–7407.
165 Bello L, Lucini V, Giussani C, Carrabba G, Pluderi M, Scaglione F, et al: IS20I, a specific
alphavbeta3 integrin inhibitor, reduces glioma growth in vivo. Neurosurgery 2003;52:177–185;
166 Ma HI, Guo P, Li J, Lin SZ, Chiang YH, Xiao X, et al: Suppression of intracranial human glioma
growth after intramuscular administration of an adeno-associated viral vector expressing angio-
statin. Cancer Res 2002;62:756–763.
167 Ashley DM, Sampson JH, Archer GE, Batra SK, Bigner DD, Hale LP: A genetically modified
allogeneic cellular vaccine generates MHC class I-restricted cytotoxic responses against
tumor-associated antigens and protects against CNS tumors in vivo. J Neuroimmunol 1997;78:
34–46.
168 Stewart J: Modulation of the subjective and physiological effects of drugs by contexts and expec-
tations – The search for mechanisms: Comment on Alessi, Roll, Reilly, and Johanson (2002).
[comment]. Exp Clin Psychopharmacol 2002;10:96–98; discussion 101–103.
169 Fan Z, Mendelsohn J: Therapeutic application of anti-growth factor receptor antibodies. Curr Opin
Oncol 1998;10:67–73.
170 Cobleigh MA, Vogel CL, Tripathy D, Robert NJ, Scholl S, Fehrenbacher L, et al: Multinational
study of the efficacy and safety of humanized anti-HER2 monoclonal antibody in women who
have HER2-overexpressing metastatic breast cancer that has progressed after chemotherapy for
metastatic disease. J Clin Oncol 1999;17:2639–2648.
171 Pegram MD, Lipton A, Hayes DF, Weber BL, Baselga JM, Tripathy D, et al: Phase II study of
receptor-enhanced chemosensitivity using recombinant humanized anti-p185HER2/neu mono-
clonal antibody plus cisplatin in patients with HER2/neu-overexpressing metastatic breast cancer
refractory to chemotherapy treatment. J Clin Oncol 1998;16:2659–2671.
172 Slamon DJ, Leyland-Jones B, Shak S, Fuchs H, Paton V, Bajamonde A, et al: Use of chemother-
apy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses
HER2.[comment]. N Engl J Med 2001;344:783–792.
173 Overholser JP, Prewett MC, Hooper AT, Waksal HW, Hicklin DJ: Epidermal growth factor recep-
tor blockade by antibody IMC-C225 inhibits growth of a human pancreatic carcinoma xenograft
in nude mice. Cancer 2000;89:74–82.
174 Prewett M, Rothman M, Waksal H, Feldman M, Bander NH, Hicklin DJ: Mouse-human chimeric
anti-epidermal growth factor receptor antibody C225 inhibits the growth of human renal cell
carcinoma xenografts in nude mice. Clin Cancer Res 1998;4:2957–2966.
175 McCarthy M: Antiangiogenesis drug promising for metastatic colorectal cancer. Lancet 2003;361:
1959.
176 Wikstrand CJ, Hale LP, Batra SK, Hill ML, Humphrey PA, Kurpad SN, et al: Monoclonal anti-
bodies against EGFRvIII are tumor specific and react with breast and lung carcinomas and malig-
nant gliomas. Cancer Res 1995;55:3140–3148.
177 Sampson JH, Crotty LE, Lee S, Archer GE, Ashley DM, Wikstrand CJ, et al: Unarmed, tumor-
specific monoclonal antibody effectively treats brain tumors. Proc Natl Acad Sci USA 2000;97:
7503–7508.
178 Luwor RB, Johns TG, Murone C, Huang HJ, Cavenee WK, Ritter G, et al: Monoclonal antibody
806 inhibits the growth of tumor xenografts expressing either the de2–7 or amplified epidermal
growth factor receptor (EGFR) but not wild-type EGFR. Cancer Res 2001;61:5355–5361.
179 Mishima K, Johns TG, Luwor RB, Scott AM, Stockert E, Jungbluth AA, et al: Growth suppres-
sion of intracranial xenografted glioblastomas overexpressing mutant epidermal growth factor
receptors by systemic administration of monoclonal antibody (mAb) 806, a novel monoclonal
antibody directed to the receptor.[erratum appears in Cancer Res 2001 Oct 15;61(20):7703–7705].
Cancer Res 2001;61:5349–5354.
180 Carter P: Improving the efficacy of antibody-based cancer therapies. Nature Reviews. Cancer
2001;1:118–129.
181 Brady LW: A new treatment for high grade gliomas of the brain. Bull Mem I Acad R Med de Belg
1998;153:255–61; discussion 261–262.
182 Brady LW, Miyamoto C, Woo DV, Rackover M, Emrich J, Bender H, et al: Malignant astrocytomas
treated with iodine-125 labeled monoclonal antibody 425 against epidermal growth factor recep-
tor: A phase II trial. Int J Radia Oncol Biol Phys 1992;22:225–330.
183 Emrich JG, Brady LW, Quang TS, Class R, Miyamoto C, Black P, et al: Radioiodinated (I-125)
monoclonal antibody 425 in the treatment of high grade glioma patients: Ten-year synopsis of a
novel treatment. Am J Clin Oncol 2002;25:541–546.
Glioma-Genesis 553
medwedi.ru
184 Foulon CF, Reist CJ, Bigner DD, Zalutsky MR: Radioiodination via D-amino acid peptide
enhances cellular retention and tumor xenograft targeting of an internalizing anti-epidermal
growth factor receptor variant III monoclonal antibody. Cancer Res 2000;60:4453–4460.
185 Park JW, Hong K, Kirpotin DB, Colbern G, Shalaby R, Baselga J, et al: Anti-HER2 immunolipo-
somes: Enhanced efficacy attributable to targeted delivery. [comment]. Clin Cancer Res
2002;8:1172–1181.
186 Siwak DR, Tari AM, Lopez-Berestein G: The potential of drug-carrying immunoliposomes as
anticancer agents. Commentary re: J. W. Park et al., Anti-HER2 immunoliposomes: Enhanced
efficacy due to targeted delivery. Clin Cancer Res 2002;8:1172–1181. [comment]. Clin Cancer
Res 2002;8:955–956.
187 Zhang Y, Zhu C, Pardridge WM: Antisense gene therapy of brain cancer with an artificial virus
gene delivery system. Molecular Therapy. J Am Soc Gene Ther 2002;6:67–72.
188 Siesjo P, Visse E, Sjogren HO: Cure of established, intracerebral rat gliomas induced by thera-
peutic immunizations with tumor cells and purified APC or adjuvant IFN-gamma treatment.
J Immunother Emphasis Tumor Immunol 1996;19:334–345.
189 Liau LM, Black KL, Prins RM, Sykes SN, DiPatre PL, Cloughesy TF, et al: Treatment of intracra-
nial gliomas with bone marrow-derived dendritic cells pulsed with tumor antigens. J Neurosurg
1999;90:1115–1124.
190 Yamanaka R, Zullo SA, Tanaka R, Blaese M, Xanthopoulos KG: Enhancement of antitumor
immune response in glioma models in mice by genetically modified dendritic cells pulsed with
Semliki forest virus-mediated complementary DNA. J Neurosurg 2001;94:474–481.
191 Akasaki Y, Kikuchi T, Homma S, Abe T, Kofe D, Ohno T: Antitumor effect of immunizations with
fusions of dendritic and glioma cells in a mouse brain tumor model.[comment]. J Immunother
2001;24:106–113.
192 Yu JS, Wheeler CJ, Zeltzer PM, Ying H, Finger DN, Lee PK, et al: Vaccination of malignant
glioma patients with peptide-pulsed dendritic cells elicits systemic cytotoxicity and intracranial
T-cell infiltration. Cancer Res 2001;61:842–847.
193 Kikuchi T, Akasaki Y, Irie M, Homma S, Abe T, Ohno T: Results of a phase I clinical trial of
vaccination of glioma patients with fusions of dendritic and glioma cells. Cancer Immunol
Immunother 2001;50:337–344.
194 Graf MR, Prins RM, Hawkins WT, Merchant RE: Irradiated tumor cell vaccine for treatment of
an established glioma. I. Successful treatment with combined radiotherapy and cellular vaccina-
tion. Cancer Immunol Immunother 2002;51:179–189.
195 Sampson JH, Ashley DM, Archer GE, Fuchs HE, Dranoff G, Hale LP, et al: Characterization of a
spontaneous murine astrocytoma and abrogation of its tumorigenicity by cytokine secretion.
196 Liu MA: DNA vaccines: A review. J Intern Med 2003;253:402–410.
197 Resnicoff M, Sell C, Rubini M, Coppola D, Ambrose D, Baserga R, et al: Rat glioblastoma cells
expressing an antisense RNA to the insulin-like growth factor-1 (IGF-1) receptor are nontumori-
genic and induce regression of wild-type tumors. Cancer Res 1994;54:2218–2222.
198 Yu JS, Burwick JA, Dranoff G, Breakefield XO: Gene therapy for metastatic brain tumors by vac-
cination with granulocyte-macrophage colony-stimulating factor-transduced tumor cells. Human
Gene Ther 1997;8:1065–1072.
199 Andreansky S, He B, van Cott J, McGhee J, Markert JM, Gillespie GY, et al: Treatment of
intracranial gliomas in immunocompetent mice using herpes simplex viruses that express murine
interleukins. Gene Ther 1998;5:121–130.
200 Parker JN, Gillespie GY, Love CE, Randall S, Whitley RJ, Markert JM: Engineered herpes sim-
plex virus expressing IL-12 in the treatment of experimental murine brain tumors. Proc Natl Acad
Sci USA 2000;97:2208–2213.
201 Sobol RE, Fakhrai H, Shawler D, Gjerset R, Dorigo O, Carson C, et al: Interleukin-2 gene therapy
in a patient with glioblastoma. Gene Ther 1995;2:164–167.
202 Schneider T, Gerhards R, Kirches E, Firsching R: Preliminary results of active specific immuniza-
tion with modified tumor cell vaccine in glioblastoma multiforme. J Neuro oncol 2001;53: 39–46.
203 Levitzki A: Protein tyrosine kinase inhibitors as novel therapeutic agents. Pharmacol Ther 1999;
82:231–239.
204 Noonberg SB, Benz CC: Tyrosine kinase inhibitors targeted to the epidermal growth factor receptor
subfamily: Role as anticancer agents. Drugs 2000;59:753–767.
205 Hidalgo M, Siu LL, Nemunaitis J, Rizzo J, Hammond LA, Takimoto C, et al: Phase I and phar-
macologic study of OSI-774, an epidermal growth factor receptor tyrosine kinase inhibitor, in
patients with advanced solid malignancies. J Clin Oncol 2001;19:3267–3279.
206 Shawver LK, Slamon D, Ullrich A: Smart drugs: Tyrosine kinase inhibitors in cancer therapy.
Cancer Cell 2002;1:117–123.
207 Traxler P, Bold G, Buchdunger E, Caravatti G, Furet P, Manley P, et al: Tyrosine kinase inhibitors:
From rational design to clinical trials. Med Res Rev 2001;21:499–512.
208 Druker BJ, Sawyers CL, Kantarjian H, Resta DJ, Reese SF, Ford JM, et al: Activity of a specific
inhibitor of the BCR-ABL tyrosine kinase in the blast crisis of chronic myeloid leukemia and acute
lymphoblastic leukemia with the Philadelphia chromosome. N Engl J Med 2001;344:1038–1042.
209 Kilic T, Alberta JA, Zdunek PR, Acar M, Iannarelli P, O’Reilly T, et al: Intracranial inhibition of
platelet-derived growth factor-mediated glioblastoma cell growth by an orally active kinase
inhibitor of the 2-phenylaminopyrimidine class. Cancer Res 2000;60:5143–5150.
210 Sekulic A, Hudson CC, Homme JL, Yin P, Otterness DM, Karnitz LM, et al: A direct linkage
between the phosphoinositide 3-kinase-AKT signaling pathway and the mammalian target of
rapamycin in mitogen-stimulated and transformed cells. Cancer Res 2000;60:3504–3513.
211 Neshat MS, Mellinghoff IK, Tran C, Stiles B, Thomas G, Petersen R, et al: Enhanced sensitivity
of PTEN-deficient tumors to inhibition of FRAP/mTOR. Proc Natl Acad Sci USA 2001;98:
10314–10319.
212 Vivanco I, Sawyers CL: The phosphatidylinositol 3-kinase AKT pathway in human cancer. Nature
Reviews. Cancer 2002;2:489–501.
213 Jiang K, Coppola D, Crespo NC, Nicosia SV, Hamilton AD, Sebti SM, et al: The phosphoinositide
3-OH kinase/AKT2 pathway as a critical target for farnesyltransferase inhibitor-induced apoptosis.
Mol Cell Biol 2000;20:139–148.
214 Adjei AA: Farnesyltransferase inhibitors. Cancer Chemother Biol Response Modif 2001;19:149–164.
215 Feldkamp MM, Lau N, Guha A: The farnesyltransferase inhibitor L-744,832 inhibits the growth
of astrocytomas through a combination of antiproliferative, antiangiogenic, and proapoptotic
activities. Ann NY Acad Sci 1999;886:257–260.
216 Glass TL, Liu TJ, Yung WK: Inhibition of cell growth in human glioblastoma cell lines by farne-
syltransferase inhibitor SCH66336. Neuro Oncology 2000;2:151–158.
217 Karp JE, Kaufmann SH, Adjei AA, Lancet JE, Wright JJ, End DW: Current status of clinical trials
of farnesyltransferase inhibitors. Curr Opin Oncol 2001;13:470–476.
218 Cloughesy TF, Filka E, Nelson G, Kabbinavar F, Friedman H, Miller LL, et al: Irinotecan treat-
ment for recurrent malignant glioma using an every-3-week regimen. Am J Clin Oncol 2002;25:
204–208.
219 Plate KH, Breier G, Weich HA, Risau W: Vascular endothelial growth factor is a potential tumour
angiogenesis factor in human gliomas in vivo. Nature 1992;359:845–848.
220 Veikkola T, Alitalo K: VEGFs, receptors and angiogenesis. Semin Cancer Biol 1999;9:211–220.
221 Samoto K, Ikezaki K, Ono M, Shono T, Kohno K, Kuwano M, et al: Expression of vascular
endothelial growth factor and its possible relation with neovascularization in human brain tumors.
Cancer Res 1995;55:1189–1193.
222 Fernando NH, Hurwitz HI: Inhibition of vascular endothelial growth factor in the treatment of
colorectal cancer. Semin Oncol 2003;30:39–50.
223 Kirsch M, Santarius T, Black PM, Schackert G: Therapeutic anti-angiogenesis for malignant brain
tumors. Onkologie 2001;24:423–430.
224 Taga T, Suzuki A, Gonzalez-Gomez I, Gilles FH, Stins M, Shimada H, et al: Alpha v-Integrin
antagonist EMD 121974 induces apoptosis in brain tumor cells growing on vitronectin and
tenascin. Int J Cancer 2002;98:690–697.
225 Bauerschmitz GJ, Barker SD, Hemminki A: Adenoviral gene therapy for cancer: from vectors to
targeted and replication competent agents (review). Int J Oncol 2002;21:1161–1174.
226 Lamfers ML, Grill J, Dirven CM, Van Beusechem VW, Geoerger B, Van Den Berg J, et al:
Potential of the conditionally replicative adenovirus Ad5-Delta24RGD in the treatment of malig-
nant gliomas and its enhanced effect with radiotherapy. Cancer Res 2002;62:5736–5742.
Glioma-Genesis 555
medwedi.ru
227 Paulus W, Tonn JC: Basement membrane invasion of glioma cells mediated by integrin receptors.
J Neurosurg 1994;80:515–519.
228 Griscelli F, Li H, Bennaceur-Griscelli A, Soria J, Opolon P, Soria C, et al: Angiostatin gene trans-
fer: Inhibition of tumor growth in vivo by blockage of endothelial cell proliferation associated
with a mitosis arrest. Proc Natl Acad Sci USA 1998;95:6367–6372.
229 Kirsch M, Strasser J, Allende R, Bello L, Zhang J, Black PM: Angiostatin suppresses malignant
glioma growth in vivo. Cancer Res 1998;58:4654–4659.
230 Ma HI, Lin SZ, Chiang YH, Li J, Chen SL, Tsao YP, et al: Intratumoral gene therapy of malignant
brain tumor in a rat model with angiostatin delivered by adeno-associated viral (AAV) vector.
Gene Ther 2002;9:2–11.
231 Tanaka T, Cao Y, Folkman J, Fine HA: Viral vector-targeted antiangiogenic gene therapy utilizing
an angiostatin complementary DNA. Cancer Res 1998;58:3362–3369.
232 Boehm T, Folkman J, Browder T, O’Reilly MS: Antiangiogenic therapy of experimental cancer
does not induce acquired drug resistance.[comment]. Nature 1997;390:404–407.
233 Morimoto T, Aoyagi M, Tamaki M, Yoshino Y, Hori H, Duan L, et al: Increased levels of tissue
endostatin in human malignant gliomas. Clin Cancer Res 2002;8:2933–2938.
234 Peroulis I, Jonas N, Saleh M: Antiangiogenic activity of endostatin inhibits C6 glioma growth. Intl
J Cancer 2002;97:839–845.
235 Sorensen DR, Read TA, Porwol T, Olsen BR, Timpl R, Sasaki T, et al: Endostatin reduces vascu-
larization, blood flow, and growth in a rat gliosarcoma. Neuro Oncology 2002;4:1–8.
236 Sorensen DR, Leirdal M, Iversen PO, Sioud M: Combination of endostatin and a protein kinase
Calpha DNA enzyme improves the survival of rats with malignant glioma. Neoplasia (New York)
2002;4:474–479.
237 Yokoyama Y, Dhanabal M, Griffioen AW, Sukhatme VP, Ramakrishnan S: Synergy between angio-
statin and endostatin: Inhibition of ovarian cancer growth. Cancer Res 2000;60:2190–2196.
Donald M. O’Rourke, MD
502 Stemmler Hall, Department of Neurosurgery
University of Pennsylvania School of Medicine, 36th and Hamilton Walk
Philadelphia, PA 19104 (USA)
Tel. ⫹1 215 898 2871, Fax ⫹1 215 898 9217, E-Mail orourked@mail.med.upenn.edu
Oncolytic Viral Therapy for Glioma

Martine L.M. Lamfersa, Therese Visteda, E. Antonio Chioccab
a
Molecular Neuro-Oncology Laboratories, Neurosurgery Service, Massachusetts
General Hospital, Harvard Medical School, Charlestown, Mass., bDepartment of
Neurological Surgery, The Ohio State University Medical Center, James Cancer
Hospital and Solove Research Institute, Columbus, Ohio, USA
Introduction
Despite progress in understanding the pathogenesis and molecular charac-

teristics of malignant gliomas, successful treatment for these tumors has not
been established and survival for glioma patients has not changed in the last
decades. Gene therapy has received substantial attention as a treatment option
for these aggressive tumors. During the decade of the 1990s, new molecular
techniques yielded gene therapy strategies for the treatment of gliomas using
replication-defective viral vectors. These vectors were used as a tool to deliver
a wide assortment of therapeutic genes such as apoptotic, immune stimulating,
or prodrug activating genes. These developments led to the initiation of a num-
ber of clinical trials by the end of the decade.
The majority of published clinical trials have been based on the thymidine
kinase (TK) paradigm. Cells expressing the viral TK transgene phosphorylate
the prodrug ganciclovir into a phosphorylated derivative, which inhibits the
DNA replication of tumor cells. Thirteen clinical gene therapy trials for malig-
nant glioma, involving a total of 451 patients, have been published to date
[1–13]. Taken together, the results of these studies have not lived up to the
expectations created by preclinical data with regard to therapeutic outcome.
Reasons for this discrepancy have been examined in recent years. The inability
to infect large numbers of tumor cells, the limited spread of the vector within
the tumor mass, and the inefficient infection of individual glioma cells all
appear to have contributed to the poor results obtained in clinical trials.
Prompted by these findings and by an increasing knowledge of cancer cell
genetics and tumor cell infection, a novel concept in cancer therapy was devel-
oped: viral oncolysis, or ‘virotherapy,’ using replication-competent viruses. This
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approach utilizes the inherent cytopathic effects, which result from the normal life
cycle of the virus to kill tumor cells. Once inside the cell, the virus expresses
proteins that interfere with cellular processes to prevent apoptosis or induce cell-
cycle progression. Viral replication and viral protein production eventually cause
lysis of the infected cells and release progeny virus that can infect neighboring
cells, upon which the cycle is repeated. In this way, the virus is capable of
disseminating into a solid tumor mass. The terminology ‘oncolytic virus’ refers to
viruses, which preferentially replicate in tumor cells as opposed to normal cells.
Some naturally attenuated oncolytic viral strains exist that appear to replicate
more efficiently in cancer cells than normal tissue, whereas others acquire
oncolytic qualities through genetic engineering (e.g., herpes simplex virus or
HSV and adenovirus; Ad). The tumor selectivity of oncolytic viruses occurs either
during infection or replication. Oncolytic viral vectors based on Ad, herpes virus,
polio virus, and reovirus have been described as therapeutic agents for malignant
glioma [14–18]. Current research in this field is mainly focused on improving
tumor selectivity and oncolytic potency of these agents. In this chapter the latest
developments and future directions in research for this type of glioma therapy are
described, focusing on selectively replicating herpes- and adeno-viruses.
Herpes Virus as a Tumor-Selective Oncolytic Agent
Herpes Simplex Virus type I (HSV-1) is an enveloped, double-stranded

linear DNA virus with a well-defined segmented genome (unique long and
short segments) of 152 kb which encodes more than 80 genes. About half the
genes are essential for viral replication while the other nonessential genes
encode proteins which support the viral life cycle within the host cell.
HSV-1 is a good candidate oncolytic virus for cancer therapy for the fol-
lowing reasons: extensive knowledge of the HSV-1 genome allows the virus to
be modified to improve safety and efficacy; high-titer production is possible
(typically ⬎1010 infectious particles/ml); the HSV-1 genome contains nonessen-
tial genes that can be replaced with up to 30 kb of therapeutic genes without
significantly affecting virus titers or replication [19]; many nonessential genes
are associated with neurovirulence; neurotropism makes HSV-1 appropriate for
targeted brain tumor therapy [20]; anti-herpetic agents exist which can terminate
uncontrolled viral replication [21]; and insertional mutagenesis which is associ-
ated with retroviral vectors [22] is prevented since the viral genome does not
appear to integrate into the host cell’s DNA [23].
Nevertheless, the HSV-1 and oncolytic viral therapy faces some major chal-
lenges. First, genetic manipulation of the large HSV-1 genome is difficult.
Second, pre-existing immunity in the population must be taken into consideration.
Lamfers/Visted/Chiocca 558
HSV-1 is ubiquitous in the adult population and 50–90% of people over 30 years
of age have HSV antibodies. Neurotoxicity of HSV-1 also represents a challenge
since the wild-type virus is capable of propagating in neurons and glia, resulting
in necrotizing and potentially fatal encephalitis. Finally, HSV-1 replicates in both
dividing and nondividing cells, such as postmitotic neurons, while cancer therapy
aims for selective targeting of tumor cells. In this respect, selective oncolytic viral
therapy for brain tumors offers the advantage of targeting islands of replicating
tumor and endothelial cells (tumor angiogenesis), amidst the nonreplicating tissue
of the central nervous system.
Several different strategies have been applied to generate tumor-selective
HSV-1 vectors. These vectors are designed to target cells, which have altered
signal-transduction pathways that promote tumorigenesis. Mutations or deletions
in the viral genome are commonly introduced to eliminate the expression of
specific viral proteins, which render the mutant vector dependent on the tumor
cells for viral propagation. Some of these viral proteins are associated with neu-
rotoxicity and a dual beneficial effect is obtained by mutating these genes.
TK and Ribonucleotide Reductase HSV-1 Mutants

Many viruses, including HSV-1, encode enzymes, which are needed for the
virus to propagate in quiescent cells. Some of these proteins are similar to cel-
lular enzymes, which are expressed in dividing and thus in cancer cells, but not
in normal nondividing cells. This is the case for the enzymes TK and ribonu-
cleotide reductase. The first attenuated HSV-1 vector designed for brain tumor
therapy was targeted to the p16/Retinoblastoma (Rb) pathway by deleting the
viral TK gene, making viral replication dependent on the presence of TK in
tumor cells [24]. This so-called replication-conditional HSV-1 vector prolonged
survival in animal brain tumor models. Neurotoxicity was retained, however, and
combined with the loss of susceptibility to anti-viral therapy with acyclovir or
ganciclovir, the mutants were unsuitable for clinical application. The value of
attenuating HSV-1 vectors for a tumor targeting was nevertheless demonstrated.
The HSV-1 equivalent to cellular RR is the ICP6 protein. HSV-1 with
mutations in the ICP6 gene are targeted to tumor cells with a defect in the
p16/Rb pathway due to their up-regulation of cellular RR [25]. The ICP6-
mutant vector has been shown to replicate with a 100-fold greater viral titer in
cancer cells as compared to normal cells [26]. The vector exhibited anti-tumoral
efficacy and decreased neurovirulence in animal glioma models, and is one of
the two most intensively studied single mutant HSV-1 vectors.
g1–34.5 Mutant HSV-1 Vectors

The ␥1–34.5 gene is a major determinant of HSV-1 neurotoxicity. Further,
␥1–34.5 serves two functions which are important for viral replication. The
Oncolytic Viral Therapy for Glioma 559
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protein complexes with proliferating cell nuclear antigen and thus allows for
viral replication [27]. The cellular counterpart is GADD34, which inactivates
proliferating cell nuclear antigen in dividing cells. The second function of
␥1–34.5 is to override host cell defence mechanisms elicited as a response to
viral infection. Mutation in the ␥1–34.5 gene renders the HSV-1 vector repli-
cation conditional and at the same time neurovirulence is reduced. The
␥1–34.5 mutant is the second of the two most studied single HSV-1 mutants
and shows the lowest neurovirulence of all the single mutants. A ␥1–34.5-
deleted mutant (designated 1716) has progressed to phase 1 clinical trial in
two studies including 21 patients altogether [28, 29]. In both studies, patients
with recurrent malignant glioma, refractory to conventional therapy, were
treated. The HSV-1 vector was well tolerated at doses up to 105 infectious par-
ticles after stereotactic intratumoral injection, and no adverse events were
reported. In the first trial, multiple injections of 1 ml total volume were
performed. No viral DNA was detected by PCR in serum and buccal swab
samples taken from five patients, four out of nine patients were alive at 14
months after the treatment [28]. In the second trial, patients were injected with
the virus followed by tumor resection after 4–9 days, and virus replication was
detected in the tumor tissue. In some patients the amount of recovered virus
exceeded the input dose [29]. One thousand seven hundred and sixteen
patients have also been clinically tested for advanced melanoma [30]. In 3
patients receiving multiple intranodular injections, histopathological analyses
revealed tumor necrosis and HSV antigen presence in the tumor only.
HSV-1 Vectors with Dual Mutations

Single mutant HSV-1 vectors are potentially associated with the risk of
restoring a wild-type phenotype [31]. Concern about this risk led to the design
of vectors with dual mutations. The most studied combination virus is a condi-
tionally replicating derivative of HSV-1 with both deletions of the ␥1–34.5 gene
and the ICP6 gene inactivated by insertion of the ␤-galactosidase gene [32].
G207 has been tested in a phase I clinical trial in patients with recurrent glioma
[33]. Twenty-one patients were included and single injections of doses up to
3 ⫻ 109 infectious particles were administered. No adverse events were
observed and the virus was well tolerated to the extent that no maximum toler-
ated dose was achieved. Promisingly, 2 patients were still alive 4 years after the
treatment.
Another attenuated HSV-1 vector currently being studied intensively for
cancer therapy is NV1020, which originally was developed as a herpes vaccine
that never proved successful. A phase I clinical trial with NV1020 is ongoing
for colorectal liver metastases, with the virus administered through the hepatic
artery [34].
Tumor-Specific Promoters for Driving HSV-1 Replication
Tumor-selective replication of viral vectors can be achieved by linking viral
gene transcription to promoters being expressed only in dividing or malignant
cells. Myb34.5 represents a transcriptionally potentiated vector. This mutant
vector places the ␥1–34.5 gene under the control of the promoter of B-Myb,
a cell-cycle regulatory transcription factor which is overexpressed in cancer cells
with mutations in the p16/Rb tumor suppressor pathway. Myb34.5 also carries
a deletion in the ICP6 gene.
Another HSV-1 mutant carries a deletion in one of the viral genes which are
essential for replication, ICP4. The ICP4 gene is reintroduced into the HSV-Tk
gene, which is then inactivated, and expression of ICP4 is regulated by the
albumin promoter. In adults, albumin is only expressed in hepatocytes and hepa-
tocellular carcinomas. The mutant has shown selective replication in hepatocel-
lular carcinomas in mice, with sparing of the normal hepatocytes [35, 36]. More
recently, a second ICP4-deleted vector has been generated using the calponin
promoter to drive HSV-1 replication in soft tissue and bone tumors [37]. A sim-
ilar transcriptionally regulated replication strategy has been described for Ad
vectors (see below).
Adenovirus as Tumor-Selective Oncolytic Agent
Adenoviruses (Ad) are attractive candidates as oncolytic viruses due to

their low pathogenicity and their efficiency in infecting target cells. In addition,
an extensive knowledge of the Ad genome allows genetic engineering to mod-
ify wild-type viruses to improve their safety and efficacy. The Ad replicative
process (fig. 1) is made up of the following steps. First, primary attachment of
the Ad particle to the surface of the host cell via its fiberknob to the coxsackie
and Ad receptor (CAR). The CAR-docked particles interact with ␣v integrins,
which promote virus internalization by endocytosis. Inside the cell the Ad
escapes from the endosome and is transported toward the cell nucleus, during
which the virus particle is partially broken down. In the nucleus the viral DNA
remains episomal and is transcribed. The Ad proteins encoded by the early
regions of the Ad genome are expressed first, upon which the Ad genome is
replicated, followed by the expression of Ad proteins encoded by the late
regions. Progeny Ad particles are assembled and the induction of cell death
leads to the release of Ad progeny from the cell [38–40].
For application of replication-competent Ad as oncolytic agents, efforts
have been aimed at improving the safety of the virus by constructing genetically
modified Ad mutants. Three approaches have been described to render Ad
propagation selective for tumor cells: deletions or mutations in viral genes
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Fig. 1. Life cycle of the adenovirus.
essential for replication in nonmalignant cells; insertion of tumor-specific pro-

moters driving viral replication (transcriptional targeting); modification of viral
tropism to limit infection to tumor cells (transductional targeting).
Ad E1 Gene Modifications
For the first approach, it is necessary to make mutations or deletions in the
E1 region of the Ad genome. This region contains two genes, E1A and E1B,
which encode proteins required for a productive Ad lytic cycle [41]. E1 proteins
induce cell cycle activation and inhibit apoptosis by binding regulatory proteins
such as p53 and the retinoblastoma gene product (pRb) in the host cell. In the-
ory, Ad mutants unable to inactivate p53 or pRb would propagate poorly in cells
expressing these proteins, but more efficiently in tumor cells where p53 or pRb
is in most cases already inactive. Using this approach, an E1B-mutated Ad,
termed ONYX-015, was constructed which lacked the E1B p53-binding viral
protein, intended to enable selective replication of this virus in p53-deficient
cells [42].
Some controversy exists as to whether or not the deletion of E1B p53-
binding viral protein is the sole mechanism by which ONYX-015 has selective
replication capacity in malignant cells [43, 44]. Nevertheless, this oncolytic
Ad proved to be very effective in a wide range of preclinical models for vari-
ous tumor types, including glioma, cervical carcinoma, laryngeal carcinoma,
colorectal carcinoma, and head and neck cancer, with reports on complete
regression of xenograft tumors and long-term survival [42, 45–47]. Based on
these studies ONYX-015 entered phase I clinical trials and was the first Ad to
reach clinical testing. Multiple trials of ONYX-015 in over 300 cancer
patients, using both intratumoral and intravascular delivery techniques, have
established safety and demonstrated selective activity of the virus within
malignant tissue [48–50]. However, despite biological activity of ONYX-015,
clinical benefit was not seen in most patients until viral treatment was also
combined with chemotherapy in patients with head and neck cancer [51].
Currently, ONYX-015 is also under investigation in a phase I dose-escalating
study in which patients with recurring malignant glioma are treated by intra-
tumoral injections. Preliminary results indicated that there were no serious
adverse events attributable to ONYX-015 administration in the brain [Chiocca
et al., unpubl. observations].
On a similar theoretical basis, Ad were engineered to replicate selectively
in tumor cells with lesions in the pRb pathway. This was accomplished by delet-
ing 24 bp from the E1A gene, which abolishes the pRb-binding capacity of the
E1A protein [52, 53]. This Ad mutant, known as Ad⌬24 or Addl922–947,
demonstrated reduced replication in nonproliferating normal cells relative to
other gene-deleted and wild type Ad. The anti-cancer efficacy of these agents
was confirmed in vitro and in heterotopic xenograft animal models of human
glioma and breast cancer [52, 53]. Strict tumor selectivity of E1A-mutated Ad
has, however, been challenged and additional mutations may be needed to
enhance pRb-selectivity [54].
Tumor-Specific Promoters
Another approach to constructing tumor-selective Ad involves the substitu-
tion of a viral promoter with a promoter of a tumor-associated antigen to drive
viral replication in tumor cells. The tumor-specific promoters are generally
inserted into the E1A promoter region of the Ad. The rationale behind these
vectors is that expression of the early viral gene E1A and therefore the whole Ad
transcription program will depend on the activity of these tumor specific pro-
moters. A number of tumor-selective oncolytic Ad have been developed utilizing
this approach for a number of indications: for prostate carcinoma using the
prostate-specific antigen (PSA) [55]; for breast cancer using estrogen-responsive
elements [56]; and for hepatocellular carcinoma using the ␣-fetoprotein pro-
moter [57]. Both specificity and efficacy of these vectors were demonstrated in
vitro and in vivo. The PSA promoter-driven Ad, CV-706, was tested in a phase I
clinical trial for prostate cancer and demonstrated clinical activity as reflected by
changes in serum PSA levels [58]. Recently, Post et al. [59] described the devel-
opment of a hypoxia-dependent replicative Ad using the hypoxia-inducible
factor promoter to drive E1A. This virus-induced cytolysis of tumor cell lines
including glioma in a hypoxia-dependent manner in cell culture. Other putative
tumor-specific promoters which have been described in the context of repli-
cation-competent Ad, and which may be of interest for testing in glioma are
Midkine [60], telomerase reverse transcriptase [61], and osteocalcin [62] pro-
moters. Astrocyte-specific promoters such as glial fibrillary acidic protein or
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nestin also have been reported to drive transgenes delivered by nonreplicating
Ad vectors to glioma [63, 64]. However, these approaches have thus far not been
described for replication-competent Ad.
Modification of Ad Tropism
The third strategy to achieve tumor-targeted Ad involves the modification
of Ad tropism in order to limit Ad infection to certain cell types and to improve
the infection efficiency. For glioma, the first limitation for an efficient Ad
infection is low levels of Ad receptor CAR expression on the tumor cell mem-
brane [65–68]. To overcome this barrier, Ad can be retargeted towards other
molecules highly expressed on the glioma cell membranes such as epidermal
growth factor (EGFR) and ␣v integrins. This concept was demonstrated using
bispecific antibodies directed towards EGFR on one end and the Ad fiber knob
on its other end. Preincubation of the Ad vector with the bispecific antibodies
resulted in CAR-independent glioma cell infection and improved the infection
of primary glioma cell cultures obtained from tumor material [65, 67]. In the
context of replication-competent Ad, the use of bispecific antibodies to redirect
Ad cell binding will only improve the first infection round, as the progeny Ad
will not have access to the bispecific antibody. This limitation may be overcome
by the insertion of an expression cassette encoding the bispecific antibody into
the genome of the Ad.⌬24 Ad, thereby allowing the progeny viruses to become
retargeted. Using this method, the EGFR-targeted Ad.⌬24 efficiently killed
primary human CAR-deficient brain tumor specimens that were refractory to
the parent control virus [69].
Another approach used to target Ad to glioma cells involves the insertion
of an integrin-binding peptide, arg-gly-asp into the fiber of the virus, allowing
it to make its primary attachment to integrins. Using replication-deficient Ad
vectors encoding luciferase, it was demonstrated that arg-gly-asp modifications
drastically enhanced the infection efficiency of various tumor cells including
glioma [65, 70, 71]. Arg-gly-asp-modified Ad⌬24 virus demonstrated that
improved infection efficiency translates to markedly enhanced oncolysis in pri-
mary glioma cell lines and impressive anti-glioma activity in subcutaneous and
intracranial glioma xenografts [66, 72].
Retargeting of oncolytic Ad to glioma cells was demonstrated by Shinoura
et al. [73], where an increased cytopathic effect was found when ONYX-015
was modified with a stretch of twenty lysine residues at the C-terminus of the
fiber, both on glioma cells and in subcutaneous xenografts. Alternative mole-
cules that have proven useful for redirecting Ad attachment and entry and which
may be of interest to targeting glioma are the fibroblast growth factor receptor
[74], folate receptor [75], transferrin receptor [76] and vascular endothelial
growth factor receptor [77].
An alternative strategy to genetic retargeting of Ad involves the substitu-
tion of Ad fibers with those from a different Ad serotype or host range that is
capable of binding to a cell surface receptor other than CAR. It has been
demonstrated that such chimerics can enhance the infection of various tumor
cell lines and reduce liver transduction after intravenous injections in mice
[78–81].
All the above-described targeting approaches were designed to overcome
low CAR expression and improve the infectability of tumor cells. However, for
achieving true tumor selective infection, it is essential to eliminate binding of the
virus to nontarget cells by abolishing the native tropism of Ad. Einfeld et al. [82]
described the so-called doubly ablated Ad vectors which lacked both CAR-
binding as well as the ␣v integrin-binding capacities, and showed a dramatic
reduction in the infection of healthy organ tissues. Combining this approach with
tumor-specific targeting strategies is expected to greatly enhance the tumor-
specificity of Ad vectors. Support for this approach comes from studies in which
EGFR targeting technology was combined with doubly ablated Ad vectors.
Infection of organotypic glioma spheroids and normal brain explants from
the same patients with EGFR-targeted doubly ablated vectors exhibited up to
38-fold-improved tumor-to-normal brain targeting index compared to native
vectors [83]. Studies in immune-competent rats with doubly ablated vectors sup-
port in vitro findings of abolished brain transduction using these vectors.
However, while transduction was reduced by more than 95%, inflammation
was not reduced compared to wild-type vectors, demonstrating that brain
inflammation occurs independently of Ad binding and infection of cells;
alternative strategies are required to circumvent this problem [84].
Replication-competent Ad with ablated CAR and integrin binding in
combination with tumor-specific targeting moieties have thus far not been
described; this approach would be expected to greatly improve tumor-specific
infection and oncolysis of malignant tissues.
Improving Oncolytic Potency
Although preliminary data from ongoing clinical trials with oncolytic

viruses provide evidence for an anti-tumor effect, it appears that often the tumor
proliferation out-paces vector replication. This phenomenon can, at least in
part, be explained by the fact that the viruses used have a relatively slow life
cycle and are quite immunogenic. Therefore, investigators are attempting to
bolster the therapeutic potential of these agents by interfering with the immune
response or improving the oncolytic potency of the viruses. The latter can be
achieved by combining gene transfer with other modes of anti-tumor therapy
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such as chemotherapy and radiotherapy or by insertion of other genes into the
viral genomes.
Evading the Anti-Viral Immune Response

The immune response of the body to Ad and herpes virus follows a simi-
lar pattern, consisting of an immediate innate response and a slower adaptive
response. The nonspecific early occurring immune response contributes the
largest effect to the elimination of the viruses [85]. This innate response
includes the activation of the complement cascade [86] and recruitment and
activation of macrophages, neutrophils, and natural killer cells, which kill
infected cells either directly or indirectly by secreting anti-viral cytokines and
chemokines [87, 88]. The recruitment and activation of antigen-presenting cells
are essential for the development of an adaptive immune response [89, 90]. The
adaptive immune response is elicited when the viral antigens are presented to
T-helper cells, resulting in the activation and secretion of cytokines by the
T-helper cells and the maturing of B-cells into plasma cells. Plasma cells pro-
duce large amounts of high-affinity antibodies directed against the infected
cells and viral proteins. Together, these responses can result in a rapid inactiva-
tion of the oncolytic viruses and impede their therapeutic efficacy.
For this reason, several strategies have been developed to circumvent early
inactivation of the virus by the immune system. Initially these studies were per-
formed using replication-deficient vectors in the interest of improving and pro-
longing transgene expression.
Partial immune ablation using cytokines or CTL4A-Ig can lead to persistent
Ad gene expression in mouse lung and liver [91, 92]. The anti-cancer drugs
etoposide and cyclophosphamide (CPA) also were shown to enhance intratu-
moral transgene expression in immunocompetent mice [93]. Production of neu-
tralizing antibodies to Ad and cytotoxic T lymphocyte mediated lysis of virally
transduced cells was significantly suppressed in these animals. In a study using
herpes vectors for gene transfer to neurons in the spinal cord, the coadministra-
tion of cyclosporine A led to a more persistent transgene expression in the
infected cells [94]. The inactivating role of the complement system is under-
scored by studies of Ikeda et al. [95] who demonstrated that rodent plasma
inhibits cell transduction by Ad and herpes vectors. In vitro inactivation of com-
plement with mild heat treatment of the serum restored transduction efficiency.
In vivo, complement depletion was achieved by administering cobra venom
factor prior to the intra-arterial delivery of replication-conditional (oncolytic)
HSV in a rat model for multiple intracerebral tumors. Complement inactivation
led to a strong increase in the initial infection efficiency of the tumors. In earlier
reports, it had been demonstrated that administration of CPA enhanced the prop-
agation of the oncolytic virus [96]. Combined treatment of oncolytic HSV with
cobra venom factor and CPA inhibited both the innate and anti-HSV neutraliz-
ing antibody response, and their concerted action prolonged survival of rodents
bearing intracerebral tumors [95].
A completely different approach with regard to the anti-viral immune
response postulates that an intact immune system may actually be of benefit by
eliminating virus infected tumor cells and contributing to tumor regression
[97]. Various strategies to exploit this mechanism have been undertaken,
including the concomitant overexpression of cytokines or growth factors (see
below). Whether the immune response to the virus is helpful or harmful to the
therapeutic potency of oncolytic viruses requires additional investigations into
underlying mechanisms and especially into the precise time course of events. It
is not inconceivable that an initial immune suppression which allows viral
infection and propagation to take place, should be followed by the immune
boosting to evoke an optimal vaccination response.
Armed Therapeutic Viruses

The next approach to improving oncolysis by replicative viruses involves
the insertion of therapeutic transgenes into the viral genome. Such ‘armed’
replication-competent viruses allow the action of therapeutic proteins to be com-
bined with anti-tumor properties of the viral infection. This approach has several
possible advantages. First, due to the amplification of virus within the tumor,
transgene production and spread are highly increased as compared to infection
with the replication-defective vector counterparts. This concept was demonstrated
for Ad using the marker gene Luciferase [98, 99] as well as for HSV using the
␤-galactosidase gene [100–102]. Marked increases in transgene expression were
noted using replication-competent compared to the replication-defective vectors.
In addition, transgenes that have a nonoverlapping toxicity range with the viral-
induced oncolytic effects can be selected in order to maximize their therapeutic
benefit. Transgenes have been inserted that encode prodrug converting enzymes,
immune stimulatory molecules, and apoptosis-enhancing proteins.
Several groups have constructed genetically engineered oncolytic viruses
encoding a prodrug-converting enzyme. These enzymes convert nontoxic pro-
drugs into cytotoxic metabolites and are often soluble to allow spreading within
the tumor. Using this approach, a tumor-selective herpes virus was engineered
encoding the rat cytochrome P450 (CYP2B1) transgene [103]. This liver
enzyme activates the prodrug CPA into an active anti-cancer and immunosup-
pressive metabolite [104]. Addition of CPA potentiated oncolytic effects on this
HSV mutant against cultured tumor cells and subcutaneous tumor xenografts
established in athymic mice [103].
The addition of Ad oncolysis to the HSV-Tk/ganciclovir (enzyme-prodrug)
strategy resulted in a striking improvement in the treatment efficacy in various
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human cancer xenografts, and led to the initiation of a phase I study in patients
with malignant melanoma [105–107]. In human glioma xenografts, the oncolytic
effects of replicating Ad vectors and HSV-Tk/GCV also demonstrated comple-
mentary therapeutic efficacy [98]. The addition of ganciclovir to the treatment
with HSV mutants, which already express HSV-Tk, enhanced the regression of
syngeneic rat gliosarcomas [108]. However, contradictory evidence exists with
regard to the efficacy of combined therapy with HSV-Tk, ganciclovir, and
oncolytic viruses. In fact, when applied with HSV in colorectal cancer cells
[109, 110] and with Ad in lung carcinoma cells [111], ganciclovir actually
decreased the anti-cancer efficacy. Administration of the prodrug appears to
eradicate the infected cells, thereby counteracting further virus production and
viral-induced oncolysis. Therefore, this approach may require optimization of
the administration schedule of prodrug to ensure that sufficient viral replication
and enzyme production is maintained. In addition, the gap junctional ability of
the target cells determines the contribution of the bystander effect of phospho-
rylated ganciclovir to the final anti-cancer efficacy [109, 110].
Multiple prodrug-activating gene therapies have been used simultaneously
in combination with oncolytic Ad. A replicating Ad with double enzyme/prodrug
gene therapy containing the cytosine deaminase and HSV-Tk fusion gene
markedly enhanced oncolysis relative to the isolated viral effect in cancer cells
[112]. The combination of HSV-Tk and CYP21 gene transfer mediated by an
oncolytic HSV provided anti-tumor effects that were more significant than all
other treatment combinations [113].
The second type of genes that have been inserted into the oncolytic viruses
were selected to boost the immune response to the infected tumor cells by stim-
ulating localized inflammatory and/or immune responses. In the context of Ad,
this was first demonstrated using a tumor-selective virus engineered to express
interferon, which strongly enhanced the anti-tumor activity compared to relevant
control Ad in immune-deficient mice bearing breast carcinoma xenografts [114].
Moreover, a number of replication-competent recombinant HSVs that encode
immunostimulatory molecules have been constructed. Andreansky et al. [115]
demonstrated that survival of immunocompetent mice bearing intracerebral
tumors could be prolonged when treated with tumor-selective HSV encoding
interleukin 4 (IL-4) compared to controls and immunohistochemical analysis
demonstrated the marked accumulation of inflammatory cells [115]. Similarly,
oncolytic HSV mutants expressing IL-12, IL-2, or soluble B7–1 immunomodu-
latory molecule were found to produce survival benefit compared to control
viruses in various tumor models including glioma in immunocompetent mice, by
combining oncolytic and immunostimulatory effects [116–119]. Moreover, inser-
tion of the potent immune stimulator granulocyte macrophage colony stimulating
factor into an oncolytic HSV backbone improved the shrinkage or clearance of
tumors compared to control virus. These mice were also protected against rechal-
lenge with tumor cells [120]. These data suggest that not only can the expression
of immunomodulatory molecules potentiate oncolysis, but they may also induce
a level of anti-tumor immunity.
Finally, insertion of transgenes may improve the oncolytic potential of the
replication-competent virus itself. Opportunities for enhancing the anti-tumor
potential of oncolytic viruses are at the final stage of the viral reproductive
cycle that involves lysis of the host cell and release of viral progeny [121].
Oncolysis of cancer cells when compared to lysis of the natural host cells may
be suboptimal due to cancer cell-specific genetic alterations. These alterations
mainly affect pro- and anti-apoptotic pathways that regulate the cell cycle.
Coordinated and timely overexpression of apoptotic factors concomitant with
the viral replicative cycle is expected to enhance the anti-tumor potential of the
oncolytic virus. This concept was demonstrated using the Ad⌬24 oncolytic Ad
engineered to express p53 during late stages of viral replication and which
exhibited up to more than 100-fold enhanced the oncolytic potency on human
cancer cell lines of various tissue origins [122]. In another study, expression of
a dominant-negative I-␬B from a selectively replicating Ad sensitized tumor
cells to recombinant human tumor necrosis factor-␣ (TNF-␣)-mediated apop-
tosis. Using this approach it could be demonstrated that the induction of apop-
tosis during viral DNA replication compromised virus production, whereas
apoptosis induced after virion assembly enhanced viral release from infected
cells and dissemination [123].
Combination Therapy with Conventional Treatments

Combination treatment with conventional therapies offers a number of
advantages. First, enhanced therapeutic efficacy of dual treatments will allow
the administration of lower viral doses to achieve a therapeutic effect, which is
important given the fact that sufficient virus delivery to tumors remains one of
the major hurdles in clinical viral (gene) therapy strategies. Furthermore,
combined treatment allowing lower viral doses may also lower toxic side
effects.
The first studies to describe the effects of dual treatment with an oncolytic
virus and conventional therapy were performed with ONYX-015 Ad. The effi-
cacy of this agent combined with cisplatin and 5-fluorouracil was significantly
greater than either agent alone in nude mouse tumor xenografts [46]. These
results led to the design of a phase II trial, using these agents in patients with
squamous cell carcinoma of the head and neck [51]. The results of that study
mirrored the preclinical data, including the frequent occurrence of complete
remissions in patients treated with combination therapy. Synergy with
chemotherapeutic agents paclitaxel and docetaxel was demonstrated with the
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prostate cancer-specific oncolytic Ad, CV706, in a xenograft model of prostate
cancer [124].
Synergy with chemotherapeutic agents also has been described with herpes
viruses. The oncolytic effect of HSV-1716 in combination with mitomycin was
synergistic in two of five non-small cell lung cancer cell lines in vitro and inhib-
ited tumor growth more efficiently than either agent alone [125]. The combina-
tion treatment of the HSV mutant G207 and vincristine led to strongly enhanced
in vitro cytotoxicity without affecting infection efficiency and replication of
G207 in rhabdomyosarcoma cells. In vivo combination treatment of alveolar
rhabdomyosarcoma using intravenous G207 and vincristine resulted in complete
tumor regression without evidence of regrowth in 5 of 8 animals, whereas none
of the animals receiving either monotherapy were cured [126].
In the context of malignant brain tumors, the combination of oncolytic
viruses with radiotherapy is perhaps more relevant than with chemotherapy con-
sidering the efficacy of standard treatments. Rogulski et al. [127] have studied
the anti-tumor activity of ONYX-015 in combination with radiation in colon car-
cinoma xenografts. ONYX-015 viral therapy combined with radiation improved
tumor control beyond that of either monotherapy. Studies with PSA promoter-
driven oncolytic Ad CV706 in combination with radiotherapy demonstrated an
improvement in the therapeutic response in prostate cancer xenografts without
increasing toxicity; a phase I study was initiated based on those data [58, 128].
In glioma, synergistic oncolytic activity of ONYX-015 with radiotherapy was
demonstrated in subcutaneous xenografts [129]. Also the strong anti-tumor
activity of Ad5-⌬24RGD, the integrin-targeted Ad⌬24 variant, in malignant
glioma could be further enhanced with low-dose irradiation such that the same
therapeutic effect was achieved with a 10-fold lower viral dose [66].
The combination therapy of oncolytic HSV and irradiation has produced
varying results. Whereas a potentiating effect of irradiation on G207 viral
oncolysis in cervical and colorectal cancer xenografts was found [130, 131], no
enhancement of anti-tumor activity was seen when these treatment modalities
were combined in subcutaneous tumor models of human and murine prostate
cancer [132]. Spear et al. [133] also found complementary toxicity between
irradiation and the oncolytic HSV-1 mutant for the ICP6, regardless of cell type,
time, MOI, irradiation dose, or culture conditions, without any evidence of aug-
mented apoptosis or viral replication. In human glioma xenografts, on the other
hand, dual treatment with the HSV-1 ␥134.5 mutant caused a significantly
greater reduction in the volume or the total regression of tumors than either
irradiation or infection alone. This enhanced oncolytic effect of the combined
treatment correlated with 2- to 5-fold enhanced viral replication in irradiated
tumor cells compared to tumors receiving virus only [134]. These results were
extended to a second study in mice bearing intracerebral tumors which received
␥134.5-mutant virus in combination with fractionated radiotherapy. Analysis of
survival data revealed that the interaction between these treatment modalities
was synergistic [135].
In conclusion, results from studies of combination therapy, demonstrating
an enhanced therapeutic efficacy of oncolytic viral therapy over single modality
treatment, are very encouraging. Investigators are successfully combining the
above described strategies with armed therapeutic viruses. A trimodal approach
(lytic virus, double enzyme/prodrug gene therapy, radiation) was found to be
superior to any other combination in carcinoma xenografts. Significant tumor
regression and ultimately 100% tumor cure were reported [136].
Future Perspectives
Future development of oncolytic virus therapy for glioma will rely on the
outcome of multiple lines of ongoing research and merging of the optimal strate-
gies which have evolved from them. Viral engineering is leading to the deve-
lopment of a new generation of genetically engineered truly tumor-selective
and potent oncolytic viruses. A multimodal treatment can be envisioned in which
the optimal vector is combined with optimal dosing and scheduling regime of
conventional therapies.
Combining cytotoxic agents with differing mechanisms of action is an
attractive approach to the treatment of malignant glioma. It allows independent
tumor debulking by specific selectivity of each agent, resulting in broader
spectrum of the oncolytic action. This broader spectrum of oncolysis is
expected to be particularly more efficacious in tumors with strong hetero-
geneity and mutagenic potential, such as glioblastoma multiforme.
The design of novel imaging techniques for oncolytic viruses which mon-
itor the transgene expression, viral distribution, and/or replication of the virus
within the tumor [137] will provide a direct insight into the in vivo biology of
virus-induced tumor oncolysis and can identify potential targets for the
improvement of the vectors or the route of administration.
The route of administration also constitutes an important field of research.
Results from clinical trials have demonstrated the limitations of stereotactic or
direct intratumoral injection of vectors. Hence, investigators are studying the
efficacy of alternative delivery routes to the brain such as convection-enhanced
bulk-flow interstitial delivery, intrathecal and intraventricular injection, and
intravascular infusion with or without the modification of the blood-tumor
barrier [138]. Such new surgical approaches may improve the transduction of
tumor cells, and most importantly aim to reach the infiltrated tumor cells in the
surrounding normal brain.
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Ultimately, the field of oncolytic virus therapy will provide a variety of
agents with anti-glioma efficacy. Similar to the conventional drug-testing pro-
grams, different oncolytic viruses may have different ranges of efficacy and
toxicity. Phase I clinical trials will provide us with the answers regarding such
ranges.
References
1 Izquierdo M, Martin V, de Felipe P, Izquierdo JM, Perez-Higueras A, Cortes ML, Paz JF, Isla A,
Blazquez MG: Human malignant brain tumor response to herpes simplex thymidine kinase
(HSVtk)/ganciclovir gene therapy. Gene Ther 1996;3:491–495.
2 Ram Z, Culver KW, Oshiro EM, Viola JJ, DeVroom HL, Otto E, Long Z, Chiang Y, McGarrity GJ,
Muul LM, Katz D, Blaese RM, Oldfield EH: Therapy of malignant brain tumors by intratumoral
implantation of retroviral vector-producing cells. Nat Med 1997;3:1354–1361.
3 Puumalainen AM, Vapalahti M, Yla-Herttuala S: Gene therapy for malignant glioma patients. Adv
Exp Med Biol 1998;451:505–509.
4 Klatzmann D, Valery CA, Bensimon G, Marro B, Boyer O, Mokhtari K, Diquet B, Salzmann JL,
Philippon J: A phase I/II study of herpes simplex virus type 1 thymidine kinase ‘suicide’ gene
therapy for recurrent glioblastoma. Study Group on Gene Therapy for Glioblastoma. Hum Gene
Ther 1998;9:2595–2604.
5 Shand N, Weber F, Mariani L, Bernstein M, Gianella-Borradori A, Long Z, Sorensen AG, Barbier N:
A phase 1–2 clinical trial of gene therapy for recurrent glioblastoma multiforme by tumor trans-
duction with the herpes simplex thymidine kinase gene followed by ganciclovir. GLI328
European-Canadian Study Group. Hum Gene Ther 1999;10:2325–2335.
6 Packer RJ, Raffel C, Villablanca JG, Tonn JC, Burdach SE, Burger K, LaFond D, McComb JG,
Cogen PH, Vezina G, Kapcala LP: Treatment of progressive or recurrent pediatric malignant
supratentorial brain tumors with herpes simplex virus thymidine kinase gene vector-producer cells
followed by intravenous ganciclovir administration. J Neurosurg 2000;92:249–254.
7 Trask TW, Trask RP, Aguilar-Cordova E, Shine HD, Wyde PR, Goodman JC, Hamilton WJ, Rojas-
Martinez A, Chen SH, Woo SL, Grossman RG: Phase I study of adenoviral delivery of the HSV-tk
gene and ganciclovir administration in patients with current malignant brain tumors. Mol Ther 2000;
1:195–203.
8 Harsh GR, Deisboeck TS, Louis DN, Hilton J, Colvin M, Silver JS, Qureshi NH, Kracher J,
Finkelstein D, Chiocca EA, Hochberg FH: Thymidine kinase activation of ganciclovir in recur-
rent malignant gliomas: A gene-marking and neuropathological study. J Neurosurg 2000;92:
804–811.
9 Sandmair AM, Loimas S, Puranen P, Immonen A, Kossila M, Puranen M, Hurskainen H, Tyynela K,
Turunen M, Vanninen R, Lehtolainen P, Paljarvi L, Johansson R, Vapalahti M, Yla-Herttuala S:
Thymidine kinase gene therapy for human malignant glioma, using replication-deficient retroviruses
or adenoviruses. Hum Gene Ther 2000;11:2197–2205.
10 Rainov NG: A phase III clinical evaluation of herpes simplex virus type 1 thymidine kinase and
ganciclovir gene therapy as an adjuvant to surgical resection and radiation in adults with previ-
ously untreated glioblastoma multiforme. Hum Gene Ther 2000;11:2389–2401.
11 Floeth FW, Shand N, Bojar H, Prisack HB, Felsberg J, Neuen-Jacob E, Aulich A, Burger KJ,
Bock WJ, Weber F: Local inflammation and devascularization – In vivo mechanisms of the
‘bystander effect’ in VPC-mediated HSV-Tk/GCV gene therapy for human malignant glioma.
12 Smitt PS, Driesse M, Wolbers J, Kros M, Avezaat C: Treatment of relapsed malignant glioma with
an adenoviral vector containing the herpes simplex thymidine kinase gene followed by ganciclovir.
Mol Ther 2003;7:851–858.
13 Lang FF, Bruner JM, Fuller GN, Aldape K, Prados MD, Chang S, Berger MS, McDermott MW,
Kunwar SM, Junck LR, Chandler W, Zwiebel JA, Kaplan RS, Yung WK: Phase I trial of adenovirus-
mediated p53 gene therapy for recurrent glioma: Biological and clinical results. J Clin Oncol
2003;21:2508–2518.
14 Antonio Chiocca E: Oncolytic viruses. Nat Rev Cancer 2002;2:938–950.
15 Alemany R, Gomez-Manzano C, Balague C, Yung WK, Curiel DT, Kyritsis AP, Fueyo J: Gene
therapy for gliomas: Molecular targets, adenoviral vectors, and oncolytic adenoviruses. Exp Cell
Res 1999;252:1–12.
16 Markert JM, Gillespie GY, Weichselbaum RR, Roizman B, Whitley RJ: Genetically engineered
HSV in the treatment of glioma: A review. Rev Med Virol 2000;10:17–30.
17 Gromeier M, Wimmer E: Viruses for the treatment of malignant glioma. Curr Opin Mol Ther
2001;3:503–508.
18 Coffey MC, Strong JE, Forsyth PA, Lee PW: Reovirus therapy of tumors with activated Ras path-
way. Science 1998;282:1332–1334.
19 Roizman B: The function of herpes simplex virus genes: A primer for genetic engineering of novel
vectors. Proc Natl Acad Sci USA 1996;93:11307–11312.
20 Nishiyama Y: Herpesvirus genes: Molecular basis of viral replication and pathogenicity. Nagoya
J Med Sci 1996;59:107–119.
21 Balfour HH Jr: Antiviral drugs. N Engl J Med 1999;340:1255–1268.
22 Hacein-Bey-Abina S, von Kalle C, Schmidt M, Le Deist F, Wulffraat N, McIntyre E, Radford I,
Villeval JL, Fraser CC, Cavazzana-Calvo M, Fischer AA: Serious adverse event after success-
ful gene therapy for X-linked severe combined immunodeficiency. N Engl J Med 2003;348:
255–256.
23 Mellerick DM, Fraser NW: Physical state of the latent herpes simplex virus genome in a mouse
model system: Evidence suggesting an episomal state. Virology 1987;158:265–275.
24 Martuza RL, Malick A, Markert JM, Ruffner KL, Coen DM: Experimental therapy of human
glioma by means of a genetically engineered virus mutant. Science 1991;252:854–856.
25 Goldstein DJ, Weller SK: Herpes simplex virus type 1-induced ribonucleotide reductase activity
is dispensable for virus growth and DNA synthesis: Isolation and characterization of an ICP6 lacZ
insertion mutant. J Virol 1988;62:196–205.
26 Yoon SS, Nakamura H, Carroll NM, Bode BP, Chiocca EA, Tanabe KK: An oncolytic herpes sim-
plex virus type 1 selectively destroys diffuse liver metastases from colon carcinoma. FASEB
J 2000;14:301–311.
27 Brown SM, MacLean AR, McKie EA, Harland J: The herpes simplex virus virulence factor
ICP34.5 and the cellular protein MyD116 complex with proliferating cell nuclear antigen through
the 63-amino-acid domain conserved in ICP34.5, MyD116, and GADD34. J Virol 1997;71:
9442–9449.
28 Rampling R, Cruickshank G, Papanastassiou V, Nicoll J, Hadley D, Brennan D, Petty R,
MacLean A, Harland J, McKie E, Mabbs R, Brown M: Toxicity evaluation of replication-
competent herpes simplex virus (ICP 34.5 null mutant 1716) in patients with recurrent malignant
glioma. Gene Ther 2000;7:859–866.
29 Papanastassiou V, Rampling R, Fraser M, Petty R, Hadley D, Nicoll J, Harland J, Mabbs R,
Brown M: The potential for efficacy of the modified (ICP 34.5(-)) herpes simplex virus
HSV1716 following intratumoural injection into human malignant glioma: A proof of principle
study. Gene Ther 2002;9:398–406.
30 MacKie RM, Stewart B, Brown SM: Intralesional injection of herpes simplex virus 1716 in
metastatic melanoma. Lancet 2001;357:525–526.
31 Mohr I, Gluzman Y: A herpesvirus genetic element which affects translation in the absence of the
viral GADD34 function. Embo J 1996;15:4759–4766.
32 Mineta T, Rabkin SD, Yazaki T, Hunter WD, Martuza RL: Attenuated multi-mutated herpes sim-
plex virus-1 for the treatment of malignant gliomas. Nat Med 1995;1:938–943.
33 Markert JM, Medlock MD, Rabkin SD, Gillespie GY, Todo T, Hunter WD, Palmer CA,
Feigenbaum F, Tornatore C, Tufaro F, Martuza RL: Conditionally replicating herpes simplex virus
mutant, G207 for the treatment of malignant glioma: Results of a phase I trial. Gene Ther 2000;7:
867–874.
medwedi.ru
34 Fong Y, Kemeny N, Jarganin W: Phase 1 study of a replication-competent herpes simplex oncolytic
virus for treatment of hepatic colorectal metastases; in Am Soc Clin Oncol Ann Meeting, 2002.
35 Miyatake S, Martuza RL, Rabkin SD: Defective herpes simplex virus vectors expressing thymi-
dine kinase for the treatment of malignant glioma. Cancer Gene Ther 1997;4:222–228.
36 Nakamura H, Kasuya H, Mullen JT, Yoon SS, Pawlik TM, Chandrasekhar S, Donahue JM,
Chiocca EA, Chung RY, Tanabe KK: Regulation of herpes simplex virus gamma(1)34.5 expres-
sion and oncolysis of diffuse liver metastases by Myb34.5. J Clin Invest 2002;109:871–882.
37 Yamamura H, Yoshikawa H, Takahashi K: Aberrant methylation and silencing of the calponin gene
in human sarcoma cells. Anticancer Res 2003;23:107–114.
38 Philipson L, Pettersson U, Lindberg U: Molecular biology of adenoviruses. Virol Monogr 1975;14:
1–115.
39 Marechal V, Piolot T: Lytic infection by double-strand DNA viruses and cell cycle alterations.
Pathol Biol (Paris) 2000;48:289–300.
40 Nemerow GR: Cell receptors involved in adenovirus entry. Virology 2000;274:1–4.
41 Shenk T: Adenoviridae: The viruses and their replication; in Fields BN, Knipe DM, Howley PM
(eds): Fields Virology. Philadelphia, Lippincott-Raven, 1996, vol 2, pp 2111–2148.
42 Bischoff JR, Kirn DH, Williams A, Heise C, Horn S, Muna M, Ng L, Nye JA, Sampson-Johannes A,
Fattaey A, McCormick F: An adenovirus mutant that replicates selectively in p53-deficient human
tumor cells. Science 1996;274:373–376.
43 Hall AR, Dix BR, O’Carroll SJ, Braithwaite AW: p53-dependent cell death/apoptosis is required
for a productive adenovirus infection. Nat Med 1998;4:1068–1072.
44 Goodrum FD, Ornelles DA: p53 status does not determine outcome of E1B 55-kilodalton mutant
adenovirus lytic infection. J Virol 1998;72:9479–9490.
45 Heise CC, Williams AM, Xue S, Propst M, Kirn DH: Intravenous administration of ONYX-015,
a selectively replicating adenovirus, induces antitumoral efficacy. Cancer Res 1999;59:2623–2628.
46 Heise C, Sampson-Johannes A, Williams A, McCormick F, Von Hoff DD, Kirn DH: ONYX-015,
an E1B gene-attenuated adenovirus, causes tumor-specific cytolysis and antitumoral efficacy that
can be augmented by standard chemotherapeutic agents. Nat Med 1997;3:639–645.
47 Geoerger B, Grill J, Opolon P, Morizet J, Aubert G, Terrier-Lacombe MJ, Bressac De-Paillerets B,
Barrois M, Feunteun J, Kirn DH, Vassal G: Oncolytic activity of the E1B-55 kDa-deleted adeno-
virus ONYX-015 is independent of cellular p53 status in human malignant glioma xenografts.
Cancer Res 2002;62:764–772.
48 Post LE: Selectively replicating adenoviruses for cancer therapy: An update on clinical development.
Curr Opin Investig Drugs 2002;3:1768–1772.
49 Reid T, Warren R, Kirn D: Intravascular adenoviral agents in cancer patients: Lessons from clini-
cal trials. Cancer Gene Ther 2002;9:979–986.
50 Kirn D: Clinical research results with dl1520 (Onyx-015), a replication-selective adenovirus for
the treatment of cancer: What have we learned? Gene Ther 2001;8:89–98.
51 Khuri FR, Nemunaitis J, Ganly I, Arseneau J, Tannock IF, Romel L, Gore M, Ironside J,
MacDougall RH, Heise C, Randlev B, Gillenwater AM, Bruso P, Kaye SB, Hong WK, Kirn DH:
A controlled trial of intratumoral ONYX-015, a selectively-replicating adenovirus, in combination
with cisplatin and 5-fluorouracil in patients with recurrent head and neck cancer. Nat Med 2000;
6:879–885.
52 Fueyo J, Gomez-Manzano C, Alemany R, Lee PS, McDonnell TJ, Mitlianga P, Shi YX, Levin VA,
Yung WK, Kyritsis AP: A mutant oncolytic adenovirus targeting the Rb pathway produces anti-
glioma effect in vivo. Oncogene 2000;19:2–12.
53 Heise C, Hermiston T, Johnson L, Brooks G, Sampson-Johannes A, Williams A, Hawkins L,
Kirn D: An adenovirus E1A mutant that demonstrates potent and selective systemic anti-tumoral
efficacy. Nat Med 2000;6:1134–1139.
54 Johnson L, Shen A, Boyle L, Kunich J, Pandey K, Lemmon M, Hermiston T, Giedlin M,
McCormick F, Fattaey A: Selectively replicating adenoviruses targeting deregulated E2F activity
are potent, systemic antitumor agents. Cancer Cell 2002;1:325–337.
55 Rodriguez R, Schuur ER, Lim HY, Henderson GA, Simons JW, Henderson DR: Prostate attenu-
ated replication competent adenovirus (ARCA) CN706: A selective cytotoxic for prostate-specific
antigen-positive prostate cancer cells. Cancer Res 1997;57:2559–2563.
56 Hernandez-Alcoceba R, Pihalja M, Wicha MS, Clarke MF: A novel, conditionally replicative
adenovirus for the treatment of breast cancer that allows controlled replication of E1a-deleted
adenoviral vectors. Hum Gene Ther 2000;11:2009–2024.
57 Hallenbeck PL, Chang YN, Hay C, Golightly D, Stewart D, Lin J, Phipps S, Chiang YL: A novel
tumor-specific replication-restricted adenoviral vector for gene therapy of hepatocellular carci-
noma. Hum Gene Ther 1999;10:1721–1733.
58 DeWeese TL, van der Poel H, Li S, Mikhak B, Drew R, Goemann M, Hamper U, DeJong R,
Detorie N, Rodriguez R, Haulk T, DeMarzo AM, Piantadosi S, Yu DC, Chen Y, Henderson DR,
Carducci MA, Nelson WG, Simons JW: A phase I trial of CV706, a replication-competent, PSA
selective oncolytic adenovirus, for the treatment of locally recurrent prostate cancer following
radiation therapy. Cancer Res 2001;61:7464–7472.
59 Post DE, Van Meir EG: A novel hypoxia-inducible factor (HIF) activated oncolytic adenovirus for
cancer therapy. Oncogene 2003;22:2065–2072.
60 Adachi Y, Reynolds PN, Yamamoto M, Wang M, Takayama K, Matsubara S, Muramatsu T,
Curiel DT: A midkine promoter-based conditionally replicative adenovirus for treatment of pedi-
atric solid tumors and bone marrow tumor purging. Cancer Res 2001;61:7882–7888.
61 Huang TG, Savontaus MJ, Shinozaki K, Sauter BV, Woo SL: Telomerase-dependent oncolytic
adenovirus for cancer treatment. Gene Ther 2003;10:1241–1247.
62 Matsubara S, Wada Y, Gardner TA, Egawa M, Park MS, Hsieh CL, Zhau HE, Kao C, Kamidono S,
Gillenwater JY, Chung LW: A conditional replication-competent adenoviral vector, Ad-OC-E1a, to
cotarget prostate cancer and bone stroma in an experimental model of androgen-independent
prostate cancer bone metastasis. Cancer Res 2001;61:6012–6019.
63 Vandier D, Rixe O, Besnard F, Kim M, Rikiyama T, Goldsmith M, Brenner M, Gouyette A,
Cowan KH: Inhibition of glioma cells in vitro and in vivo using a recombinant adenoviral vec-
tor containing an astrocyte-specific promoter. Cancer Gene Ther 2000;7:1120–1126.
64 Kurihara H, Zama A, Tamura M, Takeda J, Sasaki T, Takeuchi T: Glioma/glioblastoma-specific
adenoviral gene expression using the nestin gene regulator. Gene Ther 2000;7:686–693.
65 Grill J, Van Beusechem VW, Van Der Valk P, Dirven CM, Leonhart A, Pherai DS, Haisma HJ,
Pinedo HM, Curiel DT, Gerritsen WR: Combined targeting of adenoviruses to integrins and epi-
dermal growth factor receptors increases gene transfer into primary glioma cells and spheroids.
Clin Cancer Res 2001;7:641–650.
66 Lamfers ML, Grill J, Dirven CM, Van Beusechem VW, Geoerger B, Van Den Berg J, Alemany R,
Fueyo J, Curiel DT, Vassal G, Pinedo HM, Vandertop WP, Gerritsen WR: Potential of the condi-
tionally replicative adenovirus Ad5-Delta24RGD in the treatment of malignant gliomas and its
enhanced effect with radiotherapy. Cancer Res 2002;62:5736–5742.
67 Miller CR, Buchsbaum DJ, Reynolds PN, Douglas JT, Gillespie GY, Mayo MS, Raben D, Curiel DT:
Differential susceptibility of primary and established human glioma cells to adenovirus infection:
Targeting via the epidermal growth factor receptor achieves fiber receptor-independent gene transfer.
Cancer Res 1998;58:5738–5748.
68 Fuxe J, Liu L, Malin S, Philipson L, Collins VP, Pettersson RF: Expression of the coxsackie and
adenovirus receptor in human astrocytic tumors and xenografts. Int J Cancer 2003;103:723–729.
69 van Beusechem VW, Mastenbroek DC, van den Doel P, Lamfers MLM, Grill J, Würdinger T,
Haisma H, Pinedo HM, Gerritsen WR: Conditionally replicative adenovirus expressing a target-
ing adapter molecule exhibits enhanced oncolytic potency on CAR-deficient tumors. Gene Ther
2003; in press.
70 Staba MJ, Wickham TJ, Kovesdi I, Hallahan DE: Modifications of the fiber in adenovirus vectors
increase tropism for malignant glioma models. Cancer Gene Ther 2000;7:13–19.
71 Dmitriev I, Krasnykh V, Miller CR, Wang M, Kashentseva E, Mikheeva G, Belousova N, Curiel DT:
An adenovirus vector with genetically modified fibers demonstrates expanded tropism via utiliza-
tion of a coxsackievirus and adenovirus receptor-independent cell entry mechanism. J Virol
1998;72:9706–9713.
72 Fueyo J, Alemany R, Gomez-Manzano C, Fuller GN, Khan A, Conrad CA, Liu TJ, Jiang H,
Lemoine MG, Suzuki K, Sawaya R, Curiel DT, Yung WK, Lang FF: Preclinical characterization
of the antiglioma activity of a tropism-enhanced adenovirus targeted to the retinoblastoma path-
way. J Natl Cancer Inst 2003;95:652–660.
medwedi.ru
73 Shinoura N, Yoshida Y, Tsunoda R, Ohashi M, Zhang W, Asai A, Kirino T, Hamada H: Highly aug-
mented cytopathic effect of a fiber-mutant E1B-defective adenovirus for gene therapy of gliomas.
Cancer Res 1999;59:3411–3416.
74 Gu DL, Gonzalez AM, Printz MA, Doukas J, Ying W, D’Andrea M, Hoganson DK, Curiel DT,
Douglas JT, Sosnowski BA, Baird A, Aukerman SL, Pierce GF: Fibroblast growth factor 2 retar-
geted adenovirus has redirected cellular tropism: Evidence for reduced toxicity and enhanced anti-
tumor activity in mice. Cancer Res 1999;59:2608–2614.
75 Douglas JT, Rogers BE, Rosenfeld ME, Michael SI, Feng M, Curiel DT: Targeted gene delivery
by tropism-modified adenoviral vectors. Nat Biotechnol 1996;14:1574–1578.
76 Xia H, Anderson B, Mao Q, Davidson BL: Recombinant human adenovirus: Targeting to the
human transferrin receptor improves gene transfer to brain microcapillary endothelium. J Virol
2000;74:11359–11366.
77 Fisher KD, Stallwood Y, Green NK, Ulbrich K, Mautner V, Seymour LW: Polymer-coated adeno-
virus permits efficient retargeting and evades neutralising antibodies. Gene Ther 2001;8:
341–348.
78 Kanerva A, Mikheeva GV, Krasnykh V, Coolidge CJ, Lam JT, Mahasreshti PJ, Barker SD,
Straughn M, Barnes MN, Alvarez RD, Hemminki A, Curiel DT: Targeting adenovirus to the
serotype 3 receptor increases gene transfer efficiency to ovarian cancer cells. Clin Cancer Res
2002;8:275–280.
79 Stevenson SC, Rollence M, Marshall-Neff J, McClelland A: Selective targeting of human cells by
a chimeric adenovirus vector containing a modified fiber protein. J Virol 1997;71:4782–4790.
80 Vigne E, Dedieu JF, Brie A, Gillardeaux A, Briot D, Benihoud K, Latta-Mahieu M, Saulnier P,
Perricaudet M, Yeh P: Genetic manipulations of adenovirus type 5 fiber resulting in liver tropism
attenuation. Gene Ther 2003;10:153–162.
81 Havenga MJ, Lemckert AA, Ophorst OJ, van Meijer M, Germeraad WT, Grimbergen J, van Den
Doel MA, Vogels R, van Deutekom J, Janson AA, de Bruijn JD, Uytdehaag F, Quax PH,
Logtenberg T, Mehtali M, Bout A: Exploiting the natural diversity in adenovirus tropism for ther-
apy and prevention of disease. J Virol 2002;76:4612–4620.
82 Einfeld DA, Schroeder R, Roelvink PW, Lizonova A, King CR, Kovesdi I, Wickham TJ: Reducing
the native tropism of adenovirus vectors requires removal of both CAR and integrin interactions.
J Virol 2001;75:11284–11291.
83 van Beusechem VW, Grill J, Mastenbroek DC, Wickham TJ, Roelvink PW, Haisma HJ, Lamfers ML,
Dirven CM, Pinedo HM, Gerritsen WR: Efficient and selective gene transfer into primary human
brain tumors by using single-chain antibody-targeted adenoviral vectors with native tropism abol-
ished. J Virol 2002;76:2753–2762.
84 Thomas CE, Edwards P, Wickham TJ, Castro MG, Lowenstein PR: Adenovirus binding to the
coxsackievirus and adenovirus receptor or integrins is not required to elicit brain inflammation but
is necessary to transduce specific neural cell types. J Virol 2002;76:3452–3460.
85 Worgall S, Wolff G, Falck-Pedersen E, Crystal RG: Innate immune mechanisms dominate
elimination of adenoviral vectors following in vivo administration. Hum Gene Ther 1997;8:
37–44.
86 Da Costa XJ, Brockman MA, Alicot E, Ma M, Fischer MB, Zhou X, Knipe DM, Carroll MC:
Humoral response to herpes simplex virus is complement-dependent. Proc Natl Acad Sci USA
1999;96:12708–12712.
87 Wakimoto H, Johnson PR, Knipe DM, Chiocca EA: Effects of innate immunity on herpes sim-
plex virus and its ability to kill tumor cells. Gene Ther 2003;10:983–990.
88 Ginsberg HS, Prince GA: The molecular basis of adenovirus pathogenesis. Infect Agents Dis
1994;3:1–8.
89 Guidotti LG, Chisari FV: Noncytolytic control of viral infections by the innate and adaptive
immune response. Annu Rev Immunol 2001;19:65–91.
90 Horwitz MS, Sarvetnick N: Viruses, host responses, and autoimmunity. Immunol Rev 1999;
169:241–253.
91 Kay MA, Holterman AX, Meuse L, Gown A, Ochs HD, Linsley PS, Wilson CB: Long-term
hepatic adenovirus-mediated gene expression in mice following CTLA4Ig administration. Nat
Genet 1995;11:191–197.
92 Yang Y, Trinchieri G, Wilson JM: Recombinant IL-12 prevents formation of blocking IgA anti-
bodies to recombinant adenovirus and allows repeated gene therapy to mouse lung. Nat Med 1995;
1:890–893.
93 Bouvet M, Fang B, Ekmekcioglu S, Ji L, Bucana CD, Hamada K, Grimm EA, Roth JA:
Suppression of the immune response to an adenovirus vector and enhancement of intratumoral
transgene expression by low-dose etoposide. Gene Ther 1998;5:189–195.
94 Mabon PJ, Weaver LC, Dekaban GA: Cyclosporin A reduces the inflammatory response to
a multi-mutant herpes simplex virus type-1 leading to improved transgene expression in sympa-
thetic preganglionic neurons in hamsters. J Neurovirol 1999;5:268–279.
95 Ikeda K, Wakimoto H, Ichikawa T, Jhung S, Hochberg FH, Louis DN, Chiocca EA: Complement
depletion facilitates the infection of multiple brain tumors by an intravascular, replication-conditional
herpes simplex virus mutant. J Virol 2000;74:4765–4775.
96 Ikeda K, Ichikawa T, Wakimoto H, Silver JS, Deisboeck TS, Finkelstein D, Harsh GRT,
Louis DN, Bartus RT, Hochberg FH, Chiocca EA: Oncolytic virus therapy of multiple tumors
in the brain requires suppression of innate and elicited antiviral responses. Nat Med 1999;5:
881–887.
97 Geutskens SB, van der Eb MM, Plomp AC, Jonges LE, Cramer SJ, Ensink NG, Kuppen PJ,
Hoeben RC: Recombinant adenoviral vectors have adjuvant activity and stimulate T cell responses
against tumor cells. Gene Ther 2000;7:1410–1416.
98 Nanda D, Vogels R, Havenga M, Avezaat CJ, Bout A, Smitt PS: Treatment of malignant gliomas
with a replicating adenoviral vector expressing herpes simplex virus-thymidine kinase. Cancer
Res 2001;61:8743–8750.
99 Grill J, Lamfers ML, van Beusechem VW, Dirven CM, Pherai DS, Kater M, Van der Valk P,
Vogels R, Vandertop WP, Pinedo HM, Curiel DT, Gerritsen WR: The organotypic multicellular
spheroid is a relevant three-dimensional model to study adenovirus replication and penetration in
human tumors in vitro. Mol Ther 2002;6:609–614.
100 Ichikawa T, Chiocca EA: Comparative analyses of transgene delivery and expression in tumors
inoculated with a replication-conditional or -defective viral vector. Cancer Res 2001;61:
5336–5339.
101 Boviatsis EJ, Scharf JM, Chase M, Harrington K, Kowall NW, Breakefield XO, Chiocca EA:
Antitumor activity and reporter gene transfer into rat brain neoplasms inoculated with herpes sim-
plex virus vectors defective in thymidine kinase or ribonucleotide reductase. Gene Ther 1994;
1:323–331.
102 Boviatsis EJ, Chase M, Wei MX, Tamiya T, Hurford RK Jr, Kowall NW, Tepper RI, Breakefield XO,
Chiocca EA: Gene transfer into experimental brain tumors mediated by adenovirus, herpes simplex
virus, and retrovirus vectors. Hum Gene Ther 1994;5:183–191.
103 Chase M, Chung RY, Chiocca EA: An oncolytic viral mutant that delivers the CYP2B1 transgene
and augments cyclophosphamide chemotherapy. Nat Biotechnol 1998;16:444–448.
104 Wei MX, Tamiya T, Rhee RJ, Breakefield XO, Chiocca EA: Diffusible cytotoxic metabolites con-
tribute to the in vitro bystander effect associated with the cyclophosphamide/cytochrome P450
2B1 cancer gene therapy paradigm. Clin Cancer Res 1995;1:1171–1177.
105 Wildner O, Morris JC, Vahanian NN, Ford H Jr, Ramsey WJ, Blaese RM: Adenoviral vectors capa-
ble of replication improve the efficacy of HSVtk/GCV suicide gene therapy of cancer. Gene Ther
1999;6:57–62.
106 Wildner O, Blaese RM, Candotti F: Enzyme prodrug gene therapy: Synergistic use of the herpes
simplex virus-cellular thymidine kinase/ganciclovir system and thymidylate synthase inhibitors
for the treatment of colon cancer. Cancer Res 1999;59:5233–5238.
107 Morris JC, Ramsey WJ, Wildner O, Muslow HA, Aguilar-Cordova E, Blaese RM: A phase I study
of intralesional administration of an adenovirus vector expressing the HSV-1 thymidine kinase
gene (AdV.RSV-TK) in combination with escalating doses of ganciclovir in patients with cuta-
neous metastatic malignant melanoma. Hum Gene Ther 2000;11:487–503.
108 Boviatsis EJ, Park JS, Sena-Esteves M, Kramm CM, Chase M, Efird JT, Wei MX, Breakefield XO,
Chiocca EA: Long-term survival of rats harboring brain neoplasms treated with ganciclovir and
a herpes simplex virus vector that retains an intact thymidine kinase gene. Cancer Res 1994;54:
5745–5751.
medwedi.ru
109 Carroll NM, Chase M, Chiocca EA, Tanabe KK: The effect of ganciclovir on herpes simplex
virus-mediated oncolysis. J Surg Res 1997;69:413–417.
110 Yoon SS, Carroll NM, Chiocca EA, Tanabe KK: Cancer gene therapy using a replication-
competent herpes simplex virus type 1 vector. Ann Surg 1998;228:366–374.
111 Lambright ES, Amin K, Wiewrodt R, Force SD, Lanuti M, Propert KJ, Litzky L, Kaiser LR,
Albelda SM: Inclusion of the herpes simplex thymidine kinase gene in a replicating adenovirus
does not augment antitumor efficacy. Gene Ther 2001;8:946–953.
112 Freytag SO, Rogulski KR, Paielli DL, Gilbert JD, Kim JH: A novel three-pronged approach to kill
cancer cells selectively: Concomitant viral, double suicide gene, and radiotherapy. Hum Gene
Ther 1998;9:1323–1333.
113 Aghi M, Chou TC, Suling K, Breakefield XO, Chiocca EA: Multimodal cancer treatment medi-
ated by a replicating oncolytic virus that delivers the oxazaphosphorine/rat cytochrome P450 2B1
and ganciclovir/herpes simplex virus thymidine kinase gene therapies. Cancer Res 1999;59:
3861–3865.
114 Zhang JF, Hu C, Geng Y, Selm J, Klein SB, Orazi A, Taylor MW: Treatment of a human breast
cancer xenograft with an adenovirus vector containing an interferon gene results in rapid regres-
sion due to viral oncolysis and gene therapy. Proc Natl Acad Sci USA 1996;93:4513–4518.
115 Andreansky S, He B, van Cott J, McGhee J, Markert JM, Gillespie GY, Roizman B, Whitley RJ:
Treatment of intracranial gliomas in immunocompetent mice using herpes simplex viruses that
express murine interleukins. Gene Ther 1998;5:121–130.
Sci USA 2000;97:2208–2213.
117 Carew JF, Kooby DA, Halterman MW, Kim SH, Federoff HJ, Fong Y: A novel approach to cancer
therapy using an oncolytic herpes virus to package amplicons containing cytokine genes. Mol
Ther 2001;4:250–256.
118 Wong RJ, Patel SG, Kim S, DeMatteo RP, Malhotra S, Bennett JJ, St-Louis M, Shah JP, Johnson PA,
Fong Y: Cytokine gene transfer enhances herpes oncolytic therapy in murine squamous cell carci-
noma. Hum Gene Ther 2001;12:253–265.
119 Todo T, Martuza RL, Dallman MJ, Rabkin SD: In situ expression of soluble B7–1 in the context of
oncolytic herpes simplex virus induces potent antitumor immunity. Cancer Res 2001;61:153–161.
120 Liu BL, Robinson M, Han ZQ, Branston RH, English C, Reay P, McGrath Y, Thomas SK,
Thornton M, Bullock P, Love CA, Coffin RS: ICP34.5 deleted herpes simplex virus with
enhanced oncolytic, immune stimulating, and anti-tumour properties. Gene Ther 2003;10:
292–303.
121 Kruyt FA, Curiel DT: Toward a new generation of conditionally replicating adenoviruses: Pairing
tumor selectivity with maximal oncolysis. Hum Gene Ther 2002;13:485–495.
122 van Beusechem VW, van den Doel PB, Grill J, Pinedo HM, Gerritsen WR: Conditionally repli-
cative adenovirus expressing p53 exhibits enhanced oncolytic potency. Cancer Res 2002;62:
6165–6171.
123 Mi J, Li ZY, Ni S, Steinwaerder D, Lieber A: Induced apoptosis supports spread of adenovirus
vectors in tumors. Hum Gene Ther 2001;12:1343–1352.
124 Yu DC, Chen Y, Dilley J, Li Y, Embry M, Zhang H, Nguyen N, Amin P, Oh J, Henderson DR:
Antitumor synergy of CV787, a prostate cancer-specific adenovirus, and paclitaxel and docetaxel.
Cancer Res 2001;61:517–525.
125 Toyoizumi T, Mick R, Abbas AE, Kang EH, Kaiser LR, Molnar-Kimber KL: Combined therapy
with chemotherapeutic agents and herpes simplex virus type 1 ICP34.5 mutant (HSV-1716) in
human non-small cell lung cancer. Hum Gene Ther 1999;10:3013–3029.
126 Cinatl J Jr, Cinatl J, Michaelis M, Kabickova H, Kotchetkov R, Vogel JU, Doerr HW,
Klingebiel T, Driever PH: Potent oncolytic activity of multimutated herpes simplex virus G207
in combination with vincristine against human rhabdomyosarcoma. Cancer Res 2003;63:
1508–1514.
127 Rogulski KR, Freytag SO, Zhang K, Gilbert JD, Paielli DL, Kim JH, Heise CC, Kirn DH: In vivo
antitumor activity of ONYX-015 is influenced by p53 status and is augmented by radiotherapy.
Cancer Res 2000;60:1193–1196.
128 Chen Y, DeWeese T, Dilley J, Zhang Y, Li Y, Ramesh N, Lee J, Pennathur-Das R, Radzyminski J,
Wypych J, Brignetti D, Scott S, Stephens J, Karpf DB, Henderson DR, Yu DC: CV706, a prostate
cancer-specific adenovirus variant, in combination with radiotherapy produces synergistic antitu-
mor efficacy without increasing toxicity. Cancer Res 2001;61:5453–5460.
129 Geoerger B, Grill J, Opolon P, Morizet J, Aubert G, Lecluse Y, van Beusechem VW, Gerritsen VW,
Kirn DH, Vassal G: Potentiation of radiation therapy by the oncolytic adenovirus dl1520 (ONYX-
015) in human malignant glioma xenografts. Br J Cancer, in press.
130 Blank SV, Rubin SC, Coukos G, Amin KM, Albelda SM, Molnar-Kimber KL: Replication-selective
herpes simplex virus type 1 mutant therapy of cervical cancer is enhanced by low-dose radiation.
Hum Gene Ther 2002;13:627–639.
131 Stanziale SF, Petrowsky H, Joe JK, Roberts GD, Zager JS, Gusani NJ, Ben-Porat L, Gonen M,
Fong Y: Ionizing radiation potentiates the antitumor efficacy of oncolytic herpes simplex virus
G207 by upregulating ribonucleotide reductase. Surgery 2002;132:353–359.
132 Jorgensen TJ, Katz S, Wittmack EK, Varghese S, Todo T, Rabkin SD, Martuza RL: Ionizing radi-
ation does not alter the antitumor activity of herpes simplex virus vector G207 in subcutaneous
tumor models of human and murine prostate cancer. Neoplasia 2001;3:451–456.
133 Spear MA, Sun F, Eling DJ, Gilpin E, Kipps TJ, Chiocca EA, Bouvet M: Cytotoxicity, apoptosis,
and viral replication in tumor cells treated with oncolytic ribonucleotide reductase-defective her-
pes simplex type 1 virus (hrR3) combined with ionizing radiation. Cancer Gene Ther 2000;7:
1051–1059.
134 Advani SJ, Sibley GS, Song PY, Hallahan DE, Kataoka Y, Roizman B, Weichselbaum RR:
Enhancement of replication of genetically engineered herpes simplex viruses by ionizing radia-
tion: A new paradigm for destruction of therapeutically intractable tumors. Gene Ther 1998;5:
160–165.
135 Bradley JD, Kataoka Y, Advani S, Chung SM, Arani RB, Gillespie GY, Whitley RJ, Markert JM,
Roizman B, Weichselbaum RR: Ionizing radiation improves survival in mice bearing intracranial
high-grade gliomas injected with genetically modified herpes simplex virus. Clin Cancer Res
1999;5:1517–1522.
136 Rogulski KR, Wing MS, Paielli DL, Gilbert JD, Kim JH, Freytag SO: Double suicide gene ther-
apy augments the antitumor activity of a replication-competent lytic adenovirus through enhanced
cytotoxicity and radiosensitization. Hum Gene Ther 2000;11:67–76.
137 Jacobs A, Tjuvajev JG, Dubrovin M, Akhurst T, Balatoni J, Beattie B, Joshi R, Finn R, Larson SM,
Herrlinger U, Pechan PA, Chiocca EA, Breakefield XO, Blasberg RG: Positron emission tomography-
based imaging of transgene expression mediated by replication-conditional, oncolytic herpes sim-
plex virus type 1 mutant vectors in vivo. Cancer Res 2001;61:2983–2995.
138 Rainov NG, Kramm CM: Vector delivery methods and targeting strategies for gene therapy of
brain tumors. Curr Gene Ther 2001;1:367–383.
E. Antonio Chiocca, MD, PhD

Chairman, Department of Neurological Surgery
Dardinger Family Professor of Oncologic Neurosurgery
Director of Neurosurgical Services
The Ohio State University Medical Center
James Cancer Hospital and Solove Research Institute
N-1017 Doan Hall, 410 W. 10th Avenue, Columbus, OH 43210 (USA)
Tel. ⫹1 614 293 9312, Fax ⫹1 614 293 4024, E-Mail chiocca-1@medctr.osu.edu
medwedi.ru
Molecular Neurosurgery in the

Pituitary Gland
Gene Transfer as an Adjunctive Treatment Strategy
Maria G. Castroa,b, Nelson Jovel a,b, Shyam Goverdhana a,b,

Jinwei Hu a,b, John Yu c, Moneeb Ehteshamc, Xiangpeng Yuana,b,
Diana Greengold a,b, Weidong Xiong a,b, Pedro R. Lowensteina,b
a
Gene Therapeutics Research Institute, Cedars-Sinai Medical Center, Research
Pavilion, bDepartment of Medicine, UCLA David Geffen School of Medicine,
c
Maxine Dunitz Neurosurgical Institute, Cedars-Sinai Medical Center,
Los Angeles, Calif., USA
Introduction
Current treatments for pituitary tumors encompass medical, surgical, and

radiotherapeutic methods. These therapies should not be thought to be in com-
petition; optimum results usually are obtained when these three approaches are
combined. It is important to be aware of advantages, disadvantages, and risks of
each therapeutic modality. The aim of pituitary tumor treatment is the eradication
of the tumor, restoration of biochemical and visual abnormalities, and preser-
vation of pituitary function. Treatments should not pose risks, complications or
the possibility of recurrence. Because no single method of treatment can attain
these ideals, choice of treatments will depend on the pathology of the tumor
and the availability of the appropriate skills and facilities. For example, the best
surgical results occur in centers where one surgeon has particular experience
with this type of surgery.
In most cases, pituitary tumors are benign and respond very well to current
treatment modalities, but on some occasions either the patient does not respond
to the treatment, becomes intolerant to the medical therapy, or the tumor recurs
or it becomes invasive. In these instances, new treatment modalities are needed.
Gene therapy, the use of DNA as a drug, is an emerging modality to treat resistant
pituitary tumors, tumors that have become invasive, or tumors which recur and
are difficult to treat. Expression of transgenes within the anterior pituitary (AP)
gland in vitro and in vivo is possible with viral vectors. The ability to induce
or repress transgene expression using small molecules such as tetracyclines
with tet-responsive regulatory elements has enabled experimental paradigms to
uncover the function of newly discovered genes, allow the regulation of trans-
genes expressed during adulthood, and specifically affect production of mRNA
molecules in a cell-type specific and temporal fashion. Several molecular targets
are amenable to genetic therapies to treat pituitary tumors. Once the vectors
(delivery systems) are proven safe and the therapeutic targets undergo additional
testing in preclinical models, it will be possible to introduce gene therapies to treat
human patients harboring nonresponsive and difficult to treat pituitary tumors.
Tumors of the Pituitary Gland
The pituitary gland is responsible for regulating almost every major organ
system in the body. The AP secretes hormones that control hormone production
in other glands/tissues of the body such as the thyroid gland, pancreas, adrenal
cortex, and gonads. Examples include thyroid-stimulating hormone which stimu-
lates the thyroid gland; luteinizing and follicle-stimulating hormones (FSH)
which stimulate the gonads and initiating sexual differentiation in the fetus and
sexual development during puberty; adrenal-corticotropic hormone (ACTH)
which stimulates the adrenal cortex; and prolactin (PRL) which stimulates the
mammary glands and also plays a role in modulating the immune system. The
posterior pituitary gland produces and secretes hormones that regulate water
and salt metabolism. The posterior pituitary, an extension of the neuronal tissue
of the hypothalamus, stores vasopressin and oxytocin hormones produced by the
hypothalamus until they are ready to be released. Because the pituitary gland
regulates such a vast array of functions within the body, diseases of the pituitary
gland can have dramatic body-wide adverse effects.
Tumors are one of the most common diseases affecting the AP gland.
A pituitary tumor can cause damage in two ways: it may cause an endocrine
malfunction or it may damage the surrounding brain tissue through mass effect.
Endocrine malfunctions occur because pituitary tumors can retain the hormone
production capability of their cells of origin, and tumor cells may cause a clinical
syndrome by oversecretion of these hormones. They can also inhibit secretion of
other pituitary hormones by compression effects. Tumors overproducing PRL,
growth hormone (GH), ACTH, gonadotrophins (FSH, luteinizing hormone), or
thyrotrophin often cause a typical clinical syndrome, whereas nonfunctioning
tumors tend to exhibit eventual hypopituitarism, although symptoms may not be
Neurosurgery of Pituitary Gland 581
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readily apparent. For most tumors, the degree of hypersecretion correlates with
the severity of their clinical syndrome. For nonfunctioning tumors, clinical syn-
dromes are caused by tumor mass compressing the surrounding pituitary tissue
and sometimes invading brain structures. Tumors can grow large enough to exert
dangerous pressure effects on surrounding tissue, causing headaches, nausea, or
blindness. Functioning tumors are usually identified while in the microadenoma
stage, because they present with endocrine syndromes. Nonfunctioning tumors,
on the other hand, present no clinical endocrine syndromes, except for even-
tual hypopituitarism, and they are generally not identified until they become
macroadenomas and extend outside the pituitary fossa, becoming locally inva-
sive and causing compression and mass effects. Although endocrine syndromes
often denote functioning microadenomas and pressure effects often denote
nonfunctioning macroadenomas, this does not always hold true.
Autopsy studies suggest that pituitary tumors affect as much as 11% of the
population. While they are often described as ‘benign’ adenomas, this classifi-
cation hides the extent to which these tumors can affect normal bodily function
by way of inducing endocrine abnormalities and compressing surrounding
pituitary and/or brain tissue. Pituitary tumors are classified by their size, with
microadenomas being ⬍10 mm in diameter and macroadenomas being ⬎10 mm
in diameter, by hormones they produce, and by their invasiveness into the sur-
rounding tissue.
Pituitary tumor initiation and progression are not fully understood. While
brain tumor occurrence can sometimes be traced to specific genomic alterations,
no such molecular abnormalities have been found to account for pituitary
tumor formation. Potential causes for tumor formation include abnormalities in
many types of genes, such as genes regulating growth and development, tumor-
suppressor genes which inhibit cell growth and proliferation, and genes con-
trolling programmed cell death. In addition to genomic abnormalities, tumor cell
genesis may be promoted by endocrine and hypothalamic factors as well as
hereditary disposition. All of these potential causes for pituitary tumor formation
are being examined.
Two hypotheses have been developed to address the origin of pituitary
tumors. One is the hypothalamic hypothesis which proposes that pituitary ade-
nomas are caused by abnormalities in the hypothalamus. This possibility is sup-
ported by the fact that genomic alterations seem to be nonsignificant, having
only been found in a minority of cases. The other hypothesis emphasizes local
factors within the pituitary, proposing that pituitary adenomas are caused by an
intrinsic pituitary defect within a particular pituitary cell type, which then by
clonal expansion gives rise to a pituitary adenoma/tumor. The pituitary hypoth-
esis is the more supported of the two, with two main pieces of evidence: lack
of peritumoral hyperplasia in association with pituitary tumors, and the fact that
Castro/Jovel/Goverdhana/Hu/Yu/Ehtesham/Yuan/Greengold/Xiong/Lowenstein 582
it is possible to effectively cure pituitary cancer by way of a complete tumor
excision. Neither of these features would be expected, if hypothalamic over-
stimulation were the dominant tumorgenic mechanism [1].
Below we review current treatment modalities, which in the majority of
cases, are very effective for treating pituitary tumors. Although most pituitary
tumors are clinically well managed, there are some cases in which conventional
treatments fail, and it is for these tumors that we propose the use of gene ther-
apy as a novel approach that could be used in combination with other forms of
treatment.
Treatment
Pituitary tumor treatment, if it is to be effective, must address both the
endocrine malfunctions and oncological concerns. Both issues are adequately
addressed by current treatments such as surgical therapy, pharmacological therapy,
and radiation therapy. These treatments can be used alone or in conjunction with
each other: surgical therapy to eliminate tumor mass, medical therapy to treat
endocrine symptoms and shrink tumor mass, and radiation therapy to offset both
oncological and endocrine concerns. This interdisciplinary endeavor combines
aspects of each treatment into a combined therapeutic strategy. As effective as this
interdisciplinary treatment strategy has been, there is room for improvement.
Current strategies are limited in cases where symptoms cannot be managed. It is
therefore necessary to seek out new therapies capable of managing symptoms
which current treatments cannot, one that incurs fewer side effects, and which
seeks to cure the disease instead of simply treating its symptoms.
Surgical Management of Pituitary Adenomas

Surgery is indicated in cases where there is impingement of the optic chiasma
secondary to mass effect, or where medical management has failed to control the
symptoms of hyperprolactinemia, hypercortisolism, or acromegaly. The choice
for surgical management as well as the specific route of surgical access should
be carefully evaluated in each patient. Overall, surgical therapy provides a symp-
tomatic relief, with postoperative radiotherapy playing an important role in
eliminating residual tumor and preventing recurrence.
Trans-sphenoidal Approach. This is usually the preferred route of access to
the sella turcica and is generally recognized as the optimal method for remov-
ing the intrasellar component of pituitary neoplasms (fig. 1). Initially described
using a transnasal approach by Schloffer in 1907, this method was refined by
Harvey Cushing, who utilized a sublabial incision to obtain surgical access.
Subsequently, Guiot [2] and Hardy [3] introduced fluoroscopy and the opera-
tive microscope for pituitary resection, resulting in widespread popularization
of this approach. More recently, the use of intraoperative navigational devices
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B Pterional (frontotemporal)
C Subfrontal
D Transethmoidal
E Transnasal
A
trans-sphenoidal
Subtemporal
Fig. 1. Surgical approaches to the pituitary gland. Arrows illustrate the main routes
of access to the pituitary. (A) Subtemporal, (B) pterional/frontotemporal, (C) subfrontal,
(D) transethmoidal, (E) transnasal/trans-sphenoidal.
and intraoperative magnetic resonance imaging have added to the efficacy of

this route [4]. In addition to the surgical utility, trans-sphenoidal hypophysec-
tomy has proven to have notable therapeutic benefits over alternate transcranial
approaches, particularly with regard to improvement in tumor-related visual
disturbances [5, 6] and recovery of pituitary function in cases with pre-existing
hypopituitarism [7, 8]. Trans-sphenoidal surgery has proven to be a highly
effective therapy, with certain reports describing greater than 90% tumor control
rates in selected patients with pituitary microadenomas, although success with
larger neoplasms has been more limited [9, 10]. Morbidity associated with this
procedure may involve transient or permanent diabetes insipidus, CSF fistulas,
CSF rhinorrhea, visual loss, stroke, and cranial nerve palsy. Potentially fatal
complications can occur as a result of hypothalamic injury, sepsis secondary
to CSF leak, or by intraoperative damage to carotid arteries. The occurrence of
these complications is, however, very low in the hands of an experienced sur-
geon, with operative mortality reported at between 0.27 and 0.86%. Nonfatal
minor and major complications are estimated to occur in approximately 7% of
patients [11]. Given the relative safety of trans-sphenoidal hypophysectomy, it
has become the surgical procedure of choice. However, there are certain, albeit
rare, cases where the trans-sphenoidal route is contraindicated. In these scenar-
ios, detailed below, a transcranial approach is preferred to obtain optimal tumor
control and for reasons of safety.
Transcranial Approach. In certain settings, the transcranial route may be
preferable to the generally preferred trans-sphenoidal route (fig. 1). In partic-
ular, cases where there is extensive herniation of tumor into the middle fossa
may require primary transcranial approaches to ensure adequate visualization
and resection. Additionally, severely ectatic carotid arteries impinging on the
midline would also preclude trans-sphenoidal access. Hence, it is very important
to obtain proper MRI imaging of the major vessels in proximity to the tumor
prior to deciding on a surgical approach. In some cases, the consistency of a
tumor compressing the optic chiasma may not allow for adequate resection and
decompression. In these cases, transcranial intervention is indicated to ensure
adequate relief of pressure on the optic chiasma. One of the most common trans-
cranial routes adopted is the pterional or frontotemporal approach. Good access
is provided for the removal of laterally extending tumor, and generally the optic
chiasma and carotid artery are in the line of vision. Extreme care must be taken
to preserve the integrity of the carotid artery, and its ophthalmic branch which
can easily be damaged during mobilization of the tumor mass. Overall, the goal
with pterional access, should be adequate and judicious decompression of the
optic apparatus without exposing neural or vascular structures to injury. Other
transcranial routes (fig. 1) that have been utilized for access to pituitary neo-
plasms include the subfrontal, transethmoidal, and subtemporal approaches,
which are generally less popular as they do not provide optimal access to tumor
and prohibit adequate visualization of critical structures.
Radiotherapy for Pituitary Adenomas

It is well established that pituitary adenomas, irrespective of cellular ori-
gin, are generally sensitive to radiotherapy. Given this susceptibility, radiation
therapy may be the primary treatment of choice in certain patients, especially
in those cases where disease is confined to the intrasellar space with no compres-
sion of surrounding structures. In addition, radiotherapy is frequently employed
postoperatively to target residual tumor and minimize tumor recurrence. The
efficacy of radiotherapy in preventing tumor recurrence has been substantiated
in the recent literature with a comprehensive report from Brada et al. [12] describ-
ing an 88% progression-free survival rate at 10 years following the surgery with
routine use of radiotherapy in patients with nonsecretory tumors. In contrast,
Turner et al. [13] reported up to 44% recurrence rates at 10 years postopera-
tively in a cohort of 65 patients with functionless pituitary adenomas, who did
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not receive radiotherapy. Radiation therapy has, however, not been found to be
as effective in preventing the progression of functional adenomas, most likely
as a result of easier detection of recurrence/progression in these cases secondary
to highly sensitive measurement of a secretory marker.
Conventional radiotherapy for pituitary neoplasms utilizes an external beam
approach that concentrates radiation on a significant target area in the brain.
Although effective at achieving tumor control, standard external beam radio-
therapy has been associated with significant side effects. Up to 50% of patients
develop panhypopituitarism following surgery and radiotherapy, with the rapidity
of onset directly related to the radiation dose [14–16]. Additionally, the optic
chiasma is sensitive to radiation, and external beam radiotherapy-induced blind-
ness has been well documented, occurring in up to 1–2% of cases [17–20].
Given the danger of iatrogenic injury with external beam radiation, the
use of precisely directed stereotactic radiosurgery has gained increasing atten-
tion [21]. This technique involves the delivery of a high dose of necrotizing
radiation to a delimited target area, with a sharp fall of radiation at the target
margins resulting in relatively little irradiation to surrounding structures. Current
stereotactic radiosurgical approaches utilize either proton beam therapy, gamma
knife surgery, or linear proton acceleration. Recent reports on the efficacy of
stereotactic radiosurgery for pituitary adenomas have been mixed. Initial stud-
ies have generally focused on the use of radiosurgery on small functional
adenomas [22–25]. Although significant improvements in the outcome were
not documented in these studies, a recent report clearly indicates that the treat-
ment of functional pituitary tumors with stereotactic radiosurgery results in an
accelerated decline in hormone levels compared to that of conventional exter-
nal beam radiotherapy [26]. The role of stereotactic radiosurgery in the man-
agement of nonfunctional adenomas is less clear, as there is currently little data
describing its use in this setting. However, given the efficacy that stereotactic
radiosurgery has demonstrated in the treatment of other intracranial neoplasms,
and as more data related to its use in pituitary tumors becomes available, it is
hoped that there will be an expanded role for this approach in the treatment of
pituitary neoplasms.
Medical Therapy
The aim of any pituitary tumor therapy is to reduce tumor mass and nor-
malize hormone production. Surgery, with the aim of excising tumor mass, is
most often the primary treatment. After tumor mass has been reduced by
surgery, medical therapy is implemented to normalize hormone production.
Normalization of endocrine abnormality is important, as the oversecretion of
hormones continues to effect long term health and can, in some cases, increase
mortality.
Medical therapy is tumor-specific, with certain medications being appro-
priate to treat a given tumor and its associated increased hormone hypersecre-
tion. Dopamine agonists are used to treat prolactinomas (hypersecretion of PRL),
and somatostasin analogues and GH antagonists are implemented to treat
acromegaly (hypersecretion of GH). These tumor-specific medical therapies are
based on the knowledge of releasing and inhibiting hormones and neurotrans-
mitter pathways that regulate pituitary hormone secretion as well as the exis-
tence of particular receptors on pituitary cells that respond to such hormones
and neurotransmitters.
Prolactinomas are the only pituitary tumors for which medical therapy
is used as a primary therapeutic modality. Dopamine agonists used to treat
prolactinomas can reduce tumor mass by triggering shrinkage and reducing
serum PRL levels by reducing PRL release from the pituitary. These drugs can
be so effective as to be the only therapy implemented, without the need of
surgery [27].
In addition to dopamine agonists, the other major class of medical agents
used to treat pituitary tumors is somatostatin analogs. Somatostatin is a physi-
ological inhibitor of GH release, making it ideal for treating patients with
acromegaly. Somatostatin analogs also help to minimize tumor size, but never
to a clinically useful degree. Their clinical use has been enhanced by the intro-
duction of depot preparations of octreotide and lanreotide that can be given at
2–4 week intervals, but they must still be used long term and treatment can
become expensive.
Most patients have favorable responses to these drugs, but responses can
vary. In treating prolactinomas, medical therapy successfully reduces tumor
mass and ameliorates hormone production in about 85–90% of patients [28],
but these therapies are known to elicit a high rate of side effects including, nau-
sea and vomiting, postural hypotension and dizziness, headache, constipation,
and depression. Some patients cannot tolerate these side effects, and so despite
their effectiveness in treating symptoms, these drugs cannot always be used.
Dopamine agonists and somatostatin analogs are not tumoricidal agents.
They act by inhibiting target cells and hormone release. In doing so they can
reduce, in some cases, the tumor size. Therefore, in order to be effective they
must be taken indefinitely. Interruption of the dose ensures an immediate return
of increased hormone levels and tumor size to pretreatment levels. These drugs
may help manage symptoms by reducing tumor size and normalizing hormone
production, but do not cure the disease.
Gene Therapy for Pituitary Tumors

There is as yet no treatment that results in a complete cure for any of the
different pituitary tumor types. Current progress in endocrine assays, imaging
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techniques, trans-sphenoidal microsurgery, receptor-mediated pharmacology, and
radiotherapy has led to most pituitary tumors being manageable. Nevertheless for
some pituitary tumors such as large and locally invasive endocrine tumors, only
partial success has been made possible.
For trans-sphenoidal surgery, even if initial remission rates are positive
there is a recurrence in prolactinomas, acromegaly, Cushing syndrome, and non-
functional adenomas in 24, 8, 12 and 16% of patients respectively [29].
Furthermore, 7% of patients treated for GH-secreting tumors were left with
permanent diabetes insipidus [1]. Furthermore, in terms of the correction of
endocrine abnormality in functional tumors, tiny amounts of tumor residue may
continue to hypersecrete hormones.
Radiation therapy is the widely accepted treatment for residual tumor [30].
Although radiotherapy has been shown to reduce the risk of further tumor
progression, it often damages normal pituitary tissue, frequently leading to
irreversible progressive hypopituitarism [14]. Thus patients require regular
screening and eventual substitution therapy with corticosteroids, GH, thyrox-
ine, and sex steroids. The impact of stereotaxic radiosurgery remains to be
determined, but it is likely that the long-term endocrine consequences will be
similar.
The use of dopamine agonists in reducing serum PRL levels and causing
shrinkage of prolactinomas (drugs such as bromocriptine and cabergoline, or
somatostasin analogs such as octreotide for the treatment of acromegaly) has a
debatable success rate. Despite success in 85–90% of patients, these treatments
are not curative and require lifelong administration. There is also a high rate of
side effects, notably nausea and vomiting, postural hypotension and dizziness,
headache and constipation [28]. Furthermore, in some cases there is resistance
to dopamine agonist therapy with tumors not responding [31].
There is a niche in the treatment of pituitary tumors in which a therapy is
both effective at removal of the tumor mass and corrects the endocrine dys-
function, without the need for lifelong treatment or side effects. Gene therapy
strategies may provide a novel flexible way of engineering not only tumor cells
to eliminate their growth [either by the delivery of ‘suicide’ genes such as
HSV1-TK or by the delivery of enzymes such as tyrosine hydroxylase (TH) to
agonise dopamine], but also the possibility of modifying normal surrounding
cells to inhibit tumor regrowth (fig. 2). Furthermore, the development of regu-
latable gene therapy transcription units under the control of cell-type specific
promoters may have applications in the treatment of pituitary disorders. For
example, the treatment of hypopituitarism could rely upon the controlled pul-
satile release of AP hormones, or regression of tumors by increasing dopamine
levels using TH, where excessive expression of TH may have the same effects
as conventional drug-based dopamine agonists.
Fig. 2. Gene therapy strategy to treat pituitary tumors in humans. MRI scan shows a
lobulated gonadotroph adenoma. Gene therapy could be delivered intratumorally or in the
tumor bed at the time of surgical resection. symbolizes the gene therapy vectors within
the tumor mass itself.
Viral Vectors for Gene Delivery
The aim of gene therapy is to introduce therapeutic genes into tissues, lead-
ing to an efficient and stable expression of the therapeutic molecules and mini-
mizing adverse side effects. Viruses can enter cells and express genetic material
in the nucleus, therefore they are generally much more efficient vectors than non-
viral delivery systems. The most commonly used viral vectors for gene therapy
are adenovirus (Ad), adeno-associated virus (AAV), herpes simplex virus type 1
derived vectors (HSV-1), and retrovirus/lentivirus vectors (table 1). Important
parameters to be considered when comparing gene therapy vectors include: size
limitations for transgene insertion, ease production of virus at high titers, trans-
duction efficiency, ability to infect dividing and/or quiescent cells, stability of
transgene expression, potential to integrate into the host chromosomes, cell-type
specificity, vector-associated toxicity and immunogenicity (tables 2, 3).
Ad Vectors
Ads are a family of DNA virus characterized by an icosohedral, nonen-
veloped capsid containing a linear double-stranded genome. The human Ad,
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Table 1. Gene transfer vehicles used in gene therapy applications
Castro/Jovel/Goverdhana/Hu/Yu/Ehtesham/Yuan/Greengold/Xiong/Lowenstein
Adenovirus HD-Ad HSV-1/r HSV-1/a AAV Retrovirus Vaccinia virus Microinjection Transfection
Size 36 30–36 152 10–30 4.68 3.5–9.2 186 Unlimited Unlimited

Cloning 7.5 ⬃30 30 10–30 2–4.5 ⬃8 30 Unlimited Unlimited
capacity (kb)
Transduction
In vivo? Yes Yes Yes Yes Yes Yes Yes Yes No
In vitro? Yes Yes Yes Yes Yes Yes Yes Yes Yes
Long-term No Yes Yes Yes Yes Yes No ? No
expression
Vaccination Yes Yes Yes Yes Yes Yes Yes – –
Vector titers 1012 1011 108 108 109 107 106–108 – –
(pfu/ml)
HSV-1/r, herpes simplex type 1 recombinant vector; HSV-1/a, herpes simplex type 1 amplicon; pfu/ml, plaque-forming units per ml; HD-Ad,
gutless helper-dependent adenovirus vector.
590
Table 2. Advantages and disadvantages of viral vectors for gene therapy
Virus Maximum Advantages Disadvantages

capacity
Adenovirus 8 kb Broad cell tropism, infection of dividing Inflammatory and immune

and nondividing cells, easy to produce responses, transient
at high titer expression
HD-Ad 36 kb Broad cell tropism, infection of dividing Difficulty in large-scale
and nondividing cells, less production
inflammatory and cellular immune
response, longer term transgene
expression
AAV 5 kb, 10 kb Broad cell tropism, infection of dividing Difficulty in producing pure
(concatamers) and nondividing cells, integration preparations at high titer,
into host genome discrete immune response
HSV 30 kb Broad cell tropism, latency in neurons, Highly toxic
very stable
Lentivirus 10 kb Infection of dividing and nondividing Risk of inserting mutagensis,
cells, integration into host genome risk of seroconversion
serotype 2 and serotype 5, both of subclass C, are approximately 36 kb and encode

genes that are classified into early (E1–E4) and late (L1–L5) viral functions,
depending on whether they are expressed before or after DNA replication [33].
At one end of its genome, the nominal left end, it has an inverted terminal
repeat (ITR) necessary for the initiation of viral DNA replication, and an adja-
cent DNA-packaging signal, while a second ITR is found at the right end.
Virus infection initiates with the Ad fiber protein binding to the coxsackie
virus and Ad receptor on the cell surface [33], followed by a secondary inter-
action between Ad penton and integrins [34]. Ad is internalized by endocytosis,
triggered by the penton-integrin interaction, and escapes from the early endo-
some prior to the formation of lysosome. The virion translocates to the nucleus
along the microtubule network, during which time there is a sequential disas-
sembly of the Ad virion and Ad hexon remains at the nuclear membrane, while
the DNA is released into the nucleus and remains as an episome [35].
The most common first-generation Ad vectors developed for the gene ther-
apy are based on the Ad2 and Ad5 serotypes, replication made defective through
deletions in the E1 region, where the transcriptional cassette can be inserted.
Thus 90% of the wild-type Ad genome is retained in the vector [36]. The
recombinant E1-deleted Ad vector genomes are then transfected into human
293 cells, which express the E1 proteins in trans, allowing for E1-deleted Ad
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Table 3. Possible disadvantages among gene transfer vehicles in gene therapy
Vectors
Adenovirus • Host immune responses: inflammatory and cytotoxic reactions in

patients and depletion of transduced cells
• Host’s humoral immune responses may neutralize adenoviral
vector particles during, or even before, the gene transfer processes
• Not suitable for long-term expression of the transgene due to the
lack of integration into host genome
• Complicated vector genome
Helper-dependant • These vectors can only be grown in the presence of a helper virus
adenovirus and are not economic for production of low level contamination of
helper virus
• To compare with first generation adenovirus, virus titers are much
lower
AAV • High titers of pure virus are difficult to obtain
• AAV requires a helper adeno or herpesvirus
• This vector system is still not well characterized
• Limited capacity for foreign genes (about 2–4.5 kb)
• Lack of specific integration for recombinant AAV vectors, which
may possibly result in cell mutagenesis
HSV-1 • Host immune responses, inflammatory cytopathogenicity and
neurotoxicity reactions in patients
• Complicated vector genome
• Difficult to produce
• HSV-1-derived vectors could potentially reactivate latent
wild-type HSV-1
Retrovirus • Random insertion of viral genome, which may possibly result in
mutagenesis and activate oncogenes
• Possibility of replication-competent virus formation by
homologous recombination. Possible recombination with human
endogenous retroviruses (HERVs)
• Retroviral vector particles are rapidly degraded by the complement
• Infects only dividing cells, small insert capacity (6.5 kb)
Vaccinia virus • Widespread use of vaccinia as a live vaccine, however, depends on
improving safety while achieving an even higher immune response
to the recombinant protein
Microinjection • It’s difficult to introduce DNA on a scale large enough for
biochemical analysis
Table 3. (continued)
Vectors
Transfection • Targeting is not specific

(cationic • Low transfection efficiency
liposomes or • Only transient expression
DNA protein • Difficult in vivo applications
complexes) • Host immune responses, inflammatory reactions in patients if they
express chimerical cell receptors on their surface, or in the presence
of unmethylated CpG sequences of bacterial plasmid DNA
AAV: Adeno-associated virus, HSV: herpes simplex virus, HERVs: human endogenous
retroviruses.
vector replication and packaging. E1-deleted Ad vectors have a number of

positive characteristics, one of the most important ones is their relative ease
for scale up at very high titers, above 1012 IU/ml (Infectious Units/ml). Other
attractive features include the ability to infect many different cell types, both
dividing and nondividing differentiated cells; having an extremely low proba-
bility of random integration into the host chromosomes [37]; and having a large
cloning capacity, theoretically around 8 kb with full E1 and E3 deletions.
In spite of the E1 deletion, first-generation Ad vectors have residual
expression of viral genes that lead to a strong host immune response, resulting
in the generation of high titer, neutralizing anti-capsid antibodies that inhibit
re infection with the same serotype of Ad vector [38]. In addition, at high viral
doses, residual virus gene expression leads to cellular cytotoxicity, which can
result in an immune-mediated loss of the Ad vector transduced cells [39].
Injection of first-generation recombinant Ad vectors into the brain parenchyma
causes acute cellular- and cytokine-mediated inflammatory responses. This
does not effect transgene expression. In the presence of Ad immune responses,
transgene expression for first-generation Ad is rapidly established [40, 41]. Ad
induced cytotoxicity is only seen when at high vector doses of ⬎108 IU/ml are
used to transduce the target tissue [40].
To overcome this limitation, a series of Ad vectors with multiple dele-
tions have been developed. In order to propagate multiply-deleted Ad vectors,
packaging cell lines must be developed that trans-complement the growth and
packaging of these vectors (fig. 3). Cell lines coexpressing Ad E1 and E4 genes,
the E1 and E2a (single-strand DNA-binding protein, ssDBP) genes, the EI and
pre-terminal protein genes, the E1 and protease genes have been generated and
used to package the E1, E4 deleted Ad vectors [42], the E1, E2a deleted Ad
vectors [43], the EI, E2b-deleted Ad vectors [44], the E1 and protease-deleted
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HD-Ad vector Helper virus
ITR ␺⫹ ITR ITR ␺⫹ ITR
Stuffer DNA Transcription cassette Stuffer DNA ␺⫹ Luc

loxP loxP
or
FRT FRT
293 Cre or
293 FLPe cells
ITR ␺⫹ ITR ITR ␺⫺ ITR
Stuffer DNA Transcription cassette Stuffer DNA Luc
0.01% contamination
with helper virus
Packaged Not
packaged
HD-Ad vector
ITR ␺⫹ HD-Ad vector ITR
Stuffer DNA Stuffer DNA

Promoter Therapeutic poly A
gene
Fig. 3. Schematic representation for the production of helper-dependent adenovirus

vectors. The helper virus is an E1-deleted Ad that contains a packaging signal (␺) flanked
by loxP or Frt recombination sites. The HD-Ad vector is constructed as a plasmid with trans-
gene, stuffer DNA and the Ad cis-elements, mainly the inverted terminal repeats (ITR)
and packaging signal. Upon cotransfection of the HD-Ad vector and, the helper virus into a
293-derived cell line that stably expresses the Cre and FLPe recombinase, the packaging
signal of the helper virus is excised, rendering the helper virus DNA unpackagable. But the
helper virus provides the Ad functions that are required for replicating the vector DNA, for
producing viral structural protein, and for packaging of the vector DNA into virions. The titer
of HD-Ad vectors is increased by serial passages through helper virus-infected 293-derived
cell line. A purification step by CsCl centrifugation can further reduce the contamination of
vector with helper virus to 0.01%.
Ad vectors [45] respectively. Deleted Ad vectors further reduced the acute toxi-
city and inflammatory responses and increased the transgene capacity, but had
other disadvantages, such as replication accompanied by expression of Ad pro-
teins, which reduced the length of transgene expression.
New helper-dependent Ad vectors (also known as high-capacity, ‘gutless’ or
‘gutted’ vectors; HD-Ad) are devoid of all viral coding sequences [46–49]. These
vectors have a minimum requirement for the extreme termini of the linear Ad
genome, containing only those cis-acting elements for viral DNA replication and
packaging, mainly the ITR sequences and packaging signal. Since these elements
are contained with ⬃500 bp from the ends of the genome [50], helper-dependent
vectors have the potential to range in size from a few hundred base pairs to carry
up to ⬃36 kb of the foreign DNA, which is close to the size of the native Ad
genome. HD-Ads are copropagated with an E1-deleted helper virus, which pro-
vides in trans all of the proteins required for the propagation of the vector.
Several systems have been developed to prevent packaging of the helper viral
genomes during the HD-Ad vector rescue/amplification process in order to min-
imize the helper virus contamination. The Cre/loxP-based system for the
generation of HD-Ad involves the use of a first-generation helper virus, where
the packaging signal is flanked by loxP recognition sites [51]. Infection of Cre-
expressing 293 cells with the helper virus results in excision of the viral packaging
signal, rendering the helper virus DNA unpackagable, but still able to replicate
and provide helper functions for HD-Ad vector propagation [52]. Purification by
caesium chloride centrifugation is necessary to reduce the titer of the helper virus
to negligible levels, typically ranging from 0.1 to 0.01% of the HD-Ad vector titer
[53]. Recently, another Flp/FRT-based system has been developed. The Flp
recombinase was used in place of Cre, and shown to excise the FRT-flanked pack-
aging signal in helper virus efficiently [48, 54]. The most recent improvement to
this system is the development of a new Cre-expressing cell line based on E2T, an
E1 and E2a-complementary cell line. Thus an E1 and E2a double-deleted helper
virus can be used with the new cell line to produce HD-Ad vector with low helper
contamination, further improving the HD vector safety [55].
Compared with first-generation Ad vectors, the HD-Ad vector can efficiently
transduce a wide variety of cell types from numerous species in a cell cycle-
independent manner. HD-Ad vectors have the added advantage of increased
cloning capacity, reduced toxicity and immune responses, and prolonged stable
transgene expression in vivo [40, 56]. Limitations of HD-Ad vectors are difficul-
ties in large-scale production and helper virus contamination. If these shortcom-
ings can be overcome by improved viral vector production technique, the HD-Ad
vector could become a key viral vector for gene therapy (table 4).
Adeno-Associated Virus Vector

Adeno-associated virus (AAV) is a simple, linear, single-stranded DNA
parvovirus, which is nonpathogenic to humans. It is currently being developed
as a gene therapy vector for the treatment of numerous diseases, such as diabetes,
obesity, lactose intolerance, hemophilia B and blindness [57–59].
AAV has two genes: rep, which codes for replication and integration func-
tions of the virus; and cap, which codes for the structural components of the
virus. On either side of rep and cap are two ITRs, which define the beginning
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Table 4. How to improve existing gene therapy vectors for use in clinical trials
• Increase efficiency of expression to allow lower doses of vector as required

• Prolong the duration of transgene expression
• Generate vectors with reproducible high titers and no contaminants
• Increase the area of transduction
• Target infection and transgene expression to allow systemic delivery
• Generate transcriptional switches to allow to turn therapeutic transgene expression ‘on’
or ‘off’
and the end of the virus and contain DNA sequences needed to pack the viral
genome into the capsids [60]. Any sequence flanked by AAV ITR can be con-
sidered as a recombinant AAV genome for the purposes of gene therapy vector
development. Wild-type AAV has the capacity to establish latency in the host
cells and to integrate DNA into a 4-kb region of human chromosome 19, des-
ignated AAVS1 [61]. Recombinant AAV (rAAV) vectors achieve long-term
transgene expression without major immunogenic toxic or toxicity responses,
although neutralizing antibodies are generated. They can infect and integrate in
a wide range of cells including dividing and resting cells.
The main disadvantage of AAV is the relatively small packaging capacity,
approximately 4.7 kb. Because of this size limitation, rep and cap genes were
removed from first-generation AAV based vectors to make room for the thera-
peutic or marker genes. It was later discovered that the rep gene, or at least one
of its products, the Rep68 or Rep78 protein, is required for the preferential
integration of AAV [62]. Recent developments in AAV gene therapy vector con-
struction allow the inclusion of the rep gene into an AAV vector. The packaging
capacity of these vectors has been extended by harnessing the observation that
AAV genomes concatemerize after transduction [63]. When two vectors, one
encoding for the first half and the other encoding the second half of a protein,
were transduced into cells, head-to-tail stitching of the viral genomes resulted in
the reconstitution of a functional gene, effectively increasing the size of the gene
that can be delivered [64]. A recombinant AAV vector carrying the angiostatin
gene has been constructed and developed as an anti-angiogenesis strategy to treat
malignant brain tumors in a C6 glioma rat model [65]. Intratumoral injection of
a high titer AAV-angiostatin vector has yielded efficacious tumor suppression
and resulted in long-term survival in 40% of the treated rats [65]. Another chal-
lenge with AAV vectors is that it has been difficult to scale up production.
Herpes Simplex Virus-1 Derived Vector

Herpes simplex virus-1 (HSV-1) is an enveloped double-stranded DNA
virus, with a genome of 152-kb size, containing unique coding sequences
flanked by several repeat regions. HSV-1 has been exploited for gene transfer
in different in vitro and in vivo models. It has been especially useful in the CNS
because of its ability to persist in a latent state in neurons. Its genome structure
allows large inserts (up to 30 kb). HSV-1 vectors express transgenes stably for
up to 18 months; whether similar vectors could also be used for long-term
transgene expression in forebrain neurons of the striatum or neocortex remains
to be determined. The major drawback, which limits their application, is vector
toxicity [66]. Novel, safe and robust HSV-1 vectors called HSV amplicons,
deleted of almost all genomic sequences, have been developed [67].
Retrovirus Vectors
Retroviruses are enveloped single-stranded RNA viruses, which have a
genome of about 7–10 kb composed of four gene regions termed gag, pol, pro,
env. These gene regions encode for structural capsid protein, viral integrase and
transcriptase, viral protease and envelope glycoproteins respectively. This genome
also has a packaging signal and cis-acting sequences, termed long terminal
repeats, which have a role in transcriptional control and integration. Current vec-
tor system based on type C retroviruses, such as the Moloney murine leukemia
virus has proven to be very popular as they are relatively nonpathogenic.
Upon binding to its extracellular receptor, a conformational change within
its envelope glycoprotein enables the retroviral envelope to fuse with the cell
membrane and allows the release of the capsid core into the cytoplasm. Once
inside the cytoplasm, the single-stranded RNA genome is reverse-transcribed
into a double-stranded DNA proviral genome by the viral reverse transcriptase
inside the capsid. Then a preintegration complex is formed and transported into
the cellular nucleus. The viral integrase can randomly integrate the proviral
genome into the host chromosomal genome, where the host’s transcriptional
system gives rise to the expression of viral genes [68].
Recombinant retroviral vectors are devoid of all viral genes, are replication-
defective, can carry upto 8 kb of foreign DNA, and can be engineered to have
varying cell tropism. The long terminal repeats and packaging sequence are the
only viral sequences that remain in the vector. To propagate recombinant retro-
viruses, it is necessary to provide the viral genes by packaging cell lines. With
this system, it is possible to produce viral titers of 105–107 CFU/ml [69].
The primary advantage of retrovirus is their ability to stably introduce genes
into cells. Genes integrated into host cell DNA, are regulated as if belonging to
the cell, which enables stable transfer of the gene to subsequent daughter cells
following mitosis. Levels of gene expression, however, will depend on the site
of vector insertion. In a clinical application, where long-term correction of
genetic defect is required, retroviral vectors offer a means to achieve this goal.
The ability of retroviral vectors to easily transduce cells in vitro has led to the
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development of ex vivo therapeutic strategies, whereby retroviral vectors are
used to infect peripheral blood lymphocytes or bone marrow cells. In phase-I
clinical trials using such a procedure, long-term gene expression has been
demonstrated [70]. The strategy of generating safe and transcriptionally regulat-
able vectors was the development of self-inactivating vectors, in which regu-
latory elements have been deleted. Upon integration, all viral promoter/enhancer
activity is lost and the transcription of the transgene is under the control of a
heterologous promoter [71].
The main limitation of retroviral vectors has been their inability to infect
nondividing cells, meaning that tissues such as brain, eye, and pancreas are not
amenable to direct in vivo gene delivery. Furthermore, on transplantation of
transduced cells in the host, transcription of transgenes is often extinguished [69].
These limitations have lead researchers to find new strategies for developing
vectors that can infect nondividing cells, as well as integrate into the host
chromosomes.
Lentivirus Vector
Lentiviruses, such as human immunodeficiency virus, are part of the
retrovirus family, but have acquired the capability of transducing nondividing
cells, and therefore can be used to transduce cells within the nervous system [72].
The first-generation lentiviral vectors relied largely on the substitution of the
viral Env protein with vesicular stomatitis virus G protein, which relieved them
of their dependence on CD4, the T-cell receptor protein required for lentivirus
infection. Instead, the vectors showed a wider tropism by infecting cells not
known to express CD4 protein, including neurons, hepatocytes, muscle fibers,
and retinal cells. Although first-generation vectors fulfilled many of the criteria
of an ideal vector, they were viewed with some caution because of the possi-
bility of recombination and generation of infectious human immunodeficiency
virus. To minimize those concerns, lentiviral vectors have been made devoid of
as many viral accessory genes as possible, while maintaining the key feature of
infection of nondividing cells [73]. An extra feature that has improved these
vectors includes the central polypurine tract, which allows internal initiation
of second-strand DNA synthesis and probably aids in the transport of the prein-
tegration complex to the nucleus [74].
Some nonhuman lentiviruses, such as simian immunodeficiency virus,
feline immunodeficiency virus, and equine infectious anemia virus, have been
used to generate efficient vectors capable of transducing nondividing cells. There
are no clinical trials with these vectors or any other lentiviral vectors at present.
Like other integrating vectors, the lentiviral vectors will have the disadvantage of
nonspecific integration in the chromosomes. The duration of expression of the
transgene in lentiviral vector also needs further testing [75, 76].
Table 5. Implementation of clinical gene therapy
Efficacy Efficient delivery and expression of therapeutic transgenes which should

enable revertion of the disease phenotype. Adequate persistence of
transgene expression. Transgene expression should be turned ‘on’ and
‘off’ as and when it is needed. Cell-type specific expression of
therapeutic genes
Safety Elimination of adverse immune responses and other cytotoxic reactions,
both local and/or systemic, within the patient. Limit spread in vivo
Clinical trials Large scale production and quality control of vectors. Compliance
with local and national gene therapy advisory committee’s regulations.
The cost of the therapy versus size of patient population to be treated.
Benefits to health care provision. Effects on long-term survival and
overall quality of life
Among the viral vectors described, advantages and disadvantages of each

vector are summarized in table 5. Several approaches have been aimed at manip-
ulating the vectors to maximize the efficiency, safety and duration of transgene
expression. With the aim of combining the most beneficial properties of several
vector systems, chimeric vectors have been developed such as AAV/Ad [77] or
AAV/HSV-1 [67].
Significant progress in vector development is occurring in the area of tissue-
or cell-specific expression. Targeting can be achieved by two strategies. The first
one involves engineering viral capsids for binding specific cellular receptors that
subsequently mediate viral entry. This type of engineering has been reported for
Ad [78] and for AAV vectors [79]. The second strategy relies on choosing an
appropriate promoter to confine the expression of the transgene to a particular
cell type. Examples of this later approach include the use of the used prostate-
specific antigen (PSA) or tyrosinase promoters/enhancers in HD-Ad vectors to
maintain strict tissue-specific expression [80]. A dual Ad vector system, encod-
ing for cell-type-specific and regulatable tetracycline-dependent transcription
elements, have been developed. The results demonstrated that the GFAP promoter
is able to restrict tetracycline-dependent transgene expression to glial cells in
cell lines, primary cultures and in the CNS in vivo. The neuronal specific enolase
promoter did not show neuronal restricted transgene expression in vitro, but it
did so in the CNS in vivo [81].
Although tissue- or cell-specific expression will continue to command
interest, we suspect that regulated expression of transgenes will become an impor-
tant focus for practitioners of gene therapy. The most widely used regulation sys-
tem is based on the bacterial tetracycline resistance regulation (Tet system),
where transgene expression can be switched ‘on’ and ‘off’ in the absence or
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presence of tetracycline [83]. Recently, a dual Ad encoding a tetracycline-
regulatable expression system was generated to control the production of TH
in vivo and in an experimental therapeutic setting [83]. In the absence but not in
the presence of the tetracycline analog doxycycline, TH expression was observed
in AP tumor cell lines, i.e., at T20, GH3 and MMQ. In both primary AP cell
cultures and the AP gland, in situ expression of TH was seen in lactotrophs,
somatotrophs, corticotrophs, thyrotrophs and gonadotrophs in the absence but
not in the presence of doxycycline. Furthermore, expression of TH was able to
normalize the circulating PRL levels and reduce the mass of the enlarged pitu-
itary gland [83]. These results indicated that the Tet system could be a useful tool
for the regulation of the therapeutic transgene expression in vivo [81, 83].
Gene Therapy Strategies for Tumor Treatment
The majority of pituitary tumors are clinically well managed, and normally
long-term survival of affected patients can be achieved. In spite of this, for
some very large and locally invasive pituitary tumors, treatments are far from
ideal. Gene therapy has utilized several strategies to achieve tumor cell killing.
The most widely used approaches are conditional cytotoxic cell killing, inhibi-
tion of angiogenesis, expression of proapoptotic and/or tumor-suppressor genes
and immune stimulation by ectopic cytokine expression and/or engineering the
immune recognition of tumor cells.
Conditional Cytotoxicity Approaches for Tumor Gene Therapy

Conditional cytotoxicity utilizes the conversion of an inert prodrug to a
pharmacologically active cytotoxic drug at the tumor site. The most commonly
used approach is gene-directed enzyme prodrug therapy (GDEPT). A ‘suicide’
gene encoding the enzyme is selectively delivered to tumor cells. This enzyme
must be capable of metabolizing a non or weakly toxic prodrug to an active and
highly toxic drug. The toxic drug is produced either within the tumor cells or
the tumor mass, allowing its local diffusion to non-tranduced tumor cells, pro-
ducing a ‘bystander effect’.
Thymidine kinase (TK) gene from HSV1 is the most intensely studied and
used conditional cytotoxic gene [84–87]. Its substrates include the anti-herpes
drug ganciclovir (GCV) which HSV1-TK monophosphorylates to an inter-
mediate, which is subsequently phosphorylated by cellular kinases to a di- or
triphosphate form that can be incorporated into DNA as nucleoside analog, and
therefore cause cell death of the proliferating cells (fig. 4). Phosphorylated
GCV can only kill dividing cells, as tumor cells are usually the most actively
dividing cells in tissues, it can be argued that this gives the therapy a certain
level of selectivity. HSV1-TK/GCV exhibits a strong bystander effect both in
vitro and in vivo. Mouse glioma tumor model studies showed that only 10% of
tumor cells needed to be transduced to achieve total tumor regression [88]. The
HSV1-TK/GCV pairing has been used in human gene therapy trials depending
on the basis of good preclinical data [84, 87]. In vivo gene transfer using both
retroviral and Ad delivery of HSV1-TK in clinical trials have been well toler-
ated by the patients with recurrent brain tumors, with extension of patient sur-
vival reported [87, 89, 90].
Cytosine deaminase in combination with 5-fluorocytosine (CD/5-FC) has
also been moved into human gene therapy clinical trials. Cytosine deaminase is
ubiquitous within bacteria and fungi but absent from mammalian cells. This
enzyme deaminates the anti-fungal drug 5-fluorocytosine into 5-fluorouracil
which induces cell death by inhibiting both DNA and RNA synthesis; therefore,
it only kills dividing cells. Huber et al. [91] infected a colorectal tumor cell line
ex vivo, then implanted the transduced cells into mice demonstrating antitumor
effects as a result of local 5-FU production. A bystander effect was also observed
when mixtures of transduced and nontransduced cells were implanted. These
experiments suggest that as few as 2% of the tumor cells need to be transduced
to eliminate the tumor. In vivo experiments demonstrated that the combination
of CD/5-FC gene therapy and radiation possessed superior anti-tumor effect in
comparison to single therapy [92].
Nitroreductase is an enzyme from the E. coli strain K12 and is used in
combination with the prodrug CB1954, a weak alkylating agent. Nitroreductase
converts its prodrug into the 4-hydroxylamino derivative, which after acetylation
via thioesters becomes a powerful alkylating agent capable of cross-linking DNA.
The active drug kills both quiescent and dividing cells and shows promise for
in vivo tumor cell killing [93].
Carboxypeptidase G2 is an enzyme isolated from the bacteria
Pseudomonas strain RS-16, which cleaves the C-terminal glutamate moiety
from benzoic acid mustard prodrugs. This cleavage, results in the release of
cytotoxic nitrogen mustards, which are toxic to both quiescent and dividing
cells by DNA alkylation. This enzyme was first used in antibody-directed
enzyme prodrug therapies (ADEPT), targeting carboxypeptidase G2 to a col-
orectal tumor xenograft in athymic mice, by conjugation of the enzyme to an
antibody recognizing the carcinoembryonic antigen [94], thus achieving
significant antitumor activity upon the administration of prodrug. Clinical
trials using this antibody targeting approach are ongoing in patients with
advanced, drug resistant colorectal cancer. Recently, Ad vector-mediated
delivery of the prodrug-converting enzyme carboxypeptidase G2 in a secreted
or glycosylphosphatidylinositol (GPI)-anchored form was developed to
strengthen the efficacy of the prodrug-activating system, and more than 50%
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Hypothalamus
Pituitary stalk
Promoter
Posterior pituitary
Cell type
A tumor
Suicide gene Anterior pituitary Intermediate pituitary

Cell type
B tumor
Tumor cell
Tumor cell A Normal cell B HSV1-TK/GCV system
E
E No prodrug-
activating Thymidine kinase
Specific endocrine Cell lacking enzyme
cell or tumor cell Prodrug endocrine-specific transcribed GCV GCV-MP GCV-TP
transcription genes activating or tumor-specific
enzyme transcription
elements Tri-
phosphorylated
ganciclovir
Cell toxicity
E Cell death of HSV1-TK

transduced and adjacent
Cell type B untransduced tumor cells
alive (Bystander effect)
Active toxic Nontoxic

drug drug
Reduction in tumor size
Cell type A dead
Fig. 4. Cytotoxic gene therapy approach for specific anterior pituitary adenomas. The
diagram illustrates a conditional cytotoxic approach to eliminate tumor cells through deliv-
ery of suicide genes via a recombinant Ad vector. Within tumor cell A in the presence of a
prodrug and specific endocrine or tumor transcription factors, transcription of the prodrug
activating enzyme (E) takes place. The subsequent exposure to a nontoxic prodrug alters it
to its active toxic form resulting in excessive production of toxic metabolites which ulti-
mately leads to cell death. Within normal cell B, lacking these specific transcription factors,
the expression of the conditional cytotoxic gene does not take place (i.e., HSV1-TK) and
therefore the nontoxic prodrug remains innocuous to the cell. Expression of HSV1-TK will
only occur within the cells that can activate transcription from the promoter, i.e., in specific
cell kill was achievable in all of the cell lines tested following only a single
exposure to the prodrug, ZD2767P [95].
Dual enzyme/prodrug combinations are currently being investigated with
promising results. HSV1-TK and cytosine deaminase have been simultaneously
expressed from stably transfected gliosarcoma cells implanted into nude mice
and after the prodrug administration, toxicity of the double suicide system was
shown to be 2–3 times higher than if the cytotoxic effects of each prodrug was
purely additive [96]. These enzyme prodrug combinations are also being
assessed for their effects on radiosensitization, and for their use in conjunction
with radiotherapy [97].
Inhibition of Angiogenesis
The inhibition of angiogenesis is thought to be a promising strategy that
could lead to the development of novel anti-neoplastic therapies. The process of
new blood vessel formation in growing pituitary tumors offers a potential target
for gene therapy. It has been demonstrated experimentally that it is possible to use
gene transfer methods in vivo to disrupt the angiogenic process in a solid tumor.
The main target would be vascular endothelial growth factor (VEGF) and its
receptors (VEGFR-1 or flt-1, and VEGFR-2 or flk-2), basic fibroblast growth
factor and its receptor, epidermal growth factor and its receptor, and also trans-
forming growth factor ␤ and ␣. Both antisense and dominant negative variants
could be used as gene therapy approaches to target angiogenesis. Replication-
defective retroviral vectors encoding both the soluble, truncated VEGF-R2 and
flk-1 have been used to study anti-angiogenesis processes. The results from that
work indicate that tumors from flk-1 expressing neuroblastoma cells were less
than 33% of the average volume of normal tumors after 23 days. Engineered
expression of flk-1, a competitive inhibitor of VEGF, results in the production of
an inhibitor of endothelial cell proliferation and migration that greatly restricts
the growth of the tumor cells in vivo [98]. Selective toxicity could be achieved
using this approach if the molecular targets were located preferentially within the
tumor blood vessels; blocking these pathways should mainly affect proliferating
cells in need of continuous blood flow for oxygen and nutrients.
anterior pituitary tumor cells. In the presence of HSV1-TK, the nontoxic nucleoside analog
GCV is converted to ganciclovir monophosphate (GMP), which is further phosphorylated by
mammalian kinases to ganciclovir triphosphate (GTP). These phosphorylated metabolites
are incorporated into replicating DNA leading to cell death of actively replicating tumor
cells. This approach is further effective as adjacent nontransduced tumor cells are also
destroyed through the transfer of toxic GCV metabolites. This ‘bystander’ effect produced
with the HSV1-TK/GCV model has important therapeutic implications. This tumor-specific
suicide gene therapy approach would be amenable to treat pituitary tumors without having a
negative effect on adjacent normal anterior pituitary cells.
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The discovery of angiostatin [99] and endostatin [100] provided an anti-
angiogenic therapeutic strategy that not only could regress large tumors in
vivo, but also maintain them quiescent at microscopic size for as long as the
therapy was present. These anti-angiogenic inhibitors proved neither to be
toxic, nor did they induce drug resistance. Gene therapy could provide a
means for delivering endostatin and/or angiostatin locally or systematically,
providing a constant supply of angiogenesis inhibitors. Ma et al. [65] have
utilized a recombinant AAV vector carrying the angiostatin gene to treat
the malignant brain tumor in a C6 glioma/Wistar rat model. Intratumoral
injection of a high titer AAV-angiostatin vector rendered efficacious tumor
suppression and resulted in long-term survival of the animals. Using this
approach, only migrating vascular endothelial cells in the tumor bed should
be inhibited; the remaining endothelium in the body, which is quiescent,
should not be affected. This therapeutic approach will, therefore, pose a low
risk to normal tissue.
Proapoptotic and Tumor Suppressor Genes

Other promising targets for cancer gene therapy are proapoptotic and tumor
suppressor genes [101, 102]. Apoptosis is regulated by a complex cascade of
proteases of the interleukin-1␤-converting enzyme family, also known as caspases.
Overexpression of interleukin-1␤-converting enzyme proteases is sufficient to
induce apoptosis and cell death. Abnormalities in the apoptotic cascade, such
as gene deletions, mutations or aberrant gene expression, are almost always pre-
sent in tumor cells, including gliomas [103].
Examples of proapoptotic-targeted therapy include the use of the potent
proapoptotic molecule Fas-L(CD95L). Fas-L is a 40-kDa type II transmem-
brane protein, a member of the tumor necrosis factor family of cytokines. When
bound to its receptor CD95 (Fas, APO-1), Fas-L, a glycosylated 45 kDa trans-
membrane protein that belongs to the tumor necrosis factor receptor family, it
induces very quick apoptosis [105]. The mechanism by which the Fas/Fas-L
system induces apoptosis has recently been elucidated and has suggested some
potential therapeutic targets. Binding of Fas-L to Fas induces the formation of
a death-inducing signaling complex containing the proteins FADD or RIP and
RAIDD, leading to the recruitment and activation of caspase 8 or caspase 2
depending on the components of the death inducing signaling complex [105].
The activation of either caspase ultimately leads to apoptotic cell death. Up-
regulation of Fas/Fas-L system results in cell suicide by the cross-linking of
these molecules between neighboring cells [106]. Transcriptionally targeted
Ads expressing Fas-L have been recently constructed [107, 108] and used as a
gene therapy approach for the treatment of intracranial glioblastoma tumor
model in rats [109]. Thus, gene therapy strategies may be developed that
directly act on the Fas/Fas-L sigal transduction pathway. Such an approach
should also be developed for designing gene therapy strategies for the treatment
of pituitary tumors.
Mutation of the tumor suppressor gene p53 is the most common genetic
alteration in cancers. Inactivation of p53 occurs early in glial tumorigenesis
and mutations are commonly found in low-grade astrocytomas. Replacement
of a defective p53 gene had been described for many tumor models. A recom-
binant Ad containing the wild-type p53 cDNA has been constructed to trans-
fer the wild-type p53 gene into the glioblastoma cell lines expressing either a
wild type or mutated p53 gene. In this experiment, the wild type p53 cells
showed inhibition of proliferation and mutant p53 cells underwent apoptotic
cell death [110]. In anaplastic astrocytomas, the mutations of the retinoblas-
toma (Rb) gene and/ or p16 gene are most common. To mimic the therapy of
cancer more closely, spontaneous pituitary melanotroph tumors arising in
immunocompetent Rb⫾ mice were treated with a recombinant Ad carrying
RB cDNA. Intratumoral RB gene transfer decreased tumor cell proliferation,
re-established innervation by growth-regulatory dopaminergic neurons, inhib-
ited the growth of tumors, and prolonged the life span of treated animals [111].
Recently, a tumor suppressor gene, mutated in multiple advanced cancers-
1/phosphatase and tensin homolog (MMAC1/PTEN) was identified within
chromosome 10 that is commonly mutated in human glioblastoma multiforme
and several other cancer types [112]. Cheney et al. [113] have constructed a
replication-defective Ad encoding MMAC1/ PTEN, and infection of MMAC1
mutated cells with this virus rendered them almost completely nontumori-
genic, as compared to untreated and control cells, suggesting that in vivo
gene transfer of MMAC1/PTEN could potentially be useful in cancer therapy
for aggressive gliomas. A similar strategy also could be used for aggressive
pituitary tumors.
The pathogenesis of pituitary tumors has been extensively studied to
identify proapoptotic and tumor suppressor genes, which could be used as
effective transgenes for gene therapy. Mutations have been described in the
G protein, Gs␣, which occurs in approximately 40% of GH secreting tumors.
Rarely occurring ras mutations in invasive tumors, loss of heterozygosity on
chromosome 11, and near the Rb locus on chromosome 13 have also been
implicated, but not reproducibly identified. The pituitary tumor transforming
gene (PTTG) was isolated from rat GH4 pituitary tumors and recently
the human homolog was cloned. Human PTTG is abundantly expressed in
pituitary tumors and potently transforms cells both in vitro and in vivo.
Furthermore, PTTG can be used as a marker for the invasiveness of hormone
secreting tumors, with more invasive tumors expressing the highest amounts
of PTTG [114, 115].
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Activation of the Immune Response
The role of the immune system from a therapeutic viewpoint is that once
appropriately activated against tumor-specific determinants, a small activation
signal can produce a long lasting body wide protection. These properties make
up-regulating the immune system, an attractive target for developing gene ther-
apy strategies for cancer. No gene delivery system or chemotherapeutic drug
available has the required specificity to target all tumor cells throughout the
tumor mass in cancer patients. Studies of mechanisms involved in recognition
and elimination of tumor cells have shown a key role for T lymphocytes in
conferring the specificity of tumor rejection. In particular CD8⫹ cytotoxic T
lymphocytes were identified as an important effector cell population in the
elimination of tumor cells. While responses are mainly mediated by CD8⫹
cytotoxic T lymphocytes, induction of these responses is dependent on the pres-
ence of CD4⫹ helper T cells. In addition to antigen-specific effectors, roles
have also been identified for natural killer cells and nonspecific effector cells
such as macrophages and eosinophils. It is now believed that both the innate
and adaptive immune responses act in concert through specific signaling path-
ways to generate anti-tumor immune responses.
The two most commonly used gene therapy approaches involve the enhance-
ment of the host’s immune response against tumors, i.e., cytokine-based gene
therapy and immune targeting of tumor cells. The underlying rationale for using
cytokines in cancer patients is to increase the patient’s immune response to the
autologous tumor. Cytokines may act to enhance local antigen presentation by
the tumor by means of inducing expression of MHC-I antigen on the surface of
tumor cells, MHC-II antigen on antigen-presenting cells (APCs) or by increasing
the expression of costimulatory molecules, or the tumor antigens themselves.
Local expression of cytokines also may act to enhance antigen uptake and
processing by tissue APCs. These APCs subsequently migrate to secondary lym-
phoid tissue and induce presentation to resident T and B cells. Cytokines induce
Th1 and Th2 subpopulations of helper T cells, direct activation of natural killer
and cytotoxic T cells, promote differentiation of granulocyte and macrophage
progenitors, and expansion of dendritic cells.
The effects mediated by cytokines could result in tumor cells which are
better APCs themselves, or the body’s normal APCs could become better at pre-
senting tumor antigens and tumor cells will become targets of immune activated
antitumor CTLs. The potential role of cytokines as adjuvants of the immune
response has been demonstrated by various studies, which illustrate the regres-
sion of human tumors following their systemic administration. Subcutaneous
implantation of genetically modified cells secreting cytokines, including IL-2,
IL-4, IL-6, IL-12, INF-␣, GM-CSF and tumor necrosis factor-␣, outside of the
CNS has been successful in generating cellular mediated immune response,
while at the same time, minimizing the toxic effects associated with high dose
systemic administration.
To increase the immune response against cancer cells, several approaches
are being developed. Tumor cells can be allogenized by the expression of a
highly immunogenic molecule on the surface. Nonself MHC I antigens act as
strong allo-antigens and be recognized as distinct targets by CTLs. Expression
of nonself antigens on the surface of tumor cells has been achieved by coupling
purified protein derivatives to the surface of cells. Another approach, recognition
of pre-existing tumor antigens, can be increased by enhancing T-cell activition.
The mechanisms involved in antitumor immunity are extremely complex, but it
is known that in order to activate resting T cells, the antigen must be seen by
the T cell in association with a MHC (class I or II). Despite the fact that many
tumors express MHC molecules to present antigen, an immune response, which
is capable of eliminating the tumor is rarely raised.
Further interaction with a co stimulatory molecule and/or certain cytokines
is still required to push the T-cell into active proliferation and differentiation.
Once the T-cell is pushed from resting G0, this activated cell no longer requires
costimulation to react with the target antigen. The binding of the T cells with
the target cells now depends upon the upregulation of accessory binging mole-
cules, such as CD2 and LFA-1, resulting in cytokine production. Several
costimulatory molecules such as B7–1 (CD80) and B7–2 (CD-86) are involved
in a pathway to both positively and negatively regulate the T cell response.
Immunogene therapy utilizing the B7 gene expression is one of the most
promising approaches to inhibit the tumor growth. Ando et al. [116] have inves-
tigated the difference between B7–1 and B7–2 with regard to B7 gene therapy
CNS. Their findings strongly suggest that B7–1 gene therapy could effectively
introduce CD (4⫹) TIL activation compared with B7–2 gene therapy. This
approach could also be investigated in preclinical animal models as a preclude
to its use for the treatment of aggressive pituitary tumors, which do not respond
to classical treatment strategies.
Animal Models to Study Pituitary Adenomas
The development and implementation of animal models for pituitary

tumors characterized over the years have proven to be an indispensable con-
stituent in the advancement of treatment strategies and gene therapy approaches
for pituitary disorders. The relevance of animal models was initially demon-
strated with studies on the characterization of experimentally induced pituitary
tumors in rodents [117, 118]. The establishment of a range of pituitary tumor
cell lines and innovations in transgenic technology have been crucial elements
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in the development of effective animal models in understanding various types
of adenomas. The most common animal models employed for in vivo experi-
mental studies related to pituitary tumor genesis are rats and mice. However,
the sheep has also proven to be a very useful in vivo model to study pituitary
physiology and pathogenesis and to assess the efficiency and safety of novel
exploited treatments for gene therapy. The normal sheep pituitary gland is com-
parable in structure and size to the human pituitary gland, thereby permitting
evaluation of transgene expression upon the administration of viral mediated
gene therapy. Below we discuss a number of animal models developed over the
years and their prospective uses in understanding and improving treatment
strategies for pituitary adenomas.
Prolactinomas
Existing animal models employed to study PRL-secreting adenomas include
three major types: hormonally stimulated, implantation of tumor cells, and pro-
lactinomas that are hereditary in nature. Stimulatory effects on lactotrophic
proliferation within the AP gland were first demonstrated using estrogen, a
powerful steroid hormone [119]. A range of estrogenic-related compounds such
as subcutaneous diethylstilboestrol implants, subcutaneous estrone (E1) implants
and subcutaneous implants or injections of oestradiol-17␤ were successfully
shown to induce lactotroph-hyperplasia. This paradigm was established and
widely utilized as an animal model to examine the pathophysiology of pro-
lactinomas, but the drawback of this approach is that estradiol-induced hyper-
plasia is hormone dependent unlike neoplasms of the AP gland [120]. It is also
feasible that the sustained delivery of estrogen to induce hyperplasia may abate
the effectiveness of treatment.
Among the conventional pituitary cell lines, the PRL/ACTH secreting
7315a cell line has led to the development of two frequently employed trans-
plantable tumor cell lines [121]. Implantation of the MMQ cell line into adult
female buffalo rats caused marked elevations in PRL levels, resulting in aug-
mented spleen weight and noticeable white pulp hyperplasia [121]. The 235–1
cell line demonstrated specific enhanced secretion of PRL and produced
tumors upon implantation into buffalo rats and nude athymic mice. The buffalo
rat implanted with the PRL-only MMQ tumor cells has been demonstrated to
be an effective model for the assessment of chronic hyperprolactinemia [121].
As compared to estrogen-induced ‘tumors’ and the induced transplantable
tumors, spontaneous transplantable human prolactinoma of the rat has been
shown to be an effective animal model that very much resembles in pathology
to the human prolactinoma [122]. Transplantable tumors, SmtTW and SmtTW2
implanted by serial passages were shown to exhibit 100% transplantation suc-
cess beneath the kidney capsule but only 20% subcutaneously. The perpetual
proliferation of embedded tumors was identical to those of the primary tumors,
and expected rise in PRL levels were observed while the other hormone levels
remained normal. SmtTW tumors have also demonstrated their potential appli-
cation in understanding adenoma pathology. Polysialylated neural cell adhesion
molecule (PSA-NCAM), a vastly expressed molecule during brain and pituitary
development, exhibited high expression levels in rat transplantable SmtTW
pituitary tumors [124]. Peak PSA-NCAM expression was detected in SmtTW4
malignant tumors transplanted subcutaneously and below the kidney capsule
that were maintained by successive tumor grafts. Expression of PSA-NCAM
was not identified in low growth rate benign SmtTW2, but low expression
levels were seen among high growth rate SmtTW3 transplantable tumors,
suggesting that PSA-NCAM expression is an indication of the proliferation rate
and malignancy of the tumor. Studies from this model raise the possibility of
employing the PSA-NCAM molecule as a diagnostic marker for malignant
pituitary adenomas. As this model provides features that are very similar to the
human pituitary adenoma pathology, the SmtTW tumors can be of practical use
in assessing the likely outcomes of novel approaches to modulate PSA-NCAM
expression. Also these models could be used to test the novel gene therapy
approaches.
A model for prolactinomas was also developed by cross-breeding the
Okamoto spontaneously hypertensive rat strain and the Koletsky rat strain
[124, 125]. These strains are normally used to study hypertension and obesity. This
newly developed rat strain designated as spontaneously hypertensive rat/N:Mcc-cp
had consequential complications of cardiomyopathy and congestive heart failure.
Despite these adverse side effects, assessment of the pituitaries revealed that 70%
of rats developed prolactinomas. Aged female and male Sprague Dawley and
Wistar strain rats have been shown to develop PRL-secreting pituitary tumors,
with an occurrence as high as 80% in Sprague Dawley rats, identified by distinct
augmented PRL levels.
GH Tumors
Tumors of somatotroph cells have been developed in transgenic mice.
These mice encompass the SV40 T antigen under the regulation of the bovine
arginine vasopressin promoter [126]. Few cases were reported to have tumor
occurrences within the intermediate lobe that were immunoreactive for ACTH
and proopiomelanocortin (POMC) mRNA. Further animal models that have
utilized transplantable cell lines that release GH comprise the MtT/W15 cells
[122] and GH3 cells [127]. Ad expressing the HSV1-TK under the control of
GH promoter was also effectively utilized in a transplantable tumor model
using GH3 cells in nude mice [127]. Subcutaneous transplantation of human
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GH adenoma tissue into athymic mice was also proven to be a dependable
model [128].
Cushing’s Disease
Studies with transgenic murine models have demonstrated to be very useful
in the generation and characterization of models of Cushing’s disease. ACTH
releasing pituitary tumors developed in transgenic mice which were produced by
engineering the polyoma early region promoter, coupled to a complementary
DNA (cDNA) encoding polyoma large T antigen [129]. Normal morphology
was recorded at 4 months of age, followed by microadenoma development
at 9 months and distinct macroadenomas, up to 5 mm in dimension at 13–16
months of age. Nontransgenic immunocompetent mice with subcutaneous trans-
plants of pituitary tumor cells produced tumors with indistinguishable mor-
phology and ACTH immunoreactivity comparable to the parent tumor [129].
Hypercorticotropism, a hallmark of the disease, was augmented with a brief
latency in mice carrying transgene pituitary transplants than in the polyoma
large T antigen-1 transgenic mice alone. Considerable increases in ACTH levels
were observed in transplanted mice than in clinically ill transgenic mice, indi-
cating the effectiveness of the murine model for studying this disease.
Leukemia inhibitory factor, a pleiotropic cytokine plays a key role in the
regulation of the mature hypothalamic-pituitary-adrenal axis in vivo. Leukemia
inhibitory factor influences corticotroph cell growth and stimulates POMC tran-
scription in vitro. Features of Cushing’s disease were observed when transgenic
mice expressing leukemia inhibitory factor under the regulation of the pituitary
glycoprotein hormone ␣-subunit (␣ GSU) promoter were studied. Mice pituitary
glands revealed corticotroph hyperplasia, evident somatotroph and gonadotroph
hypoplasia, and numerous Rathke-like cysts edged by ciliated cells [130].
Another model that has also shown to give rise to Cushing’s disease is the over-
expression of the mutated form of the neuroendocrine protein 7B2 in transgenic
knockout mice [131]. Mice expressing the null mutated form of 7B2 displayed
augmented levels in ACTH and corticosterone, with adrenocortical expansion
and eventual acute Cushing’s syndrome. The Rb gene, a tumor suppressor gene,
has been shown to causel the suppression of tumor cells reconstituted with Rb ex
vivo and implanted into immunodeficient mice as well as with germline trans-
mission of a human RB transgene into tumor-prone Rb⫾ mice. Heterozygous
immunocompetent Rb⫾ mice with an intermediate lobe tumor have been used to
assess spontaneously arising melanotroph tumors [111]. Tumors of this nature
treated with an Ad containing the Rb cDNA suppressed tumor cell proliferation
and extended the lifespan of treated animals. A feasible strategy for adenomas
with nonfunctional expression of tumor suppression genes could be, to reinstate
normal genes via Ad gene therapy encompassing the Rb cCDNA gene.
Null Cell Adenoma
Adenomas of this type do not synthesize AP hormones in vivo, but have
shown to express gonadotrophin-related genes. Models for these adenomas
were derived using transgenic mice bearing the temperature sensitive mutant
of the SV40 T antigen TSA58, under the regulation of the human Follicle stim-
ulating hormone-␤ (FSH-␤) regulatory elements [132]. Animals expressing the
mutated antigen developed progressive multifocal pituitary nodules with
decreasing immunoreactivity for FSH-␤ and (leutinizing hormone-␤). These
animals displayed features that are comparable to those observed in human null
cell adenoma, and these studies provide a good model for assessing the patho-
physiology of these tumors and testing novel therapeutic modalities.
Uncontrolled hormonal induction and molecular deficiencies are major
factors in the consequential formation of pituitary adenomas. Animal models
have provided evidence that hormonal stimulation is among the elements lead-
ing to tumor genesis. Development of cell lines have provided the backbone and
still remain a crucial element in the design of effective animal models in help-
ing us to further advance and improve our treatment strategies for adenomas.
Further and better insights into pituitary adenomas are imperative, as the nor-
mal gland possess complex qualities in terms of function and structure. Some
animal models may have certain drawbacks, thus additional studies on these
and other tumor models would allow us to decide the optimum in vivo model
in which to test novel therapeutic approaches for specific adenoma types.
The Preclinical Experience
Several methods of gene therapy have been explored for the treatment of
pituitary tumors, with particular focus directed at using Ads as delivery vectors
[87, 133]. Studies which aimed to use gene therapy approaches to deliver genes
into AP cells include recombinant Ad vectors and HSV-1 recombinant viral
vectors, which were both successfully used to deliver and target transgenes to AP
cells in vitro [134, 135]. The HSV-TK gene has since been targeted at pituitary
tumor cells as a conditional toxic approach to kill proliferating tumor cells in con-
junction with glaciclovir (GCV). In vivo studies modeling Cushing’s disease
showed that targeting TK gene expression to the ACTH-producing tumor cells
yielded considerable regression of the tumors [136]. High-level expression of the
TK gene was observed under control of the POMC promoter (AdPOMCGal;
AdPOMCTK). This promoter also yielded good cell-type specificity, in that non-
pituitary cells were unaffected by infection and expression of the therapeutic
gene. In another study, Windeatt et al. [86] used recombinant Ad vectors express-
ing HSV-1 thymidine kinase to treat estrogen-induced lactotroph hyperplasias
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Needle
Bregma Lambda
Needle Cerebral cortex
Superior
colliculus
Bevel Mid-
Hypo- brain
thalamus
Pituitary
Lateral 10.40mm
Fig. 5. Delivery of recombinant adenovirus to the anterior pituitary gland in vivo.

Following anesthesia with halothane and placement of the rat in a stereotaxic frame, the skull
is exposed and a hole is drilled posterior to the bregma revealing the superior sagittal sinus,
and the surrounding brain. Intrapituitary injections are made using a 26-gauge-modified
Hamilton syringe needle, with its tip previously ground until the opening of the needle is at
the base of the tip. The needle is lowered until touching the sphenoidal bone, and making
contact with the bottom of the rat equivalent of the sella turcica. This leaves the opening of
the needle within the pituitary gland and adequate amounts of recombinant vector are then
injected. Under these conditions of injection, the pituitary can be transduced by recombinant
Ad in 100% of surgical attempts [86].
within the AP gland (fig. 5). In addition to the observation of reduced tumor size
and nontoxicity of AP, a bystander effect which also destroyed untransduced
tumor cells was also documented [86]. Transgene expression of HSV-1-thymidine
kinase (HSV1-TK) was also restricted to lactotrophic cells using the human PRL
promoter [137]. Again, the importance of cell type specificity was underscored
by the resulting selective apoptosis induced in lactotrophic GH3 tumor cells. The
promoters used in each of these applications have played important roles in
achieving specific and efficient expression: while generally stronger promoters
tend to lack cell type specificity, on the other hand, cell type-specific promoters
generally achieve less efficient transgene expression [137].
Further along these lines, Lee et al. [138] used Ad vectors to target the
expression of the diphtheria toxin gene to GH-producing pituitary tumor cells.
Their success in this targeted suicide gene therapy approach may be useful for
treating GH-secreting adenomas. In another similar approach [139], gene ther-
apy was used to induce apoptosis of lactotropic tumor cells and suppress tumor
formation by infecting the GH4 cells with dominant negative estrogen response
mutant using an Ad vector. The regulation of gene expression and cell growth
by endogenous ER was interrupted, and the cells were rendered unable to dif-
ferentiate or proliferate [139]. This strategy presents another possibility for the
targeted treatment of pituitary lactotroph adenomas. An alternative modality for
treating PRL-secreting adenomas has also been developed. This approach har-
nesses the overproduction of dopamine as an inhibitor of PRL secretion and
lactotroph growth, thereby reducing the growth and hypersecretion of pro-
lactinomas. A gene therapy strategy enhanced the production of dopamine in
situ by augmenting expression of tyrosine hydroxylase in the AP gland in order
to speed up this rate limiting step in dopamine synthesis [83].
In addition to targeting specific cells for treatment, gene therapy tech-
niques have developed regulatable therapeutic transgene expression (fig. 6).
Smith-Arica et al. [81] devised a dual Ad system in which the expression could
be regulated using an inducible tetracycline system and the transcription could
also be targeted to specific brain or AP cell types. The astrocyte-specific, glial
fibrillary acidic protein (GFAP) and the neuronal specific enolase promoter
used were able to restrict the transgene expression to glial cells and neuronal
cells in vivo, respectively. Such restriction was not so apparent for the neuronal
specific enolase promoter in vitro. The most efficient transgene expression was
accomplished by using an excess of the transactivator in the dual Ad system,
which could be completely shut off with the administration of doxycycline
(fig. 6). In 2001, Smith-Arica et al. applied this concept to lactotrophic cells in
the AP gland, by reusing tetracycline-responsive transcriptional elements.
Under the control of the human lactotroph-specific PRL promoter, they again
achieved cell-type specificity and system inducibility using a dual Ad vector
system.
Besides targeting tumor cells for treatment, gene therapy has the capacity
for replacing gene deficiencies. Sarac et al. [140] focused on the replacement
of the chaperone protein, 7B2, the absence of which can reduce the effective-
ness of the prohormone convertase PC2 and lead to effects similar to Cushing’s
disease [140]. It was shown that stereotactic injection of recombinant Ad vector
encoding 7B2 into a pituitary deficient in 7B2 raised 7B2 levels in the pituitary
and blood, increased PC2 activity, lowered ACTH levels in the blood, and
slightly increased circulating ␣ MSH levels. Additionally, blood glucose levels
increased, corticosterone decreased, and survival times increased. Such results
indicate that the Ad administration of 7B2 can help support 7B2 null mice and
illustrate the potential for gene replacement therapy.
Since Ads have been established as viable vectors for gene therapy, assess-
ment of their safety profile is necessary. Southgate [141] studied the physio-
logical and therapeutic implications of their use to transfer genes into the AP
medwedi.ru
Tet ‘on’ regulated expression
Transcription
Ad Promoter rtTA Ad
Repressor proteins bind

Transactivators
to the operator site,
Tc Tc
preventing the binding
of transactivators Tc
⫺Tc Tc
Tc
Tc
Transcription ⫹Tc Tc
Ad TRE Transgene Ad
To binds to the repressor
proteins, releasing them
from the operator site
Tc
Tc Tc
Tc
Tc Tc Tc Transcription
Transactivators are able to bind Ad TRE Transgene Ad

to the new open operator site,
allowing transcription to occur
Transcription
a Ad TRE Transgene Ad
Tet ‘off’ regulated expression
Transcription
Ad Promoter tTA (nls) Ad
Transactivators
Tc
Tc Tc
Tc
Tc
⫺Tc ⫹Tc Tc Tc
Transcription
Ad TRE Transgene Ad
tetO tetO tetO tetO tetO tetO tetO pminCMV
3'TCTCTATCACTGATAGAGA 5' Transcription
b Ad TRE Transgene Ad
Fig. 6. Tet ‘on’ and Tet ‘off’ regulated expression. a Tet-off regulated expression. In the
absence of Tetracycline (Tc), tTA transactivators bind to the TRE operator site and activate
transcription. When present, Tc inhibits transactivators from binding to the TRE operator site
gland as well as safety issues associated with such interventions. An investi-
gation into the duration of transgene expression, local immune responses and
consequences on circulating pituitary hormone levels was conducted, as well as
a review of the levels of circulating anti-Ad neutralizing antibodies and AP hor-
mones in sera and the presence of vector genome and cellular immune infiltrates
within the AP gland. Though reduced transgene expression was seen over time
and virus-induced immune responses were observed, results showed no major
cytotoxicity or disruption of AP hormonal functions. Additionally, endocrine
function in a large animal model does not seem to be affected by stereotaxic
intrapituitary delivery of such cell type-specific recombinant Ads [142]. Such
findings indicate that Ad-mediated delivery to the AP gland may be a safe and
effective method of treating pituitary diseases. However, in 2002, Davis [142]
did report evidencing that injection of high doses of recombinant Ads into
the sheep AP can lead to severe inflammation of the pituitary, and warned that
histological investigations and vector dosage evaluations are important for devel-
oping safe and effective endocrine gene therapy.
The development of gene therapy as a useful treatment for pituitary dis-
eases has shown very promising results. The versatility of this technique has
allowed for a wide range of applications, including tumor reduction and
supplementation of deleted genes. Though further investigation of in vivo
effects of gene delivery and its efficiency and safety are needed, this area of
medical research offers tremendous potential for increasing new and powerful
therapeutic modalities.
Conclusions
Pituitary adenomas are usually benign and are well managed with classi-
cal therapies, i.e., receptor-mediated medical therapy, surgery, and in some
instances, radiotherapy. In spite of this, there are occasions where the tumors do
not respond to these forms of therapy, the therapies are not well tolerated by the
patients, or the tumors become invasive or recur and become very difficult to
treat. This usually depends on the nature of the tumor, for example whether it
is secretory or nonfunctioning, its size and also the type of hormonal cell-type
giving rise to the tumor. In these instances, gene therapy can provide a novel
modality to treat these tumors.
and transcription does not occur. b Tet-on regulated expression. Repressor proteins bind to
the TRE operator site, preventing transactivator binding and initiation of transcription. When
present, Tc binds to the repressor proteins, releasing them from the operator site. rtTA trans-
activators are then able to bind to the open operator site and initiate transcription.
medwedi.ru
Gene therapy could be used by itself or in combination with existing thera-
pies, depending on the presentation of the tumor and also on the medical history
of the patient. As discussed above, there are several gene therapeutic approaches,
which could be very attractive for treating these tumors. New therapeutic targets
together with novel, improved vector systems and genetic switches to turn ther-
apeutic gene expression ‘on’ or ‘off’ as needed, will render these therapies safe
for human trials.
In spite of enormous progress in the fields of vector development, thera-
peutic targets and improved preclinical models, much work still needs to be
done with respect to improving efficacy and safety of these gene therapies
before they can be implemented to treat human patients (tables 4, 5). This field
is exciting and we hope that this review will inspire scientists and clinicians to
embark in the basic and translational research needed to bring these treatments
to the clinic.
Acknowledgements
We thank the generous funding our institute receives from the Board of Governors at
Cedars-Sinai Medical Center and the encouragement and support of its members. We wish
to thank the unparalleled support and academic leadership of Dr. Shlomo Melmed. We are
grateful to Mr. Richard Katzman for his superb administrative organizational skills and to
Mr. Danny Malaniak for his encouragement, support, and commitment. Work is funded by:
National Institutes of Health/National Institute of Neurological Disorders & Stroke Grants
#NS42893.01, NS047298–01, U54 4NS 04–5309 to Pedro R. Lowenstein, M.D., Ph.D.;
National Institutes of Health/National Institute of Neurological Disorders & Stroke Grant
#NS44556.01 and TW006273–01A1 to Maria Castro, Ph.D.; the European Community
Framework V; the Parkinson’s Disease Foundation; and the Linda Tallen & David Paul Kane
Annual Fellowship in Cancer Gene Therapy.
References
1 Thapar K, Kovacs K, Laws ER: Pituitary tumors; in Black PM, Loeffler JS (eds): Cancer of the
Nervous System. Cambridge, Blackwell Science, 1997, pp 363–401.
2 Guiot G: Considerations on the surgical treatment of pituitary adenomas; in Werder K: Treatment
of Pituitary Adenomas. Stuttgart, Thieme 1978, p 202.
3 Hardy J: Transphenoidal microsurgery of the normal and pathological pituitary. Clin Neurosurg
1969;16:185–217.
4 Hardy J: Transsphenoidal approach to the pituitary gland neurosurgery; in Rengachary S (ed).
New York, McGraw-Hill, 1996, pp 1375–1384.
5 Ciric I, Mikhael M, Stafford T, Lawson L, Garces R: Transsphenoidal microsurgery of pituitary
macroadenomas with long-term follow-up results. J Neurosurg 1983;59:395–401.
6 Cohen AR, Cooper PR, Kupersmith MJ, Flamm ES, Ransohoff J: Visual recovery after transsphe-
noidal removal of pituitary adenomas. Neurosurgery 1985;17:446–452.
7 Arafah BM: Reversible hypopituitarism in patients with large nonfunctioning pituitary adenomas.
J Clin Endocrinol Metab 1986;62:1173–1179.
8 Ober KP, Kelly DL: Return of gonadal function with resection of nonfunctioning pituitary ade-
noma. Neurosurgery 1988;22:386–387.
9 Tyrrell JB, Brooks RM, Fitzgerald PA, Cofoid PB, Forsham PH, Wilson CB: Cushing’s disease.
Selective trans-sphenoidal resection of pituitary microadenomas. N Engl J Med 1978;298:753–758.
10 Post KD, Biller BJ, Adelman LS, Molitch ME, Wolpert SM, Reichlin S: Selective transsphenoidal
adenomectomy in women with galactorrhea-amenorrhea. JAMA 1979;242:158–162.
11 Couldwell W, Weiss M: Pituitary macroadenomas; in Apuzzo M (ed): Brain surgery. Churchill
Livingstone, 1993, pp 295–308.
12 Brada M, Rajan B, Traish D, Ashley S, Holmes-Sellors PJ, Nussey S, Uttley D: The long-term effi-
cacy of conservative surgery and radiotherapy in the control of pituitary adenomas. Clin Endocrinol
(Oxf) 1993;38:571–578.
13 Turner HE, Stratton IM, Byrne JV, Adams CB, Wass JA: Audit of selected patients with nonfunc-
tioning pituitary adenomas treated without irradiation – A follow-up study. Clin Endocrinol (Oxf)
1999;51:281–284.
14 Littley MD, Shalet SM, Beardwell CG, Ahmed SR, Applegate G, Sutton ML: Hypopituitarism
following external radiotherapy for pituitary tumours in adults. Q J Med 1989;70:145–160.
15 Littley MD, Shalet SM, Beardwell CG, Robinson EL, Sutton ML: Radiation-induced hypopitu-
itarism is dose-dependent. Clin Endocrinol (Oxf) 1989;31:363–373.
16 Tsang RW, Brierley JD, Panzarella T, Gospodarowicz MK, Sutcliffe SB, Simpson WJ: Radiation
therapy for pituitary adenoma: Treatment outcome and prognostic factors. Int J Radiat Oncol Biol
Phys 1994;30:557–565.
17 Kline LB, Kim JY Ceballos R: Radiation optic neuropathy. Ophthalmology 1985;92:1118–1126.
18 al-Mefty O, Kersh JE, Routh A, Smith RR: The long-term side effects of radiation therapy for
benign brain tumors in adults. J Neurosurg 1990;73:502–512.
19 Tachibana O, Yamaguchi N, Yamashima T, Yamashita J: Radiation necrosis of the optic chiasm,
optic tract, hypothalamus, and upper pons after radiotherapy for pituitary adenoma, detected by
gadolinium-enhanced, T1-weighted magnetic resonance imaging: Case report. Neurosurgery
1990;27:640–643.
20 Boelaert K, Gittoes NJ: Radiotherapy for non-functioning pituitary adenomas. Eur J Endocrinol
2001;144:569–575.
21 Marcou Y, Plowman PN: Stereotactic radiosurgery for pituitary adenomas. Trends Endocrinol
Metab 2000;11:132–137.
22 Degerblad M, Rahn T, Bergstrand G, Thoren M: Long-term results of stereotactic radiosurgery to
the pituitary gland in Cushing’s disease. Acta Endocrinol (Copenh) 1986;112:310–314.
23 Pollock BE, Kondziolka D, Lunsford LD, Flickinger JC: Stereotactic radiosurgery for pituitary
adenomas: Imaging, visual and endocrine results. Acta Neurochir Suppl (Wien) 1994;62:
33–38.
24 Park YG, Chang JW, Kim EY, Chung SS: Gamma knife surgery in pituitary microadenomas.
Yonsei Med J 1996;37:165–173.
25 Voges J, Sturm V, Deuss U, Traud C, Treuer H, Schlegel W, Winkelmann W, Muller RP: LINAC-
radiosurgery (LINAC-RS) in pituitary adenomas: Preliminary results. Acta Neurochir Suppl (Wien)
1996;65:41–43.
26 Landolt AM, Haller D, Lomax N, Scheib S, Schubiger O, Siegfried J, Wellis G: Stereotactic radio-
surgery for recurrent surgically treated acromegaly: Comparison with fractionated radiotherapy.
J Neurosurg 1998;88:1002–1008.
27 Bevan JS, Webster J, Burke CW, Scanlon MF: Dopamine agonists and pituitary tumor shrinkage.
Endocr Rev 1992;13:220–240.
28 Castro MG, Davis JR, Xiong W, Lowenstein PR: ‘Recent developments in gene therapy:
Applications for the treatment of pituitary tumours.’ Baillieres Best Pract Res Clin Endocrinol Metab
1999;13:431–449.
29 Laws JE, Chenelle A, Thapar K: Recurrence after transsphenoidal surgery for pituitary adenomas:
Clinical outcomes and basic science aspects; in Fahlbusch R: Pituitary Adenomas: From Basic
Research to Diagnosis and Therapy. The Netherlands, Elsevier Science BV, 1996, pp 3–9.
30 Plowman PN: Pituitary adenoma radiotherapy – When, who and how? Clin Endocrinol (Oxf)
1999;51:265–271.
medwedi.ru
31 Pellegrini I, Rasolonjanahary R, Gunz G, Bertrand P, Delivet S, Jedynak CP, Kordon C, Peillon F,
Jaquet P, Enjalbert A: Resistance to bromocriptine in prolactinomas. J Clin Endocrinol Metab
1989;69:500–509.
32 Shenk T: Adenoviridae and Their Replication; in Fields B, Knipe D, Howley P (eds): Virology, 4th
ed, Philadelphia, Lippincott-Raven Publishers, 1996, pp 2111–2148.
33 Bergelson JM, Cunningham JA, Droguett G, Kurt-Jones EA, Krithivas A, Hong JS, Horwitz MS,
Crowell RL, Finberg RW: Isolation of a common receptor for Coxsackie B viruses and aden-
oviruses 2 and 5. Science 1997;275:1320–1323.
34 Wickham TJ, Mathias P, Cheresh DA, Nemerow GR: Integrins alpha v beta 3 and alpha v beta 5
promote adenovirus internalization but not virus attachment. Cell 1993;73:309–319.
35 Leopold PL, Ferris B, Grinberg I, Worgall S, Hackett NR, Crystal RG: Fluorescent virions:
Dynamic tracking of the pathway of adenoviral gene transfer vectors in living cells. Hum Gene
Ther 1998;9:367–378.
36 Danthinne X, Imperiale MJ: Production of first generation adenovirus vectors: A review. Gene
Ther 2000;7:1707–1714.
37 Harui A, Suzuki S, Kochanek S, Mitani K: Frequency and stability of chromosomal integration of
adenovirus vectors. J Virol 1999;73:6141–6146.
38 Morral N, O’Neal W, Rice K, Leland M, Kaplan J, Piedra PA, Zhou H, Parks RJ, Velji R, Aguilar-
Cordova E, Wadsworth S, Graham FL, Kochanek S, Carey KD, Beaudet AL: Administration of
helper-dependent adenoviral vectors and sequential delivery of different vector serotype for long-
term liver-directed gene transfer in baboons. Proc Natl Acad Sci USA 1999;96:12816–12821.
39 Christ M, Louis B, Stoeckel F, Dieterle A, Grave L, Dreyer D, Kintz J, Ali Hadji D, Lusky M,
Mehtali M: Modulation of the inflammatory properties and hepatotoxicity of recombinant aden-
ovirus vectors by the viral E4 gene products. Hum Gene Ther 2000;11:415–27.
40 Thomas CE, Schiedner G, Kochanek S, Castro MG, Lowenstein PR: Peripheral infection with
adenovirus causes unexpected long-term brain inflammation in animals injected intracranially
with first-generation, but not with high-capacity, adenovirus vectors: Toward realistic long-
term neurological gene therapy for chronic diseases. Proc Natl Acad Sci USA 2000;97:
7482–7487.
41 Thomas CE, Schiedner G, Kochanek S, Castro MG, Lowenstein PR: Preexisting antiadenoviral
immunity is not a barrier to efficient and stable transduction of the brain, mediated by novel high-
capacity adenovirus vectors. Hum Gene Ther 2001;12:839–846.
42 Grave L, Dreyer D, Dieterle A, Leroy P, Michou AI, Doderer C, Pavirani A, Lusky M, Mehtali M:
Differential influence of the E4 adenoviral genes on viral and cellular promoters. J Gene Med
2000;2:433–443.
43 Brann T, Kayda D, Lyons RM, Shirley P, Roy S, Kaleko M, Smith T: Adenoviral vector-mediated
expression of physiologic levels of human factor VIII in nonhuman primates. Hum Gene Ther
1999;10:2999–3011.
44 Hu H, Serra D, Amalfitano A: Persistence of an [E1-, polymerase-] adenovirus vector despite
transduction of a neoantigen into immune-competent mice. Hum Gene Ther 1999;10:355–364.
45 Hodges BL, Evans HK, Everett RS, Ding EY, Serra D, Amalfitano A: Adenovirus vectors with the
100K gene deleted and their potential for multiple gene therapy applications. J Virol 2001;75:
5913–5920.
46 Kochanek S, Clemens PR, Mitani K, Chen HH, Chan S, Caskey CT: A new adenoviral vector:
Replacement of all viral coding sequences with 28 kb of DNA independently expressing both
full-length dystrophin and beta-galactosidase. Proc Natl Acad Sci USA 1996;93:5731–5736.
47 Parks RJ, Chen L, Anton M, Sankar U, Rudnicki MA, Graham FL: A helper-dependent adenovirus
48 Umana P, Gerdes CA, Stone D, Davis JR, Ward D, Castro MG, Lowenstein PR: Efficient FLPe
recombinase enables scalable production of helper-dependent adenoviral vectors with negligible
helper-virus contamination. Nat Biotechnol 2001;19:582–585.
49 Lowenstein PR, Thomas CE, Umana P, Gerdes CA, Verakis T, Boyer O, Tondeur S, Klatzmann D,
Castro MG: High-capacity, helper-dependent, ‘gutless’ adenoviral vectors for gene transfer into
brain. Methods Enzymol 2002;346:292–311.
50 Grable M, Hearing P: cis and trans requirements for the selective packaging of adenovirus type 5
DNA. J Virol 1992;66:723–731.
51 Hardy S, Kitamura M, Harris-Stansil T, Dai Y, Phipps ML: Construction of adenovirus vectors
through Cre-lox recombination. J Virol 1997;71:1842–1849.
52 Chen L, Anton M, Graham FL: Production and characterization of human 293 cell lines express-
ing the site-specific recombinase Cre. Somat Cell Mol Genet 1996;22:477–488.
53 Morsy MA, Gu M, Motzel S, Zhao J, Lin J, Su Q, Allen H, Franlin L, Parks RJ, Graham FL,
Kochanek S, Bett AJ, Caskey CT: An adenoviral vector deleted for all viral coding sequences
results in enhanced safety and extended expression of a leptin transgene. Proc Natl Acad Sci USA
1998;95:7866–7871.
54 Ng P, Beauchamp C, Evelegh C, Parks R, Graham FL: Development of a FLP/frt system for gen-
erating helper-dependent adenoviral vectors. Mol Ther 2001;3:809–815.
55 Zhou H, Zhao T, Pastore L, Nageh M, Zheng W, Rao XM, Beaudet AL: A Cre-expressing cell line
and an E1/E2a double-deleted virus for preparation of helper-dependent adenovirus vector. Mol
Ther 2001;3:613–622.
56 Schiedner G, Morral N, Parks RJ, Wu Y, Koopmans SC, Langston C, Graham FL, Beaudet AL,
Kochanek S: Genomic DNA transfer with a high-capacity adenovirus vector results in improved
in vivo gene expression and decreased toxicity. Nat Genet 1998;18:180–183.
57 Murphy JE, Zhou S, Giese K, Williams LT, Escobedo JA, Dwarki VJ: Long-term correction of
obesity and diabetes in genetically obese mice by a single intramuscular injection of recombinant
adeno-associated virus encoding mouse leptin. Proc Natl Acad Sci USA 1997;94:13921–13926.
58 Acland GM, Aguirre GD, Ray J, Zhang Q, Aleman TS, Cideciyan AV, Pearce-Kelling SE, Anand V,
Zeng Y, Maguire AM, Jacobson SG, Hauswirth WW, Bennett J: Gene therapy restores vision in a
canine model of childhood blindness. Nat Genet 2001;28:92–95.
59 Chao H, Monahan PE, Liuy, Samulski RJ, Walsh CE: Sustained and complete phenotype correc-
tion of hemophilia B mice following intramuscular injection of AAV1 serotype vectors. Mol Ther
2001;4:217–222.
60 Surosky RT, Urabe M, Godwin SG, McQuiston SA, Kurtzman GJ, Ozawa K, Natsoulis G: Adeno-
associated virus Rep proteins target DNA sequences to a unique locus in the human genome.
J Virol 1997;71:7951–7959.
61 Kearns WG, Afione SA, Fulmer SB, Pang MC, Erikson D, Egan M, Landrum MJ, Flotte TR,
Cutting GR: Recombinant adeno-associated virus (AAV-CFTR) vectors do not integrate in a site-
specific fashion in an immortalized epithelial cell line. Gene Ther 1996;3:748–755.
62 Balague C, Kalla M, Zhang WW: Adeno-associated virus Rep78 protein and terminal repeats
enhance integration of DNA sequences into the cellular genome. J Virol 1997;71:3299–3306.
63 Nakai H, Storm TA, Kay MA: Increasing the size of rAAV-mediated expression cassettes in vivo
by intermolecular joining of two complementary vectors. Nat Biotechnol 2000;18:527–532.
64 Yan Z, Zhangy, Duan D, Engelhardt JF: Trans-splicing vectors expand the utility of adeno-
associated virus for gene therapy. Proc Natl Acad Sci USA 2000;97:6716–6721.
65 Ma HI, Lin SZ, Chiang YH, Li J, Chen SL, Tsao YP, Xiao X: Intratumoral gene therapy of malig-
nant brain tumor in a rat model with angiostatin delivered by adeno-associated viral (AAV) vec-
tor. Gene Ther 2002;9:2–11.
66 Laquerre S, Anderson DB, Stolz DB, Glorioso JC: Recombinant herpes simplex virus type 1 engi-
neered for targeted binding to erythropoietin receptor-bearing cells. J Virol 1998;72:9683–9697.
67 Costantini LC, Jacoby DR, Wang S, Fraefel C, Breakefield XO, Isacson O: Gene transfer to the
nigrostriatal system by hybrid herpes simplex virus/adeno-associated virus amplicon vectors.
Hum Gene Ther 1999;10:2481–2494.
68 Roe T, Reynolds TC, Yu G, Brown PO: Integration of murine leukemia virus DNA depends on
mitosis. Embo J 1993;12:2099–2108.
69 Palmer TD, Rosman GJ, Osborne WR, Miller AD: Genetically modified skin fibroblasts persist
long after transplantation but gradually inactivate introduced genes. Proc Natl Acad Sci USA
1991;88:1330–1334.
70 Bordignon C, Notarangelo LD, Nobili N, Ferrari G, Casorati G, Panina P, Mazzolari E, Maggioni D,
Rossi C, Servida P, et al: Gene therapy in peripheral blood lymphocytes and bone marrow for
ADA-immunodeficient patients. Science 1995;270:470–475.
medwedi.ru
71 Yu SF, von Ruden T, Kantoff PW, Garber C, Seiberg M, Ruther U, Anderson WF, Wagner EF,
Gilboa E: Self-inactivating retroviral vectors designed for transfer of whole genes into mammalian
cells. Proc Natl Acad Sci USA 1986;83:3194–3198.
72 Naldini L: Lentiviruses as gene transfer agents for delivery to non-dividing cells. Curr Opin
Biotechnol 1998;9:457–463.
73 Zufferey R, Nagy D, Mandel RJ, Naldini L, Trono D: Multiply attenuated lentiviral vector
achieves efficient gene delivery in vivo. Nat Biotechnol 1997;15:871–875.
74 Zennou V, Petit C, Guetard D, Nerhbass U, Montagnier L, Charneau P: HIV-1 genome nuclear
import is mediated by a central DNA flap. Cell 2000;101:173–185.
75 Stone D, David A, Bolognani F, Lowenstein PR, Castro MG: Viral vectors for gene delivery and
gene therapy within the endocrine system. J Endocrinol 2000;164:103–118.
76 Thomas CE, Ehrhardt A, Kay MA: Progress and problems with the use of viral vectors for gene
therapy. Nat Rev Genet 2003;4:346–358.
77 Sandalon Z, Gnatenko DV, Bahou WF, Hearing P: Adeno-associated virus (AAV) Rep protein
enhances the generation of a recombinant mini-adenovirus (Ad) utilizing an Ad/AAV hybrid virus.
J Virol 2000;74:10381–10389.
78 Wickham TJ: Targeting adenovirus. Gene Ther 2000;7:110–114.
79 Girod A, Ried M, Wobus C, Lahm H, Leike K, Kleinschmidt J, Deleage G, Hallek M: Genetic cap-
sid modifications allow efficient re-targeting of adeno-associated virus type 2. Nat Med 1999;5:1438.
80 Shi CX, Hitt M, Ng P, Graham FL: Superior tissue-specific expression from tyrosinase and
prostate-specific antigen promoters/enhancers in helper-dependent compared with first-genera-
tion adenoviral vectors. Hum Gene Ther 2002;13:211–224.
81 Smith-Arica JR, Morelli AE, Larregina AT, Smith J, Lowenstein PR, Castro MG: Cell-type-specific
and regulatable transgenesis in the adult brain: Adenovirus-encoded combined transcriptional
targeting and inducible transgene expression. Mol Ther 2000;2:579–587.
82 Kafri T, van Praag H, Gage FH, Verma IM: Lentiviral vectors: Regulated gene expression. Mol
Ther 2000;1:516–521.
83 Williams JC, Stone D, Smith-Arica JR, Morris ID, Lowenstein PR, Castro MG: ‘Regulated,
adenovirus-mediated delivery of tyrosine hydroxylase suppresses growth of estrogen-induced
pituitary prolactinomas.’ Mol Ther 2001;4:593–602.
84 Dewey RA, Morrissey G, Cowsill CM, Stone D, Bolognani F, Dodd NJ, Southgate TD, Klatzmann D,
Lassmann H, Castro MG, Lowenstein PR: Chronic brain inflammation and persistent herpes sim-
plex virus 1 thymidine kinase expression in survivors of syngeneic glioma treated by adenovirus-
mediated gene therapy: Implications for clinical trials. Nat Med 1999;5:1256–1263.
85 Cowsill C, Southgate TD, Morrissey G, Dewey RA, Morelli AE, Maleniak TC, Forrest Z,
Klatzmann D, Wilkinson GW, Lowenstein PR, Castro MG: Central nervous system toxicity of two
adenoviral vectors encoding variants of the herpes simplex virus type 1 thymidine kinase: reduced
cytotoxicity of a truncated HSV1-TK. Gene Ther 2000;7:679—685.
86 Windeatt S, Southgate TD, Dewey RA, Bolognani F, Perone MJ, Larregina AT, Maleniak TC,
Morris ID, Goya RG, Klatzmann D, Lowenstein PR, Castro MG: Adenovirus-mediated herpes
simplex virus type-1 thymidine kinase gene therapy suppresses oestrogen-induced pituitary pro-
lactinomas. J Clin Endocrinol Metab 2000;85:1296–1305.
87 Castro M, Goverdhana S, Hu J, Jovel N, Yuan X, Lowenstein P: Gene therapy for pituitary tumors:
From preclinical models to clinical implementation. Front Neuroendocrinol 2003;24:62–77.
88 Chen SH, Shine HD, Goodman JC, Grossman RG, Woo SL: Gene therapy for brain tumors:
regression of experimental gliomas by adenovirus-mediated gene transfer in vivo. Proc Natl Acad
Sci USA 1994;91:3054–3057.
89 Klatzmann D, Valery CA, Bensimon G, Marro B, Boyer O, Mokhtari K, Diquet B, Salzmann JL,
Philippon J: A phase I/II study of herpes simplex virus type 1 thymidine kinase ‘suicide’ gene
therapy for recurrent glioblastoma. Study Group on Gene Therapy for Glioblastoma. Hum Gene
Ther 1998;9:2595–2604.
90 Sandmair AM, Loimas S, Puranen P, Immonen A, Kossila M, Puranen M, Hurskainen H,
Tyynela K, Turunen M, Vanninen R, Lehtolainen P, Paljarvi L, Johansson R, Vapalahti M,
Yla-Herttuala S: Thymidine kinase gene therapy for human malignant glioma, using replication-
deficient retroviruses or adenoviruses. Hum Gene Ther 2000;11:2197–2205.
91 Huber BE, Austin EA, Good SS, Knick VC, Tibbels S, Richards CA: In vivo antitumor activity of
5-fluorocytosine on human colorectal carcinoma cells genetically modified to express cytosine
deaminase. Cancer Res 1993;53:4619–4626.
92 Kambara H, Tamiya T, Ono Y, Ohtsuka S, Terada K, Adachi Y, Ichikawa T, Hamada H, Ohmoto T:
Combined radiation and gene therapy for brain tumors with adenovirus-mediated transfer of
cytosine deaminase and uracil phosphoribosyltransferase genes. Cancer Gene Ther 2002;9:
840–845.
93 Searle PF, Weedon SJ, McNeish IA, Gilligan MG, Ford MJ, Friedlos F, Springer CJ, Young LS,
Kerr DJ: Sensitisation of human ovarian cancer cells to killing by the prodrug CB1954 following
retroviral or adenoviral transfer of the E. coli nitroreductase gene. Adv Exp Med Biol 1998;451:
107–113.
94 Blakey DC, Davies DH, Dowell RI, East SJ, Burke PJ, Sharma SK, Springer CJ, Mauger AB,
Melton RG: Anti-tumour effects of an antibody-carboxypeptidase G2 conjugate in combination
with phenol mustard prodrugs. Br J Cancer 1995;72:1083–1088.
95 Cowen RL, Williams JC, Emery S, Blakey D, Darling JL, Lowenstein PR, Castro MG: Adenovirus
vector-mediated delivery of the prodrug-converting enzyme carboxypeptidase G2 in a secreted or
GPI-anchored form: High-level expression of this active conditional cytotoxic enzyme at the
plasma membrane. Cancer Gene Ther 2002;9:897–907.
96 Rogulski KR, Kim JH, Kim SH, Freytag SO: Glioma cells transduced with an Escherichia coli
CD/HSV-1 TK fusion gene exhibit enhanced metabolic suicide and radiosensitivity. Hum Gene
Ther 1997;8:73–85.
97 Kim JH, Kolozsvary A, Rogulski K, Khil MS, Brown SL, Freytag SO: Selective radiosensitiza-
tion of 9L glioma in the brain transduced with double suicide fusion gene. Cancer J Sci Am 1998;
4:364–369.
98 Davidoff AM, Leary MA, Ng CY, Vanin EF: Gene therapy-mediated expression by tumor cells of
the angiogenesis inhibitor flk-1 results in inhibition of neuroblastoma growth in vivo. J Pediatr
Surg 2001;36:30–36.
99 O’Reilly MS, Holmgren L, Shing Y, Chen C, Rosenthal RA, Moses M, Lane WS, Cao Y, Sage EH,
Folkman J: Angiostatin: A novel angiogenesis inhibitor that mediates the suppression of metas-
tases by a Lewis lung carcinoma. Cell 1994;79:315–328.
100 O’Reilly MS, Boehm T, Shing Y, Fukai N, Vasios G, Lane WS, Flynn E, Birkhead JR, Olsen BR,
Folkman J: Endostatin: An endogenous inhibitor of angiogenesis and tumor growth. Cell 1997;
88:277–285.
101 Lowenstein PR, Southgate TD, Smith-Arica JR, Smith J, Castro MG: Gene therapy for inherited
neurological disorders: Towards therapeutic intervention in the Lesch-Nyhan syndrome. Prog
Brain Res 1998;117:485–501.
102 Watson AJM, Lowenstein PMR: Therapeutic Manipulation of Apoptosis in Cancer and
Neurological Disease; in Wilson JW, Booth C, Potten CS (eds): Apoptosis Regulatory Genes,
Boston/Dordecht/London, Kluwer Academic Publisher, 1999, pp 281–303.
103 Ueki K, Ono Y, Henson JW, Efird JT, von Deimling A, Louis DN: CDKN2/p16 or RB alterations
occur in the majority of glioblastomas and are inversely correlated. Cancer Res 1996;56:150–153.
104 Nagata S: Apoptosis by death factor. Cell 1997;88:355–365.
105 Strasser A, O’Connor L: Fas ligand – Caught between Scylla and Charybdis. Nat Med 1998;4:21–22.
106 Friesen C, Herr I, Krammer PH, Debatin KM: Involvement of the CD95 (APO-1/FAS) receptor/
ligand system in drug-induced apoptosis in leukemia cells. Nat Med 1996;2:574–577.
107 Larregina AT, Morelli AE, Dewey RA, Castrov MG, Fontana A, Lowenstein PR: FasL induces
Fas/Apo1-mediated apoptosis in human embryonic kidney 293 cells routinely used to generate
E1-deleted adenoviral vectors. Gene Ther 1998;5:563–568.
108 Morelli AE, Larregina AT, Smith-Arica J, Dewey RA, Southgate TD, Ambar B, Fontana A,
Castro MG, Lowenstein PR: Neuronal and glial cell type-specific promoters within adenovirus
recombinants restrict the expression of the apoptosis-inducing molecule Fas ligand to predeter-
mined brain cell types, and abolish peripheral liver toxicity. J Gen Virol 1999;80:571–583.
109 Ambar BB, Frei K, Malipiero U, Morelli AE, Castro MG, Lowenstein PR, Fontana A: Treatment
of experimental glioma by administration of adenoviral vectors expressing Fas ligand. Hum Gene
Ther 1999;10:1641–1648.
medwedi.ru
110 Gomez-Manzano C, Fueyo J, Kyritsis AP, Steck PA, Roth JA, McDonnell TJ, Steck KD, Levin VA,
Yung WK: Adenovirus-mediated transfer of the p53 gene produces rapid and generalized death of
human glioma cells via apoptosis. Cancer Res 1996;56:694–699.
111 Riley DJ, Nikitin AY, Lee WH: Adenovirus-mediated retinoblastoma gene therapy suppresses
spontaneous pituitary melanotroph tumors in Rb⫹/⫺ mice. Nat Med 1996;2:1316–1321.
112 Rasheed BK, Stenzel TT, McLendon RE, Parsons R, Friedman AH, Friedman HS, Bigner DD,
Bigner SH: PTEN gene mutations are seen in high-grade but not in low-grade gliomas. Cancer
Res 1997;57:4187–4190.
113 Cheney IW, Johnson DE, Vaillancourt MT, Avanzini J, Morimoto A, Demers GW, Wills KN,
Shabram PW, Bolen JB, Tavtigian SV, Bookstein R: Suppression of tumorigenicity of glioblastoma
cells by adenovirus-mediated MMAC1/PTEN gene transfer. Cancer Res 1998;58:2331–2334.
114 Zhang X, Horwitz GA, Heaney AP, Nakashima M, Prezant TR, Bronstein MD, Melmed S:
Pituitary tumor transforming gene (PTTG) expression in pituitary adenomas. J Clin Endocrinol
Metab 1999;84:761–767.
115 McCabe C: Genetic targets for the treatment of pituitary adenomas: Focus on the pituitary tumor
transforming gene. Curr Opin Pharmacol 2001;1:620–625.
116 Ando H, Saio M, Ohe N, Tamakawa N, Yuh, Nakayama T, Yoshimura S, Kaku Y, Iwama T, Shinoda J,
Sakai N, Takami T: B7.1 immunogene therapy effectively activates CD(4⫹) tumor-infiltrating
lymphocytes in the central nervous system in comparison with B7.2 gene therapy. Int J Oncol
2002;20:807–812.
117 Ueda G, Takizawa S, Moy P, Marolla F, Furth J: Characterization of four transplantable mam-
motropic pituitary tumor variants in the rat. Cancer Res 1968;28:1963–1975.
118 Furth J, Nakane P, Pasteels JL: Tumours of the pituitary gland. IARC Sci Publ1976;6:201–237.
119 Lloyd RV: Estrogen-induced hyperplasia and neoplasia in the rat anterior pituitary gland. An
immunohistochemical study. Am J Pathol 1983;113:198–206.
120 Treip CS: The regression of oestradiol-induced pituitary tumours in the rat. J Pathol 1983;141:
29–40.
121 Adler RA, Krieg RJ, Farrell ME, Deiss WP, MacLeod RM: Characterization of a new animal
model of chronic hyperprolactinemia. Metabolism 1991;40:286–291.
122 Trouillas J, Girod C, Claustrat B, Joly-Pharaboz MO, Chevallier P: Spontaneous prolactin trans-
plantable tumor in the Wistar/Furth rat (SMtTW): A new animal model of human prolactinoma.
Cancer Res 1990;50:4081–4086.
123 Daniel L, Trouillas J, Renaud W, Chevallier P, Gouvernet J, Rougon G, Figarella-Branger D:
Polysialylated-neural cell adhesion molecule expression in rat pituitary transplantable tumors
(spontaneous mammotropic transplantable tumor in Wistar-Furth rats) is related to growth rate
and malignancy. Cancer Res 2000;60:80–85.
124 Kojima A, Kubota T, Sato A, Yamada T, Yamori Y, Okamoto K: Congenital abnormality of
pituitary-thyroid axis in spontaneously hypertensive rats (SHR) and stroke-prone rats (SPR). Proc
Soc Exp Biol Med 1975;150:571–573.
125 Kojima A, Takahashi Y, Ohno SI, Sato A, Yamada T, Kubota T, Yamori Y, Okamoto K: An eleva-
tion of plasma ISH concentration in spontaneously hypertensive rats (SHR). Proc Soc Exp Biol
Med 1975;149:661–663.
126 Stefaneanu L, Rindi G, Horvath E, Murphy D, Polak JM, Kovacs K: Morphology of adeno-
hypophysial tumors in mice transgenic for vasopressin-SV40 hybrid oncogene. Endocrinology
1992;130:1789–1795.
127 Lee EJ, Anderson LM, Thimmapaya B, Jameson JL: Targeted expression of toxic genes directed
by pituitary hormone promoters: A potential strategy for adenovirus-mediated gene therapy of
pituitary tumors. J Clin Endocrinol Metab 1999;84:786–794.
128 Puchner MJ, Ludecke DK, Saeger W, Herrmann HD: Use of athymic nude mice for in vivo stud-
ies of human growth-hormone-secreting pituitary adenomas. Horm Res 1991;35:198–204.
129 Helseth A, Siegal GP, Haug E, Bautch VL: Transgenic mice that develop pituitary tumors. A model
for Cushing’s disease. Am J Pathol 1992;140:1071–1080.
130 Yano H, Readhead C, Nakashima M, Ren SG, Melmed S: Pituitary-directed leukemia inhibitory
factor transgene causes Cushing’s syndrome: Neuro-immune-endocrine modulation of pituitary
development. Mol Endocrinol 1998;12:1708–1720.
131 Westphal CH, Muller L, Zhou A, Zhu X, Bonner-Weir S, Schambelan M, Steiner DF, Lindberg I,
Leder P: The neuroendocrine protein 7B2 is required for peptide hormone processing in vivo and
provides a novel mechanism for pituitary Cushing’s disease. Cell 1999;96:689–700.
132 Kumar TR, Graham KE, Asa SL, Low MJ: Simian virus 40 T antigen-induced gonadotroph ade-
nomas: A model of human null cell adenomas. Endocrinology 1998;139:3342–3351.
133 Castro MG, Southgate T, Lowenstein PR: Molecular therapy in a model neuroendocrine disease:
Developing clinical gene therapy for pituitary tumours. Trends Endocrinol Metab 2001;12:58–64.
134 Castro MG, Goya RG, Sosa YE, Rowe J, Larregina A, Morelli A, Lowenstein PR: Expression of
transgenes in normal and neoplastic anterior pituitary cells using recombinant adenoviruses: Long
term expression, cell cycle dependency, and effects on hormone secretion. Endocrinology 1997;
138:2184–2194.
135 Goya RG, Rowe J, Sosa YE, Tomasec P, Lowenstein PR, Castro MG: Use of recombinant herpes
simplex virus type 1 vectors for gene transfer into tumour and normal anterior pituitary cells. Mol
Cell Endocrinol 1998;139:199–207.
136 Lee EJ, Martinson F, Kotlar T, Thimmapaya B, Jameson JL: Adenovirus-mediated targeted expres-
sion of toxic genes to adrenocorticotropin-producing pituitary tumors using the proopiome-
lanocortin promoter. J Clin Endocrinol Metab 2001;86:3400–3409.
137 Southgate TD, Windeatt S, Smith-Arica J, Gerdes CA, Perone MJ, Morris I, Davis, JR, Klatzmann D,
Lowenstein PR, Castro MG: Transcriptional targeting to anterior pituitary lactotrophic cells using
recombinant adenovirus vectors in vitro and in vivo in normal and estrogen/sulpiride-induced
hyperplastic anterior pituitaries. Endocrinology 2000;141:3493–3505.
138 Lee EJ, Jameson JL: Cell-specific Cre-mediated activation of the diphtheria toxin gene in pituitary
tumor cells: Potential for cytotoxic gene therapy. Hum Gene Ther 2002;13:533–542.
139 Lee EJ, Duan WR, Jakacka M, Gehm BD, Jameson JL: Dominant negative ER induces apoptosis
in GH(4) pituitary lactotrope cells and inhibits tumor growth in nude mice. Endocrinology 2001;
142:3756–3763.
140 Sarac MS, Windeatt S, Castro MG, Lindberg I: Intrapituitary adenoviral administration of 7B2 can
extend life span and reverse endocrinological deficiencies in 7B2 null mice. Endocrinology
2002;143:2314–2323.
141 Southgate TD, Stone D, Williams JC, Lowenstein PR, Castro MG: Long-term transgene expres-
sion within the anterior pituitary gland in situ: Impact on circulating hormone levels, cellular and
antibody-mediated immune responses. Endocrinology 2001;142:464–476.
142 Davis JR, McVerry J, Lincoln GA, Windeatt S, Lowenstein PR, Castro MG, McNeilly AS: Cell
type-specific adenoviral transgene expression in the intact ovine pituitary gland after stereotaxic
delivery: An in vivo system for long-term multiple parameter evaluation of human pituitary gene
therapy. Endocrinology 2001;142:795–801.
Dr. M.G. Castro

Gene Therapeutics Research Institute
Cedars-Sinai Medical Center, Research Pavilion
Room R-5090, 8700 Beverly Boulevard, Los Angeles, CA 90048–1860 (USA)
Tel. ⫹1 310 423 7302, Fax ⫹1 310 423 7308, E-Mail castromg@cshs.org
medwedi.ru
Stem Cells for Targeting

CNS Malignancy
Stephen Yipa, Richard L. Sidmanb, Evan Y. Snyderb,c
a
Division of Neuropathology, Department of Pathology and Laboratory Medicine,
Vancouver General Hospital, University of British Columbia, Vancouver, Canada;
b
Department of Neurology, Harvard Medical School, Harvard Institutes of Medicine,
Beth Israel-Deaconess Medical Center & Children’s Hospital, Boston, Mass., and
c
The Burnham Institute, Program in Developmental & Regenerative Cell Biology,
La Jolla, Calif., USA
The Neural Stem Cell as a Model Somatic Stem Cell
In the early 1990s, a handful of investigators interested in fundamental neural

development began to identify, within cultures obtained from the developing and
mature central nervous system (CNS), cells with surprising plasticity, multipo-
tency, and a propensity for dynamically shifting their fates [1–4]. The existence
of such cells – if, indeed, they represented a population normally resident in the
brain – challenged the prevailing dogma that the nervous system was rigidly and
immutably constructed. Neural stem cells (NSCs), as these plastic cells came to be
termed, began to garner the interest of not just the developmental community but
also that of the neural repair, gene therapy, and transplant communities when it was
recognized that they could be expanded in culture and reimplanted into the mam-
malian brain where they would reintegrate appropriately and stably express foreign
genes [3]. Their abundance, multipotency, ease of manipulation and engraftability
made this strategy a powerful method for CNS gene therapy and repair.
In comparison to extant techniques, NSCs presented certain advantages:
they were a homogeneous and relatively well-defined neural cell population that
could be stored and expanded on demand, and, if necessary, genetically mani-
pulated ex vivo to express a wide variety of foreign transgenes. These transduced
genes, as well as their inherent genetic repertoire, could be imported into
the CNS following transplantation almost anywhere into the developing and
mature host brain. Furthermore, NSCs and their progeny possessed a capacity to
integrate not only locally at their site of implantation, and competing with and
interdigitating seamlessly with endogenous cells [3, 5–8], but also more broadly
[9–13]. These cells were quite migratory – particularly if implanted into germi-
nal zones – permitting cell and gene therapy to be contemplated for disseminated,
even global, CNS disease processes. In that sense, NSCs had a distinct advantage
over fetal tissue and non-neural cells for cell replacement and over most viral
vectors for gene delivery. Even such alternative cellular vectors as hematopoietic
cells, when used for protein delivery in bone marrow transplantation paradigms
[14, 15], could not efficiently circumvent the restrictions of the blood-brain
barrier and integrate throughout the CNS as effectively as NSCs. A single bona
fide NSC clone could take up residence in, and accommodate to, any nervous
system region, permitting an economy of resources.
In addition, NSCs appear to be attracted by degenerating neural tissue by
local cues [7, 16, 17], replacing dead or dysfunctional cells in those regions. In
pathological niches, multipotent NSCs, in response to signals still poorly under-
stood (though probably linked to inflammatory cytokines), would shift their
fate toward neural lineages most in need of repletion, even if beyond the clas-
sical developmental window for genesis of that cell type. These observations
gave birth to the hypothesis that certain neurodegenerative environments reca-
pitulate developmental cues, because NSCs responded to neurogenic cues not
only during their normal embryological expression, but also when recreated
by particular types of cell death. NSCs, in other words, could sense niches of
neurogenesis or small areas of pathology in the brain [5, 6, 11, 16].
These observations in the CNS served as an impetus to investigators in
other solid organ systems to search for ‘stem-like’ cells even within tissues gen-
erally held to be more regenerative, more forgiving, and/or more redundant than
the CNS. Hence, the NSC, in effect the first solid organ stem cell isolated and
exploited, served as a model for other somatic stem cells.
Importantly, despite the spotlight of therapeutic promise the NSC has
thrown upon itself (and other stem cells), it is critical to remember that its exis-
tence was unveiled in the course of understanding development and that, in the
end, it is simply one player in a broad and exceedingly complex, interdepen-
dent, finely-tuned developmental system, one that requires fundamental devel-
opmental understanding [18, 19]. In this endeavor, the CNS continues to serve
as an instructive model for the stem cell field in general [20, 21].
NSCs as a Therapeutic Tool
Knowledge of the fundamental biology of NSCs has grown significantly,

which in conjunction with advances in molecular biological techniques, has
Stem Cells for Targeting CNS Malignancy 625
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promoted their use in the treatment of various neurological disorders [13].
NSCs can migrate over long distances to areas of injuries in the CNS followed
by developmentally appropriate (temporally and regionally) differentiation
[22]. Experimental evidence has demonstrated that NSCs can populate as well
as repopulate developing and injured/regenerating areas of the immature and
mature CNS [16, 23]. For example, transplanted NSCs can effectively integrate
into abnormal brains and yield either neurons or glia (e.g., myelinating oligo-
dendrocytes in myelin-impoverished mutants) [5, 6, 11, 24, 25]. NSCs also can
serve as effective gene delivery vehicles, promoting effective changes in the
phenotype of genetically based loss-of-function models of neurodegeneration
such as lysosomal storage diseases [9]. Other examples abound for the multi-
potentiality and migratory potential of NSCs in experimental models of the dis-
eased or traumatically injured CNS [5, 6, 24, 26]. In view of their innate ability
to home in on sites of injury or dysregulated tissue, one of the newer and most
promising applications is the therapeutic use of NSCs to target malignancies of
the CNS [27, 28].
Sources of NSCs
NSCs may be prepared for use by a number of strategies, which have been
well summarized elsewhere [29]. The most straightforward method is simply to
isolate them directly from the neuroectoderm or from neuroectoderm-derived
structures. Whether it is better to abstract such cells from the embryo, fetus,
newborn, juvenile, or adult, remains to be determined empirically. Clearly, the
younger the age of the region and the more active its neurogenic potential, the
easier the process becomes.
Cells isolated from the inner cell mass of the blastocyst, known as embry-
onic stem cells, can presumably be instructed to yield cells of neuroectodermal
lineage (i.e., NSCs) in vitro when given the appropriate stimuli [30]. Several
groups have argued that there exists plasticity among germ layers such that
NSCs can be obtained from, for example, bone marrow mesenchyme or umbil-
ical cord cells of mesodermal origin [31, 32]. The fundamental ability of the
latter sources to yield bona fide neural elements remains quite controversial
and, at best, can do it only inefficiently [29].
The goal in all stem cell-based therapy is to generate a stable, self-
renewing line of such cells that can provide an inexhaustible source of cells
for experimentation. One line that has proven instructive for studying and
dissecting the potential and pitfalls of somatic stem cell-based therapies in
general and NSC-based therapies in particular is the murine clone C17.2.
These cells, initially derived from the developing murine cerebellum and
Yip/Sidman/Snyder 626
maintained as a single clone, have been transduced with the oncogene myc in
order to insure that their stem-like state would persist in vitro. These NSCs
self-renew efficiently and for prolonged passages in vitro; yet when intro-
duced to the CNS environment, they exit the cell cycle, constitutively down-
regulate the ‘stem’ state-promoting gene myc (along with other genes) and
differentiate into appropriate daughter lineages. The cells have been shown to
be nontumorigenic even in non-immunocompetent nude mice. C17.2 cells
provide an abundant and reproducible cell source for experiments that can be
well controlled from passage-to-passage, condition-to-condition, and animal-
to-animal over prolonged periods of time [3, 17].
CNS Tumors
Gliablastoma multiforme (GBM) are highly infiltrative and invasive glial

cell tumors. Median survival is one year or less for GBM and three years for
anaplastic astrocytoma, an intermediate form [33]. Low-grade gliomas can
transform into GBM as a result of specific genetic changes. Since Harvey
Cushing’s era, surgical treatment for GBM has been largely ineffective. Radical
resection, even hemispherectomy, has consistently failed due to recurrence of
tumor in the contralateral, normal-looking, hemisphere [34]. Spread of tumor
cells along white matter tracts and perineuronal, perivascular, or subpial spaces
was initially described in the 1940s in high-grade gliomas by Scherer. These
routes of dissemination are responsible for the highly infiltrative nature of
glioma and also the many challenges in their surgical management [35]. In fact,
Silbergeld and Chicoine [36] demonstrated the presence of neoplastic cells in
histological benign brain away from the tumor mass using tissue culture tech-
nique. Taking this biological destiny to an extreme, gliomatosis cerebri is a
form of glioma characterized by diffuse global infiltration and is associated
with a very poor prognosis [37]. Despite advances in every facet of neurosur-
gical oncology, from the wide use of neuronavigational systems, advancement
in real time intraoperative magnetic resonance imaging (MRI)-assisted surgery
and neurofunctional imaging coupled with better intraoperative neurophysio-
logical monitoring that have permitted almost routine use of awake craniotomy,
glioma surgical outcome still remains very poor [38, 39]. In summary, the
inability to effect surgical cure seems to be inherent in the underlying biology
of gliomas and new approaches must be found to combat the disease in a bio-
logically rational fashion.
The potential of de-differentiated tumor cells to migrate and infiltrate sur-
rounding normal brain structures resembles that of NSCs during normal neural
development. Brain tumor cells express and secrete a variety of proteases, that
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help in breaking down the intracerebral extracellular matrix to facilitate tumor
invasion and to propagate a microenvironment that is favorable for survival of
the tumor [40]. Glioma cells may arise from a small existing pool of neural
progenitors that have undergone neoplastic transformation. On the other hand,
de-differentiation of mature glial cells could give rise to gliomas exhibiting
many, but not all, of the characteristics of normal NSCs [41].
Many similarities as well as subtle differences exist between normal neuro-
development and glioma-genesis. In fact, normal glial development and glial
neoplasm formation share similar molecular genetic profiles [42]. Dai et al.
[43] showed platelet-derived growth factor-induced de-differentiation of astro-
cytes with the resultant formation of oligodendroglioma. Singh et al. [44] found
evidence of a population of CD133 positive brain tumor stem cells in primary
human glioma. These cells can undergo self-renewal as well as differentiation
similar to normal NSCs. Interestingly, the authors found a positive correlation
between the proportion of brain tumor stem cell in tumors and their biological
aggressiveness. The discovery of this population of tumor stem cells has impli-
cations in understanding the biology and clinical behavior of glioma.
Glioma-genesis is associated with characteristic genetic alterations, many
of which are well characterized [45–47]. Knowledge of the specifics of these
alterations has permitted the re-creation of the different glioma phenotypes in
rodents [48]. For example, enforced expression of the constitutively active
epidermal growth factor receptor (EGF-RvIII) under the control of the S100
promoter has generated a murine model of oligodendroglioma [49]. Haplo-
insufficiency for either p53 or ink4/arf significantly augmented the pathogenic-
ity and aggressiveness of the tumor, recapitulating glioma genetics in human
patients. Gene mutations specific to other types of human brain tumors have also
allowed for the creation of the respective animal models. Meningioma develop-
ment is recapitulated in mice with nf2 gene mutation in arachnoid cells [50].
Lineage specific knockout of the nf1 gene in Schwann cells in conjunction with
loss of heterozygosity of nf1 in non-neoplastic cells generated neurofibromas in
mice whereas the nf1 null/inactivated p53 phenotype triggered the formation of
soft tissue sarcoma resembling human neurofibromatosis 1 tumors [51, 52].
These murine brain tumor models offer a test bed for novel therapeutics as well
as a system for investigating the molecular events of brain tumorigenesis [53].
Questions still remain as to whether NSC may home in on brain tumors
other than gliomas. For example, chordoma is a histologically benign tumor
that nevertheless carries a high degree of morbidity and mortality due to its
insidious nature and the prohibitive recurrence rate despite aggressive surgi-
cal resection [54]. The question has been posed if NSCs could home in on to
chordoma and play a role in its overall management, and experiments are
underway to test this hypothesis. In addition, experimental evidence is still
pending in terms of NSC migration towards intracranial metastases from
extracranial tumors (e.g., lung, breast, melanoma). The therapeutic potential
is vast if, indeed, NSCs exhibit tropism for these intracranial tumors of
non-neural origin.
Therapeutic Uses of NSCs against Primary Brain Tumors
In 2000, Aboody et al. [17] reported that transplanted exogenous murine

and human NSCs were capable of ‘homing in’ over long distances to intracere-
bral xenogeneic brain tumors deposited into rodent brains. The authors also
demonstrated the ability of NSCs to ‘track’ tumor cells escaping from the orig-
inal inoculated tumor mass and invading normal brain. Specially tagged NSCs
were found adjacent to invading tumor cells that appeared to be infiltrating
normal brain along white matter tracts, tumor-related endothelium, and inter-
stitial spaces. There appeared to be a particular predilection of NSCs for tumor-
associated endothelium. NSCs could be introduced either intraparenchymally
or into the lateral ventricles with equal homing ability. Whether introduced into
the ipsilateral or contralateral cerebral ventricles or into the ipsilateral or
contralateral cerebral parenchyma, NSCs were able to migrate toward the
implanted tumor and appose themselves initimately to escaping, infiltrating
tumor cells even at far distances from the main tumor mass.
Indeed, even NSCs injected into the tail vein demonstrated successful
intracranial tumor ‘homing,’ establishing a paradigm since used by other inves-
tigators for other intracranial pathologies, e.g., models of multiple sclerosis
[55]. The blood-brain barrier normally acts as an efficient barrier to the suc-
cessful delivery of many therapeutic agents and dictates the limited repertoire
of brain tumor chemotherapeutic agents [56, 57]. In this instance, the blood-
brain barrier did not appear to affect the migration and homing of NSCs toward
intracranial pathology (as modeled by the neoplasms).
This ‘glioma-tropic’ capability of NSCs bodes well for their use in clinical
applications. NSCs could serve as a tumor-directed homing device expressing a
variety of anti-tumor genes, including those encoding cytotoxic, anti-angiogenic,
anti-mitotic, anti-migratory, immunomodulatory, pro-differentiating, and/or
pro-apoptotic agents. Ultimately the goal would be for NSCs to be used to in
conjunction with, and optimize, other therapeutic modalities by providing the
ability to track invading cells, heretofore, the weak point of radiation, surgical,
and pharmacological strategies, and complete eradication of the glioma cells
[58]. Aboody et al. [17] tested this hypothesis with the use of cytosine deami-
nase (CD)-expressing murine NSCs. This enzyme converts the non-toxic pre-
cursor 5-fluorocytosine (5-FC) into the toxic compound 5-fluorouracil.
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Injection of 5-FC into tumor-bearing mice that had been inoculated with
NSCs engineered to express CD was followed by dramatic reduction of intra-
cranial tumor burden. An impressive bystander effect on tumor cells was noted;
Barresi et al. [59] replicated this phenomenon by showing in vivo regression of
implanted C6 glioma cells after co-inoculation of an immortalized neural prog-
enitor cell line ST14A expressing CD followed by administration of 5-FC. This
work gave further credence to the utility of exploiting NSCs to effect the well
established but hitherto unsuccessful ‘prodrug/prodrug-converting enzyme’
strategy for destroying intracerebral malignancies.
The virtue of the prodrug approach is two-fold. First, NSCs need only
kill a small percentage of tumor cells to have a large impact on other tumor
cells by virtue of the ‘bystander effect.’ This effect can be explained by anal-
ogy to an exploding hand-grenade around the site of dying tumor cells, which
send out toxic factors that are inimical to other surrounding tumor cells. The
action of CD on 5-FC creates one of the greatest bystander effects of all the
pro-drug converting enzymes. Second, while no adverse effects or contribu-
tion to tumor growth from NSCs have ever been detected in these models,
should such an unlikely situation arise, the CD within the NSC would cause
it to self-eliminate were it to become mitotic, providing a built-in safety
mechanism.
As an extension of using NSCs to deliver cytolytic genes, and based on the
report by Lynch et al. [60] that NSCs could be used as engraftable, mobile intra-
cranial viral packaging lines, Herrlinger et al. [61] showed that murine NSCs,
engineered to release replication-conditional herpes simplex virus thymidine
kinase, were efficient in the destruction of an intracranial tumor mass as well
as isolated escaping tumor microdeposits. In preliminary studies, Dr. William
Weiss and colleagues at UCSF have validated the glioma-tropism of murine
NSCs in a transgenic oligodendroglioma-bearing mouse model. This double
transgenic mouse model is the product of mating a transgenic mouse in which
a mutant EGF receptor is transcribed from the S100 promoter (S100-v-erbB)
with an INK4aNull mouse, yielding progeny that almost uniformly develop
tumors by 6–12 months of age [49]. These investigators showed in this model
that NSCs homed in on spontaneously developing tumors. With the use of
CD-expressing NSCs (clone C17.2) in pilot studies, host survival was
improved. Because NSCs seemed to home in on even small ‘subclinical’ tumors
whose presence was unsuspected by the investigators prior to histological
examination, NSCs also may be used in tumor diagnosis if armed with tags that
can be imaged in the living state. It warrants mentioning that transgenic mice
that spontaneously develop brain tumors appear to provide models for studying
tumor biology and therapies – including the glioma-specific migratory poten-
tial of NSCs – that far more faithfully emulate the true clinical situation in
human and are, therefore, superior to older models that depend on the artificial
implantation of glioma cell lines.
Immunomodulation via the judicious use of cytokines has been shown to
be extremely effective in the destruction of experimental brain tumors, either
via direct cytotoxicity or the activation of immune-mediated anti-tumor effect
[62–64]. Benedetti et al. [65] showed that intratumoral injection of interleukin
4 expressing murine NSCs effected radiographic tumor regression and prolon-
gation of host survival. In addition, the authors detected the presence of NSCs
several weeks post injection in the recipient animals. This evidence implied
persistence of implanted NSCs and possibly persistence of anti-tumor effect
in vivo. The authors also described an inherent tumor inhibitory effect exhibited
by the injected stem cells. This phenomenon was first observed many years
ago using murine NSC clone C17.2. Gliastatin, a membrane-associated factor
isolated from these NSCs was capable of converting C6 glioma cells to pheno-
typically normal astrocytes simply via cell contact [66].
Exploiting the specific homing capability of NSCs to intracranial tumors,
Ehtesham et al. [67] transfected murine neural progenitors ex vivo with the
IL-12-gene using an adenoviral vector. Stable expression of the IL-12 protein
was demonstrated in vitro and in vivo. Implantation of IL-12 expressing NSCs
into tumor-bearing syngeneic mice effected tumor destruction and improved
host survival. In addition, the authors showed enhanced tumor infiltration by
T lymphocyte as a result of the expression of IL-12 in close proximity to the
tumor mass. In a separate study, the authors transfected similarly derived
murine neural progenitors with the human TRAIL gene using a replication-
deficient adenoviral vector [68]. TRAIL belongs to the tumor necrosis factor
superfamily of pro-apoptotic proteins, which had previously been shown to
induce apoptosis in experimental tumor models [69]. Introduction of TRAIL-
expressing neural stem/progenitor cells into nude mice bearing GBM
xenografts was followed by eradication of tumor via induction of apoptotic
cell death.
Glioma-Tropism: Molecular ‘Breadcrumbs’
The exquisite tumor-homing capability of NSCs is likely a result of multi-

ple mechanisms. Expression of a similar repertoire of adhesion molecules by
NSCs and glioma cells could effect migration to the same trophic source
[70–72]. Alternatively, ‘molecular breadcrumbs’ left by the tumor cells could
lead the migratory NSCs toward escaping tumor cells. Tumors could potentially
secrete chemotactic factors that encourage the homing of NSCs. Brain inflam-
mation secondary to tumor necrosis could attract NSCs directly or indirectly via
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factors secreted by microglia and leukocytes [73]. We have previously reviewed
some potential tumor-homing mechanisms of NSC [28].
Chemokines are involved in the normal development of the CNS and are
also implicated in various CNS pathologies [74]. The chemokine receptor
CXCR4 and its ligand SDF-1 have been implicated in glioma biology [75].
Rempel et al. [76] showed that this receptor/ligand pair is over-expressed and is
involved in glioma migration and neoangiogenesis. Zhou et al. [77] demon-
strated expression of CXCR4 and a functional CXCR4 signaling pathway in
primary glioma specimens, effecting cell survival during serum-deprivation. In
addition, the authors showed CXCR4-mediated glioma chemotaxis toward a
source of SDF-1. The same receptor/ligand combination has been shown to
be expressed and to function in the development of early neural and glial cells
[78, 79] and in migration of cells in vivo during formation of the cerebellum
and cortex [80]. Recently, Rubin et al. [81] showed that a small molecule
inhibitor of CXCR4 abrogates the growth of implanted glioblastoma multi-
forme and medulloblastoma in rodents. We have also observed, in preliminary
studies, that both murine and human NSCs (the same cells that are drawn to
intracranial tumors) bear CXCR4 receptors. We have found that NSCs will pro-
liferate and migrate toward a source of SDF-1 in a Boyden chamber [E.Y.
Snyder, J. Imitola, S. Kouhry, unpubl. data] and therefore we have hypothesized
that one mechanism for NSC homing to intracranial gliomas is mediated by
chemotaxis toward a source of SDF-. Ehtesham et al. recently showed that
CXCR4 mediates glioma-topic migration of neural stem cells [124].
Malignant gliomas are among the most angiogenic of all solid tumors.
Glioblastomas have elevated and deregulated expression of the highly pro-
angiogenic cytokines vascular endothelial growth factor and basic fibroblast
growth factor [82–84]. Preliminary data from J. Allport and R. Weissleder (in col-
laboration with us) suggest that human and murine NSCs, the same cells shown
to home in to tumors, express cell surface receptors with preferential binding
to tumor-derived endothelium over normal endothelium. This binding appears
to be independent of CD49d binding to endothelial 4-integrin, suggesting
other binding mechanisms [Allport, Weissleder, Snyder, unpubl. data]. NSCs can,
therefore, migrate toward areas with active tumor-associated neoangiogenesis.
Of the molecular markers selectively associated with angiogenic blood
vessels in tumors, several are cell membrane-associated proteinases. With a
powerful selection system in which circulating ligands that home to specific
vascular beds in vivo are isolated from a phage display random peptide library,
the Arap/Pasqualini team, with our participation, has identified aminopeptidase
A (APA) to be upregulated and enzymatically active in new blood vessels of
human tumor xenografts [126]. An APA-binding 9-amino acid peptide was
identified and shown to bind specifically to APA, inhibit its enzymatic activity,
suppress migration and proliferation of endothelial cells, inhibit in vitro and in
vivo angiogenesis, home to tumor vasculature in vivo and inhibit growth of
human xenografted tumors in vivo. Prolonged delivery of this and similar ther-
apeutic peptide ligands might be achieved with NSCs.
Boockvar et al. [85] observed that enhanced activation of the EGF-R, even
in the absence of ligand binding, was associated with increased motility of NSCs.
They showed that EGF-R activation triggered signaling events that resulted in
cytoskeletal rearrangements that might explain this facilitated stem cell migra-
tion. EGF is a known mitogen in primary brain neoplasm. Over-expression of
EGF and constitutive EGF-R signaling is a relatively common occurrence in
high-grade glioma [86, 87]. In addition, human glioma motility and invasion is
enhanced by the interaction between EGF and its receptor [88]. Relative cytokine
concentration gradients in the tumor microenvironment could cause NSC migra-
tion in the direction of the EGF source i.e., location of brain tumor cells.
Cellular adhesion is mediated by multiple cell surface molecules of which
the cell surface glycoprotein CD44 is noteworthy because it plays an important
role in normal cell migration and tumor metastasis [89]. Hyaluronic acid (HA)
is a predominant extracellular component in the brain and is also a major lig-
and for CD44 [90]. CD44 expression is up-regulated in glioma and this has
been shown to be instrumental in the migration and invasion of the neoplastic
cell [91–93]. Interestingly, Delpech et al. [94] showed over-expression of HA
by glioma cells, thereby creating a favorable substrate environment for glioma
motility. Preliminary studies also have demonstrated the expression of CD44 in
murine NSC clone C17.2 [Allport, Snyder, Weissleder, unpubl. observations].
CD44 expression appears to be regulated by the developmentally crucial
cytokine leukemic inhibitory factor in human NSCs [95]. Therefore, glioma-
tropism exhibited by NSCs could be, in part, mediated by a CD44-HA interac-
tion, especially in the HA-rich environment of the tumor.
Recent preliminary evidence has suggested a role for NOGO-A in glioma
migration [125]. NOGO-A, along with myelin-associated glycoprotein and
oligodendrocyte myelin glycoprotein, binds to the common cognate receptor
NOGO receptor resulting in inhibition of axonal regeneration. The inhibitory
antibody IN-1 partially restores axonal regeneration in animal models [96].
Recently, a NOGO-A knockout mouse model was demonstrated to have
enhanced nerve regeneration post experimental trauma [97], although results
from other NOGO-A knock out mice were contradictory, suggesting possible
redundancy (given the other two ligands for NOGO receptor) [98]. However,
there do not appear to be deleterious effects on neural development in the
NOGO-A knockout mice. It is not certain whether NOGO-A or its fellow ligands
play a role in mediating glioma formation or, more provocatively, gliomatropism
by NSCs.
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Smart Imaging of Intracranial Neoplasms
Instead of utilizing NSCs as carriers of therapeutic packages, Weissleder

and Ntziachristos [99] have lead research in the ‘smart imaging’ of brain
neoplasms using specially tagged NSCs. Tang et al. [100] demonstrated the
in vivo gliomatropic migration of NSCs (clone C17.2) stably transfected with
the firefly luciferase gene in a rodent model. Using bioluminescence imaging
of living mice in real time, the authors showed persistent luciferase activity
up to 4 weeks postinoculation of luciferase-expressing NSCs. While this
expression bodes well for the persistent expression of therapeutic genes in
similar situations, the technique had the further advantage of permitting the
real time tracking of migratory NSCs, offering powerful insight into the in vivo
behavior of NSCs in living animals.
C17.2 luciferase-tagged NSCs could be visualized tracking from one side
of the cerebrum to the other in order to home in and appose the implanted tumor.
MRI, a commonly used imaging modality for brain tumors in humans, also can
be scaled down for use in experimental animals as small as a mouse. Lewin
et al. [101] labeled the same murine NSCs described above (clone C17.2) with
tat peptide-derivatized magnetic nanoparticles, which renders the cells visible
under MRI. Using this imaging technique the authors could track-off the move-
ment of as few as one thousand labeled NSCs of in living rodents. One can
foresee the use of MRI or bioluminescence to follow the migration of labeled
therapeutic NSCs in brain tumor patients to monitor both safety and efficacy.
Future Directions and Application of NSCs in Brain Tumors
The Therapeutic Package: New Candidates

NSCs offer a way to deliver a broad range of therapeutic molecules to
intracranial glioma cells in a specific fashion. Immunomodulatory factors hold
particular promise for use with stem cells. Ehtesham et al. [67, 68] have shown
successful and efficacious delivery of IL-12 and TRAIL to implanted brain
tumors. Other cytokines with demonstrated promise in the treatment of glioma
could similarly be delivered by NSCs [102]. For example, IL-24 or melanoma
differentiation-associated-7 induces expression of the pro-apoptotic protein
BAX and suppresses the growth of glioma cells [103]. Aoki et al. [104] showed
that interleukin-7 reduced the tumorigenicity of glioma cells in vivo via a T
cell-mediated mechanism. Parker et al. [105] also showed immune-mediated
suppression of in vivo glioma with IL-12 delivered by a herpes simplex virus
vector. Utilizing NSCs as gliomatropic cytokine/drug delivery vehicles would
likely significantly increase the therapeutic potential of these agents.
NSCs can also prolong the bioavailability of a therapeutic agent in vivo.
For example, the potent anti-angiogenic molecule endostatin has a relatively
short half-life in vivo and intracranial bioavailability is restricted by the blood-
brain barrier. Encapsulation of cells from an embryonal kidney cell line engi-
neered to produce endostatin encased in alginate gel beads significantly
prolonged the sustained release of the protein intracranially resulting in mea-
surable reduction in tumor-related neoangiogenesis [106]. NSCs transfected
with the endostatin gene or genes for other anti-angiogenic molecules can likely
serve a similar role but with the added advantage of a migratory delivery sys-
tem that is specific for glioma and tumor-related endothelium, that is transcrip-
tionally and translationally active for several weeks postintroduction, and that
can track invading cells throughout the brain that might well set up new tumor
foci.
Herrlinger et al. [61] reported the use of NSCs in the focused delivery of
engineered herpes simplex virus expressing thymidine kinase into implanted
brain tumors. The authors delayed cell cycle entry and hence viral replication
in the NSCs with mimosine prior to transplantation into the tumor-bearing
rodent host. This, however, had no deleterious effects on the migratory poten-
tial of the NSCs. Once the cytostatic effect of mimosine had expired, viral pro-
duction reinitiated in close proximity to the tumor bulk as well as escaping
glioma cells with effective tumor destruction following the administration of
gangciclovir to trigger the thymidine kinase-mediated cytolysis.
Oncolytic viruses have recently garnered much attention in neuro-oncology,
and are discussed elsewhere in this volume [107]. Multiple naturally occurring
as well as engineered viruses are being considered for glioma-specific treatment
[108]. Taking advantage of the prevalence of ras mutations in brain tumors,
reovirus has been shown to effect selective oncolysis of glioma and medul-
loblastoma cells [109, 110]. Clinical trials are ongoing to assess the effectiveness
of the virus in patients with GBM. Advances in the understanding of the mole-
cular genetics of vesicular stomatitis virus and isolation of unique attenuated
strains has recently propelled the field of tumor oncolytic therapy forward [111].
VSV, AV1, and AV2 effect tumor-specific lysis by taking advantage of the
mutated abrogation of the interferon / signaling pathway in tumor cells.
The attenuated vesicular stomatitis virus strains have favorable therapeutic indices
and demonstrate the selective killing of cancer cells (including CNS tumors)
from the NCI-60 tumor cell panel.
Can we perhaps exploit NSCs as carriers of these lethal viruses and enhance
their delivery to infiltrative glioma cells? This concept is intriguing and has yet
to be attempted. Assuming NSCs have none of the genetic mutations that endow
the glioma cells with growth advantage (but also susceptibility to viral-mediated
oncolysis), then NSCs should be able to survive viral infection and deliver the
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lethal package to glioma cells. It is beyond the scope of this review to provide a
comprehensive and up-to-date review on novel glioma therapeutics, but one can
appreciate that NSCs offer a way to deliver therapeutically relevant molecules
and drugs effectively to diffuse glioma in vivo.
Biopolymer Scaffolds
Biodegradable polymer scaffolds, composed of poly-glycolic acid or poly
lactic acid or combinations of the two, offer a ‘backbone’ for the growth, differ-
entiation, and three-dimensional structure of tissues from stem cells [112]. They
also permit the stem cells and degenerating CNS environment to have prolonged
contact and communication with each other. Furthermore, they may fix in space
various molecules emanating both from the stem and from the tissue such that
the exposure of the various cells and tissue to each of these is protracted. Teng
et al. [113] used NSCs supported by a poly-glycolic acid scaffold to promote the
functional recovery of severely spinal cord injured adult rats. Park et al. [25]
used NSCs supported by a poly-glycolic acid scaffold to promote the growth of
new, reinnervated and revascularized cerebral parenchyma within a region that
otherwise would have undergone cystic cavity formation following severe
hypoxic-ischemic injury and its resultant liquefactive necrosis.
The scaffold, acting as a template for the implanted NSCs as well as a plat-
form for enhancing stem cell-host interactions, significantly aided the reconsti-
tution of the injured brain. One could imagine that NSCs engineered variously
to express anti-glioma and/or regenerative cytokines could be seeded onto
biosynthetic scaffolds and placed within a tumor resection cavity to enhance
killing of residual tumor cells and facilitate brain repair. This is somewhat anal-
ogous to the use of carmustine-impregnated biodegradable polymer wafers or
GLIADEL in brain tumor, as discussed in this volume, but is significantly more
biologically dynamic and adaptive. Tagging stem cells with ‘smart imaging’
agents will allow for their monitoring in the patient post-tumor resection or
biopsy. In fact, this could be used as a sensitive method to assess the amount of
residual tumor and the degree of recurrence.
Image Guidance and Enhanced Photodynamic Therapy

Shinoda et al. [114] showed recently that vital staining of glioma with flu-
orescein sodium introduced intravenously during dural opening at craniotomy
helped to define the stained tumor and facilitated radiographically guided total
resection. Photosensitizers preferentially accumulate in neoplastic cells due to
a variety of mechanisms [115]. Light at an appropriate excitation wavelength
when shone on the tissue results in fluorescence within areas of photosensitizer
accumulation. Photodynamic therapy (PDT) using photosensitizers already has
a long history in brain tumor therapy [116, 117].
PDT for brain tumor is still not widely used clinically for several reasons.
Even though PDT offers a binary approach to relatively preferential drug
uptake and photic activation, and therefore constitutes a relatively safe treat-
ment modality, systemic side effects, such as skin photosensitivity, still persist.
Yet more ominous is the problem with PDT-induced cerebral edema. These
problems are exacerbated by the uptake of the photosensitizer by non-
neoplastic tissues [118]. The delivery of photosensitizers could be made much
more selective and efficient for the detection and destruction of glioma by
exploiting the exquisite gliomatropic effect of NSCs. For example, NSCs could
be preloaded with a photosensitizer or smart imaging agent (see above) prior to
intracavitary implantation postresection or even intravenous injection [17].
Delivery of the photosensitizer could be made much more selective and effi-
cient for the detection and destruction of glioma by exploiting the exquisite
gliomatropic effect of NSCs.
Differentiating Radiation Necrosis from Tumor Recurrence

Radiation therapy of brain tumors is usually followed by the recurrence of
tumor. However, tumor recurrence due to treatment failure can be confused with
radiation-induced neoplasm or radiation necrosis [119]. These three entities have
similar radiographical appearances and can often present a diagnostic dilemma.
Rock et al. [120] recently confirmed the utility of magnetic resonance spec-
troscopy in differentiating pure radiation necrosis from pure tumor recurrence.
The authors, however, had trouble distinguishing tissues with intermediate
amounts of tumor and necrosis. Could NSCs discriminate between recurrences of
primary tumor, radiation-induced neoplasm and radiation necrosis? There could
be differences in the migratory pattern of NSCs to each of the above intracranial
pathologies that could be made apparent with specially tagged NSCs and appro-
priate imaging techniques. This concept is awaiting further experimental proof.
Brain Reconstruction Post-Therapy

Glioma growth distorts and destroys surrounding normal neural structures.
Patients who survive the initial treatment are often left with permanent neuro-
logical deficits with significant effects on their activities of daily living. This
damage to adjacent structures has long-term clinical consequences especially in
rare cases of curable gliomas such as pediatric pilocytic astrocytomas. NSCs
might repopulate areas of injured brain, differentiate on cue in response to local
environmental signals, and promote the reformation of functional interactions
with the host [121]. NSCs, therefore, might have dual roles in glioma therapy:
(1) delivery of anti-tumor agents to glioma cells and (2) repair of the injured
brain [122]. Park et al. [25] indeed showed that injected NSCs promote and
interact with endogenous repair pathways to facilitate brain repair.
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Clinical Applications
How might the biology described above someday be applied in actual clini-
cal situations? Some scenarios are envisioned below (see also tables 1, 2.)
Gliomatosis cerebri
Consider a young man with a suspected radiological diagnosis of
gliomatosis cerebri who is about to undergo a biopsy and/or resection [123].
A stable, readily available line of NSCs has been transfected with genes for
anti-tumor and prodifferentiation cytokines, perhaps tailored to the specific
tumor in this patient. These altered NSCs are administered to the patient. The
engineered cells are injected either intraventricularly or into the vicinity of
the tumor following biopsy. The injected cells will then target the main lesion
as well as infiltrating glioma cells followed by the delivery of the therapeu-
tic payload. There need not be concern for immunological matching of the
NSCs to the patient because immune rejection of the stem cells would prob-
ably enhance their tumor-killing ability, especially if already within the
tumor mass or juxtaposed to tumor cells. The physician has the choice of
transducing the cells with a therapeutic gene or tagging the cells with smart-
imaging agents, or both. Migration of the cells, in this way, could be followed
by either bioluminescence or MRI. NSCs might also discover unsuspected
small islands of tumor cells that may already have left the main tumor mass
and migrated to more distant locations. The patient may undergo multiple
treatments with NSCs carrying different therapeutic packages. For example,
initial delivery of proapoptotic genes to effect tumor destruction can be fol-
lowed by immunomodulatory genes that can elicit persistent anti-glioma
immunity.
The most compelling transplantation work to date has been performed
with NSCs and it remains unclear whether stem cells from other organs, even
if they were somehow directed to transdifferentiate into NSCs (a controversial
notion), would be equally effective in tracking and destroying very infiltrative
brain tumors. If they were, it is also conceivable that such stem cells could be
collected from the patient (e.g., via apheresis to harvest circulating stem cells,
an inefficient process, or via bone marrow aspiration to obtain hematopoi-
etic/mesenchymal stem cells) that might then be purified and expanded ex vivo
with a specially tailored cytokine cocktails and custom extracellular matrix
[123]. In fact, ex vivo stimulation of the stem cells could be tailored specifi-
cally to the individual patient depending on his/her age and the robustness of
the harvested stem cells. At present, only NSCs seem to be on the brink of being
a promising clinical application.
GBM
Consider a woman in her late fifties who presents with a contrast-enhancing
left frontal mass lesion with significant mass effect and edema. The lesion is con-
sistent with a high-grade intra-axial tumor. NSCs undergo ex vivo expansion,
therapeutic packages are loaded into the cells, and the engineered cells are seeded
in a biopolymer matrix. The patient undergoes awake craniotomy for surgical
debulking of the tumor with the aid of advanced neuronavigation and intraopera-
tive neurophysiological monitoring followed by placement of the cell-laden
biopolymer matrix into the resection cavity. NSCs within the matrix will seek out
and destroy glioma cells; in addition, using the matrix as a physical scaffold, the
NSCs will start to initiate repair pathways in conjunction with endogenous stem
cells. The patient is released from the hospital 2 days after the procedure.
Will We Truly See These Approaches Used in the Clinic?

One has to admit that the above two scenarios seem ‘far-fetched,’ but many
of the technical elements are already in place. Carefully performed preclinical
and then clinical trials are essential before realization of the above pictures
becomes routine practice. Initial trials should focus on confirming the in vivo
gliomatropic migration of NSCs in patients with high-grade terminal brain
tumors such as GBM and pediatric diffuse intrinsic glioma. Safety of NSCs
should also be rigorously addressed in these trials before the introduction of
therapeutic genes. Realistically, many technical hurdles have to be surpassed
before clinical translation ensues. Nevertheless, NSCs offer an intriguing and
promising therapeutic option. Importantly, they should be viewed not as a
substitute for other approaches but as one additional weapon in a multifaceted,
biologically rational arsenal for attacking brain tumors. The unique biology of
the stem cell may permit them to be the ‘glue’ that holds together and helps
coordinate the application of these many approaches.
References
1 Ryder EF, Snyder EY, Cepko CL: Establishment and characterization of multipotent neural cell
lines using retrovirus vector-mediated oncogene transfer. J Neurobiol 1990;21:356–375.
2 Renfranz PJ, Cunningham MG, McKay RD: Region-specific differentiation of the hippocampal stem
cell line HiB5 upon implantation into the developing mammalian brain. Cell 1991;66:713–729.
3 Snyder EY, Deitcher DL, Walsh C, Arnold-Aldea S, Hartwieg EA, Cepko CL: Multipotent
neural cell lines can engraft and participate in development of mouse cerebellum. Cell 1992;68:
33–51.
4 Reynolds BA, Weiss S: Generation of neurons and astrocytes from isolated cells of the adult mam-
malian central nervous system. Science 1992;255:1707–1710.
5 Snyder EY, Yoon C, Flax JD, Macklis JD: Multipotent neural precursors can differentiate toward
replacement of neurons undergoing targeted apoptotic degeneration in adult mouse neocortex.
medwedi.ru
6 Rosario CM, Yandava BD, Kosaras B, Zurakowski D, Sidman RL, Snyder EY: Differentiation of
engrafted multipotent neural progenitors towards replacement of missing granule neurons in
meander tail cerebellum may help determine the locus of mutant gene action. Development 1997;
124:4213–4224.
7 Park KI, Liu S, Flax JD, Nissim S, Stieg PE, Snyder EY: Transplantation of neural progenitor and
stem cells: Developmental insights may suggest new therapies for spinal cord and other CNS dys-
function. J Neurotrauma 1999;16:675–687.
8 Zlomanczuk P, Mrugala M, de la Iglesia HO, et al: Transplanted clonal neural stem-like cells
respond to remote photic stimulation following incorporation within the suprachiasmatic nucleus.
Exp Neurol 2002;174:162–168.
9 Snyder EY, Taylor RM, Wolfe JH: Neural progenitor cell engraftment corrects lysosomal storage
throughout the MPS VII mouse brain. Nature 1995;374:367–370.
10 Ourednik V, Ourednik J, Park KI, Snyder EY: Neural stem cells – A versatile tool for cell replace-
ment and gene therapy in the central nervous system. Clin Genet 1999;56:267–278.
11 Yandava BD, Billinghurst LL, Snyder EY: ‘Global’ cell replacement is feasible via neural stem cell
transplantation: Evidence from the dysmyelinated shiverer mouse brain. Proc Natl Acad Sci USA
1999;96:7029–7034.
12 Auerbach JM, Eiden MV, McKay RD: Transplanted CNS stem cells form functional synapses
in vivo. Eur J Neurosci 2000;12:1696–1704.
13 Park KI, Ourednik J, Ourednik V, et al: Global gene and cell replacement strategies via stem cells.
Gene Ther 2002;9:613–624.
14 Thomas ED: A history of haemopoietic cell transplantation. Br J Haematol 1999;105:330–339.
15 Burt RK, Traynor AE, Craig R, Marmont AM: The promise of hematopoietic stem cell transplan-
tation for autoimmune diseases. Bone Marrow Transplant 2003;31:521–524.
16 Flax JD, Aurora S, Yang C, et al: Engraftable human neural stem cells respond to developmental
cues, replace neurons, and express foreign genes. Nat Biotechnol 1998;16:1033–1039.
17 Aboody KS, Brown A, Rainov NG, et al: Neural stem cells display extensive tropism for path-
ology in adult brain: Evidence from intracranial gliomas. Proc Natl Acad Sci USA 2000;97:
12846–12851.
18 Rosenthal N: Prometheus’s vulture and the stem-cell promise. N Engl J Med 2003;349:
267–274.
19 Hochedlinger K, Jaenisch R: Nuclear transplantation, embryonic stem cells, and the potential for
cell therapy. N Engl J Med 2003;349:275–286.
20 McKay R: Stem cells in the central nervous system. Science 1997;276:66–71.
21 Gage FH: Mammalian neural stem cells. Science 2000;287:1433–1438.
22 Vescovi AL, Snyder EY: Establishment and properties of neural stem cell clones: Plasticity in vitro
and in vivo. Brain Pathol 1999;9:569–598.
23 Pincus DW, Goodman RR, Fraser RA, Nedergaard M, Goldman SA: Neural stem and progenitor
cells: A strategy for repair. Neurosurgery 1998;42:858–867.
24 Ourednik J, Ourednik V, Lynch WP, Schachner M, Snyder EY: Neural stem cells display an inher-
ent mechanism for rescuing dysfunctional neurons. Nat Biotechnol 2002;20:1103–1110.
25 Park KI, Teng YD, Snyder EY: The injured brain interacts reciprocally with neural stem cells sup-
ported by scaffolds to reconstitute lost tissue. Nat Biotechnol 2002;20:1111–1117.
26 Riess P, Zhang C, Saatman KE, et al: Transplanted neural stem cells survive, differentiate, and
improve neurological motor function after experimental traumatic brain injury. Neurosurgery
2002;51:1043–1052; discussion 1052–1054.
27 Burns MJ, Weiss W: Targeted therapy of brain tumors utilizing neural stem and cells. Front Biosci
2003;8:e228–e234.
28 Yip S, Aboody KS, Burns M, et al: Neural stem cell biology may be well suited for therapies.
Cancer J 2003;9:189–204.
29 Gottlieb DI: Large-scale sources of neural stem cells. Annu Rev Neurosci 2002;25:381–407.
30 Reubinoff BE, Itsykson P, Turetsky T, et al: Neural progenitors from human embryonic stem cells.
Nat Biotechnol 2001;19:1134–1140.
31 Kabos P, Ehtesham M, Kabosova A, Black KL, Yu JS: Generation of neural progenitor cells from
whole adult bone marrow. Exp Neurol 2002;178:288–293.
32 Jiang Y, Henderson D, Blackstad M, Chen A, Miller RF, Verfaillie CM: Neuroectodermal differ-
entiation from mouse multipotent adult progenitor cells. Proc Natl Acad Sci USA 2003;100:
11854–11860.
33 DeAngelis LM: Brain tumors. N Engl J Med 2001;344:114–123.
34 Kaye AH, Laws ER: Historical perspective; in Kaye AH, Laws ER (eds): Brain Tumors: An ency-
lopedic Approach. New York, Churchill Livngstone, 2001, pp 3–8.
35 Holland EC: Glioblastoma multiforme: The terminator. Proc Natl Acad Sci USA 2000;97:
6242–6244.
36 Silbergeld DL, Chicoine MR: Isolation and characterization of human malignant glioma cells
from histologically normal brain. J Neurosurg 1997;86:525–531.
37 Vates GE, Chang S, Lamborn KR, Prados M, Berger MS: Gliomatosis cerebri: A review of
22 cases. Neurosurgery 2003;53:261–271; discussion 271.
38 Hentschel SJ, Lang FF: Current surgical management of glioblastoma. Cancer J 2003;9:113–125.
39 Laws ER, Parney IF, Huang W, et al: Survival following surgery and prognostic factors for recently
diagnosed malignant glioma: Data from the Glioma Outcomes Project. J Neurosurg 2003;99:
467–473.
40 Rao JS: Molecular mechanisms of glioma invasiveness: The role of proteases. Nat Rev Cancer
2003;3:489–501.
41 Bachoo RM, Maher EA, Ligon KL, et al: Epidermal growth factor receptor and Ink4a/Arf:
Governing terminal differentiation and transformation of stem cell to astrocyte axis. Cancer Cell
2002;1:269–277.
42 Dai C, Holland EC: Astrocyte differentiation states and glioma formation. Cancer J 2003;9:72–81.
43 Dai C, Celestino JC, Okada Y, Louis DN, Fuller GN, Holland EC: PDGF autocrine stimulation
dedifferentiates cultured astrocytes and induces oligodendrogliomas and oligoastrocytomas from
neural progenitors and astrocytes in vivo. Genes Dev 2001;15:1913–1925.
44 Singh SK, Clarke ID, Terasaki M, et al: Identification of a cancer stem cell in human brain. Cancer
Res 2003;63:5821–5828.
45 Holland EC: Gliomagenesis: Genetic alterations and mouse models. Nat Rev Genet 2001;2:120–129.
46 Zhu Y, Parada LF: The molecular and genetic basis of neurological tumours. Nat Rev Cancer
2002;2:616–626.
47 Maher EA, Furnari FB, Bachoo RM, et al: Malignant glioma: Genetics and biology of a grave
matter. Genes Dev 2001;15:1311–1333.
48 Hesselager G, Holland EC: Using mice to decipher the molecular genetics of brain tumors.
Neurosurgery 2003;53:685–694; discussion 695.
49 Weiss WA, Burns MJ, Hackett C, et al: Genetic determinants of malignancy in a mouse model for
oligodendroglioma. Cancer Res 2003;63:1589–1595.
50 Kalamarides M, Niwa-Kawakita M, Leblois H, et al: Nf2 gene inactivation in arachnoidal cells is
rate-limiting for meningioma development in the mouse. Genes Dev 2002;16:1060–1065.
51 Vogel KS, Klesse LJ, Velasco-Miguel S, Meyers K, Rushing EJ, Parada LF: Mouse tumor model
for neurofibromatosis type 1. Science 1999;286:2176–2179.
52 Zhu Y, Ghosh P, Charnay P, Burns DK, Parada LF: Neurofibromas in NF1: Schwann cell origin
and role of tumor environment. Science 2002;296:920–922.
53 Koutcher JA, Hu X, Xu S, et al: MRI of mouse models for gliomas shows similarities to humans
and used to identify mice for preclinical trials. Neoplasia 2002;4:480–485.
54 Barnes L, Kapadia SB: The biology and pathology of selected skull base tumors. J Neurooncol
1994;20:213–240.
55 Pluchino S, Quattrini A, Brambilla E, et al: Injection of adult neurospheres induces recovery in a
chronic model of multiple sclerosis. Nature 2003;422:688–694.
56 Pardridge WM: Drug and gene targeting to the brain with molecular Trojan horses. Nat Rev Drug
Discov 2002;1:131–139.
57 Pardridge WM: Drug and gene delivery to the brain: The vascular route. Neuron 2002;36:555–558.
58 Kabos P, Ehtesham M, Black KL, Yu JS: Neural stem cells as delivery vehicles. Expert Opin Biol
Ther 2003;3:759–770.
59 Barresi V, Belluardo N, Sipione S, Mudo G, Cattaneo E, Condorelli DF: Transplantation of prodrug-
converting neural tumor therapy. Cancer Gene Ther 2003;10:396–402.
medwedi.ru
60 Lynch WP, Sharpe AH, Snyder EY: Neural stem cells as engraftable packaging lines can mediate
gene delivery to microglia: Evidence from studying retroviral env-related neurodegeneration.
J Virol 1999;73:6841–6851.
61 Herrlinger U, Woiciechowski C, Sena-Esteves M, et al: Neural precursor cells for delivery of
replication-conditional HSV-1 vectors to intracerebral gliomas. Mol Ther 2000;1:347–357.
62 Jean WC, Spellman SR, Wallenfriedman MA, Hall WA, Low WC: Interleukin-12-based immuno-
therapy against rat 9L glioma. Neurosurgery 1998;42:850–856; discussion 856–857.
63 Ehtesham M, Samoto K, Kabos P, et al: Treatment of intracranial glioma with in situ interferon-
gamma and necrosis factor-alpha gene transfer. Cancer Gene Ther 2002;9:925–934.
64 Rhines LD, Sampath P, DiMeco F, et al: Local immunotherapy with interleukin-2 delivered from
polymer microspheres combined with interstitial chemotherapy: A treatment for experimental
malignant glioma. Neurosurgery 2003;52:872–879; discussion 879–880.
65 Benedetti S, Pirola B, Pollo B, et al: Gene therapy of experimental brain tumors using neural prog-
enitor. Nat Med 2000;6:447–450.
66 Weinstein DE, Shelanski ML, Liem RK: C17, a retrovirally immortalized neuronal cell line,
inhibits the proliferation of astrocytes and astrocytoma cells by a contact-mechanism. Glia 1990;
3:130–139.
67 Ehtesham M, Kabos P, Kabosova A, Neuman T, Black KL, Yu JS: The use of interleukin 12-secreting
neural stem cells for the treatment of intracranial glioma. Cancer Res 2002;62:5657–563.
68 Ehtesham M, Kabos P, Gutierrez MA, et al: Induction of glioblastoma apoptosis using neural stem
cell-mediated delivery of tumor necrosis factor-related apoptosis-inducing ligand. Cancer Res
2002;62:7170–7174.
69 Walczak H, Miller RE, Ariail K, et al: Tumoricidal activity of tumor necrosis factor-related
apoptosis-ligand in vivo. Nat Med 1999;5:157–163.
70 Dirks PB: Glioma migration: Clues from the biology of neural progenitor cells and embryonic
CNS cell migration. J Neurooncol 2001;53:203–212.
71 Noble M: Can neural stem cells be used to track down and destroy migratory brain tumor cells
while also providing a means of repairing tumor-associated damage? Proc Natl Acad Sci USA
2000;97:12393–12395.
72 Noble M: Can neural stem cells be used as therapeutic vehicles in the treatment of brain tumors?
Nat Med 2000;6:369–370.
73 Graeber MB, Scheithauer BW, Kreutzberg GW: Microglia in brain tumors. Glia 2002;40:252–259.
74 Tran PB, Miller RJ: Chemokine receptors: Signposts to brain development and disease. Nat Rev
Neurosci 2003;4:444–455.
75 Barbero S, Bajetto A, Bonavia R, et al: Expression of the chemokine receptor CXCR4 and its
ligand stromal cell-derived factor 1 in human brain tumors and their involvement in glial prolif-
eration in vitro. Ann NY Acad Sci 2002;973:60–69.
76 Rempel SA, Dudas S, Ge S, Gutierrez JA: Identification and localization of the cytokine SDF1
and its chemokine receptor 4, to regions of necrosis and angiogenesis in glioblastoma. Clin Cancer
Res 2000;6:102–111.
77 Zhou Y, Larsen PH, Hao C, Yong VW: CXCR4 is a major chemokine receptor on glioma cells and
mediates their survival. J Biol Chem 2002;277:49481–49487.
78 Luo Y, Cai J, Liu Y, et al: Microarray analysis of selected genes in neural stem and progenitor cells.
J Neurochem 2002;83:1481–1497.
79 Lazarini F, Tham TN, Casanova P, Arenzana-Seisdedos F, Dubois-Dalcq M: Role of the alpha-
chemokine stromal cell-derived factor (SDF-1) in the developing and mature central nervous
system. Glia 2003;42:139–148.
80 Stumm RK, Zhou C, Ara T, et al: CXCR4 regulates interneuron migration in the developing neo-
cortex. J Neurosci 2003;23:5123–5130.
81 Rubin JB, Kung AL, Klein RS, et al: A small-molecule antagonist of CXCR4 inhibits intracranial
growth of primary brain tumors. Proc Natl Acad Sci USA 2003;100:13513–13518.
82 Leon SP, Folkerth RD, Black PM: Microvessel density is a prognostic indicator for patients with
astroglial brain tumors. Cancer 1996;77:362–372.
83 Brem S, Tsanaclis AM, Gately S, Gross JL, Herblin WF: Immunolocalization of basic fibroblast
growth factor to the microvasculature of human brain tumors. Cancer 1992;70:2673–2680.
84 Chan AS, Leung SY, Wong MP, et al: Expression of vascular endothelial growth factor and its
receptors in the anaplastic progression of astrocytoma, oligodendroglioma, and ependymoma. Am
J Surg Pathol 1998;22:816–826.
85 Boockvar JA, Kapitonov D, Kapoor G, et al: Constitutive EGFR signaling confers a motile phe-
notype to neural stem cells. Mol and Cell Neurosci 2003;in press.
86 Feldkamp MM, Lau N, Guha A: Signal transduction pathways and their relevance in human astro-
cytomas. J Neurooncol 1997;35:223–248.
87 Dunn IF, Heese O, Black PM: Growth factors in glioma angiogenesis: FGFs, PDGF, EGF, and
TGFs. J Neurooncol 2000;50:121–137.
88 Chicoine MR, Silbergeld DL: Mitogens as motogens. J Neurooncol 1997;35:249–257.
89 Goodison S, Urquidi V, Tarin D: CD44 cell adhesion molecules. Mol Pathol 1999;52:189–196.
90 Ruoslahti E: Brain extracellular matrix. Glycobiology 1996;6:489–492.
91 Merzak A, Koocheckpour S, Pilkington GJ: CD44 mediates human glioma cell adhesion and inva-
sion in vitro. Cancer Res 1994;54:3988–3992.
92 Akiyama Y, Jung S, Salhia B, et al: Hyaluronate receptors mediating glioma cell migration and
proliferation. J Neurooncol 2001;53:115–127.
93 Bouvier-Labit C, Liprandi A, Monti G, Pellissier JF, Figarella-Branger D: CD44H is expressed by
cells of the oligodendrocyte oligodendrogliomas in humans. J Neurooncol 2002;60:127–134.
94 Delpech B, Maingonnat C, Girard N, et al: Hyaluronan and hyaluronectin in the extracellular
matrix of human brain tumour stroma. Eur J Cancer 1993;29A:1012–1017.
95 Wright LS, Li J, Caldwell MA, Wallace K, Johnson JA, Svendsen CN: Gene expression in human
neural stem cells: Effects of leukemia inhibitory factor. J Neurochem 2003;86:179–195.
96 Filbin MT: Myelin-associated inhibitors of axonal regeneration in the adult mammalian CNS. Nat
Rev Neurosci 2003;4:703–713.
97 Simonen M, Pedersen V, Weinmann O, et al: Systemic deletion of the myelin-associated outgrowth
inhibitor Nogo-A improves regenerative and plastic responses after spinal cord injury. Neuron
2003;38:201–211.
98 Woolf CJ: No Nogo: Now where to go? Neuron 2003;38:153–156.
99 Weissleder R, Ntziachristos V: Shedding light onto live molecular targets. Nat Med 2003;9:123–128.
100 Tang Y, Shah K, Messerli SM, Snyder E, Breakefield X, Weissleder R: In vivo tracking of neural
progenitor cell. Hum Gene Ther 2003;14:1247–1254.
101 Lewin M, Carlesso N, Tung CH, et al: Tat peptide-derivatized magnetic nanoparticles allow in
recovery of progenitor cells. Nat Biotechnol 2000;18:410–414.
102 Marras C, Mendola C, Legnani FG, DiMeco F: Immunotherapy and biological modifiers for the
treatment of malignant brain tumors. Curr Opin Oncol 2003;15:204–208.
103 Yacoub A, Mitchell C, Lister A, et al: Melanoma differentiation-associated 7 (interleukin 24)
inhibits growth and enhances radiosensitivity of glioma cells in vitro and in vivo. Clin Cancer Res
2003;9:3272–3281.
104 Aoki T, Tashiro K, Miyatake S, et al: Expression of murine interleukin 7 in a murine glioma cell
line results in reduced tumorigenicity in vivo. Proc Natl Acad Sci USA 1992;89:3850–3854.
Sci USA 2000;97:2208–2213.
106 Bjerkvig R, Read TA, Vajkoczy P, et al: Cell therapy using encapsulated cells producing endo-
statin. Acta Neurochir Suppl 2003;88:137–141.
107 Chiocca EA: Oncolytic viruses. Nat Rev Cancer 2002;2:938–950.
108 Rainov NG, Ren H: Oncolytic viruses for treatment of malignant brain tumours. Acta Neurochir
Suppl 2003;88:113–123.
109 Wilcox ME, Yang W, Senger D, et al: Reovirus as an oncolytic agent against experimental human
malignant gliomas. J Natl Cancer Inst 2001;93:903–912.
110 Yang WQ, Senger D, Muzik H, et al: Reovirus prolongs survival and reduces the frequency of
leptomeningeal metastases from medulloblastoma. Cancer Res 2003;63:3162–3172.
111 Stojdl DF, Lichty BD, tenOever BR, Paterson JM, Power AT: VSV strains with defects in their
ability to shutdown innate immunity are potent systemic anti-cancer agents. Cancer Cell 2003;4:
263–275.
medwedi.ru
112 Levenberg S, Huang NF, Lavik E, Rogers AB, Itskovitz-Eldor J, Langer R: Differentiation of human
embryonic stem cells on three-dimensional polymer scaffolds. Proc Natl Acad Sci USA 2003;15:15.
113 Teng YD, Lavik EB, Qu X, et al: Functional recovery following traumatic spinal cord injury medi-
ated by a unique polymer scaffold seeded with neural stem cells. Proc Natl Acad Sci USA 2002;
99:3024–3029.
114 Shinoda J, Yano H, Yoshimura S, et al: Fluorescence-guided resection of glioblastoma multiforme
by using high-dose fluorescein sodium. Technical note. J Neurosurg 2003;99:597–603.
115 Dolmans DE, Fukumura D, Jain RK: Photodynamic therapy for cancer. Nat Rev Cancer 2003;3:
380–387.
116 Kaye AH, Morstyn G, Apuzzo ML: Photoradiation therapy and its potential in the management of
neurological tumors. J Neurosurg 1988;69:1–14.
117 Popovic EA, Kaye AH, Hill JS: Photodynamic therapy of brain tumors. Semin Surg Oncol 1995;
11:335–345.
118 Vrouenraets MB, Visser GW, Snow GB, van Dongen GA: Basic principles, applications in oncol-
ogy and improved selectivity of photodynamic therapy. Anticancer Res 2003;23:505–522.
119 Forsyth PA, Kelly PJ, Cascino TL, et al: Radiation necrosis or glioma recurrence: Is computer-
assisted stereotactic biopsy useful? J Neurosurg 1995;82:436–444.
120 Rock JP, Hearshen D, Scarpace L, et al: Correlations between magnetic resonance spectroscopy
and image-guided histopathology, with special attention to radiation necrosis. Neurosurgery 2002;
51:912–919; discussion 919–920.
121 Park KI, Lachyankar M, Nissim S, Snyder EY. Neural stem cells for CNS repair: State of the art
and future directions. Adv Exp Med Biol 2002;506:1291–1296.
122 Noble M, Dietrich J: Intersections between neurobiology and oncology: Tumor origin, treatment
and repair of treatment-associated damage. Trends Neurosci 2002;25:103–107.
123 Liu CY, Apuzzo ML, Tirrell DA: Engineering of the extracellular matrix: Working toward neural
stem cell programming and neurorestoration – Concept and progress report. Neurosurgery
2003;52:1154–1165; discussion 1165–1167.
124 Ehtesham M, Yuan X, Kabas P, Chung NH, Liu G, Akasaki Y, Black KL, Yu JS: Glioma tropic
neural stem cells consist of astrocytic precursors and their migratory capacity is mediated by
CXCR4. Neoplasia 2004;6:287–293.
125 Liao H, Duka T, Teng FY, Sun L, Bu WY, Ahmed S, Tang BL, Xiao ZC: Nogo-66 and myelin-
associated glycoprotein (MAG) inhibit the adhesion and migration of Nogo-66 receptor express-
ing human glioma cells. J Neurochem 2004;90:1156–1162.
126 Marchiò S, Lahdenranta J, Schlingemann RO, Valdembri D, Wesseling P, Arap MA, Hajitou A,
Ozawa M, Trepel M, Giordano R, Nanus DM, Dijkman HB PM, Oosterwijk E, Sidman RL,
Cooper MD, Bussolino F, Pasqualini R, Arap W: Aminopeptidase A is a functional target in angio-
genic blood vessels. Cancer Cell 2004;5:151–162.
Dr. Evan Y. Snyder, MD, PhD

Department of Neurology, Harvard Medical School
Harvard Institutes of Medicine, Beth Israel-Deaconess Medical Center &
Children’s Hospital, Boston, MA 02115 (USA)
Tel. 1 858 646 3158, Fax 1 858 713 6273, E-Mail esnyder@burnham.org
Author Index
Anderson, D.G. 5 Grauer, J.N. 52 Nakamura, M. 104

Anderson, W.F. XI Greengold, D. 580 Niranjan, A. 439
Andrews, D.W. 499 Gullans, S.R. 246
Ogawa, Y. 104
Bankiewicz, K. 213 Hadaczek, P. 213 Okano, H. 104
Barone, F.C. 336 Harshyne, L. 499 Okano, H.J. 104
Bomback, D.A. 52 Heistad, D.D. 413 O’Rourke, D.M. 521
Boulis, N.M. 65 House, P.A. 124
Brady, R.O. 202 Hu, J. 580 Rao, G. 124
Brady, R.O. Jr. 202 Read, S.J. 336
Brem, H. 458 Iwanami, A. 104
Broggi, G. 270 Samulski, R.J. 154
Burchiel, K. 284 Sawamoto, K. 104
Jensen, R.V. 246
Bussone, G. 270 Scherzer, C.R. 246
Jovel, N. 580
Schumacher, J.M. 146
Castro, M.G. 580 Janson, C. 1
Sheehan, J. 439
Celix, J. 169 Sidman, R.L. 624
Chaudhary, P. 284 Kaneko, S. 104
Simeone, F.A. 1
Chiocca, E.A. 557 Kang, J.D. 37
Snyder, E.Y. 624
Couldwell, W. 124 Kapoor, G.S. 521
Steinmetz, M.P. 65
Kim, J. 37
Daadi, M. 213 Kondziolka, D. 439 Telfeian, A. 169
Dichter, M. 169 Kurpad, S.N. 30
Toyama, Y. 104
Ehtesham, M. 580
Lamfers, M.L.M. 557 Visted, T. 557
Fink, D. 322 Leo, B.M. 5
Flomenberg, P. 499 Leone, M. 270 Walker, M.H. 5
Franzini, A. 270 Leone, P. 1 Wang, P.P. 458
Freese, A. 1 Lifshutz, J. 30 Watanabe, Y. 413
Liu, J.K. 65
Gilbertson, L.G. 37 Lowenstein, P.R. 580 Xiong, W. 580
Giles, J. 154 Lunsford, L.D. IX
Glorioso, J.C. 322 Yip, S. 624
Gouras, G.K. 258 Macdonald, R.L. 377 Yu, J. 580
Goverdhana, S. 580 Mata, M. 322 Yuan, X. 580
645
medwedi.ru
Subject Index
Acid-sensing ion channels, pain reception pain gene therapy vector 326
290, 291 Parkinson’s disease gene therapy vector
Adeno-associated virus (AAV) 216, 218, 219
advantages as gene therapy vector 154 pituitary adenoma gene therapy vector
brain gene transfer 127, 160–163 589, 591, 593–595, 611–613
Canavan’s disease gene transfer 164, replication 561
165 spinal cord injury gene therapy vector
clinical-grade vector production 155, 82, 83, 85–88, 90–92
156 Adrenergic receptors, pain reception 290,
crystal structure 158–160 291
epilepsy gene transfer 163, 164 Adriamycin, polymeric drug delivery
genome and proteins 154, 155 systems 479, 480
global gene delivery 164, 165 Akt
Huntington’s disease gene transfer 163 glioma progression signaling 531–533
Parkinson’s disease gene therapy vector small molecule inhibitors 541
164, 221–226 Alzheimer’s disease (AD)
Parkinson’s disease gene transfer 164 ␤-amyloid mutations, deposition, and
parvovirus homology 159, 160 therapeutic targeting 259–261, 266,
pituitary adenoma gene therapy vector 267
595, 596 cerebrospinal-fluid-based therapies 258,
serotypes for gene transfer 157, 158 259, 267
spinal cord injury gene therapy vector economic impact 258
83, 88, 92 epidemiology 258
Adenovirus gene therapy prospects 265, 266
intervertebral disc degeneration vector management
40, 41 antioxidants 265
oncolytic viral therapy for glioma estrogen replacement therapy studies
advantages 561, 562 263, 264
E1 gene modifications 562, 563 excitotoxicity inhibitors 265
tropism modification 564, 565 metal chelation therapy 264, 265
tumor-specific promoters for driving nonsteroidal anti-inflammatory drugs
replication 563, 564 264
646
protease augmentation 265 Autoantibodies, epilepsy 177–179
secretase inhibitors 261, 262 Autosomal dominant polycystic kidney
statins in protection 263 disease, cerebral aneurysm 383, 384
vaccination against ␤-amyloid 263
treatment difficulty 125 B7, gene therapy for immune response
␥-Aminobutyric acid (GABA) enhancement in cancer 607
modulation in epilepsy treatment 171, 172 Back pain
nociception 295, 296 economic impact 5
receptor expression in epilepsy 197 epidemiology 5, 30
Aminopeptidase A, upregulation in glioma Bcl-2
angiogenesis 632, 633 ischemic stroke gene therapy 448
Amyloidosis spinal cord injury gene therapy 85
␤-amyloid mutations, deposition, and Bcl-XL, spinal cord injury gene therapy 85
therapeutic targeting in Alzheimer’s O6-Benzylguanine, polymeric drug delivery
disease 259–261, 266, 267 systems 472
intracerebral hemorrhage risks 378–380 Bleomycin, polymeric drug delivery
Amyotrophic lateral sclerosis, treatment systems 483, 484
difficulty 125 Blood-brain barrier (BBB)
Angiogenesis antiepileptic drug delivery 183, 184
aminopeptidase A upregulation 632, 633 bypassing for drug delivery 460, 461
endogenous inhibitors 543 disruption for drug delivery 459, 460
gene therapy with inhibitors 603, 604 drug permeability 459, 460
glioma 632 Bone morphogenetic proteins (BMPs)
inhibition therapy in glioma 542–544 carotid artery stenosis gene therapy with
inhibitor delivery using polymeric drug BMP-2 42
delivery systems 478 discovery 53
therapeutic angiogenesis 426–428, 450 polymeric drug delivery systems 485, 486
Angiotensin-converting enzyme (ACE), receptors 54
gene mutations in stroke 340 regulation 54
Antigen-presenting cells (APCs) spinal fusion
glioma immune response 500, 501, 509 carriers 61
immunotherapy manipulation 512, 513 clinical studies
tumor vaccines 539 BMP-2 57–59
Antisense gene therapy BMP-7 59
constructs 126 demineralized bone matrix 59
pain management 309–311 expression 54
Apolipoprotein E, gene mutations in stroke gene therapy prospects 60–62
340, 379 preclinical studies
Apoptosis BMP-2 55, 56
hemorrhagic stroke 389 BMP-7 56, 57
intervertebral disc degeneration 19, 32, 35 demineralized bone matrix 57
ischemic stroke 389, 446, 447 preparations for use 54, 55
Arteriovenous malformation (AVM), Bradykinin (BK), pain mediation 300
intracerebral hemorrhage risks Brain
380, 381 convection-enhanced delivery of drugs
Atrial natriuretic peptide (ANP), gene 127, 128
mutations in stroke 341–343 gene transfer vectors 127, 160–163
Subject Index 647
medwedi.ru
Brain (continued) Cerebrospinal fluid infusion, drug delivery
neuroprosthetic therapies 131, 133, 134 461
pain gene therapy 333 Cerebrovascular gene therapy, see
stem cell therapy 129 Hemorrhagic stroke; Ischemic stroke
stroke, see Hemorrhagic stroke; Ischemic Cholecystokinin (CCK), pain mediation
stroke 299, 300
Brain-derived neurotrophic factor (BDNF) CI1033, epidermal growth factor receptor
brain graft neuroprotection 446 inhibition 541
spinal cord injury gene therapy 84 Cisplatin, polymeric drug delivery systems
Brain tumors, see also specific tumors 483
classification 458 Cluster headache (CH)
polymeric drug delivery systems, see diagnostic criteria 271
Polymeric drug delivery systems hypothalamic deep brain stimulation
surgical resection 458 electrode implantation in first patient
Buthionine sulfoximine (BSO), polymeric 274
drug delivery systems 479 outcomes 277–279
patient selection criteria 274–276,
CADASIL, gene mutations and stroke 279
338–340 prospects 280, 282
Calcitonin-gene-related peptide (CGRP) rationale 273
cerebral vasospasm gene therapy after surgery 276, 277
hemorrhagic stroke 419 pathophysiology 271–273, 279
pain mediation 298 pharmacotherapy 273
Calcium flux, smooth muscle contraction and trigeminal nerve surgery 273
relaxation in hemorrhagic stroke 391, 394 Complement, activation in
Camptothecin, polymeric drug delivery hemorrhagic-stroke-induced edema 388
systems 473, 474 Computed tomography (CT), intraoperative
Canavan’s disease, adeno-associated virus 134
for gene transfer 164, 165 Convection-enhanced delivery (CED), drug
Cannabinoid receptors, pain reception 289 delivery to brain 127, 128, 486, 487
Capsaicin receptors, pain reception 288, 289 Cushing’s disease, animal models 610
Carbamazepine, mechanism of action 171 Cyclophosphamide, polymeric drug
Carboplatin, polymeric drug delivery delivery systems 479
systems 482, 483 Cytosine deaminase, prodrug combination
Carboxypeptidase G2, prodrug combination in gene therapy 601, 629, 630
in gene therapy 601
Carmustine (BCNU), delivery using Deep brain stimulation (DBS)
polyanhydride fibers in glioma cluster headache
development 464, 465 electrode implantation in first patient
initial therapy trials 470–472 274
preclinical trials 465–467 outcomes 277–279
recurrent glioma trials 467–469 patient selection criteria 274–276,
Carotid artery stenosis, gene therapy targets 279
422–427 prospects 280, 282
CD44, glioma expression 633 rationale 273
Cell transplantation therapy, see specific surgery 276, 277
cells and diseases epilepsy management 190, 191
Subject Index 648

historical perspective 270 Endostatin, angiogenesis inhibition 543,
indications and mechanisms of action 544, 604
270, 271, 279, 280 Endothelin
Degenerative disc disease, see cerebral vasospasm antisense gene
Intervertebral disc degeneration therapy after hemorrhagic stroke 420
Dementia, see also Alzheimer’s disease endothelial dysfunction in hemorrhagic
differential diagnosis 258, 259 stroke 396, 397
Dexamethasone, polymeric drug delivery Enkephalin, proenkephalin gene therapy for
systems 483 pain 325–333
Differential display Enzyme replacement therapy (ERT)
hemorrhagic stroke vasospasm differential gene therapy 209, 210
gene expression studies 390, 391 historical perspective 204–206
ischemic stroke differential gene intracerebral injection of
expression studies 349 glucocerebrosidase 207–209
Disc degeneration, see Intervertebral disc overview 202
degeneration sphingolipid storage disorders 202, 204
DNA microarray substrate depletion 206, 207
disease gene identification Epidermal growth factor (EGF),
biological validation 249 intervertebral disc degeneration
error minimization 248, 249 treatment prospects 22, 35
primary screen 247 Epidermal growth factor receptor (EGFR)
secondary screen 247, 248 glioma expression 633
shotgun microarrays 248 glioma progression signaling 522–524
epilepsy gene expression and monoclonal antibody inhibition 538
pharmacogenomics 196–199 small molecule inhibitors 540, 541
hemorrhagic stroke vasospasm differential Epilepsy
gene expression studies 390, 391 adeno-associated virus for gene transfer
ischemic stroke differential gene 163, 164
expression studies 357, 358 autoantibodies 177–179
neurodegeneration modifying gene cell transplantation therapy 191, 192
analysis deep brain stimulation 190, 191
candidate modifier screen in animal DNA microarray analysis of gene
models 252, 253 expression and pharmacogenomics
cellular profiling 250, 251 196–199
extraneuronal profiling 251, 252 epidemiology 169
linkage analysis 253, 255 gamma knife radiosurgery 189
neuroprotective therapy prospects gene therapy prospects 192–195
255 ion channel defects and therapeutic
regional profiling 250 targeting 180, 181
selective vulnerability profiles 249, 250 ketogenic diet therapy 185, 186
principles 246, 247 neuroprotection in prevention 179, 180
pharmacotherapy
Ehlers-Danlos syndrome, cerebral carbamazepine 171
aneurysm 383, 384 drug delivery advances 181–185
EKB-569, epidermal growth factor receptor drug discovery 172, 173
inhibition 541 efficacy 169, 170
Endoglin, gene mutations in stroke 379 ethosuximide 172
Subject Index 649
medwedi.ru
Epilepsy (continued) pain mediation 302, 303
pharmacotherapy (continued) spinal cord injury gene therapy 84, 90–93
gabapentin 172, 173 Glioblastoma multiforme (GBM)
mechanisms of action 170, 171 gene therapy 126
pharmacogenetics and drug metabolism neural stem cell applications 639
174, 175, 196 progression 521
phenobarbital 172 survival 499, 627
phenytoin 171 treatment difficulty 124, 125, 499, 545
prospects 174 Glioma, see also Glioblastoma multiforme
resistance mechanisms 175–177 angiogenesis 632
tiagabine 173 angiogenesis inhibition therapy 542–544
valproic acid 171, 172 carmustine delivery using polyanhydride
vigabatrin 173 fibers
surgical resection of lesions 186–188 development 464, 465
vagus nerve stimulation 188 initial therapy trials 470–472
ErbB, glioma progression signaling 524–527 preclinical trials 465–467
Ethosuximide, mechanism of action 172 recurrent glioma trials 467–469
CD44 expression 633
Factor V, gene mutations in stroke 340 epidermal growth factor receptor
Farnesyltransferase inhibitors, Ras expression 633
signaling inhibition 542 grading 521
Fas ligand, gene therapy for apoptosis immune response
induction 604, 605 antigen-presenting cells 500, 501, 509
Fibrinogen, gene mutations in stroke 340 dysfunction 506–509
Fibromuscular dysplasia, cerebral T cells
aneurysm 383 cytolytic activity assays 504
5-Fluorouracil flow cytometry of cytokine
cytosine deaminase combination in gene production 504, 505
therapy 601, 629, 630 gene expression analysis 505, 506
polymeric drug delivery systems 480, 481 proliferation assay 503, 504
types in response 501–503
Gabapentin, mechanism of action 172, 173 immunotherapy
Galanin, pain mediation 299 monoclonal antibodies 537–539
Gamma knife radiosurgery, epilepsy prospects 509–513
management 189 tumor vaccines 539, 540
Gaucher’s disease neural stem cells
enzyme replacement therapy 204–209 cell tagging for imaging 634
gene therapy 209, 210 glioma-genesis 628, 629
Gene chip, see DNA microarray homing and therapeutic applications
Gene therapy, see specific diseases and genes 629–639
Gerstmann-Sträussler-Scheinker disease, prospects for therapy 634–638
clinical features 379, 380 radiation necrosis differentiation from
Gliadel, see Carmustine tumor recurrence 637
Glial-derived neurotrophic factor (GDNF) NOGO-A expression 633, 634
brain graft neuroprotection 446 oncolytic viral therapy
ischemic stroke gene therapy 447 adenovirus
nerve repair role 129, 130 advantages 561, 562
Subject Index 650

E1 gene modifications 562, 563 amyloid angiopathies 378–380
tropism modification 564, 565 arteriovenous malformation 380, 381
tumor-specific promoters for hypertension 377, 378
driving replication 563, 564 intracranial aneurysm 382–385
herpes simplex virus gene therapy
advantages and limitations 558, 559 approaches 413, 414
dual mutation vectors 560 cerebral vasospasm 418–420, 422,
␥1-34.5 mutants 559, 560 426, 427
thymidine kinase and ribonucleotide injection of vectors 415–418
reductase mutants 559 prospects 400
tumor-specific promoters for vectors 414, 415
driving replication 561 pathophysiology
potency improvement strategies apoptosis 389
arming with therapeutic transgenes cerebral vasospasm 390
567–569 direct versus indirect molecular effects
combination therapy 569–571 385–387
immune response evasion 566, 567 edema 387–389
principles 557, 558 endothelial dysfunction 395–397
prospects 571, 572 gene expression in vasospasm 390,
origins 628 391, 397, 398
photodynamic therapy 637 inflammation 389, 390, 398, 399
signaling in glioma-genesis ischemia 389
Akt 531–533 microvascular vasospasm 390
epidermal growth factor/ErbB 524–527 remodeling and fibrosis 399, 400
mitogen-activated protein kinase smooth muscle
527–531 contraction 391–394
phosphatidylinositol 3-kinase 531–533 relaxation 394, 395
phospholipase C 533–535 polymeric drug delivery systems 484, 485
platelet-derived growth factor Herpes simplex virus (HSV)
522–524 oncolytic viral therapy for glioma
prospects for study 544, 545 advantages and limitations 558, 559
receptor tyrosine kinases 521, 522 dual mutation vectors 560
small molecule inhibitors 540–542 ␥1-34.5 mutants 559, 560
STATs 535 thymidine kinase and ribonucleotide
transforming growth factor-␣ 526, reductase mutants 559
527 tumor-specific promoters for driving
replication 561
Heat shock protein 70 (HSP70), expression pain gene therapy 326–333
after hemorrhagic stroke 386 Parkinson’s disease gene therapy vector
Heme oxygenase 215, 216
cerebral vasospasm gene therapy after pituitary adenoma gene therapy vector
hemorrhagic stroke 420 596, 597, 600–602, 611, 612
expression after hemorrhagic stroke 386, spinal cord injury gene therapy vector
387 81, 82
Hemorrhagic stroke Histamine, pain mediation 302
biological therapies 400, 401 Human immunodeficiency virus, see
etiology Lentivirus
Subject Index 651
medwedi.ru
Huntington’s disease (HD) prospects 21–24, 35, 49
adeno-associated virus for gene transfer rabbit studies 41–43, 46, 47
163 transforming growth factor-␤ 22, 23,
treatment difficulty 125 35, 38, 42, 43, 45
Hypertension immune system 40
genetics 378 nutrition and oxygenation 13, 15, 40
intracerebral hemorrhage risks 377, 378 pain etiology 6, 18
pathophysiology
Ibuprofen, polymeric drug delivery systems apoptosis 19, 32, 35
484 collagen changes 32–34
Immunotherapy cytokines 20, 33, 34
glioma endplate changes 34
monoclonal antibodies 537–539 growth factors 21
prospects 509–513 inflammation 20, 33
tumor vaccines 539, 540 matrix metalloproteinases 20, 34, 37,
polymeric drug delivery systems 475, 38
477, 478 proteoglycan changes 32, 37, 38
Insulin-like growth factor-1 (IGF-1), water loss and diffusion impairment
intervertebral disc degeneration 31, 32, 40
expression 19, 21 progression characterization 5, 6
treatment prospects 38, 43 structure and anatomy 9–13, 30, 31
Intercellular adhesion molecule-1 (ICAM-1), treatment
hemorrhagic stroke gene expression 399 conventional therapy 37
Interleukin-1 (IL-1) prospects 21–24, 35, 49
inflammatory pain 296 Intracerebral hemorrhage, see Hemorrhagic
intervertebral disc degeneration stroke
expression 20, 33, 34 Intrathecal drug delivery
Interleukin-2 (IL-2), immunotherapy 475, analgesic peptides 325
477, 540 epilepsy 182, 183
Interleukin-8 (IL-8), pain role in rationale 461
intervertebral disc degeneration 18 Ischemic stroke
Interleukin-12 (IL-12), immunotherapy animal models
475, 540 clinical relevance 346–348
Intervertebral disc degeneration magnetic resonance imaging 346–348
biomechanics 15, 16 spontaneous stroke 341–345
economic impact 5 transgenic animals 362
embryology of disc development 6–9 brain injury gene expression
epidemiology 5, 30 neuroprotective and neurodestructive
etiology 16–18 genes 362, 363
gene therapy overview 345, 346
adenoviral vectors 40, 41 plasticity and recovery 364, 365
combination gene therapy 45, 46 candidate genes 340, 341
cultured cell studies 43 cell transplantation therapy
epidermal growth factor 22, 35 ex vivo gene therapy 447, 448
matrix metalloproteinase tissue historical perspective 439, 440, 442,
inhibitors 43, 45, 46 443
overview 38–40 immunosuppression 441, 442
Subject Index 652

neural stem cell transplantation Laser capture microdissection (LCM), DNA
448–450 microarray analysis of samples 250, 251
NT2 cell trials 443–445 Lentivirus
support factors for graft survival and Parkinson’s disease gene therapy vector
axonal reconnection 445–447 219–221
tissue sources and preparation for pituitary adenoma gene therapy vector
implantation 440, 441 598–600
differential gene expression studies spinal cord injury gene therapy vector
assay variation and confidence levels 80, 81, 89, 90
359, 360 Leukotrienes, pain role in intervertebral
carotid artery stenosis 422–427 disc degeneration 18
confirmation 360–362 Linkage analysis, combination with DNA
differential display 349 microarray analysis 253, 255
DNA microarray analysis 357, 358 Liposome
ex vivo gene therapy 447, 448 antiepileptic drug delivery 183, 184
overview 348, 349 DNA complexes for Parkinson’s disease
representational differential analysis gene therapy 227
356, 357 immunoliposomes 538, 539
serial analysis of gene expression 358,
359 Macrophage, stimulation for spinal cord
subtractive hybridization 356 injury treatment 76, 77
table of genes 350–355 Magnetic resonance imaging (MRI)
therapeutic angiogenesis 426–428, 450 intraoperative
economic impact 337 costs 135, 136
epidemiology 337, 442 glioma surgery 627
gene therapy prospects 138, 141
approaches 413, 414 safety 134, 135
injection of vectors 415–418 surgical outcome advantages 134, 135
principles 365 systems 136–138
target identification 365, 366 neural stem cell tagging for glioma
vectors 414, 415, 447 imaging 634
genetic heterogeneity 336, 340, 341 stroke studies in animals 346–348
heredity studies 337, 338 Mannitol, blood-brain barrier disruption for
preconditioning stress in brain tolerance drug delivery 459, 460
363, 364 Marfan’s syndrome, cerebral aneurysm 384
prevention 337 Marrow stromal cell (MSC), ischemic
single gene mutations stroke transplantation studies 448, 449
CADASIL 338–340 Matrix metalloproteinases (MMPs)
MELAS 338–340 antisense knockdown 126
thrombolytic therapy 337 inhibition in anti-angiogenesis therapy
543
Kallikrein, carotid artery stenosis gene intervertebral disc degeneration
therapy 425 expression 20, 34, 37, 38
Ketogenic diet, epilepsy management 185, tissue inhibitor treatment prospects
186 43, 45, 46
KRIT1, disruption and cavernous MELAS, gene mutations and stroke
malformation 381 338–340
Subject Index 653
medwedi.ru
Methotrexate, polymeric drug delivery history of study 624
systems 481, 482 integration potential 625, 626
N-methyl-D-aspartate (NMDA) ischemic stroke transplantation studies
nociception 294, 295 448–450
receptor role in epilepsy 173, 197, 198 isolation and culture 67, 69, 70,
Microchip, drug delivery 487 105–108, 626, 627
Minocycline, angiogenesis inhibition 478 markers 106
Mitogen-activated protein kinase (MAPK) migration 625, 626
carotid artery stenosis antisense gene persistence of grafts 631
therapy 424 pluripotency 67, 69, 104, 626
cerebral vasospasm antisense gene therapy prodrug-enzyme engineering 629–631
after hemorrhagic stroke 420, 422 spinal cord injury transplantation therapy
glioma progression signaling 527–531 aims 110
small molecule inhibitors 541, 542 animal studies 109–116, 118
smooth muscle contraction in clinical prospects 118–120
hemorrhagic stroke 393, 394 endogenous cell properties 110, 111
Mitoxantrone, polymeric drug delivery gene transfer 67, 68
systems 474 graft survival 70
Multidrug-resistance-associated protein rationale 68, 69
(MRP), antiepileptic drug resistance role timing 111–116, 118
176, 177 tissue engineering with biopolymer
Multiple sclerosis, DNA microarray studies scaffolds 636
249 Neuropeptide Y (NPY), pain mediation 299
Neurotensin (NT), pain mediation 298
Nerve growth factor (NGF) Neurotrophin-3 (NT-3)
brain graft neuroprotection 446 brain graft neuroprotection 446
spinal cord injury gene therapy 84, 95 pain mediation 302
Neural stem cell (NSC) spinal cord injury gene therapy 84, 130
adult cell localization and characterization Neurotrophin-4/5 (NT-4/5), pain mediation
107, 108 302
chemokines in tumor homing 632 Nitric oxide (NO)
endogenous stem cells cerebral aneurysm role 383
differentiation into astrocytes 105 endothelial dysfunction in hemorrhagic
manipulation 72, 73 stroke 395, 396
remyelination studies 74 pain mediation 303
glioma smooth muscle contraction in
cell tagging for imaging 634 hemorrhagic stroke 393
glioblastoma multiforme applications Nitric oxide synthase (NOS)
639 carotid artery stenosis gene therapy 425
glioma-genesis role 628, 629 cerebral vasospasm gene therapy after
gliomatosis cerebri applications 638, hemorrhagic stroke 418, 419
639 Nitroreductase, prodrug combination in
homing and therapeutic applications gene therapy 601
629–634 NOGO-A, glioma expression 633, 634
prospects for therapy 634–639 Notch, gene mutations in stroke 339
radiation necrosis differentiation from NT2 cells, transplantation trials in stroke
tumor recurrence 637 443–445
Subject Index 654

Nuclear factor-␬B (NF-␬B) galanin 299
cerebral vasospasm antisense gene therapy glial-derived neurotrophic factor 302,
after hemorrhagic stroke 420, 422 303
hemorrhagic stroke gene expression 399 histamine 302
inhibitory GABA receptors 295, 296,
Olfactory ensheathing cell (OEC), spinal 323
cord injury therapy 75, 76, 96 ion channels
Opioid peptides, pain mediation 297, 323, acid-sensing ion channels 292,
324 293
OSI-774, epidermal growth factor receptor adenosine receptors 292
inhibition 541 ATP-gated channels 291, 292
Osteoconduction, definition 52 potassium channels 293
Osteogenesis, definition 53 sodium channels 293, 294
Osteoinduction, definition 52, 53 neuropeptide Y 299
Oxidative stress neurotensin 298
intervertebral disc degeneration role 20 neurotrophins 302
neurodegenerative disease 265, 267 nitric oxide 303
opioid peptides 297, 323, 324
p53, replacement in cancer therapy 605 overview 285–287
Paclitaxel, polymeric drug delivery systems pituitary adenylyl cyclase activating
473 peptide 303
Pain prostaglandins 301
analgesics 322, 323 protein kinase C in signal transduction
cell transplantation therapy 324–326 304
definition 284, 322 serotonin 301, 302
gene expression regulation 285 somatostatin 299
gene therapy substance P 297, 298
antisense gene therapy 309–311 vasoactive intestinal peptide 303
brain gene transfer 333 neuronal receptors
ex vivo therapy 325 adrenergic receptors 290, 291
prospects 311–313 cannabinoid receptors 289
rationale and targets 304, 305 capsaicin receptors 288, 289
RNA interference 310 cholinergic receptors 291
viral vectors cold receptors 289, 290
adenovirus 326 proteinase-activated receptors 290
herpes simplex virus 326–333 nociceptors 284, 285, 323
overview 305–308 Parkinson’s disease (PD)
hypothalamic deep brain stimulation for gene therapy
management, see Deep brain dopamine synthesis enzymes 214
stimulation expression detection
intervertebral disc degeneration 6, 18 microdialysis 237
mediators positron emission tomography 236,
bradykinin 300 237
calcitonin-gene-related peptide 298 expression regulation 228–230
cholecystokinin 299, 300 ex vivo therapy 231–233
cytokines 296 in vivo therapy 214, 215
excitatory glutamate receptors 294, 295 rationale 213, 214
Subject Index 655
medwedi.ru
Parkinson’s disease (PD) (continued) Photodynamic therapy (PDT),
gene therapy (continued) photosensitizer delivery with neural stem
vectors cells 637
adeno-associated virus 164, Pituitary adenylyl cyclase activating peptide
221–226 (PACAP), pain mediation 303
adenovirus 216, 218, 219 Pituitary tumors
comparison of viral vectors 217 animal models
herpes simplex virus 215, 216 Cushing’s disease 610
hybrid vectors 226 growth hormone tumors 609, 610
lentivirus 219–221 null cell adenoma 611
liposome-DNA complexes 227 prolactinoma 608, 609
naked DNA 226, 227 conventional treatment
polyplexes 228 goals 583
targeting of tissues 230, 231 overview 580
neural progenitor cell transplantation pharmacotherapy 586, 587
history 108, 109, 235, 236 radiation therapy 585, 586, 588
stem cell therapy 129 surgery
tissue transplantation studies 108, transcranial approach 585
233–235, 439, 440 transsphenoidal approach 583–585,
treatment difficulty 125 588
xeno-neurotransplantation damage mechanisms 581, 582
embryonic porcine ventral gene mutations 605
mesencephalon tissue 149, 150 gene therapy
outcomes 150, 151 angiogenesis inhibition 603, 604
overview 146–148 apoptosis induction 604, 605
patient selection 149 conditional cytotoxicity 600, 601, 603
postoperative evaluation 150 immune response activation 606, 607
preoperative preparation 150 preclinical studies 611–613, 615
surgery 150 prospects 615, 616
Parvovirus rationale 580, 581, 587, 588
adeno-associated virus homology 159, tumor suppressor genes 604, 605
160 vectors
crystal structure 158, 159 adeno-associated virus 595, 596
Peripheral nerve injury, polymeric drug adenovirus 589, 591, 593–595,
delivery systems 485 611–613
P-glycoprotein, antiepileptic drug comparison of viral vectors 590–593
resistance role 176, 177 herpes simplex virus 596, 597,
Phenobarbital, mechanism of action 172 600–602, 611, 612
Phenytoin lentivirus 598–600
mechanism of action 171 retrovirus 597, 598
polymeric drug delivery systems 484 initiation 582, 583
Phosphatidylinositol 3-kinase (PI3K) prevalence 582
glioma progression signaling PKI166, epidermal growth factor receptor
531–533 inhibition 541
small molecule inhibitors 541 Platelet-derived growth factor (PDGF)
Phospholipase C (PLC), glioma progression glioma progression signaling 522–524
signaling 533–535 isoforms 523
Subject Index 656

receptor gene therapy for carotid artery Quinacrine, polymeric drug delivery
stenosis 424 systems 474
small molecule inhibitors 540, 541
Polymeric drug delivery systems Representational differential analysis
adriamycin delivery 479, 480 (RDA), ischemic stroke differential gene
angiogenesis inhibitor delivery 478 expression studies 356, 357
antiepileptic drugs 184 Rho, smooth muscle contraction in
O6-benzylguanine delivery 472 hemorrhagic stroke 394
buthionine sulfoximine delivery 479 RMP-7, blood-brain barrier disruption for
camptothecin delivery 473, 474 drug delivery 460
carboplatin delivery 482, 483 RNA interference, pain gene knockdown
carmustine delivery using polyanhydride 310
fibers in glioma
development 464, 465 Schwann cell, spinal cord injury therapy 76
initial therapy trials 470–472 Serial analysis of gene expression (SAGE)
preclinical trials 465–467 hemorrhagic stroke vasospasm differential
recurrent glioma trials 467–469 gene expression studies 390, 391
cisplatin delivery 483 ischemic stroke differential gene
cyclophosphamide delivery 479 expression studies 358, 359
dexamethasone delivery 483 Serotonin, pain mediation 301, 302
5-fluorouracil delivery 480, 481 Somatostatin, pain mediation 299
hemorrhagic stroke applications 484, 485 Sphingolipids
historical perspective 461–464 enzyme replacement therapy 204–210
immunotherapy 475, 477, 478 storage disorders 202, 204
methotrexate delivery 481, 482 Spinal cord injury (SCI)
mitoxantrone delivery 474 animal models 66, 75
paclitaxel delivery 473 axonal regeneration barriers 65, 66, 125,
peripheral nerve injury applications 126
485 cellular therapy
phenytoin delivery 484 endogenous stem cells
prospects 487–489 differentiation into astrocytes 105
quinacrine delivery 474 manipulation 72, 73
radiosensitizer delivery 484 remyelination studies 74
rationale 458, 459, 461 fetal tissue transplantation 74, 75
spinal fusion applications 485, 486 functionality of grafts 71, 72
Polyplex, Parkinson’s disease gene therapy macrophage stimulation 76, 77
vector 228 neural restricted precursors 70, 71
Prostaglandins, pain mediation 301 neural stem cells
Prosthetics, neuroprosthetic therapies 131, adult cell localization and
133, 134 characterization 107, 108
Proteinase-activated receptors (PARs), pain aims 110
reception 290 animal studies 109–116, 118
Protein kinase C (PKC) clinical prospects 118–120
pain signal transduction 304 endogenous cell properties 100,
smooth muscle contraction in 111
hemorrhagic stroke 392, 393 gene transfer 67, 68
Prothrombin, gene mutations in stroke 340 graft survival 70
Subject Index 657
medwedi.ru
Spinal cord injury (SCI) (continued) demineralized bone matrix 59
cellular therapy (continued) expression 54
neural stem cells (continued) gene therapy prospects 60–62
isolation and culture 67, 69, 70, preclinical studies
105–108 BMP-2 55, 56
markers 106 BMP-7 56, 57
pluripotency 67, 69, 104 demineralized bone matrix 57
rationale 68, 69 preparations for use 54, 55
timing 111–116, 118 graft substitutes 52
olfactory ensheathing cells 75, 76, 96 Spinal fusion, polymeric drug delivery
overview 66 systems 485, 486
Schwann cells 76 Spine
conventional therapy 65 disc degeneration, see Intervertebral disc
economic impact 65 degeneration
epidemiology 65 embryology of disc development 6–9
gene therapy Squalamine, angiogenesis inhibition 478
animal models 90, 91 Statins, Alzheimer’s disease protection 263
Bcl-2 85 STATs, glioma progression signaling 535
Bcl-XL 85 Stroke, see Hemorrhagic stroke; Ischemic
brain-derived neurotrophic factor 84 stroke
delivery barriers 77, 78 Subarachnoid hemorrhage, see
ex vivo gene transfer 93–96 Hemorrhagic stroke
glial-derived neurotrophic factor 84, Substance P (SP), pain mediation 297, 298
90–93 Subtractive hybridization, ischemic stroke
nerve growth factor 84, 95 differential gene expression studies 356
neurotrophin-3 84, 130 Superoxide dismutase (SOD), cerebral
nonviral vectors 78, 79 vasospasm gene therapy after
prospects 96 hemorrhagic stroke 420
viral vectors
adeno-associated virus 83, 88, 92 T cell
adenovirus 82, 83, 85–88, 90–92 gene therapy for immune response
herpes simplex virus 81, 82 enhancement in cancer 606, 607
injection 87, 88 glioma response studies
lentivirus 80, 81, 89, 90 cytolytic activity assays 504
mortality 65 flow cytometry of cytokine production
recovery assays 67 504, 505
regeneration potential 104 gene expression analysis 505, 506
surgical techniques 130, 131 immunotherapy prospects 509–513
Spinal fusion proliferation assay 503, 504
animal models 53, 54 types in immune response 501–503
autogenous iliac crest bone graft Tiagabine, mechanism of action 173
limitations 52 TIE2, mutation in venous anomalies 381
bone morphogenetic proteins Transforming growth factor-␣ (TGF-␣),
carriers 61 glioma progression signaling 526, 527
clinical studies Transforming growth factor-␤ (TGF-␤)
BMP-2 57–59 carotid artery stenosis antisense gene
BMP-7 59 therapy 424
Subject Index 658

intervertebral disc degeneration treatment Vasoactive intestinal peptide (VIP), pain
prospects 22, 23, 35, 38, 42, 43, 45 mediation 303
neural stem cell expression following Vigabatrin, mechanism of action 173
spinal cord injury 112
TRPM8, pain reception 289, 290 Xeno-neurotransplantation
TRPV1, pain reception 288, 289 historical perspective 147, 148
Tumor necrosis factor-␣ (TNF-␣) immune response and rejection 148,
inflammatory pain 296 149
pain role in intervertebral disc Parkinson’s disease
degeneration 18 embryonic porcine ventral
mesencephalon tissue 149, 150
Vagus nerve stimulation, epilepsy outcomes 150, 151
management 188 overview 146–148
Valproic acid patient selection 149
mechanism of action 171, 172 postoperative evaluation 150
prodrug development 182 preoperative preparation 150
Vascular endothelial growth factor (VEGF) surgery 150
carotid artery stenosis gene therapy 425, prospects 151, 152
426 safety 149
inhibition in anti-angiogenesis therapy
542, 543, 603 ZD-1839, epidermal growth factor receptor
therapeutic angiogenesis 426–428, 450 inhibition 541
Subject Index 659
medwedi.ru

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medwedi.

L. Dade Lunsford Pittsburgh, Pa.

Andrew Freese Minneapolis, Minn.

96 figures, 12 in color, and 25 tables, 2005

Basel · Freiburg · Paris · London · New York ·

Library of Congress Cataloging-in-Publication Data

© Copyright 2005 by S. Karger AG, P.O. Box, CH-4009 Basel (Switzerland)

XI Series Editor’s Note

The Spine and Spinal Cord

5 The Molecular Basis of Intervertebral Disc Degeneration

30 Genetics of Degenerative Disc Disease

37 Gene Therapy for Degenerative Disc Disease

65 Cellular and Gene Therapy Approaches to Spinal Cord Injury

104 Neural Stem Cell Transplantation for Spinal Cord Repair

Functional and Restorative Molecular Neurosurgery

124 Contemporary Applications of Functional and Stereotactic Techniques

154 Adeno-Associated Viral Vectors for Clinical Gene Therapy

169 Molecular Mechanisms of Epilepsy and Gene Therapy

202 Emerging Treatment of Neurometabolic Disorders

213 Gene Therapy for Parkinson’s Disease

246 Simplifying Complex Neurodegenerative Diseases by

258 Molecular Pathology of Dementia. Emerging Treatment Strategies

284 Molecular Mediators of Pain

322 Gene Transfer in the Treatment of Pain

336 Gene Discovery Underlying Stroke

377 Molecular Mediators of Hemorrhagic Stroke

413 Advances towards Cerebrovascular Gene Therapy

439 Ex vivo Gene Therapy and Cell Therapy for Stroke

458 Neurosurgical Applications for Polymeric Drug Delivery Systems

499 Immunotherapy Strategies for Treatment of Malignant Gliomas

521 Glioma-Genesis. Signaling Pathways for the Development of Molecular

557 Oncolytic Viral Therapy for Glioma

580 Molecular Neurosurgery in the Pituitary Gland. Gene Transfer as an

645 Author Index

646 Subject Index

We gratefully acknowledge the dedicated, diligent work of Jackie Alutis in

Changing the Paradigm of Neurosurgery

Series Editor’s Note XII

There are not many advantages to becoming ‘senior’, an euphemism for

Until recently, Neurosurgery has been a surgical discipline largely focused

Andrew Freese, MD, PhD

Freese A, Simeone FA, Leone P, Janson C (eds): Principles of Molecular Neurosurgery.

The Molecular Basis of Intervertebral

Low back pain is an endemic problem in Western societies leading to sig-

Embryological Development of the Disc

Fig. 1. The developing embryo has a

Molecular Basis of Intervertebral Disc Degeneration 7

fairly constant throughout development, and in lumbar discs constitutes twelve

Fig. 3. Schematic of the human spine. (Reproduced with permission [117].)

Structure and Anatomy

Molecular Basis of Intervertebral Disc Degeneration 9

Type Predominant Percent of total

‘physoliferous’ (or ‘bubble-bearing’), containing prominent cellular processes

Molecular Basis of Intervertebral Disc Degeneration 11

proteoglycan side chains predominantly of aggrecan. The connection between

Most of the normal intervertebral disc is avascular, depending on the dif-

Molecular Basis of Intervertebral Disc Degeneration 13

6 2 NAD ⫹Pi 2 NADH ⫹2 H

Fig. 6. The glycolytic pathway. Anaerobic metabolism leads to lactate as a by-product,

studies demonstrating a poor correlation between measured oxygen or lactate

Intervertebral discs in humans have evolved to withstand the significant

Molecular Basis of Intervertebral Disc Degeneration 15

Etiology of Disc Degeneration