Ocular Neuroprotection

Ocular
Neuroprotection
edited by
Leonard A. Levin
University of Wisconsin-Madison
Madison, Wisconsin, U.S.A.
Adriana Di Polo
University of Montreal
Montreal, Quebec, Canada
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Preface
Neuroprotection comprises a broad range of therapeutic strategies aimed at preventing death of neurons in trauma or disease. As applied to ocular disorders,
neuroprotection has received enormous attention in past years because of its potential for treating diseases that were heretofore untreatable or difcult to treat,
including disorders primarily involving death of photoreceptors (such as in macular degeneration, retinitis pigmentosa, and retinal detachment) or retinal ganglion
cells (such as in glaucoma).
The goal of this book is to provide the reader with up-to-date ocular neuroprotection research methods that are used in leading laboratories worldwide.
Chapters 1 through 15 focus on research methods at the laboratory level, with
emphasis on various cell culture and animal models, classes of neuroprotective
drugs, biochemical and cellular outcome measures, and applicability to human
disease. Chapters 16 through 18 focus on how clinical trials of neuroprotection
may be carried out, concentrating on issues of recruitment, outcome measures,
and requirements of regulatory agencies.
The information conveyed in this book is intended to be practical. The
chapters address the nuts and bolts of the actual methods themselves, along with
potential problems, solutions to those problems, potential shortcuts (and contraindications to shortcuts), and specic suppliers, where appropriate. The book is
not intended to be an exhaustive presentation of research data or theories, but
rather to provide a focused description of useful methods for conducting neuroprotection research.
This book will acquaint those considering research in the area with the
major techniques, tools, and issues in ocular neuroprotection, while investigators
with experience in neuroprotection research may benet from exposure to new
models, methodologies, and clinical approaches. Regardless of the readers experience, this volume will be an invaluable resource.
iii
iv
Preface
We sincerely thank all the contributorsleading scientists in their elds

who have generously shared their knowledge and expertise in each chapter.
Leonard A. Levin
Adriana Di Polo
Contents
Preface
Contributors
iii
ix
I. In Vitro Models
1. Culture of Retinal Neurons
James D. Lindsey
II. In Vivo Models of Optic Nerve Damage

2. Crush Injury of the Optic Nerve
Michal Schwartz and Eti Yoles
13
3. Intraocular Pressure Elevation: Vein Cauterization

Sansar C. Sharma
23
4. Intraocular Pressure Elevation: Injecting Hypertonic Saline

into Episcleral Veins
John C. Morrison, Elaine C. Johnson, Lijun Jia, and William
O. Cepurna
5. Intraocular Pressure Elevation: Laser Photocoagulation of the
Trabecular Meshwork
BAnn T. Gabelt, James N. Ver Hoeve, and Paul L. Kaufman
31
47
v
vi
Contents
III. In Vivo Models of Retinal Damage

6.
Light-Induced Retinal Degeneration

Daniel T. Organisciak, R. M. Darrow, and L. S. Barsalou
85
7.
Retinal Detachment
Ward M. Peterson
109
8.
Retinal Ischemia
Manuel Vidal-Sanz, Mara P. Lafuente, Inmaculada SellesNavarro, Mara E. Rodrguez, Sergio Mayor-Torroglosa,
Mara P. Villegas-Perez
129
IV. Neuroprotective Strategies

9.
10.
Drug Delivery
Robert W. Nickells and Cassandra L. Schlamp
153
Recombinant Viral Vectors

Przemyslaw Sapieha and Adriana Di Polo
167
V. Analysis of Neuronal Survival and Function

11.
Quantication of Retinal Cells

Christine A. Curcio and Kenneth R. Sloan
189
12.
Ex Vivo and Whole-Mount Retinal Preparations

Arthur J. Weber
205
13.
Detection of Single-Cell Apoptosis

William G. Tatton, Ruth M. E. Chalmers-Redman, and
Nadine A. Tatton
225
14.
Imaging of Retinal Ganglion Cells

Joshua P. Vrabec and Leonard A. Levin
241
15.
Evaluation of Retinal Function: Electroretinography

Marc Hebert and Pierre Lachapelle
249
Contents
vii
VI. Clinical Trials

16. Evaluation of Visual Outcome
Pamela A. Sample
273
17. Clinical Trials in Neuroprotection

Scott M. Whitcup
291
18. Regulatory Issues in Clinical Trials

Anthony C. Arnold
303
Index
313
Contributors
Anthony C. Arnold, M.D. Neuro-Ophthalmology Division, Department of

Ophthalmology, Jules Stein Eye Institute, David Geffen School of Medicine,
UCLA, Los Angeles, California, U.S.A.
L. S. Barsalou Wright State University, Dayton, Ohio, U.S.A.
William O. Cepurna, B.S. Department of Ophthalmology, Casey Eye Institute, Oregon Health & Science University, Portland, Oregon, U.S.A.
Ruth M. E. Chalmers-Redman
New York, U.S.A.
Mount Sinai School of Medicine, New York,
Christine A. Curcio, Ph.D. Department of Ophthalmology, School of Medicine, University of Alabama at Birmingham, Birmingham, Alabama, U.S.A.
R. M. Darrow
Wright State University, Dayton, Ohio, U.S.A.
Adriana Di Polo, Ph.D. Department of Pathology and Cell Biology, University

of Montreal, Montreal, Quebec, Canada
BAnn T. Gabelt, M.S. Department of Ophthalmology and Visual Sciences,
University of WisconsinMadison, Madison, Wisconsin, U.S.A.
Marc Hebert, Ph.D. University of Laval, Quebec City, Quebec, Canada
Lijun Jia, M.D., Ph.D. Department of Ophthalmology, Casey Eye Institute,
Oregon Health & Science University, Portland, Oregon, U.S.A.
Elaine C. Johnson, Sc.D. Department of Ophthalmology, Casey Eye Institute,
Oregon Health & Science University, Portland, Oregon, U.S.A.
Paul L. Kaufman, M.D. Department of Ophthalmology and Visual Sciences,
University of WisconsinMadison, Madison, Wisconsin, U.S.A.
ix
Contributors
Pierre Lachapelle, Ph.D. Department of Ophthalmology, McGill University,

Mara P. Lafuente, M.D., Ph.D.
de Murcia, Murcia, Spain
Department of Ophthalmology, Universidad
Leonard A. Levin, M.D., Ph.D. Departments of Ophthalmology and Visual

Sciences, Neurology, and Neurological Surgery, University of WisconsinMadison, Madison, Wisconsin, U.S.A.
James D. Lindsey, Ph.D. Hamilton Glaucoma Center and Department of Ophthalmology, School of Medicine, University of California, San Diego, La Jolla,
California, U.S.A.
Sergio Mayor-Torroglosa, O.D.
John C. Morrison, M.D. Casey Eye Institute, Oregon Health & Science University, Portland, Oregon, U.S.A.
Robert W. Nickells, Ph.D. Department of Ophthalmology and Visual Sciences, University of WisconsinMadison, Madison, Wisconsin, U.S.A.
Daniel T. Organisciak, Ph.D. Department of Biochemistry and Molecular Biology, Wright State University, Dayton, Ohio, U.S.A.
Ward M. Peterson, Ph.D. Preclinical Programs, Inspire Pharmaceuticals, Durham, North Carolina, U.S.A.
Mara E. Rodrguez, M.D., Ph.D.
dad de Murcia, Murcia, Spain
Department of Ophthalmology, Universi-
Pamela A. Sample, Ph.D. Department of Ophthalmology, University of California, San Diego, La Jolla, California, U.S.A.
Przemyslaw Sapieha Department of Pathology and Cellular Biology, University of Monteal, Monteal, Quebec, Canada
Cassandra L. Schlamp, Ph.D. Department of Ophthalmology and Visual Sciences, University of WisconsinMadison, Madison, Wisconsin, U.S.A.
Michal Schwartz The Weizmann Institute of Science, Rehovot, Israel
Inmaculada Selles-Navarro, M.D., Ph.D.
Universidad de Murcia, Murcia, Spain
Department of Ophthalmology,
Sansar C. Sharma, Ph.D. Department of Ophthalmology, New York Medical

College, Valhalla, New York, U.S.A.
Contributors
xi
Kenneth R. Sloan, Ph.D. Department of Computer and Information Sciences,

University of Alabama at Birmingham, Birmingham, Alabama, U.S.A.
Nadine A. Tatton Mount Sinai School of Medicine, New York, New York,
U.S.A.
William G. Tatton
U.S.A.
Mount Sinai School of Medicine, New York, New York,
James N. Ver Hoeve, Ph.D. Department of Ophthalmology and Visual Sciences, University of WisconsinMadison, Madison, Wisconsin, U.S.A.
Manuel Vidal-Sanz, M.D., Ph.D.
Mara P. Villegas-Perez, M.D., Ph.D.

versidad de Murcia, Murcia, Spain
Department of Ophthalmology, Uni-
Joshua P. Vrabec Department of Ophthalmology and Visual Sciences, University of WisconsinMadison, Madison, Wisconsin, U.S.A.
Arthur J. Weber, Ph.D. Department of Physiology, Michigan State University, East Lansing, Michigan, U.S.A.
Scott M. Whitcup, M.D. Allergan, Inc., Irvine, and Jules Stein Eye Institute,
David Geffen School of Medicine, UCLA, Los Angeles, California, U.S.A.
Eti Yoles
The Weizmann Institute of Science, Rehovot, Israel
1
Culture of Retinal Neurons
James D. Lindsey
Hamilton Glaucoma Center
School of Medicine
University of California, San Diego
La Jolla, California, U.S.A.
I.
INTRODUCTION
The culture of retinal tissue and cells has led to new insights regarding the inuence of neurotransmitters, growth factors, hormones, and other biological molecules on retinal neuron survival and differentiation, as well as the role of the
extracellular matrix in retinal cell function; it has also provided important screening tools to investigate potential neuroprotective treatments [17]. A particular
advantage of many culture systems is they can permit visual assessment of cell
shape, viability, and synaptogenesis of living retinal neurons [711]. Through
the use of specialized dyes, it is possible to monitor changes of intracellular
calcium, apoptosis markers, and other cytoplasmic molecules within living retinal
cells (see Chap. 14) [12,13]. Also, the effects of drug treatments can be assessed
in the absence of systemic responses or dilution that would be encountered in
vivo.
Despite these attractions, however, it can be challenging to established retinal neuron cultures based on the abbreviated methods often presented in published articles. Hence, this chapter discusses the design of successful retinal neuron culture systems, provides details for certain common procedures that often
are overly simplied or omitted in the methods of published articles, and addresses the identication and correction of problems that are occasionally encountered during the culture of retinal neurons. In addition, several resources are iden1
Lindsey
tied that provide further information useful for the design and evaluation of
experiments using retinal neuron cultures.
II. DESIGN CONSIDERATIONS

A.
Culture Type
The type of culture chosen is often constrained by the cellular response that is to
be studied. For example, the effects of experimental treatments on retinal neuron
survival and differentiation are easily studied in monolayer cell cultures. In these
cultures, a completely dissociated retinal cell suspension is placed in tissue culture dishes, and within 6 to 8 hours, the neurons attach to the dish surface and
begin to differentiate neuritic processes. Because each neuron can be directly
inspected using an inverted microscope, it is relatively easy to determine whether
the neuron is alive and whether process growth has been altered. These features
also can be studied in explant retinal culture systems in which some of the original
retinal tissue organization is retained. However, because it is often difcult to
inspect individual living neurons in explants, assessment of survival and differentiation can be better accomplished using appropriate biochemical or histological
analyses.
B.
Developmental Age
The developmental age of the retina also can be important. For example, growing
cells from embryonic retina cells are often easier to maintain in culture than cells
from adult retina. It is also possible to expand the number of embryonic retinal
cells by encouraging cell division. In contrast, cells from older embryo retina or
adult retina typically are post-mitotic. Hence, it can be challenging to obtain large
numbers of retinal cells for culture experiments from adult retina. However, such
experiments can be important because the cellular responses of embryonic retinal
cells can vary from the responses of adult retinal cells.
C.
Culture Media
The various culture media contain different types and amount of salts, amino
acids, energy sources, buffers, and vitamins. For example, Dulbeccos modied
Eagles medium (DMEM), which can be used for culturing embryonic and newborn rat or mouse retinal neurons, contains 7 salts, 13 amino acids, 8 vitamins,
glucose, and pyruvate. In contrast, Medium 199, which has been used for culturing chick retinal neurons, contains 11 salts, 21 amino acids, 17 vitamins, and
glucose, plus 17 additional ingredients, but no pyruvate. For the successful culture
of retinal neurons expressing glutamate or aspartate receptors, it may be benecial
to eliminate these amino acids from the medium because they could induce excitotoxic stress. Neurobasal medium (available from Gibco BRL) is one such medium that has been formulated without glutamate or aspartate [14]. It is important
to note that the pH of tissue culture medium often is regulated by exposing the
medium to an atmosphere containing a specic amount of carbon dioxide. For
example, DMEM is designed to buffer correctly in an atmosphere containing
10% carbon dioxide. However, media such as Hams F12 and RPMI 1640 require
5% carbon dioxide. The correct concentration of carbon dioxide for a particular
medium can be obtained from the medium supplier or from previously published
protocols. As each medium has its advantages and limitations, the best medium
for each retinal neuron type should be determined empirically.
D. Media Supplements
The performance of media is often enhanced with supplements including hormones, growth factors, cofactors, lipids, carrier proteins, and natural uids. Hormones that often are benecial include insulin, progesterone, and hydrocortisone.
Growth factors that have been helpful for the maintenance of certain retinal neurons include broblast growth factor2, nerve growth factor, brain-derived neurotrophic factor, ciliary neurotrophic factor, neurotrophin-3, and neurotrophin-4
[3,15,16]. Useful cofactors, molecules that facilitate certain biological reactions,
include compounds such as sodium selenite and copper sulfate. Lipids that have
been used in certain retinal neuron cultures include linoleic acid and docosahexaenoic acid [10,17]. Carrier proteins that have proved to be benecial include
transferrin and serum albumin. Note that the addition of unbuffered solutions of
certain supplements can change the media pH. Natural uids such as sera, amniotic uid, and cerebrospinal uid have been used as supplements. These contain
hormones, growth factors, and other potentially benecial molecules. It should
be noted that the composition of natural uids can vary among suppliers and
even among lots from the same supplier. With natural uids, as with puried
supplements, the optimal amount to add can vary among retinal neuron types
and culture systems. Hence, the best mixture of supplements and/or natural uids
needs to be determined for each case.
E.
Substrate Enhancements
The performance of retinal cultures often can be improved by application of specialized coatings to the culture dishes. The rst type of coating is the charge
modiers and includes poly-l-lysine and poly-l-ornithine. Many retinal neurons
express glycoproteins that contain negatively charged sialic acid. As a result,
they do not bind strongly to negatively charged tissue culture plastic. Poly-llysine and poly-l-ornithine are positively charged synthetic amino acid polymers.
Lindsey
Coating the culture dish with either of these compounds changes the charge of
the tissue culture surface and thereby promotes neuronal attachment [18]. The
second type of coating includes extracellular matrix molecules such as collagens,
bronectin, laminin, and heparin sulfate proteoglycan. These molecules can bind
to specic cell surface receptors, facilitate growth factor action, and can often
dramatically improve neuronal differentiation and survival [19,20]. The third type
of coating includes molecules with specic afnities such as antibodies and lectins. These molecules can facilitate or modulate binding of certain subsets of
cells present within a dissociated retinal cell suspension [18]. As with media and
supplements, the optimal coating should be determined for each retinal cell type
and culture system.
F.
Co-Cultures
The survival of certain retinal cell types has been improved by co-culturing with
other cell types such as astrocytes, or with target neurons such as lateral geniculate neurons in the case of retinal ganglion cells [6]. In the case of astrocytes, a
conuent monolayer of astrocytes is often generated prior to seeding the retinal
neurons (adding the dissociated cell suspension to the cultures). Because retinal
neurons can grow on top of the astrocytes in these cultures, their survival and
differentiation may be easily evaluated with the inverted microscope. Retinal
neuron aggregate cultures also can be generated in the presence of other cell
types. This will be further discussed below. Co-cultures have been useful for
investigating the cellular basis of benecial cell interactions.
G.
Cell Density
The density of cells placed within monolayer cell cultures can inuence cell survival and differentiation [21]. Usually, increased survival is observed with increased cell density. This is because the retinal neurons produce molecules that
are benecial for their survival in vitro. If cell density is too high, however,
differentiation can be inhibited. Also, cell clumping can become problematic in
high-density cultures. This can make it difcult to assess survival changes and
changes in cell structure.
III. SELECTED METHODS

A.
Isolation of Chick Retina
This protocol is appropriate for chick embryos ranging in age from 7 to 12 days.
All instruments must be sterilized before use. After being briey wiped with 70%
ethanol, the rounded end of the egg is opened using blunt forceps, and the shell
membrane over the embryo is removed with ne forceps. Using ne-curved forceps, the vascular connections to yolk membranes are broken. Next, the embryo
is lifted out using blunt-curved forceps and placed in a 60 mm dish. After separation of the head, the eyelid rudiments are either pulled off with ne forceps or
removed using spring-action scissors. Then the eyes are lifted from their sockets
using the curved portion of curved sharp forceps and transferred to a 60-mm dish
containing Hanks buffered saline solution (HBSS). Any orbital tissues such as
muscle and fat tissue is then removed from the surface of the eyes.
Next, the tips of sharp forceps are inserted into the globe near the optic
nerve to create a small opening. With two pairs of sharp forceps, the edge of the
sclera at the opening is grasped at opposite sides of the opening and, by spreading,
the sclera and retinal pigment epithelium (RPE) are peeled from the globelike
removing a thin glove from a hand. The lens and cornea typically come off with
the sclera/RPE. The center of the vitreous gel is grasped with blunt forceps, and
the retina is gently teased from the vitreous gel using sharp forceps. The retina
is transferred to another 60 mm dish containing HBSS by gently lifting with either
re-polished glass hooks or spread curved forceps. Nonretinal tissue present on
small portions of the retina are sliced off using sharp sterile tungsten needles that
have been glued with epoxy to glass rod handles. The cleaned retina is transferred
to another 60 mm dish containing HBSS. At this point, the retina is ready to be
sliced for sliced cultures, diced with tungsten needles and then placed in explant
cultures, or diced and then enzymatically dissociated into a single cell suspension.
This protocol can be adapted to newborn and adult rat eyes by using appropriate sterile surgical technique to remove eyes and then proceeding as described
to isolate the retina.
B. Generating Retinal Neuron Monolayer Cultures
This method is adapted from a protocol for retinal neuron cultures that promotes
the survival and differentiation of several types of retinal neurons [10]. Precoat
tissue culture dishes with a charge modier (e.g., by lling 35 mm dishes with
0.1 mg/mL 3070 kDa poly-l-ornithine dissolved in puried water and sterile
ltered; incubate at least 1 h at room temperature). Add an extracellular matrix
coating (e.g., 0.5 g/mL laminin dissolved in DMEM; incubate in 10% CO2
incubator at 37oC, overnight). Prepare media fresh, add supplements (e.g., Medium 199 containing 10% heat-inactivated fetal bovine serum [FBS], 110 g/
mL linoleic acidalbumin, 100 U/mL penicillin, and 2 mM glutamine) and place
in humidied incubator (5% CO2, 37C) to equilibrate temperature and pH.
Isolate chick retina and dice into small pieces (1 mm squares) as described
above. The retinal pieces are transferred to a test tube and allowed to settle to
the bottom. The HBSS is replaced with calcium- and magnesium-free HBSS
(CMF-HBSS) and incubated for 10 minutes in a 37C water bath. After two
Lindsey
rinses (changes) in CMF-HBSS, a solution containing 0.25% trypsin and 1.85

U/ml DNAse (dissolved in CMF-HBSS) is added and the tissue is incubated at
37C for 20 min. After trypsin is removed, the retinal pieces are rinsed with
HBSS containing 10% fetal bovine serum (FBS). After 1 mL of 10% FBS/HBSS
is added, the tissue is triturated (sucked in and out of a Pasteur pipette in which
the tip has been reduced by ame polishing) several times until it dissociates
into a single cell suspension. Avoid excessive trituration that can reduce viability.
Examine a drop of the suspension placed on a microscope slide using a microscope with phase optics. If there are cell clumps present, these can be removed
by passing the cell suspension through a sterile Nytex lter fabric afxed to a
sterile glass tube.
The concentration of cells in the suspension is determined by adding one
drop of suspension to each side of a Neubauer ruling hemacytometer. After placing the hemacytometer on a conventional (upright) phase microscope, cells visible in a 1 mm square area are counted (a group of 16 squares present at the
corners of the ruling). A button-activated counter facilitates tallying the count.
Multiply the count obtained by 10,000 to obtain cells per milliliter. Total yield
is calculated by multiplying the suspension concentration by the total suspension
volume. After appropriate dilution with nal media, the cell suspension is added
to the culture dishes. In the case of chick retinal neurons seeded in 35 mm dishes
described above, dilute the suspension to 400,000 cells/mL and add 1 mL per
dish (add to the 1 mL already present in the dish). The dishes are then placed
into the incubator.
IV. ALTERNATIVE CULTURE FORMATS

A.
Retinal Explant Cultures
Retinal explant cultures can be useful for examining neurite outgrowth as well
as neuronal differentiation [19,22,23]. Typically, isolated retina is diced into
pieces and the pieces are placed in a drop of medium containing 40 to 50% serum
on the bottom of a culture dish. After allowing the serum to clot, which holds
the explant in place, add culture medium. Within 1 to 2 days, glia are observed
growing out of the explant. Several days later, neurites are observed radiating
from the explants. These cultures have been used to evaluate interactions between
growing axons and extracellular matrix, the effects of growth factors, and the
role of glia in axon extension.
B.
Retinal Slice Cultures
Cellular contacts and interactions are typically well preserved in slice cultures
[24,25]. The usual approach is to lay the isolated retina on a cellulose lter mem-
brane and transfer it to the sterilized stage of a tissue chopper, where slices 100
400 m thick are cut. The slices, along with the attached membrane, are then
transferred into culture medium in a culture dish and placed into an incubator.
These cultures preserve the association of retinal cells with their microenvironment and neighboring cell types. Within the slice, synaptic associations also are
retained. Cells within retinal slices can be studied using electrophysiological recordings. Subsets of cells, such as amacrine cells, can be identied and studied
within these slice cultures using specic immunohistochemical markers.
C. Reaggregation Cultures
The presence of adhesion- or contact-mediated interactions can be studied in
reaggregation cultures [2628]. A retinal cell suspension is prepared as described
above. Aliquots of the suspension are transferred to reaggregation culture dishes
or small (e.g., 25 mL), sterile Erlenmeyer asks and then placed in a gyratory
shaker contained within a 37C incubator. The size of the aggregate is dependent
on cell density, gyration amplitude (1025 mm), and gyration frequency (50
80 rpm). By mixing the retinal cell suspension with other cell type suspensions
(such as from lateral geniculate), target cellular interactions can be studied. Reaggregate cultures can be evaluated by biochemical analysis, by counting, or by
histology.
D. Enriched Photoreceptor Cultures
Methods have been developed to generate mixed retinal neuron cultures from
embryonic chick or mouse eyes that contain identiable photoreceptors as well
as multipolar neurons (neurons that express multiple neurites) [10,29]. The photoreceptors are readily identied by their extension of a single neurite, their expression of opsin, and, in the case of chick photoreceptors, a cytoplasmic lipid droplet
visible by phase microscopy. Adding 2 mM kainic acid or 12 nM -bungarotoxin to the cultures can eliminate up to 70% of the multipolar neurons without
apparent effect on the photoreceptors [30,31]. Combined treatments with these
agents does not show additive effects or potentiation between the toxins [31].
Another method useful for generating cultures of puried photoreceptors separates these cells from other retinal neurons during isolation through the use of
vibratome sectioning [32].
E.
Puried Retinal Ganglion Cell Cultures
Retinal ganglion cells can be puried from retinal cell suspensions using cell
panning [33] and then cultured in chemically dened media [2]. This technique
relies on retinal ganglion cell expression of the cell surface marker protein Thy-
Lindsey
1 and the absence of expression of an antigen recognized by anti-macrophage

antibody. A retinal cell suspension is generated as described above. This suspension is incubated with monoclonal mouseanti-macrophage antibodies and then
poured into a large petri dish precoated with goatanti-mouse IgG antibodies.
Nonadherent cells are collected and then incubated in a second petri dish precoated with antiThy-1 antibody. After repeated washing with HBSS to remove
nonadherent cells, the remaining cells are greater than 95% retinal ganglion cells.
The attached cells are then released from the panning dish using mild trypsin
digestion and placed in monolayer cell culture.
V.
TROUBLESHOOTING
A.
Pathogen Contamination
The appearance of yeast, fungi, and bacteria in cultures will alter results and
eventually destroy the cultures. Yeast infection appears as a proliferation of round
or ovoid particles within the cultures. Fungi form lamentous threads (mycelia)
that often are branched. Bacteria appear as clumps of small rods or particles.
Excellent pictures of cultures infected with these microbes are present in a cell
culture manual by Freshney [34]. Mycoplasma also can infect cultures and reduce
viability. It is not possible to see mycoplasma by microscopic inspection. However, they can be detected by staining cultures using the dye Hoechst 33258 or by
use of a commercial test kit (available from Gibco BRL). Antibiotics rarely can
control an established infection. Usually, the best course of action is to discard the
contaminated cultures and suspected media, resterilize the equipment and workspaces of the culture laboratory, and then resume with new media and plates.
B.
Chemical Contamination
Abnormal performance of solutions used in tissue culture procedures can reect

chemical contamination. For example, if glassware used to prepare media is insufciently rinsed after washing, residual detergent can be left on the glassware
walls and then contaminate solutions placed in the glassware. A failure in the
performance of the laboratory water purier also can introduce chemical contamination. Other possible sources of chemical contamination include a pH electrode
that is inadvertently used for measuring nontissue culture solutions, and spilled
chemicals on the pan of the laboratory microbalance used to weigh puried media
supplements.
C.
Inactivation of Tissue Culture Reagents
Several tissue culture reagents have a relatively short shelf life. For example,
glutamine in solution becomes deactivated within several days. Hence, frozen
stock glutamine often is added to media just before use. Certain peptide supplements can become deactivated by repeated freeze-thaw. Hence, they should be
stored in a nonfrost-free freezer. Certain enzymes, such as trypsin, can become
deactivated by auto-digestion if kept at room temperature or 37C for an extended
period. Hence, frozen enzyme solutions used in tissue dissociation should be
thawed in the water bath just before they are needed.
D. Incorrect Incubator Settings or Function
Incorrect concentration of carbon dioxide within the incubator will produce incorrect pH within the media. As mentioned above, the correct carbon dioxide concentration for a particular media can be obtained from the supplier. Correct performance of the incubator controls can be conrmed using a Fyrite ue-gas
analyzer (available from Fisher Scientic). A small air sample is withdrawn from
the incubator chamber through a special port without opening the incubator door.
This air sample is exposed to an alkali solution that absorbs the carbon dioxide.
The change in the volume of the air sample is measured to determine carbon
dioxide concentration in the original sample. Regular testing of incubators will
often identify performance problems prior to the loss of valuable experiments.
E.
Phototoxicity
Examination of cultures using an inverted microscope is often the best way to

monitor the status of retinal monolayer, explant, or slice cultures. However, the
cells are easily damaged by too much light exposure. Use of a green lter can
reduce the more-harmful short wavelength light and thus protect the cultures
during examination.
VI. RECOMMENDED RESOURCES
Excellent books on basic cell culture technique are available that discuss the
culture laboratory, aseptic technique, selection of culture supplies, sterilization,
basic considerations in the generation of primary cultures, characterization of cell
cultures, and contamination control [34,35]. A classic reference for the formulation of chemically dened media for neuronal cultures is a book series edited by
Barnes, Sirbasku, and Sato [36]. Also helpful are several articles in recent editions
of Methods in Cell Biology [3745].
VII. CONCLUDING COMMENTS
First, start with an established protocol, if available. If one that accomplishes
what you want is not available, start with the most similar one you can nd and
10
Lindsey
then move toward your goal by making and evaluating changes one at a time.
Isolate your cell culture activities from other laboratory activities as much as
possible. Standardize all procedures. Meticulously document the source, age, lot
number, and usage of all media components. Minimize delay in the generation
of cultures. Regularly examine cultures after they have become attached (this
usually occurs within 8 h). Do not examine too many cultures at one time as
they are sensitive to pH and temperature shock. Finally, document your results
as completely as possible with each experiment, even when it seems that it did not
work. This will often help considerably in identifying problems and developing
improved methods for retinal cell cultures.
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cells in a chemically dened microenvironment. Invest Ophthalmol Vis Sci 1994;
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Shindler KS, Zurakowski D, Dreyer EB. Caspase inhibitors block zinc-chelator induced death of retinal ganglion cells. Neuroreport 2000;11:22992302.
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Morgan J, Caprioli J, Koseki Y. Nitric oxide mediates excitotoxic and anoxic damage in rat retinal ganglion cells cocultured with astroglia. Arch Ophthalmol 1999;
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Sherry DM, St JR, Townes-Anderson E. Morphologic and neurochemical target
selectivity of regenerating adult photoreceptors in vitro. J Comp Neurol 1996;376:
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Adler R, Lindsey JD, Elsner CL. Expression of cone-like properties by chick embryo
neural retina cells in glial-free monolayer cultures. J Cell Biol 1984;99:11731178.
Madreperla SA, Adler R. Opposing microtubule- and actin-dependent forces in the
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Brewer G. Isolation and culture of adult rat hippocampal neurons. J Neurosci Methods 1997;71:143155.
Heidinger V, Hicks D, Sahel J, Dreyfus H. Peptide growth factors but not ganglioside protect against excitotoxicity in rat retinal neurons in vitro. Brain Res 1997;
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Meyer-Franke A, Kaplan M, Pfrieger F, Barres B. Characterization of the signaling
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Rotstein N, Aveldano M, Politi L. Essentiality of docosahexaenoic acid in retina
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Adler R. Regulation of neurite growth in puried retina neuronal cultures: effects
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Carri NG, Perris R, Johansson S, Ebendal T. Differential outgrowth of retinal neurites on puried extracellular matrix molecules. J Neurosci Res 1988;19:428439.
Hewitt AT, Lindsey JD, Carbott D, Adler R. Photoreceptor survival-promoting activity in interphotoreceptor matrix preparations: characterization and partial purication. Exp Eye Res 1990;50:7988.
Tezel GM, Seigel GM, Wax MB. Density-dependent resistance to apoptosis in retinal cells. Curr Eye Res 1999;19:377388.
Rowe-Rendleman C, Mitchell CK, Habrecht M, Redburn DA. Expression and
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Mattson MP, Barger SW, Begley JG, Mark RJ. Calcium, free radicals, and excitotoxic neuronal death in primary cell culture. Methods Cell Biol 1995;46:187216.
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2
Crush Injury of the Optic Nerve
Michal Schwartz and Eti Yoles
The Weizmann Institute of Science
Rehovot, Israel
I.
INTRODUCTION
Optic neuropathies are chronic neurodegenerative diseases of the optic nerve

[13], in which degeneration of the axons leads eventually to death of their corresponding cell bodies, the retinal ganglion cells (RGCs). The mechanism underlying the progression of disease is not yet fully understood but probably
involves the activity of physiological compounds that become cytotoxic when
their normal concentrations are exceeded. In order to study the mechanisms of
RGC death, identify the nerve-derived mediators of toxicity causing the ongoing
spread of damage, and screen compounds for their neuroprotective potential
(i.e., their ability to arrest or reduce this secondary degeneration), suitable animal
models are needed. We have established a rat model in which the optic nerve
is subjected to a well-calibrated partial crush injury of the required severity
[4,5]. Use of the model has made it possible to demonstrate self-propagating
secondary degeneration [5], identify some of the mediators of degeneration
common to many neurodegenerative disorders [6], study the molecular mechanisms underlying RGC death, and discover processes of neuroprotection [712].
Molecular mechanisms can also be studied in the severely crushed optic nerve
of the mouse, an easily obtained model in which the availability of transgenic
mice can be exploited for studies of the effects of relevant genes on RGC survival
[13,14].
13
14
Schwartz and Yoles
Figure 1 Anatomy of the rat visual system.
II. ANATOMY OF THE RAT VISUAL SYSTEM

The axons of RGCs in the rat and mice, after exiting the retina through the optic
nerve head, form the optic nerve. At the optic chiasm most of the bers cross
to the contralateral optic tract to reach the optic tectum (superior colliculus).
Fewer than 10% of the bers beyond the optic chiasm are ipsilateral [15]. Nearly
all of the RGCs in each tract project to the superior colliculus on that side, and
fewer than 40% of them have collateral projections to the dorsal lateral geniculate
nucleus (LGN) [1517].
III. PARTIAL CRUSH INJURY OF THE RAT OPTIC

NERVE
A.
Surgical Exposure of the Optic Nerve

Intraorbitally
The intraorbital part of the optic nerve is longer in rodents than in other species,
making it relatively easy to carry out experimental manipulations without impinging on adjacent tissues or harming the nerve itself. All surgical procedures are
done under general anesthesia. We use a binocular operating microscope; the
conjuctiva is incised lateral to the cornea, the retractor bulbi muscles are separated
using curved blunt forceps, and the optic nerve is identied and exposed near
15
Figure 2 Cross-action forceps.
the eyeball by blunt dissection for 2.53 mm, and care is taken not to stretch
the nerve.
B. Calibrated Crush Injury
A reproducible crush injury of graded severity is inicted on the optic nerve by
the use of pre-calibrated cross-acting (self-closing) forceps, which open when
the handles are pressed and close when the handles are released [4]. The force
exerted by the grasping jaws (and thus the severity of the crush lesion inicted)
is adjusted by varying the number of revolutions of the screw attached to the
handle.
Using the forceps, a moderate, mild, or very mild crush injury is inicted
on the exposed optic nerve about 1 mm distal to the eye, for a period of 30
seconds.
IV. SEVERE CRUSH INJURY OF THE MOUSE

OPTIC NERVE
To identify and characterize the molecules participating in the process of RGC
death, it is necessary to devise an animal model that allows molecular manipulation. Establishment of the mouse model makes it possible to study the effects of
severe optic nerve injury in genetically manipulated mice. For this purpose, all
RGCs must be labeled 72 h before optic nerve crush. With the aid of a binocular
operating microscope, the conjunctiva over the posterior pole of the eye of the
anesthetized mouse is incised. The optic nerve is exposed by gentle blunt dissection between the surrounding muscle and the retrobulbar region, as described
above for the rat. Using cross-action forceps and taking care not to interfere with
the blood supply, we then crush the nerve for 2 s.
16
V.
Schwartz and Yoles
RETROGRADE LABELING OF RETINAL

GANGLION CELLS
Because of the anatomical construction of the visual system, retinal ganglion cell
(RGC) survival at any time after axonal injury can be quantied by the use of
retrograde neuronal tracers. Properties of the tracers selected for this purpose
should include (1) lack of any effect on neuronal viability and activity; (2) intense
uorescence; (3) resistance to fading; (4) absence of diffusion from labeled cells;
and (5) relatively prolonged survival time.
The RGCs that survive an optic nerve crush injury, and are potentially
capable of being rescued by neuroprotective therapy, are the cell bodies of damaged bers and of intact bers that escaped the injury. To determine the total
number of surviving RGCs, the protocol of choice is of the labeling prior to the
injury. To assess the number of surviving RGCs with still-intact bers the protocol of choice is the post-injury labeling. These two protocols are done by employing the retrograde labeling procedures, as described in the following section.
A.
Labeling of All Retinal Ganglion Cells Prior

to Injury
The total number of RGCs in the retina is determined after stereotactic injection
of a uorescent dye to the superior colliculus of both hemispheres, where almost
all of the optic axons form synapses.
1. The Rat Model
The anaesthetized rat is placed in a stereotactic device, the skull is exposed and
kept dry and clean, and the bregma is identied and marked (see diagram below).
The designated point of injection is 6 mm rostral to the bregma and 1.2 mm lateral
to the midline. A window is drilled in the scalp above the designated coordinates
in both hemispheres. Using a Hamilton syringe, 2 L of the neurotracer dye FluoroGold [18] (5% solution in saline; Fluorochrome, Denver, CO), which meets all of
the criteria mentioned above, is injected into the superior colliculus 3.8 mm, 4 mm,
and 4.2 mm below the bony surface, at a rate of 1 L/min at each of the three depths.
The needle is then slowly withdrawn and the skin is sutured.
2. The Mouse Model
Anesthetized mice are placed in a stereotactic device, the skull is exposed and
kept dry and clean, and the bregma is identied and marked. The designated
point of injection is at a depth of 2 mm from the brain surface, 2.92 mm posterior
to the bregma and 0.5 mm lateral to the midline. A window is drilled in the scalp
above the designated coordinates in both hemispheres. With a Hamilton syringe,
17
Figure 3 The rat skull.
the neurotracer dye FluoroGold (5% solution in saline) is then applied (1 L, at

a rate of 0.5 L/min) and the skin over the wound is sutured.
B. Post-injury Labeling of Cell Bodies of Rescude
Fibers
Post-injury application of the uorescent lipophilic dye 4-(4-(didecylamino)styryl)-N-methylpyridinium iodide (4-Di-10-Asp) [19] (Molecular Probes, Europe
BV) distal to the site of optic nerve lesion results in the labeling of RGCs with
intact axons, as only axons whose continuity is preserved across the site of injury
are capable of transferring the dye to RGC bodies.
At different times after crush injury, the optic nerve is reexposed intraorbitally as described above. With the use of a 27-G syringe, a small hole is made
in the dura 1 mm from the distal border of the site of injury, and the axons are
cut to allow dye uptake. Solid crystals (0.20.4 mm diameter) of the dye are
deposited at the cut edge of the optic nerve. Five days after dye application, the
number of labeled RGCs is determined. The dye application procedure has no
effect on RGC survival during the period until retinal excision [5].
Figure 4 Application of a dye distally to the lesion site.
18
Schwartz and Yoles
Figure 5 Whole-mounted attened retina.
VI. COUNTING OF LABELED RGCs

At the end of the experimental period, the rats or mice are killed and their eyes
are excised into petri dishes containing phosphate-buffered saline (PBS). The
retina is detached from the eye without the vitreous body and xed in freshly
prepared 4% paraformaldehyde. Four cuts are made in the xed retina to allow
attening of the retina onto a nitrocellulose lter.
Labeled RGCs are counted using the uorescent microscope. It should be
noted that RGC density across the rat retina ranges from about 1000 cells/mm2
at the periphery to 6000 cells/mm2 in the center. However, over most of the
retina, except at the outer periphery, the average density is about 3000 cells/mm2
[5,15]. Nevertheless, after optic nerve injury the rate of RGC death is higher at
the periphery than at the center of the retina [5]. Accordingly, labeled RGCs are
counted in four to six elds at the same distance from the center of the retina,
at a magnication of 250. The numbers of labeled RGCs per eld are averaged,
and the mean number of RGCs per square millimeter is calculated.
VII. ELECTROPHYSIOLOGICAL MEASUREMENTS

The visual evoked potential (VEP) response to light indicates the integrity of an
animals visual system and can be used to assess the effects of injury and treatment on the systems functional integrity. Only those axons that escaped the
primary lesion and remain intact, with or without protection from secondary de-
19
generative processes, are capable of conducting action potentials. The pattern of

eld potentials in response to light stimulation is recorded from the primary visual
cortex. The potential evoked by the light originates in the retina and is propagated
along the surviving axons to reach the superior colliculus, their target in the brain.
Electrodes are implanted above the primary visual cortex as follows: The anesthetized rat is placed in a small stereotactic instrument and two holes are drilled in
the skull. Through each hole an electrode is implanted epidurally, with the dura
kept intact to minimize cortical damage. The electrodes are gold contact pins
(Wire-Pro, Salem, NJ) soldered to stainless steel screws, which are screwed into
the holes and cemented to the skull with acrylic cement. An electrode inserted
through a hole drilled in the nasal bone is used as a reference point. The second
hole is in area V1 (primary visual cortex), with coordinates bregma 8 mm and
lateral 3 mm.
Field potentials, before and after injury, are recorded from the visual cortex
in response to stroboscopic light stimulation (xenon ash tube 4 W/s, 12 ms
duration, 0.3 Hz). The signal evoked in the cortex is amplied 1000 times with
a microelectrode AC amplier, model 1800 (AM Systems) and digitized (12 bits,
5000 samples/s) with an MIO169 board and the LabView 2.2.1 data acquisition
and management system (National Instruments). Potentials should be presented
as the means of six recordings, three with and three without light, each involving
60 light ashes.
VIII. SOME PRACTICAL TIPS
1. When incising the conjunctiva, make sure that you do it as far as possible from the limbal area, which has abundant vasculature. An incision
at this site might cause massive bleeding, obscuring the area close to
the optic nerve.
2. While exposing the optic nerve, separate it as much as possible from
the adjacent fat and fascia.
3. While labeling the RGCs with a lipophilic uorescent dye such as 4Di-10-Asp, make sure that the dye is completely immersed in the hole
you have made in the dura sheath by injecting a drop of incomplete
Freunds adjuvant.
4. If the view of the retina under the microscope is too blurred to count
the cells, the problem might be caused by one or more of the following:
The vitreous body is still attached to the retina.
The paraformaldehyde solution in which the retina is soaked is not
fresh.
Dye application process went wrong.
20
Schwartz and Yoles
REFERENCES
1.
2.
3.
4.
5.
6.
7.
8.
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Schwartz M, Belkin M, Yoles E, Solomon A. Potential treatment modalities for

glaucomatous neuropathy: neuroprotection and neuroregeneration. J Glaucoma
1996; 5:427432.
Schwartz M, Yoles E, Levin LA. Axogenic and somagenic neurodegenerative
diseases: denitions and therapeutic implications. Mol Med Today 1999; 5:470
473.
Schwartz M, Yoles E. Optic nerve degeneration and potential neuroprotection: implications for glaucoma (abst). Eur J Ophthalmol 1999; 9 (suppl 1): S911.
Assia E, Rosner M, Belkin M, Solomon A, Schwartz M. Temporal parameters of
low energy laser irradiation for optimal delay of post-traumatic degeneration of rat
optic nerve. Brain Res 1989; 476:205212.
Yoles E, Schwartz M. Degeneration of spared axons following partial white matter
lesion: implications for optic nerve neuropathies. Exp Neurol 1998; 153:17.
Yoles E, Schwartz M. Elevation of intraocular glutamate levels in rats with partial
lesion of the optic nerve. Arch Ophthalmol 1998; 116:906910.
Schwartz M, Moalem G, Leibowitz-Amit R, Cohen IR. Innate and adaptive immune responses can be benecial for CNS repair. Trends Neurosci 1999; 22:295
299.
Schwartz M, Cohen IR. Autoimmunity can benet self-maintenance. Immunol Today 2000; 21:265268.
Yoles E, Belkin M, Schwartz M. HU-211, a nonpsychotropic cannabinoid, produces
short- and long-term neuroprotection after optic nerve axotomy. J Neurotrauma
1996; 13:4957.
Yoles E, Muller S, Schwartz M. NMDA-receptor antagonist protects neurons
from secondary degeneration after partial optic nerve crush [published erratum
appears in J Neurotrauma 1999 Apr;16(4):345]. J Neurotrauma 1997; 14:665
675.
Moalem G, Yoles E, Leibowitz-Amit R, Muller-Gilor S, Mor F, Cohen IR, Schwartz
M. Autoimmune T cells retard the loss of function in injured rat optic nerves. J
Neuroimmunol 2000; 106:189197.
Kipnis J, Yoles E, Porat Z, Cohen A, Mor F, Sela M, Cohen IR, Schwartz M.T
cell immunity to copolymer 1 confers neuroprotection on the damaged optic nerve:
possible therapy for optic neuropathies. Proc Natl Acad Sci USA 2000; 97:7446
7451.
Levkovitch-Verbin H, Harris-Cerruti C, Groner Y, Wheeler LA, Schwartz M, Yoles
E. RGC death in mice after optic nerve crush injury: oxidative stress and neuroprotection. Invest Ophthalmol Vis Sci 2000; 41:41694174.
Fisher J, Levkovitch-Verbin H, Schori H, Yoles E, Butovsky O, Kaye JF, Ben-Nun
A, Schwartz M.Vaccination for neuroprotection in the mouse optic nerve: implications for optic neuropathies. J Neurosci 2001; 21:136142.
Linden R, Perry VH. Massive retinotectal projection in rats. Brain Res 1983; 272:
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Dreher B, Sefton AJ, Ni SY. Nisbett G. The morphology, number, distribution and
central projections of Class I retinal ganglion cells in albino and hooded rats. Brain
Behav Evol 1985; 26:1048.
Martin PR. The projection of different retinal ganglion cell classes to the dorsal
lateral geniculate nucleus in the hooded rat. Exp Brain Res 1986; 62:7788.
Schmued LC, Fallon JH. Fluoro-Gold: a new uorescent retrograde axonal tracer
with numerous unique properties. Brain Res 1986; 377:147154.
Fritzsch B, Wilm C. Dextran amines in neuronal tracing. Trends Neurosci 1990;
13: 14.
3
Intraocular Pressure Elevation:
Vein Cauterization
Sansar C. Sharma
New York Medical College
Valhalla, New York, U.S.A.
I.
INTRODUCTION
Glaucoma is an optic neuropathy in which damage primarily occurs to the optic

nerve axons and the retinal ganglion cells. The death of neurons and damage to
the axons is related to the increase in intraocular pressure. The need to develop
a suitable animal model stems from the fact that experimentation on monkeys
(the ideal animal to study progression of a pathology) is expensive. In an ideal
situation, an animal model should provide results that are consistent and cost
effective. Our model is based on the concept that obstructing the outow of aqueous humor would mimic the disease process, thereby providing conditions suitable for studying the effect of various neuroprotective agents.
In the following sections we describe a method by which chronic elevation
of intraocular pressure is induced in the rat eye. The procedure is simple and
reproducible, with little or no inammatory response.
II. ANTERIOR DRAINAGE PATHWAYS

OF THE RAT EYE
One of the major pathways is via the anterior chamber angle and into the
Schlemms canal. The Schlemms canal is in communication with the venous
23
24
Sharma
plexus via the collector channels. The venous plexus drains into the episcleral
veins, which are radially oriented and are called radial veins. They run posteriorly
and merge with the large vessels near the extraocular muscle insertion. The large
vessels join posteriorly with the inferior and superior ophthalmic veins and enter
the cavernous sinus. Any interruption of the episcleral drainage will cause a rise
in IOP and, possibly, secondary glaucoma. It is therefore post-trabecular glaucoma. Because the outow is affected, this procedure may lead to open-angle
glaucoma [1,2]. If one increases the post-trabecular meshwork resistance by cauterizing the deep episcleral veins, one would expect an increase in IOP.
Described below are the essential steps in creating elevation of IOP in adult
rat eyes. This method is reliable and reproducible. The range of elevated IOP
pressure correlates with the number of cauterized veins. This model does not
involve introduction of exogenous material in the eye.
III. PROCEDURE
1. Adult Wistar rats (250300 g) are anesthetized with intraperitoneal
injection of 0.1 to 0.15 mL mixture of acepromazine maleate (1.2
mg/kg), xylazine (8 mg/kg), and ketamine (40 mg/kg).
Anesthetic injection can be given intramuscularly; however, it
takes between 15 and 20 min to induce deep anesthetic condition to
perform surgery. Certain hyperactive rats may require a second or
third injection of the anesthetic mixture administered in much smaller
doses than the initial dose.
2. To maintain the animals body temperature during the procedure, they
should be placed on a heated pad.
3. For each animal, the contralateral eye should serve as a comparative
control and should either have a sham operation or be left undisturbed.
Because of individual variations in IOP, pre- and postoperative IOP
should be compared between normal and experimental eyes for each
animal.
4. In order to keep the eye open, place the sutures on the eyelids.
5. Four to ve aqueous-containing radial veins emerge from the circumferential venous plexus in the rat eye. The supranasal vein should be
located rst. In order to stabilize the globe and expose the veins, make
an incision 23 mm long within the limbal periphery using sterile
microscissors. Then make two radial relaxing incisions at the edges
of the initial incision and recess the tissue posteriorly to expose the
underlying extraocular muscle.
6. Isolate the muscle (superior rectus) and anchor with a suture to expose
the underlying episcleral-radial vein. Special attention should be paid
Intraocular Pressure Elevation: Vein Cauterization
25
Figure 1 The photograph shows the location of the episcleral radial (arrow) merging caudally with the ciliary vein. Following the incision to conjunctiva in the rat
eye, superior rectus muscle is anchored by a suture (black) and pulled forward to
expose the circumferential veins.
to minimize the blood loss and damage to the conjunctiva and, especially, the sclera. Aqueous-containing radial veins lie slightly deeper
to the ocular muscles. The radial veins can be distinguished by their
darker color. The radial veins travel on the surface of the sclera (Fig.
1).
7. The radial veins merge with the ciliary veins at about one-third the
distance between the episcleral venous plexus and the optic nerve
head. Near the junction, isolate the radial vein with the least damage
to the sclera and put an open number-ve forcep under it to create a
bridge. Apply the cautery at the center of the isolated vein. The cautery tip should be about the same size as the outer diameter of the
vein. The purpose of this procedure is to minimize thermal damage
to the sclera during cauterization. Different material, such as wood
or metal, can be placed under the isolated vein to protect from heat.
As the vein is cauterized, a global mass appears on both ends of the
severed vein. At the proximal portion of the cauterized vein, there is
a distention of the radial vein, as the drainage is blocked. Even a
26
Sharma
8.
9.
10.
11.
12.
minor leak of the uid at the cauterized end will encourage neovascularization. Incidentally, this happens in 10 to 15% of the surgeries.
In each of these cases, we have observed formation of microvessels
within a week. Measure IOP 2 to 3 days after surgery and check the
sites of cauterization; if any are leaky, recauterize them. A successful
operation requires that vessels not be reconnected. Another method
is to isolate the vein, ligate it, and then cauterize.
In Wistar rats there are four to ve major venous trunks that run
posteriorly in each eye. Usually they are equidistant around the circumference of the globe. On the dorsal aspect of the globe there are
two major vessels equidistant from the superior oblique and the superior rectus muscles. Near the superior rectus muscle, the radial vein
is located closer to the temporal border. Similarly, there are two episcleral venous trunks located on the ventral aspect of the eye. One
trunk is located between inferior rectus and inferior oblique and the
other located at the inferior border of the lateral rectus muscle. Occasionally, some rats have a fth venous trunk that is located deeper
than the medial rectus. In order to elevate IOP to approximately double the value of normal IOP, it is advisable to cauterize a minimum
of two and a maximum of three vessels. If all venous trunks are cauterized, the eye becomes necrotic within a week.
Expose the second and third venous trunks by manipulating the anchored superior rectus muscle or superior oblique muscle. The isolation procedure is similar to that described above. After the venous
occlusion, ush the eyes with saline and apply antibiotic ophthalmic
ointment.
The veins can be cauterized at the junction of the episcleral vein and
ciliary vein (Fig. 2, incision 2) which leads to elevated IOP. This
procedure does not lead to any change in constriction and or dilation
of the pupil. No inammation was noticed in the anterior aspect of
the eye.
Some practitioners of this model have utilized neovascularizaton inhibitors such as 5-uorouracil applied subconjunctively for the rst
few days after venous occlusion. It is highly advisable not to measure
the IOP on the rst day after surgery, as chances of dislodging the
cauterized plug are very high. Monitoring IOP can begin within 2
3 days, when conjuctival incisions are healed.
Following the occlusion of three veins, the IOP is usually in the range
of 28 to 30 mmHg (normal average being 13.0 mmHg). Within 2 to
3 days following surgery, there is a small drop in the IOP. Subsequently, the elevated IOP remains high (22 to 25 mmHg) for the longest period of study (120 days).
27
Figure 2 Diagramatic representation of the rat eye showing circumferential venous plexus (ven. plex.) and the Schlemm canal (Schl. can.). Notice the anchoring
of superior rectus (sup. rec.) muscle. Collector channels (coll. cha.) connect the
Schlemm canal with the venous plexus, which subsequently exit via radial veins
on the scleral surface, and carry aqueous (rad. vein. aqu.). Interruptions 1 and 2
on the scleral vessel mark the point where cauterization leading to the elevation
of IOP can be made.
IV. METHODS FOR MEASURING IOP

For rats in the awake state, animals are held on a at surface gently with minimal
pressure applied to the shoulder. Press gently on the head. Apply topical anesthesia (a drop or two of proparacaine), and measure the IOP. Using Tonopen X-L
(Mentor Ophthalmics) applanation is made and a reading is noted. The Tonopen
probe usually requires three to four applications to the cornea before its processor
is activated. Routinely, four readings are obtained and averaged and the mean
values recorded. The IOP of each experimental eye is compared with the contralateral unoperated eye. For the sake of consistency and to exclude variations in
IOP due to diurnal cycle, measurements should be made at one set time. We
measure IOP between 9 and 10 a.m. Measurements are made twice a week in
28
Sharma
the rst 2 weeks following the surgery and once a week thereafter for the duration
of the experiments.
When using Mentor 1 pneumotonometer, we mildly anesthetize the rat,
with the same anesthetic used for the initial surgery. Three consecutive IOP measurements are recorded. Mentor 1 pneumotonometer provides consistent IOP
preferences, and experience of each investigator will play a greater role in choosing the method of IOP measurement. In anesthetized Wistar rats the mean IOP
of the normal eye is 12.5 mmHg, whereas in restrained but awake rats it is 13.5
mmHg. The range of normal IOP in Wistar rats is between 10 and 16 mmHg.
V.
ADVANTAGES AND DISADVANTAGES

1.
2.
3.
This procedure requires careful surgical manipulations and experience.

In initial trials, our success rate was 25%. After realizing the formation
of new vessels at the occluded end and being exceedingly careful in
all surgical procedures, we reached a success rate of 80 to 90%.
If more than one rat is transferred to a cage, this usually leads to some
aggressive behavior, which amplies the chance of reopening the
wound. It is preferable to keep rats isolated, one per cage.
In less than 5% of cases, corneal lesions or hemorrhaging of the globe
occurred. Affected animals should be excluded from any studies.
The elevated IOP can be maintained consistently in a large number of animals for a considerable period of time. Therefore, this procedure leads to the
development of an excellent experimental system for studying the mechanisms
of cell death of retinal ganglion cells and the effects of various neuroprotective
agents. Using this model system, we have studied the effects of neurotrophins
and gene transfer to the retinal ganglion cells in glaucomatous animals and animals with optic nerve transections [38]. Prelabeling of all retinal ganglion cells
exclusively in the retina was achieved by use of Fluorogold (Fluorochrome Inc.,
Englewood, CO) following protocols described in the above references.
VI. CONCLUSIONS
The procedure described above serves as a simplied means of inducing chronic
elevation of intraocular pressure and experimental glaucoma in rodents. This procedure clearly allows viewing of the optic disc [9] and does not induce a strong
inammatory response within the eye [10]. This procedure has furthered our understanding of the relationship between IOP and optic neuropathy on one hand
29
and the evaluation of various neuroprotective agents on the otherand will continue to do so.
REFERENCES
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
Minas TF, Podos SM. Familial glaucoma associated with elevated episcleral venous
pressure. Arch Ophthalmol 1968; 80:201208.
McGovern RA. Glaucoma associated with raised episcleral venous pressure. In:
Cairns JE, ed. Glaucoma. Vol. 2. Florida: Grune & Stratton. 1986:711728.
Chaudhary P, Ahmed FAK, Sharma SC. MK801A neuroprotectant in rat hypertensive eye. Brain Res 1998; 792:154158.
Chaudhary P, Ahmed FAK, Quebada P, Sharma SC. Caspase inhibitors block the
retinal ganglion cell death following optic nerve transection. Mol Brain Res 1999;
67:3645.
Garcia-Valenzuela E, Sharma SC. Rescue of retinal ganglion cells following axotomy induced apoptosis through TRK oncogene transfer. Neuroreport 1998; 9:
31653170.
Garcia-Valenzuela E, Shareef S, Walsh J, Sharma SC. Programmed cell death of
retinal ganglion cells during experimental glaucoma. Exp Eye Res 1995; 61:33
44.
Ko ML, Hu DN, Ritch R, Sharma SC. The combined effect of brain derived neurotrophic factor and a free radical scavanger in experimental glaucoma. Invest Ophthalmol Vis Sci 2000; 47:29672971.
Shareef SR, Garcia-Valenzuela E, Salierno A, Walsh J, Sharma SC. Chronic ocular
hypertension following episcleral venous occlusion in rats. Exp Eye Res 1995; 61:
379382.
Sawada A, Neufeld AR. Conrmation of the rat model of chronic moderately elevated intraocular pressure. Exp Eye Res 1999; 69:525531.
Mittag TW, Danias J, Pohorenec G, Yuan HY, Burakgazi E, Chalmers-Redman R,
Podos SM, Tatton WG. Retinal damage after 3 to 4 months of elevated intraocular
pressure in a rat glaucoma model. Invest Ophthalmol Vis Sci 2000; 41:34513459.
4
Injecting Hypertonic Saline
into Episcleral Veins
John C. Morrison, Elaine C. Johnson, Lijun Jia,
William O. Cepurna
Casey Eye Institute
Oregon Health & Science University
Portland, Oregon, U.S.A.
I.
INTRODUCTION
Recent studies have afrmed the effectiveness of aggressively treating intraocular

pressure (IOP) for stabilizing visual eld loss in patients with advanced glaucomatous optic nerve damage [1]. However, many patients will still suffer progressive visual eld loss despite what may appear to be adequate lowering of IOP.
It is for these patients that effective neuroprotection offers the best hope for preserved vision.
Some of these patients already have advanced glaucomatous optic nerve
damage. For others, it may not be possible to achieve maximal pressure lowering
because of medication side effects, surgical failure, or unacceptable risks of surgery. All of these considerations, as well as the observation that patients with
normal tension glaucoma can also benet from aggressive IOP control [2,3],
suggest that understanding the mechanisms of pressure-induced optic nerve damage may be important for developing effective neuroprotection. This would be
particularly true if some of these mechanisms are irreversible, rendering the remaining optic nerve bers vulnerable to otherwise normal IOP.
It is clear that modeling optic nerve damage by creating chronically elevated intraocular pressure (IOP) offers an important opportunity to develop meth31
32
Morrison et al.
ods to protect or preserve the optic nerve in glaucoma. The optic nerve head is
a highly complicated structure, consisting of axons surrounded by glial cells passing through a pressure gradient, with a unique vascular supply. Because of this,
such models must be created in the intact, functioning eye. Once such a model
is in place, it provides a reproducible system for testing the effectiveness of potential neuroprotective compounds.
II. OVERVIEW OF METHODS IN THE CONTEXT
OF NEUROPROTECTION
The primate model of trabecular meshwork sclerosis from argon laser treatment
has proven highly useful over the past 3 decades [46]. The advantages of this
approach are that the primate optic nerve anatomy is very similar to that of the
human, and the pathology of chronically elevated IOP in monkeys bears many
similarities to that of human glaucoma. However, the cost and difculty of working with this model precludes its use for studies assessing the cellular response
to IOP and other experimental manipulations, where individual variability necessitates the use of large numbers of animals. This is also true for studies designed
to test the effect of potential neuroprotective agents.
The development of chronic IOP elevation in laboratory rats provides an
alternative model, which has many advantages [79]. First, these animals are
easy to handle, allowing frequent measurement of IOP with the animal awake.
Second, their relatively low cost makes it possible to use them in studies requiring
large numbers of animals. Third, a large body of knowledge based primarily on
the rat already exists with regard to the cellular and molecular biology of central
nervous system and optic nerve damage. This provides opportunities for understanding the cellular mechanisms of pressure-induced optic nerve damage. Finally, the anatomy of the rat optic nerve head bears several features in common
with that of the human [10,11] and the pathology of elevated pressure in the
model described here has many similarities to that of human glaucoma [12].
A reliable model of pressure-induced optic nerve damage must have three
components: a method of creating elevated IOP; a reliable, unbiased method of
measuring IOP that will provide objective understanding of the pressure experienced by the eye and the optic nerve head; and a rapid, reproducible system for
assessing the resulting optic nerve damage. By using all of these features such a
model can be used to develop a reliable understanding of the mechanism of pressureinduced optic nerve damage and to assess potential neuroprotective therapies.
Three basic methods of creating chronically elevated IOP in rats have been
described. These include cauterization of large episcleral veins [13], laser photocoagulation of the angle vessels [14], and episcleral vein injection of hypertonic saline
[7]. The former two methods are described and discussed in other chapters of this
book. This chapter will describe our method of producing scarring of the aqueous
humor outow pathways using hypertonic saline injection of the episcleral veins.
Intraocular Pressure Elevation: Hypertonic Saline
33
In addition to the method itself, we will present our experience with measuring IOP
and determining the extent of optic nerve damage produced by this technique.
III. CREATING AQUEOUS OUTFLOW

OBSTRUCTION BY HYPERTONIC SALINE
INJECTION OF EPISCLERAL VEINS
A. Procedure Objectives
The major objective in creating a chronic model of elevated IOP is to selectively
obstruct aqueous outow. In the rat, this is complicated by the close proximity of
the aqueous outow pathways within the angle of the eye and the blood supply to
the ciliary body that comes from the major arterial circle of the iris. A too vigorous
destruction of the outow system can easily compromise the blood supply to the
ciliary body and produce unwanted hypotony. Our method produces selective injury
to this outow system, based on the anatomy of aqueous humor outow.
In the rat, the primary route of aqueous humor outow is through the trabecular meshwork and into Schlemms canal (Fig. 1). Following this, it escapes
through numerous aqueous collector channels and into the circular venous plexus
Figure 1 Schematic of the anatomy of aqueous humor outow in the rat eye.
Aqueous humor (dots) moves into Schlemms canal (SC), through trans-scleral
collector channels and into the limbal venous plexus. From here, ow occurs posteriorly within radial aqueous-containing veins (which also contain blood) (AV). The
arterial supply to the anterior segment is also illustrated, showing arterial supply
from anterior ciliary arteries (ACA) and long posterior ciliary arteries (LPCA). These
arterioles interconnect via a circular limbal artery. The iris (I) and ciliary process
(CP) arterial blood supply arise from the major arterial circle of the iris. (From Ref. 15.)
34
Morrison et al.
Table 1
Injection Equipment
Operating microscope (at least 163 with 103 oculars) with foot-drive
focus
Dumont #5 forceps (0.05 3 0.01 mm)
Curved Vannas scissors (Storz)
Curved Mosquito hemostatic forceps
Heavy tissue (iris) scissors
Weck cell sponges
Dumont #7, reverse action curved forceps (blunted tip)
Pump (settings to microliters per minute)
Plastic (delrin) ring
Hypertonic saline (0.22 Fm millipore lter)
Microneedle
that encircles the limbus. Numerous radial veins drain aqueous (and blood) away
from this plexus, posteriorly within the episclera [15].
We create isolated scarring of the trabecular meshwork by injecting a sclerosing agent (hypertonic saline) into the episcleral vein in retrograde fashion. By
applying a resilient plastic ring around the equator of the eye, with a gap straddling the vein to be injected, the other episcleral veins are temporarily blocked
off. The injection, via a microneedle into episcleral veins, is conned to the limbal
plexus, and the saline is forced into Schlemms canal and the trabecular meshwork. We have found that the needle insertion is best done by hand, as a micromanipulator is too cumbersome and not easily adaptable to the anatomical variations in limbal vasculature.
B.
Equipment
The necessary equipment for the entire procedure is listed in Table 1. Methods
of manufacturing the microneedle used for injecting hypertonic saline and a description of the plastic ring are presented in detail below.
C.
Microneedle Construction
The equipment needed to construct the microneedle is listed in Table 2. The steps
in constructing the microneedle consist of heating the polyethylene tubing over
a bunsen burner and stretching it to an even taper. This produces a segment
leading from normal diameter to a thinnest dimension that is approximately 15
to 20 cm long. A 56 mm section of glass microneedle (pulled out on a needle
puller) is then inserted into the tip of the tubing and a drop of the glue is applied

Table 2
35
Equipment for Manufacturing the Microneedle
P50 tubing
10 Fl borosilicate micropipettes
Bunsen burner
Epoxy glue
Needle puller (to pull micropipette to approx 50 m outer diameter and 30 m
inner diameter
23-G needle (tip broken off)
1 cc syringe
Abrasive wheel (Dremel tool)
to the junction. This microneedle must have little or no taper, so that the needle
will provide an effective seal of the vessel wall where it is inserted. A tapered
needle, as seen with a standard micropipette, will leak if the needle is not held
perfectly still within the vessel, and it is allowed to slide back and forth. A small
amount of the glue will naturally migrate between the tubing and the needle, so
an equal length segment of glass must be inside the tubing to prevent the glue
from covering and plugging up the internal opening of the glass needle. The large
end of the tubing is then swedged onto the 23-G needle shaft (with the tip broken
off) and secured with a drop of glue (Fig. 2). Once this is entirely dry (24 hs), the
needle is beveled using the ne abrasive wheel on a dremel tool. Water applied to
the stone provides good lubrication and produces a smooth, sharp bevel. When
a needle is no longer usable, it can be cut off the end of the tubing and a new
one glued in its place. This allows the tubing, which can be difcult to pull to
a taper, to be reused several times.
D. Plastic Ring
The plastic ring is essential for isolating the injection to the limbus. It compresses
all of the veins draining aqueous away from the limbus, except the one being
injected, and connes the saline to the limbal vessels and Schlemms canal. This
ring is manufactured from Delrin stock using a mini lathe. Dimensions and
appearance of the current ring are shown in Figure 3. There is a 1 mm wide
groove machined in the inner aspect of the ring. This gives the ring a U shape
when cut in cross section and improves its stability when applied to the equator
of the eye. A small gap is cut in this ring, allowing the ring to straddle the vein
to be injected. We have found that this gap is most easily cut with a razor blade,
with the sides beveled away from each other. This allows better access to the
vessel with the microinstruments, both for dissection and for the needle placement.
36
Morrison et al.
Figure 2 Microneedle and syringe construction (above), with detail of needle attachment to tubing (below). (From Ref. 7.)
E.
Steps of Episcleral Vein Injection

1.
2.
3.
A lateral canthotomy is made to improve access to the eye following

a 5 s compression with a hemostat. This heals quickly and does not
require suturing.
The best position for the ring is at the equator of the eye, and it should
be placed under the dissecting or surgical microscope. The ring is
spread, with a ne mosquito forceps, just large enough to slip over the
equator, then slid off the forceps with a nger. This should immediately
blanch out the limbal vessels (except possibly the artery) for a few
minutes, due to aqueous being forced out of the anterior chamber. The
plexus and episcleral veins will rell with blood once IOP falls enough
to allow blood to rell the limbal plexus, either from lling of the long
posterior ciliary artery, the anterior ciliary artery, or both. At this point,
any possible routes of saline escape from the limbus should be identied and the ring shifted to occlude them, still leaving the injection
vein unobstructed.
Generally, the most accessible and largest veins are located superiorly.
(a)
37
(b)
Figure 3 Delrin ring used to isolate saline injection to the limbal vasculature and
aqueous outow pathways. (a) Appearance of the ring, both whole and when cut
in cross section; (b) overall dimensions of the ring, as well as the gap cut in the
ring, with beveled sides to improve access to the vessel.
Thus, the animal is best positioned on his chest, with the superior aspect of the globe pointed up. Excellent illumination is essential, particularly when working with the higher magnications. An assistant, who
is operating and timing the injection pump, can rotate the eye down
to improve access to the vein, approximately at the equator.
4. The vessel wall is rst exposed through a conjunctival incision. This
is performed using the Dumont forceps to put traction on the conjunctiva or Tenons just over the vessel. Complete removal of connective
tissue improves the chances of inserting the needle into the vessel lumen, without creating a false passage between the outside of the vessel
wall and perivascular adventitia.
5. Once the vessel is exposed, the surgeon grasps the needle, bevel up,
with curved, reverse action forceps. The best place to grasp the needle
is at the bead of glue that forms at the junction of the needle and the
tubing. The glue protects the tubing and the glass from being crushed
by the forceps. It is also helpful to place a groove in the inner edges
38
Morrison et al.
6.
7.
8.
9.
of the forceps blades. This provides a more positive grasp of the needle
and keeps the needle from pivoting, since the sides of the glue joint
are usually curved.
Needle insertion is performed by grasping the vein with Dumont forceps posterior to the intended insertion site. This stabilizes the vein
and increases its size by temporarily obstructing the venous blood as
it ows away from the limbal plexus. It is best to choose a straight
section of the vein to decrease the chance that the needle will be passed
out of the vein accidentally.
The technique for needle insertion involves placing the tip of the
needle (which is held nearly parallel to the vein) against the vein wall.
By advancing the needle toward the limbus slightly, the tip will engage
the vessel wall. This is usually aided by slightly loosening tension on
the vein, thus allowing it to gain its maximum size. Once engaged, the
needle is passed forward into the lumen of the vein. Generally, this
requires increasing tension on the vein slightly, so that it will not move.
The needle can then be advanced into the vessel lumen, approximately
2 to 3 mm. Often, this is accompanied by a slight reux of blood into
the needle. Tipping the needle up slightly after entering the vessel lumen will also decrease the chance of accidentally passing the tip out
of the opposite wall of the vein.
The injection is begun once the needle is safely in the vessel. Accidental extravasation usually prevents successful cannulation and injection,
although it can still be attempted. A failed injection can sometimes be
remedied by nding and injecting an alternative vessel, or going to the
fellow eye. Usually, the eye has to be abandoned, but injection can be
reattempted in 1 to 2 weeks.
The injection is timed by noting when the pressure in the needle
builds enough to ll the limbal vessels. This is the point at which timing
starts. At the end of the injection time, the needle is removed and the
vessel clamped with the Dumont forceps for several seconds to prevent
reux of the saline.
Typically, good signs of a successful injection are immediate blanching
of the limbal vasculature and deepening of the anterior chamber. A
temporary lens opacity usually develops due to osmotic effects of the
hyertonic saline in the anterior chamber. All of these are consistent
with the saline being forced into Schlemms canal and across the trabecular meshwork. Following the injection, the limbal vessels will rell slowly, usually beginning with the limbal artery.
Antibiotic ointment is instilled without suturing and the animal allowed
to recover from the anesthesia. We do not typically use post-operative
steroids or repeated dosing with antibiotics.
39
The typical postoperative course involves a mild amount of inammation,

usually manifested by slight corneal haze for a day or two. This almost always
is self-limited. Following the injection, the IOP may initially show a brief elevation, but generally is normal or low for a few days. This is then followed by a
gradual increase. Most eyes will demonstrate a rise in IOP by 10 days to 2 weeks
after the injection. This is most likely due to gradual scarring of the trabecular
meshwork and angle, producing a variable degree of angle closure. Once aqueous
humor formation returns to normal, the closed angle is not able to accommodate
the increased ow, resulting in elevated IOP.
We have found that, when performed on animals housed in a 12-h light:
dark cycle, the scarring of the aqueous ouow pathways produces elevated pressures that can vary markedly with the circadian cycle [16]. This can range from
the low 20s to the mid 40s from the light to the dark phase of the cycle, representing a doubling of the normal circadian rhythm [17]. We have also found that
placing the animals in a constant, low level light environment will increase the
mean IOP to approximately 27 mm Hg and minimize the extent of circadian
uctuation. In this situation, the typical range of mean IOP following the procedure varies from the low 30s to low 40s, as determined by the Tonopen in awake
animals (see below).
The success rate of producing an elevated pressure with this method is quite
high. Review of a recent group of 40 eyes shows that pressures were signicantly
elevated in 33 eyes following a single injection. A second injection is always
possible in eyes that fail to respond, and it can increase the success rate even
further [7].
F.
Factors That Inuence the Success of a Given

Injection in Producing Elevated IOP
Three main factors inuence the success of saline injection. These factors appear
to affect each other, and experience, correlated with success in producing elevated
IOP, is required to nd the proper combination of saline concentration, injection
force, and duration of injection.
1. Molarity of the saline solution. We have found that a 1.752.5 M solution produces a reliable increase in IOP. Concentrations higher than this
will result in excessive inammation and, often, long-lasting hypotony.
2. Force of injection. This inuences the ability of the saline to gain access to the trabecular meshwork. Too low a force will fail to perfuse
the angle structures sufciently. An excessive force may produce too
much inammation, with a result similar to that of using too high a
concentration of the saline. This may be due to subtle interconnections
that exist between the episcleral vasculature and emissary veins that
40
Morrison et al.
3.
G.
drain venous blood from the ciliary body to the episclera. A high injection force may also cause perfusion of the ciliary processes with the
saline. We have found that using a pump for this injection, aiming for
a force sufcient to inject at a rate of 50 L over 30 s, helps achieve
more reproducible injections.
Duration of injection. We generally try to inject for 15 to 30 s. Longer
times will produce excessive inammation.
Reasons for Failure of Injection to Produce

Elevated IOP
These generally correspond to inadequate delivery of the saline into the outow
system. They often correlate with a lack of anterior chamber deepening and lens
opacity, and with poor blanching of the limbal vasculature. The most common
cause of this is if the ring fails to adequately occlude all vessels leading away
from the limbus. This may be due to improper placement of the ring, or anatomic
peculiarities of vessels that make it impossible to adequately occlude all of these
vessels. Occasionally there are large, deep veins that appear to drain directly from
the ciliary body, emptying into the limbal plexus. We generally avoid these if it
is not possible to occlude the vessel with the ring, as these vessels are large
enough to allow much of the saline to drain away from the limbal plexus. Accidental injection of these vessels may also produce inadvertent injection of the
ciliary body vasculature, which could produce ciliary body shutdown and hypotony.
IV. IOP MEASUREMENT

IOP measurement must be done in an unbiased fashion, using a method that is
objective and calibrated to actual IOP [18,19]. We use the Tonopen XL tonometer
for measuring IOP. This is done using topical anesthesia, taking 10 consecutive
readings and then using the average as the measured IOP. Acceptable readings
are those that register immediately upon contact of the tonometer tip with the
cornea, using rm but not excessive force. The eye itself should be moved posterior slightly with each applanation of the tonometer.
The operator must learn to recognize valid readings and objectively ignore
all those that do not meet these criteria. A very light touch with the corneal tear
lm may produce a low single digit reading that is not accurate. In addition,
readings that occur when the tip breaks contact with the cornea (off readings)
and the instrument generated averages are also not accurate. The key challenge
with this tonometry is that the high curvature of the rat cornea requires that the
41
tonometer be held exactly perpendicular to the corneal surface. This positioning

is acquired with practice.
Measuring IOP in the rat eye requires considerable experience and this skill
is best acquired by rst calibrating the instrument on a cannulated rat eye connected to a low pressure transducer and an extra syringe to adjust the IOP, described below. With this setup, the individual learns, through immediate feedback, how to judge an acceptable reading. With time and practice, the ability to
measure IOP in an unbiased fashion improves, and this can be seen by a steadily
diminishing standard deviation in the measurements obtained.
Finally, we have found that general anesthetics can produce a rapid and
substantial decrease in measured IOP [20]. Because of this, we now measure all
IOPs with animals in the awake state, using only topical anesthesia. This allows
the best understanding of the pressure to which the eye and optic nerve are actually exposed. This technique also requires considerable practice. However,
Brown Norway rats are very docile and rapidly become accustomed to these
measurements. Once this technique is mastered, measuring awake IOP is actually
much faster, and obviously more accurate, than under any general anesthetic.
Periodic refresher calibration sessions are useful, even after the practitioner
becomes skillful with this technique. These help the practitioner maintain the
ability to sense acceptable readings and help uncover any possible systematic
errors that may develop. Such sessions should also be done whenever the Tonopen is serviced or a new one is purchased.
V.
TONOMETER CALIBRATION
The tonometer is best calibrated in the living rat eye. The objective is to manipulate the IOP in known amounts while allowing simultaneous measurements with
the tonometer. To do this, the eye of an anesthetized rat is cannulated and connected to a manifold equipped with 3-way stopcocks. The manifold is also connected to a low pressure transducer and to a Hamilton syringe for altering IOP.
A third port is connected to a 60 cc syringe to allow periodic relling of the
system, as needed, while another is connected to a sphygmomanometer for calibrating the transducer and chart recorder. By keeping the system between the
eye, the transducer and the chart recorder open at the same time, actual IOP can
be rapidly manipulated by one operator, who also records tonometer readings
made by another directly on the chart strip as they are made. With this arrangement, IOP measurements can be obtained with the tonopen and compared immediately to actual IOP, obtained through the transducer. This provides immediate
feedback, helping the Tonopen operator improve technique and learn to recognize
valid IOP readings in an unbiased fashion.
42
Morrison et al.
The most difcult part of this technique is in cannulating the anterior chamber without damaging the lens or iris. We rst make a beveled incision into the
peripheral cornea from the temporal side using a sharp 23-G needle, so that the
beveled tip enters the anterior chamber but the needle does not go in all the way.
A blunted 23-G needle attached to the tubing that leads to the manifold is then
inserted into the eye, bevel down rst. It is helpful to have an assistant gently
inject through the needle to keep the anterior chamber inated. Once the needle
is in the eye, the bevel is rotated anterior to keep the tip from being blocked by
iris. By making the initial incision slightly small, the seal of the blunted needle
will be tight and will not leak. This can be maintained for a reasonably long time,
and the pressure range from 10 to 50 can be sampled several times, although it
is possible that higher pressures may cause these thin corneas to be even more
thin, thus altering the ne calibration of the instrument.
The correlations that result from these experiments demonstrate a linear
relationship between actual IOP and the measured, Tonopen IOP (Fig. 4). As
Figure 4 Example of an actual Tonopen calibration curve. Tonometer readings

at each IOP level are plotted against the actual IOP, as measured with a transducer
connected to the anterior chamber. Regression of this relationship demonstrates
a good t to a straight line. This indicates that Tonopen readings can be reliably
used to determine actual IOP.
43
with all tonometers in all eyes, we have found that the Tonopen readings are less
than actual pressure at the higher end of this range.
VI. ASSESSING NERVE DAMAGE AND ITS

RELATIONSHIP TO IOP LEVEL
Optic nerve damage can be assessed using many techniques. We use a qualitative
grading scale of damage determined by histological examination of a crosssection of the myelinated portion of the optic nerve. This is rapid, reproducible,
easily taught to others, and correlates well with actual numbers of axons lost, as
determined by careful ultrastructure analyses. This grading system is described
in Table 3.
When using this system to assess injury, four or ve masked graders record
their assessment of the grade, and the average grade is calculated as the score
for that nerve. When using this system to compare the nerve injury grade to IOP
level between two groups of animals, we determine the best t line for one group
(e.g., the control group) to generate a formula to predict each data point for both
groups. The difference between observed and expected values for each data point
are then calculated. The mean difference values for each group can then be compared by t-test to determine the signicance of the differences. Alternatively, data
can be linearized before statistical analysis. Sample size analyses using data like
this indicate that group sizes of 20 will provide a test with sufcient power to
detect a 10% difference in the number of optic nerve axons between groups.
Table 3
Injury
Grade
1
2
3
4
Grading System for Assessing Extent of Optic Nerve Cross-Section

Description
Normal optic nerve morphology with, at most, only a few randomly scattered
degenerating axons
Densely staining, degenerating axons appear focally, with a few axonal swellings
Numerous degenerating axons and axonal swellings appear to spread away
from the focal area. Central damage tends to exceed that in the periphery
Numerous degenerating and axonal swellings appear throughout the nerve, interspersed with apparently normal axons. Numbers of degenerating and normal axons appear to be approximately equivalent
Degenerating axons and axonal swellings make up nearly the entire mass of
the nerve, with scattered, apparently normal axons. Gliosis may appear in severe cases
44
Morrison et al.
VII. ADVANTAGES AND DISADVANTAGES

The major advantages of the method discussed here is the low cost of purchasing
and maintaining laboratory rats. This allows relatively large numbers of animals
to be used for studies, both of injury mechanism as well as for testing neuroprotection effectiveness of new agents. In this manner, it is possible to minimize
difculties imposed by individual variation in response to elevated IOP that exists
among animals. The equipment needed for the injections and for making the
microneedle is standard and readily available.
A second advantage of this method is that it clearly relies on obstructing
aqueous outow, which is also the primary mechanism by which elevated IOP
occurs in glaucoma. This is supported by our studies of the aqueous outow
system of the rat eye, histologic documentation of angle closure in these specimens, and the increased uctuation of IOP that we see in these animals [17]. In
the latter respect, this model clearly mimics human glaucoma, in which increased
IOP uctuation is a characteristic nding in many patients.
A third advantage of this model is that these animals are easy to work with
and allow us to measure IOP while they are awake. This provides a very accurate
assessment of the IOP experienced by the eye throughout a given experiment.
This presents a distinct advantage over obtaining IOP from animals that are under
the inuence of general anesthetics, commonly employed for studies of primate
glaucoma models.
Fourth, experience to date strongly suggests that the optic nerve damage
experienced by these animals results solely from the pressure elevation. This is
supported by our observation of no optic nerve damage in eyes that received an
injection but failed to develop elevated IOP. In addition, we have found a very
strong correlation between the extent of IOP elevation and optic nerve damage.
This validates the accuracy of our pressure measurements, as well as our method
of determining optic nerve damage. In addition, we have found that specic cellular responses, such as message production for Thy-1 [21] and neurolament protein are closely linked to the extent of pressure elevation.
Finally, this model provides a unique opportunity to understand the sequence of
cellular events that follows elevation of IOP. The entire knowledge of neuropathology
and cell biology of CNS injury developed in other rat CNS models is now available
to the vision researcher to understand the cell biology of pressure-induced optic nerve
damage. This provides a powerful opportunity to develop directed, rational strategies
of neuroprotection for patients with extensive glaucomatous optic nerve damage who
have progressive loss of visual function despite maximally controlled IOP.
As stated above, the primary difculty with creating elevated IOP in these
small eyes lies in the challenge of scarring the aqueous outow pathways without
damaging the immediately adjacent structures that are necessary for maintaining
aqueous humor formation. Damage to the latter will often produce hypotony,
rather than ocular hypertension.
45
A potential disadvantage, common to all methods of modeling glaucoma

in rats, lies in the difculty of obtaining accurate measurements of IOP. Due to
uctuations of IOP, a characteristic of all eyes with aqueous outow obstruction,
frequent daily measurements are essential. Measuring IOP in awake rats is an
important skill that requires considerable experience. However, once this skill is
acquired, we have found that measuring IOP in awake animals is faster and less
traumatic for the animals than using general anesthetics.
Disadvantages of our method in particular primarily lie in the surgical skill
needed to successfully cannulate these small vessels for injection and the time
and patience needed to learn to measure IOP in rats. However, with practice and
careful attention to detail, most researchers should be able to acquire this skill.
Several laboratories have already successfully reproduced this model. However,
this success requires a strong commitment, and nearly all of the individuals learning this technique have included in a personal visit, often more than once, to our
laboratory to gain rst-hand knowledge of these skills.
VIII. CONCLUSIONS
Our method of producing chronic elevation of IOP by episcleral vein injection
of hypertonic saline is both reproducible and reliable. It combines the advantages
of using laboratory rats with a simple, elegant method of scarring the aqueous
humor outow pathways, while minimizing the effects on other anterior segment
structures. Our experience with this model demonstrates that the resulting pressure elevation has many similarities to that of human glaucoma, as does the pathology of the optic nerve damage. With careful assessment of IOP and the extent
of optic nerve damage, it is possible to understand the relationship between pressure and optic nerve damage. This understanding will allow us to use this model
to test the effectiveness of potential neuroprotective agents and improve our understanding of the mechanism of pressure-induced optic nerve damage.
REFERENCES
1.
2.
3.
The AGIS Investigators: The advanced glaucoma intervention study (AGIS): 7. The
relationship between control of intraocular pressure and visual eld deterioration.
Am J Ophthalmol 2000; 130:429440.
The Collaborative Normal-Tension Glaucoma Study Group. The effectiveness of
intraocular pressure reduction in the treatment of normal-tension glaucoma. Am J
Ophthalmol 1998; 126:498505.
The Collaborative Normal-Tension Study Group. Comparison of glaucomatous progression between untreated patients with normal-tension glaucoma and patients with
therapeutically reduced intraocular pressures. Am J Ophthalmol 1999; 126:487
497.
46
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5.
6.
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8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
Morrison et al.
Gaasterland D, Kupfer C. Experimental glaucoma in the rhesus monkey. Investigative Ophthalmol 1974; 13:455457.
Quigley HA, Addicks EM. Chronic experimental glaucoma in primates: I: Production of elevated intraocular pressure by anterior chamber injection of autologous
ghost red blood cells. Invest Ophthalmol Vis Sci 1980;19:126136.
Quigley HA, Addicks EM. Chronic experimental glaucoma in primates: II: Effect
of extended intraocular pressure on optic nerve head and axonal transport. Invest
Ophthalmol Vis Sci 1980; 19:137152.
Morrison JC, Moore CG, Deppmeier LMH, Gold BG, Meshul CK, Johnson EC. A
rat model of chronic pressure-induced optic nerve damage. Exp Eye Res 1997; 63:
8596.
Morrison JC, Johnson EC, Cepurna W. Animal models in glaucoma research. Ophthalmic Practice 1998; 16:1220.
Morrison JC, Cepurna WO, Johnson EC. Modeling glaucomatous optic nerve damage. Ophthalmol Clin North Am 1999; 39:2941.
Morrison JC, Farrell SK, Johnson EC, Deppmeier LMH, Moore CG, Grossmann
E. Structure and composition of the rodent lamina cribrosa. Exp Eye Res 1995; 60:
127135.
Morrison JC, Johnson EC, Funk R. The microvasculature of the rat optic nerve
head. Invest Ophthalmol Vis Sci 1999; 40:17021709.
Johnson EC, Morrison JC, Farrell SK, Deppmeier LMH, Moore CG, McGinty MR.
The effect of chronically elevated intraocular pressure on the rat optic nerve head
extracellular matrix. Exp Eye Res 1996; 62:663674.
Shareef SR, Garcia-Valenzuela E, Salierno A, Sharma S. Chronic ocular hypertension following episcleral venous occlusion in rats. Exp Eye Res 1995; 61:379382.
Schori H, Kipnis J, Yoles E, WoldeMussie E, Ruiz G, Wheeler LA, Schwartz M.
Vaccination for protection of retinal ganglion cells against death from glutamate
cytotoxicity and ocular hypertension: implications for glaucoma. Proc Natl Acad
Sci USA 2001; 98:33983403.
Morrison JC, Fraunfelder FW, Milne ST, Moore CG. Limbal microvasculature of
the rat eye. Invest Ophthalmol Vis Sci 1995; 36:751756.
Jia L, Cepurna WO, Johnson EC, Morrison JC. Patterns of intraocular pressure
elevation after aqueous humor outow obstruction in rats. Invest Ophthalmol Vis
Sci 2000; 41:13801385.
Moore CG, Johnson EC, Morrison JC. Circadian rhythm of intraocular pressure in
the rat. Curr Eye Res 1996; 15:185-191.
Moore CG, Milne S, Morrison JC. Non-invasive measurement of rat IOP with the
TonoPen. Invest Ophthalmol Vis Sci 1993; 34:363369.
Moore CG, Epley D, Milne ST, Morrison JC. Chronic non-invasive measurement
of intraocular pressure in the rat eye. Curr Eye Res 1995; 14:711717.
Jia L, Cepurna WO, Johnson EC, Morrison JC. Effect of general anesthetics on
IOP in rats with experimental aqueous outow obstruction. Invest Ophthalmol Vis
Sci 2000; 41:34153419.
Schlamp CL, Johnson EC, Li Y, Morrison JC, Nickells RW. Changes in Thy1 gene
expression associated with damaged retinal ganglion cells. Mol Vis 2001; 7:192201.
5
Laser Photocoagulation
of the Trabecular Meshwork
BAnn T. Gabelt, James N. Ver Hoeve, and Paul L. Kaufman
University of WisconsinMadison
I.
INTRODUCTION
Glaucomas occur widely throughout the animal kingdom. However, primary

glaucomas usually involve a genetic predisposition and tend to occur most commonly in domesticated animal species (for review, see Ref. 1). Development of
animal models with different forms of spontaneous glaucoma is time consuming
and expensive. Experimental glaucoma was induced in animals as early as 1905
[2]. More current experimental animal models of glaucoma have been reviewed
by Gelatt [3] and are only briey summarized below. However, there are limited
numbers of in vivo animal models of experimental glaucoma that are useful for
evaluating the pathophysiology and potential therapy of human glaucomatous
optic neuropathy. The eye and visual system of the macaque monkey more
closely resemble that of the human, and the monkey model of ocular hypertension
with its resulting optic neuropathy is generally acknowledged to best reect the
optic neurodegeneration associated with human glaucoma. This primate model
will be reviewed in detail, including the application of current clinical and basic
science technologies.
Experimental glaucoma in nonhuman primates has permitted study of aqueous humor dynamics, glaucomatous changes in the visual pathways from the
photoreceptors to the visual cortex, and anterior and posterior ocular segment
pharmacologic effects. Nonhuman primate species used include cynomolgus
monkeys (Macaca fascicularis), owl monkeys (Aotus trivirgatus), rhesus mon47
48
Gabelt et al.
keys (Macaca mulatta), and squirrel monkeys (Saimiri sciurea), but the most
work has been done in cynomolgus and rhesus.
II. MONKEY MODELS

A.
Trabecular Laser
1. Historical
Most current studies are done with laser-induced glaucoma model in the rhesus
and cynomolgus monkey, which was rst described by Gaasterland and Kupfer
in 1974 [4]. Barany, studying large numbers of vervet monkeys in Uganda, had
noted elevated intraocular pressure (IOP) and glaucomatous cupping in an occasional monkey following apparent ocular trauma and postulated its cause as
trauma-induced damage and malfunction of the trabecular meshwork (TM) (personal communication). Gaasterland and Kupfer reasoned correctly that controlled
trauma and scarication conned to the TM could be induced by the application
of high energy laser burns delivered by standard clinical methods, and could
elevate IOP similar to human traumatic glaucoma, but without inducing other
anterior segment abnormalities that could themselves impair vision and preclude
evaluation of the posterior segment.
2. Method
Laser photocoagulation of the TM is conducted in the anesthetized monkey. Typically, ketamine (10 mg/kg, I.M.) or ketamine plus diazepam (1 mg/kg, I.M.) or
acepromazine (0.21 mg/kg, I.M.) are sufcient to minimize eye movements
during the procedure. If additional short duration sedation is necessary, methohexital sodium (10 mg/kg, I.M.) can be added and will usually last about 1 h.
Monkeys are placed prone on a wooden, plastic, or metal board with a post on
which a custom-fabricated head holder is mounted. The head holder has a metal
bar that ts in the monkeys mouth, can be adjusted to hold the head securely,
and can be further adjusted to position the eye for lasering. Alternatively, an
assistant can simply hold the head in the proper position. Topical anesthetic is
administered and a custom-fabricated [5] mirrored Goniolens (Ocular Instruments) lled with hydroxypropyl methylcellulose (Gonak, Akorn, Buffalo
Grove, IL), is placed on the eye. The custom lenses have a smaller diameter lens
to easily t through the small palpebral ssure. Also, the curvature of the lens
and angle of the mirrors are different due to the smaller eye. In some larger
animals, a standard adult or pediatric Goldmann-type lens can be used or a canthotomy can be performed to facilitate lens placement. However, in smaller monkeys, the procedure is greatly aided by the smaller and properly constructed
monkey-specic lenses.
Intraocular Pressure Elevation: Laser Photocoagulation

Table 1
49
Laser Parameters for Producing Experimental Glaucoma in Monkeys
Laser Type
Argon
(e.g., Coherent, model 900)
Red Diode
(e.g., Iris Medical Oculight SLX)
Green Diode
(e.g., Iris Medical Oculight GL)
Power
(mW)
Duration
(ms)
Spot size
(mm)
Number
of burns
1500
5001000
50
50250
1250
5001000
75
50250
1000
5001000
75
50250
A standard clinical argon laser (e.g., Coherent, model 900), or portable red
diode (Iris Medical Oculight SLX) or green diode (Iris Medical Oculight GL)
may be used. Typical settings are as follows: argon1500 mW power, 500
1000 ms duration, 50 m spot size, 50250 burns; red diode1250 mW, 500
1000 ms, 75m, 50250 burns [6]; green diode1000 mW, 5001000 ms,
75 m, 50250 burns (Table 1). Contiguous burns are placed in the mid-TM
over 180270 of the circumference of the TM in each session. Care should be
taken to avoid burn spread posteriorly over the ciliary muscle, as this will increase
the post-treatment inammatory reaction and prolong the post-treatment hypotony (see below). Scarication of the anterior ciliary muscle may obstruct uveoscleral outow and result in greater IOP elevation. These factors are very variable
and hard to control, and placing the center of the burn at the junction of the
pigmented and nonpigmented portion of the TM seems like the best compromise.
At least one quadrant is left untreated to avoid very high IOP rises. Immediately
after laser treatment, moderate iridocyclitis occurs with resultant ocular hypotony,
which usually resolves within 34 weeks. IOP will then usually return to normal,
or rise above normal if the session was effective (Fig. 1). However, additional
treatment sessions are usually necessary, and the number of sessions required
and their effectiveness can vary greatly between monkeys. The intensity and location of subsequent sessions can nonetheless be titrated to the target IOP. Unless
one is striving for a very high IOP, it is generally advisable never to treat the
entire 360 circumference with contiguous burns at any one sitting. We always
leave at least 90 untreated at each sitting, although the untreated quadrant may
have been treated at a prior session. In our experience, it is usually necessary to
treat the entire circumference at least once, however the sessions are split. If no
sustained pressure rise is achieved after the rst session and resolution of the posttreatment inammation, we will again treat 270, encompassing the previously
untreated quadrant. Third and subsequent sessions are titrated according to the
response and the desired target IOP. A nal IOP of within 10 mmHg of target
can usually be obtained by varying the treatment strategy. Only rarely does one
50
Gabelt et al.
Figure 1 IOP and its control following argon laser treatment of the TM in one
eye (experimental glaucoma) of rhesus monkey 1035. This monkey was unusual
in that only one laser session was required to elevate the IOP; usually two or three
sessions are required. Topical Timoptic-XE 0.5% was given once daily or every
other day where indicated to maintain IOP at the desired level. Lowering IOP pharmacologically (with Timoptic, Alphagan, Trusopt and PGF2-1-isopropylester) for
1 week prior to sacrice resulted in no change in the surprisingly modest disc cupping (experimental glaucoma C/D0.4; Control C/D0.2), thus suggesting that
there was actual loss of tissue, rather than simply a pressure-induced mechanical
backward bowing of the elastic lamina cribrosa. The axonal loss for experimental
glaucoma was mild (0.28). Reproduced with permission. (From Ref. 10.)
encounter an animal in which the IOP never rises with multiple treatments. Typically, 23 treatment sessions are required to achieve a sustained IOP rise. Some
animals may need to be re-treated after a sustained period of IOP elevation that
is then followed by a gradual decrease in IOP.
If IOP exceeds the desired level or if the monkey displays any signs of
discomfort (usually when IOP exceeds 60 mmHg by Goldmann applanation tonometry), standard antiglaucoma medications can be administered once or twice
a day. Monkeys can be trained to enter an iron maiden (a modied squeeze
cage) that has been adapted to immobilize the conscious monkey while tilting it
to a vertical position so that the eyelid may be retracted and the medications
dropped onto the cornea. Alternatively, for uncooperative monkeys, 510 mg/kg
ketamine I.M. can be given for sedation and the medications then administered.
51
However, some monkeys may show a decrease in appetite with frequent anesthesia so this must be assessed on a case-by-case basis. The medications that have
been used successfully alone or in combination include Timoptic-XE (0.5% timolol maleate in gel-forming vehicle, Merck & Co, West Point, PA); Alphagan
(0.2% brimonidine tartrate, Allergan, Irvine, CA); Trusopt (2% dorzolamide
hydrochloride, Merck); and PGF2-1-isopropylester (2 g in 5 l saline, Caymen
Chemical Co, Ann Arbor, MI) or Xalatan (0.005% latanoprost, Pharmacia Corp,
Peapack, NJ). If necessary, acetazolamide (5 mg/kg, Ben Venue Laboratories,
Bedford, OH) I.M. has been given once or twice daily.
3. Clinical
IOP under ketamine anesthesia is monitored weekly or more frequently if medications are being implemented to target a specic pressure range. IOP is measured
with a minied Goldmann (Haag-Streit, Koniz, Switzerland) applanation tonometer [7]. Occasionally, these are backed up by measurements with a Tonopen XL
(Mentor O&O, Norwell, MA) if corneal edema or neovascularization, or head
and eye movements under ketamine anesthesia preclude readings with the Goldmann [8]. Others have also used pneumotonometry for IOP determinations [9].
In some cases, IOP can uctuate greatly. Slit lamp biomicroscopy of the anterior
and posterior segments, including stereoscopic optic disc evaluation with a fundus lens are performed once a month when IOP has stabilized. The size, shape,
and pallor of the optic disc, the cup-to-disc ratio, and the retinal nerve ber layer
are evaluated. The pupil in the lasered eye usually becomes dilated, probably
due to damaging the parasympathetic motor nerves to the iris by the laser treatments (energy spread to the anterior ciliary muscle through which the nerves
travel to reach the iris). Iridolenticular adhesions may develop independently of
corneal changes or duration of IOP elevation. Anterior and posterior synechiae
are often observed. However, IOP may be elevated even if most or all of the
angle remains open and there is no papillary block. The TM is invariably heavily
pigmented. As the duration of IOP elevation becomes longer, some animals may
develop corneal edema followed, in some cases, by neovascularization of the
cornea.
Cupping of the optic nerve head, with posterior bowing of the lamina cribrosa is typical [4]. Optic disc cupping occurs more rapidly in the monkey than
in the human. For a given monkey, this is dependent on the IOP elevation and
duration. However, results can uctuate between monkeys. In order to accurately
assess cupping due to loss of neural/glial tissue versus posterior bowing of the
elastic monkey lamina, the IOP should be lowered. This can be done with the
combinations of pharmacological agents mentioned above to control IOP. Alternatively, I.V. mannitol (1.5 g/kg) can be administered over a 30 min period. If
52
Gabelt et al.
IOP is still too high, give I.V. acetazolamide (5 mg/kg) and wait 30 min more.
If IOP is still greater than 20 mmHg, give I.V. methohexital (5 mg/kg) or pentobarbital (10 mg/kg). If IOP after mannitol is still greater than 40 mmHg, give
both acetazolamide and methohexital or pentobarbital. Fundus stereo photography (Topcon TRC 50IA fundus camera (Topcon America Corporation, Paramus,
NJ) can be used to document the time course of the changes. An example of
glaucomatous damage is shown (Fig. 2) after 4 months of elevated pressure where
the entire optic disc surface is excavated; the disc margin is undermined; there is
substantial peripapillary atrophy; and the retinal nerve ber layer is substantially
attenuated [10]. Other instrumentation used to image the optic nerve in humans
produce results that are more difcult to interpret in the monkeys due to a lack of
corrections for the smaller eye, steeper corneal curvature, uncompensated corneal
birefringence, and poor ocular xation. Confocal scanning laser ophthalmoscopy
(TopSS Topographic Scanning System, Laser Diagnostic Technologies, Inc., San
Diego, CA) [11,12] and Heidelberg retinal tomography (HRT; Heidelberg Engineering, Heidelberg, Germany) [13,14] can be used for optic disc and peripapillary retinal contour analysis. Scanning laser polarimeter (GDx, Laser Diagnostic
Technologies, Inc., San Diego, CA) to assess retinal nerve ber layer thickness
may be used for generalized qualitative evaluation of differences between the
eyes of a given monkey. Adaptations of the HRT and GDx are being made for
more accurate and quantitative use in the monkey and even for rodents [15] (R.
Weinreb, personal communication). Optical coherence tomography (OCT) is under study for evaluation of retinal nerve ber layer in glaucoma in humans [16
18] and may be applicable to monkeys. For all these photographic/imaging procedures, pupils are dilated with 2.5% phenylephrine HCl (Mydfrin, Alcon, Ft.
Worth, TX) and 1% tropicamide (Mydriacyl, Alcon). Anesthesia for these procedures is ketamine (10 mg/kg, I.M.) acepromazine (0.21 mg/kg I.M.), methohexital sodium (15 mg/kg, I.M.) if needed to eliminate eye movements.
4. Perimetry
Behavioral perimetry in monkeys shows the same intersubject variability in the
effects of elevated IOP on visual eld sensitivities that are common with hightension glaucoma or ocular hypertension patients [19]. Perimetry regimens with
either white or monochromatic stimuli are not useful predictors of ganglion cell
loss until a substantial proportion of cells have died. The variance in ganglion
cell loss is large for mild defects, which would be diagnostic of early glaucoma,
and for visual eld locations near the fovea, where sensitivity losses occur relatively late in the disease process [20]. Monkeys with laser-induced glaucoma
exhibit the same type of Humphrey visual eld defects as do glaucomatous humans.
53
Figure 2 (A) Fundus photographs of experimental glaucoma and control (B) eyes
of a rhesus monkey at 4.5 months after unilateral laser-induced IOP elevation (experimental glaucoma 40 mmHg; control 20 mmHg). The entire experimental glaucoma disc surface is excavated, the disc margins are undermined (white
arrowhead), there is substantial peripapillary atrophy (white arrow), and the retinal
nerve ber layer (asterisks in control) is substantially attenuated (white star). (C)
Fundus photographs of ONT and of control (D) eyes of a rhesus monkey, 3.5 wk
after transection, 1 week prior to sacrice. Note normal retinal vasculature, absence of retinal or vitreous hemorrhage, and presence of pallor but absence of disc
cupping and very early, mild attenuation of nerve ber layer (absence of striations
emanating from the temporal disc margin) as a result of ONT. (Reproduced with
permission from Ref. 10.)
54
Gabelt et al.
Spectral sensitivity defects occur in experimental glaucoma similar to those

found in patients with glaucoma. Elevated IOP resulted in short wavelength sensitivity losses. The optimum condition identifying the greatest short wavelength
sensitivity reduction is a yellow background of moderate intensity. In the early
stages of experimental glaucoma, the cone mechanisms and the rod mechanism
typically showed decreased test and eld sensitivities. In advanced stages of experimental glaucoma, the largest sensitivity losses are in the longer wavelength,
red-green opponent mechanisms [21].
5. Electrophysiology
Objective, noninvasive electrophysiological measures of retinal ganglion cell
(RGC) function may be of value for monitoring glaucomatous damage in humans
and for providing comparable functional measures in experimental animal
models.
Ganzfeld ERG. The a- and b-waves of the full-eld ERG reect a massed
electrical response from the entire retina that is dominated by photoreceptor and
bipolar activity with little apparent contribution from the ganglion cell layer [22].
Most ERG studies of human patients with glaucoma found no correlation between
visual loss and scotopic or photopic full-eld ERG [2326] perhaps due to variability in severity, disease progression, and treatment regimens. This is of less
concern in laser-induced experimental glaucoma in nonhuman primates. In agreement with human investigations, several studies of experimental glaucoma have
found no effect on the scotopic a- and b-waves of the traditional full-eld ERG
[2731].
However, some recent investigations in human glaucoma patients do nd
alterations of certain features of the a- and b-waves [3236] suggesting that layers
of the retina distal to the ganglion cells may be involved in glaucoma. Support
for outer retinal changes are recent studies showing swelling of cell bodies in
the outer nuclear and outer plexiform layers in both human and experimental
glaucoma [37].
Oscillatory Potentials. The dark-adapted full-eld ERG elicited by a
bright ash can be ltered to reveal time-locked high frequency wavelets riding
on the a- and b-waves known as oscillatory potentials. Oscillatory potentials are
thought to be generated within the inner plexiform layer [38]. Several studies
have found that oscillatory potentials are reduced in human glaucoma [34,39,40].
Investigations of oscillatory potentials in nonhuman primate models of glaucoma
are mixed [28,41].
Photopic Negative Response. Another feature of the full-eld ERG that
appears to be affected in glaucoma is a negative wave that follows the b-wave
recorded under photopic conditions. In some studies the photopic negative re-
55
sponse was found to be greatly reduced in nonhuman primate experimental glaucoma [31,42], and in human patients [26,43,44] whereas other investigators found
no consistent changes in patients with advanced glaucoma [45].
Scotopic Threshold Response. The scotopic threshold response is the
dark-adapted ERG to a very dim ash of light that rst appears as a cornea
negative wave and peaks approximately 200 ms after stimulation [46]. On the
basis of pharmacologic manipulations, the scotopic threshold response is thought
to reect proximal, or inner retinal activity [46]. However, the scotopic threshold
response is not abolished in humans with long-standing optic atrophy or in cats
with optic nerve transection (ONT) [47], suggesting that a substantial part of the
scotopic threshold response may be due to amacrine cell activity. The scotopic
threshold response in human glaucoma patients is highly variable and generally
unchanged in humans with glaucomatous eld loss [48]. In contrast, the scotopic
threshold response is greatly altered or abolished in nonhuman primate experimental glaucoma [28]. Frishman [28] suggests the scotopic threshold response
may receive contributions from both rod amacrine (a short-latency component)
and ganglion cells (a long-latency component). There appears to be marked species differences in the relative contribution of the two cell types.
Pattern ERG. The pattern pattern ERG is a small amplitude ERG response to pattern reversal (e.g., exchange of the black and white checks of a
chessboard pattern) or onset of a pattern in which there is no global change in
luminance. It is not a full-eld stimulus; rather the pattern ERG is the summed
response from a large area of the central eld. Many studies have shown that the
pattern ERG reects ganglion cell activation [22,49,50].
The pattern ERG is reduced in human diseases of the optic nerve [51],
including glaucoma [5263]. Changes in the pattern ERG also have been reported
in ocular hypertension [57,58,6470]; see Korth [71] for a review of the pattern
ERG in glaucoma. The pattern ERG has also been examined in animal models
of glaucoma. Reduction of pattern ERG amplitude is correlated with the degree
of disc cupping in monkeys with chronic ocular hypertension [27,41]. The pattern
ERG is not used extensively in the diagnosis and monitoring of disease progression, perhaps due to the technical difculties with recording small amplitude
signals and the nding that substantial peripheral loss must occur before the pattern ERG is affected [71].
In addition, some of the inconsistencies among these studies have been
attributed to differences in the relative insensitivity of the full-eld ERG to ganglion cell loss and the inability of the mass ERG response to reect localized or
patchy loss of function that is characteristic in glaucoma. Several novel electrophysiological techniques have been introduced that appear promising for detecting and monitoring glaucoma because they are thought to reect inner retinal
activity.
56
Gabelt et al.
Multifocal ERG (mERG). The mERG technique derives the local electrical response of many small patches of the retina (typically 103) using a sparse
binary m-sequence cross correlation technique introduced by Sutter [72,73]. The
technique has attracted considerable attention in the study of glaucoma for two
reasons. First, it can image the patchy, localized areas of retinal dysfunction that
characterize the glaucomatous visual eld loss. Second, the binary m-sequence
method permits a single recording to be analyzed in rst-order (linear) and higherorder components, or kernels. Higher-order kernels reect complex nonlinear
dynamic responses that are presumed to originate in layers of the retina proximal
to photoreceptors, possibly including ganglion cells.
Chan [74] found reductions in the amplitudes of both the rst- and secondorder kernels in humans with glaucoma. Delays in mERG waveforms also have
been found in primary open angle glaucoma [7577]. Amplitude differences were
not evident in these studies.
The relationship between the mERG and glaucomatous eld losses as measured by standard automated perimetry has not been established [7880].
The mERG has also been examined in experimental glaucoma. In normal
eyes of rhesus monkeys, the rst-order mERG response kernel contains more
prominent oscillatory potentials ( 60 Hz) than in comparable recordings in humans [81,82]. The rhesus macaque mERG oscillatory potentials are larger in
central than in peripheral locations. The second-order kernel also contains larger
oscillatory potentials than seen in human recordings. In addition, there are prominent naso-temporal variations in the oscillatory potentials of both kernels; responses near the optic nerve head have larger amplitude oscillatory potentials
than temporal locations [81,82]. One approach to reversibly simulating glaucoma
in nonhuman primates is to pharmacologically suppress sodium-based spiking
activity of the inner retinal with tetrodotoxin and NMDA. Four effects on the
mERG have been noted following intravitreal administration of tetrodotoxin and
NMDA: (1) a marked increase in the amplitude of the mERG rst and second
order responses; (2) retinotopic changes, with the largest increased amplitudes
occurring in the foveal region; and (3) a reduction of the prominent oscillatory
potentials; and (4) removal of the naso-temporal variations. These ndings differ
from human glaucoma studies that found no effect or reduced amplitude foveal
responses.
In nonhuman primates with advanced experimental glaucoma, Frishman
found the effects were similar to those produced by suppressing inner retinal
activity with tetrodotoxin and NMDA [82]. In addition, the later portion of the
rst order kernel waveform was altered, lacking a dip after the large positive
wave, similar to the changes seen in the photopic negative response. mERG
changes increased over the time course of glaucoma and were more diffusely
distributed across the visual eld [82]. Another study of nonhuman primates with
57
IOPs elevated for over 16 months, and histologically documented ganglion cell
loss, found the amplitude of both rst and second order mERG responses were
attenuated and were highly correlated with RGC density (Fig. 3) [29,30]. A similar effect was found in nonhuman primates in the early stages of experimental
glaucoma [83]. mERG after 4 weeks of elevated IOP (25 mmHg) showed an
attenuation of the negative waveform complex at 4070 ms following the prominent positive (P1) wave. A similar alteration of these waves following ONT suggests this waveform feature of the mERG, to some extent, reects ganglion cell
activity [83].
Visual Evoked Potential (VEP). Amplitude reductions and delays in peak
latency of the VEP elicited by patterned stimuli have been reported in human
glaucoma [52,60,8494]. In addition, distortion of waveforms, rendering them
unscoreable using traditional metrics, were found in a high proportion of glaucoma patients [52,89]. The correlation with standard automated perimetry defects
has been attempted but has yielded poor results [71,84]. Pattern VEP has been
considered less sensitive than standard automated perimetry for the early detection of glaucoma.
Recently, a multifocal VEP (mVEP) method has been introduced that appears to correlate well with eld loss [80,95]. When all factors are taken into
account, the mVEP appears to capture standard automated perimetry eld losses
with exceptional accuracy. Its role in early detection remains to be established.
The mVEP has not been reported in studies of experimental glaucoma.
6. Aqueous Humor Dynamics
Outow facility can be measured by constant pressure perfusion [96], Schiotz
tonography [97,98], pneumotonography [99] and by a uorophotometric technique [9,100]. In vivo constant pressure outow facility measurement decreases
from prelaser baseline of 0.33 L/min/mmHg0.75 L/min/mmHg, to 0.02 L/
min/mmHg0.11 L/min/mmHg post-laser [4]. Toris also showed tonographic
outow facility was decreased by 71% at 3675 days and uorophotometric outow facility was decreased by 63% at least 1.7 years later [9]. Outow facility
by constant pressure perfusion in a group of 16 cynomolgus monkeys at 15
months post laser, when IOP averaged 32.9 3.5 mmHg in lasered versus
17.0 1.2 mmHg in control eyes, was 0.049 0.012 L/min/mmHg versus
0.424 0.038 L/min/mmHg, respectively (Kiland J, Kaufman PL, unpublished
data). Uveoscleral outow measured with tracers or calculated was increased at
least one year after laser treatment [9]. This could be partially due to the persistent
low-grade inammation that may be associated with chronic endogenous prostaglandin release. Also, articial openings (small cyclodialysis clefts) between the
scleral spur and the ciliary body may have resulted from the laser burns, which
58
Gabelt et al.
(1)
Figure 3 Part 1. First order mERG responses from both eyes of a monkey with
severe ocular hypertension. (A) Normotensive (OS) eye. Trace array of 61 responses, each of which represents the local retinal response corresponding to the
stimulus element at that location in the stimulus eld. The central seven traces
(shaded) represent responses from macular retina extending to approximately 8
retinal eccentricity. The surrounding 54 traces (unshaded) represent responses
from perimacular retina extending from approximately 8 to 25 retinal eccentricity.
Calibration bars 200 nV, 100 ms. Note that amplitude of individual traces is expressed in units of volts because each trace represents the response from retinal
areas of the same size. (B) Macular and perimacular responses obtained by summation of either the central seven or surrounding 54 response traces, respectively,
from (A). Amplitude measures for the ve peaks of the macular response were
made as indicated. Calibration bars, 5 nV/degree squared (deg2) and 25 ms. Note
that macular and perimacular response amplitude is expressed as response density (volts/unit retinal area), because these responses reect retinal stimulus areas
of different size. (C) Sixty-one response array obtained from the hypertensive (OD)
eye of the same animal whose responses are shown in (A) and (B). Calibration
as for (A). (D) Macular (top trace) and perimacular (bottom trace) responses obtained from (C); calibration as for (B).
Part 2. Second-order mERG responses from the same recordings that produced the rst-order responses shown in Part 1. (A) As for Part 1A. (B) As for Part
1B. A single peak-to-peak measure of second-order macular response amplitude
was made as indicated. Calibration bars, 2.5 nV/deg2 and 25 ms. (C) As for Part
1C. (D) As for Part 1D.
59
(2)
(3)
Part 3. Comparison of macular and perimacular responses from the hypertensive (OD, light traces) and normotensive (OS, heavy traces) eyes shown in Parts 1
and 2. Histological analysis showed that normalized (OD/OS) perifoveal RGC density
in the hypertensive eye was 0.11. First order response calibration bars, 5 nV/deg2
and 25 ms; second-order calibration bars, 2.5 nV/deg2 and 25 ms. (From Ref. 30.)
60
Gabelt et al.
could increase uveoscleral outow [101]. Cyclodialysis clefts can also increase
uveoscleral facility in monkeys [101]. The increased uveoscleral outow in experimental glaucoma was not sufcient to prevent the IOP rise [9].
Apparent aqueous humor ow in lasered eyes measured by uorophotometry signicantly decreased by 46% at 3675 days after laser at a time when IOP
was increased by 17.0 9.3 mmHg [9]. If real, the aqueous ow decrease may
have been caused by inammation and damage to the ciliary processes [102].
Aqueous ow returned to normal when measured at least one year after laser
treatment [9].
Accumulated protein in and around the ciliary body would likely not hinder
egress of aqueous humor. If anything, it would slightly increase osmotic pressure,
which would draw more water, although this effect would likely be small. A
breakdown of the blood-aqueous barrier should also not affect aqueous humor
production [103].
However, both increased protein concentration and bloodaqueous barrier
breakdown could affect uorophotometric measurement of ow. If uorescein
binds to albumin and is quenched, the uorophotometer would not see as much
as is actually present. Also, the increased scatter from macromolecules could
increase the uorescence signal (by multiple scatter of excitation light and uorescence into the measurement window, and by some scattered excitation light
passing the barrier lter, which is not 100% efcient) leading to an overestimate
of uorescein concentration. It is difcult to predict how both of these processes
would affect the uorophotometric estimate of ow rate. The ow estimate is
proportional to the change in mass of uorescein in the cornea and the anterior
chamber divided by the mean concentration in the anterior chamber on an interval. If uorescein concentration in the anterior chamber is over- or underestimated, ow rate would be under- or overestimated respectively. Thus, quenching
could increase the estimate of ow, whereas scatter might decrease it.
Breakdown of the bloodaqueous barrier would also allow more uorescein to diffuse directly into the blood. One assumes that this loss normally accounts for 8% or less of uorescein loss [104]. Diffusional loss in an eye with
a compromised bloodaqueous barrier has not been measured, but it would likely
be higher. This increased loss would lead to an overestimate of ow rate rather
than an underestimate and lower ow, as Toris and Pederson observed.
One might also expect that aqueous humor ow would decrease during
inammation. A diminished ow would increase concentration of macromolecules that respond to the insult and help repair the damage, and would slow the
release of any toxins into the venous blood. Reduced ow would give the TM
more time to break down and remove debris and invading substances. The increased protein concentration in the aqueous humor may reect this lower ow
rate as well as breakdown of the bloodaqueous barrier, although it would be
difcult to measure either independently.
61
Flow rate could be measured in an inamed eye by using a large radioactive

tracer, large enough not to cross the corneal endothelium or to leave the anterior
chamber by diffusional pathways. Any uorescence methods to measure ow
would likely be difcult because of the increased scatter.
7. Blood Flow
Blood ow in the retina, optic nerve head, and retrobulbar optic nerve measured
with tritiated iodoantipyrine did not differ between control and glaucomatous
eyes [105].
8. Vitreous Sampling/Injections/Glutamate
Visualization of the area of the vitreous for injections and for sampling can be
accomplished by placing the monkey on its back, dilating the pupil, and placing
a well made of tubing on the cornea and lling it with gonioscopic gel. Under
microscopic visualization, a 23-G needle attached to a tuberculin syringe can be
inserted and 0.10.2 mL of vitreous withdrawn from the desired location without
producing hemorrhage. The pressure in the eye can be restored by then injecting
viscoelastic material into the vitreous. Drugs of interest can be delivered to the
vitreous with a similar set up but using a smaller (30-G) needle and injecting,
usually, up to 50 L of material.
Vitreous glutamate was shown to be elevated in experimental glaucoma of
1832 weeks duration to concentrations potentially toxic to RGCs [106]. Anterior
and posterior vitreous levels were, respectively, 59.7 7.3 and 80.3 7.8 mol/
L in experimental glaucoma and 12.3 1.5 and 12.3 2.3 mol/L in control
eyes. However, in a group of 20 cynomolgus and rhesus monkeys with laserinduced glaucoma from 351.7 weeks, glutamate levels in the posterior vitreous
sampled from near the posterior pole were no different between the experimental
glaucoma and contraleral normal control eyes (experimental glaucoma
32.9 6.8 M; control 36.0 7.6 M) (Kaufman, unpublished data).
9. Neuroprotection
Memantine, a noncompetitive NMDA receptor antagonist (NMDA-type glutamatergic open-channel blocker), was administered orally to monkeys with experimental glaucoma for 15 months. Signicantly less loss of visually evoked cortical
potential amplitude was demonstrated in memantine-treated compared with untreated animals with experimental glaucoma [107].
Ganglion cell survival was enhanced in regions of the retina where the
photoreceptors had been focally destroyed by retinal laser photocoagulation prior
to the induction of experimental glaucoma in the monkey [108]. These ndings
demonstrate that RGCs can be protected from pressure-induced damage in the
62
Gabelt et al.
monkey and that the outer retina may be involved in the pathophysiology of
glaucomatous optic neuropathy. The monkey is a good model for such studies.
Morphological changes in the photoreceptors have been observed in experimental
glaucoma in the monkey [37]. These changes are remarkably similar to what has
been found in chronic human glaucoma [37]. In both cases, there is selective,
ischemic-like swelling of the L/M-cones (red- and green-sensitive cones), perhaps due to decreased choroidal blood ow [109,110]. (The L/M-cones are numerically the dominant cone population since the S- or blue-sensitive cones make
up only about 9% of all cones.) At the molecular level, preliminary studies have
shown decreased production of mRNA that encodes for the L/M-cone opsins in
both human glaucoma and experimental glaucoma in monkeys [111]. Rhodopsin
mRNA levels, by contrast, remain relatively unaffected. Interestingly, the L/Mcones that have the greatest reduction in mRNA in monkeys may be those in the
arcuate region. Independent evidence for outer retinal injury in human as well
as experimental glaucoma has now been obtained using the mERG [75,112].
Delays in an early feature (N1) of the mERG seem to be most prominent in the
arcuate region, as well.
10. IOP-Lowering Strategies
The experimental glaucoma monkey has been used to investigate the efcacy
and mechanism of clinical and potential glaucoma therapeutic agents. Timolol
(0.5%), epinephrine (2%), pilocarpine (4%), vanadate (1%), PGF2 (500 g),
and forskolin (1%) were all found to signicantly decrease IOP after single or
multiple treatments [113,114]. Other prostaglandin derivatives have been tested
extensively in this model [115,116], including some of the newest antiglaucoma
therapies (e.g., latanoprost) [116,117]. The combined effects of brimonidine, dorzolamide, or latanoprost with timolol were all found to decrease IOP more than
timolol alone [118].
Trabeculectomy surgery to construct a guarded stula between the anterior
chamber of the eye and the subconjunctival space is often complicated by the
ability of the surrounding tissue to scar over the stula, thus blocking the route
of exit for the aqueous and reducing the ability of the treatment to lower IOP.
Strategies to prevent wound healing and the scarring process can be studied in
experimental glaucoma in monkeys. Typically, mitomycin C has been used in
humans for this purpose, but recently studies in monkeys using a recombinant
adenovirus carrying the coding region for human p21 has shown this to be just as
effective without the other adverse effects often encountered with mitomycin C.
11. Histological Findings
Light and electron microscopic examinations of the ltration angle treated by
laser several months previously indicate blunting of the trabecular beams and
63
Figure 4 (A) Anterior chamber angle after laser photocoagulation on two occasions. IOP, 40 mmHg. Noteworthy are scarring of TM and adjacent tissue (arrow),
obliteration of canal of Schlemm, and pigment dispersion with melanin phagocytosis within iris root and TM. (B) Anterior chamber angle of control monkey eye.
Normal trabecular area (small arrow) separates anterior chamber from canal of
Schlemm (large arrow). (From Ref. 4.)
scattered peripheral anterior synechiae [119]. Scarring of the TM and obliteration of the canal of Schlemm is observed in treated eyes (Fig. 4) [4]. (Kaufman,
Carassa, Albert, Tamm, unpublished data).
In the retina, there is a selective loss of ganglion cells and thinning of the
nerve ber layer (Fig. 5) [4]. The large RGCs located in the midperiphery and
fovea appear to be preferentially damaged in laser-induced experimental glaucoma [120,121].
In the optic nerve, optic nerve bers larger than the mean axon diameter
atrophy more rapidly in the glaucomatous eye [122]. RGCs in glaucomatous eyes
undergo a pattern of degeneration that, morphologically, originates with the dendritic arbor of the cell. Both midget and parasol cells show signs of atrophy,
although parasol cells appeared to be slightly more sensitive to the degenerative
effects of prolonged IOP elevation [123]. Parafoveal cone loss was not found in
experimental glaucoma in which there was extensive damage to RGCs [124].
Small RGCs that project to the parvocellular layers of the lateral geniculate
nucleus belong to the P pathway or the color system. Large RGCs project to the
magnocellular layers and belong to the M pathway or the luminance system.
Rapid-phase axonal transport, measured by radioactive labeling, to the dorsal
lateral geniculate body in monkeys with chronic experimental glaucoma is de-
64
Gabelt et al.
Figure 5 (A) Macular retina and optic nerve head of treated monkey eye from
Figure 4A. There is selective loss of ganglion cells with thinning of the nerve ber
layer (small arrow) and cupping of the nervehead with posterior bowing of the lamina cribrosa (large arrow). Splitting of the thin retina at the juncture with optic nerve
is an artifact. (B) Macular retina and optic nervehead of control monkey eye. A thick
layer of ganglion cells is present next to the fovea (small arrow). There is no cupping
of the nervehead; the lamina cribrosa goes straight across the anterior optic nerve
(large arrow). (A and B at same low-power magnication.) (From Ref. 4.)
(1)
Figure 6 Part 1. Primate retinogeniculate pathway showing the regions of the
LGN examined and the approximate locations of their retinal inputs. Layers 1 and
2 are the M-layers, and layers 3 through 6 are the p-layers. Ganglion cells in nasal
retina project to the contralateral LGN, whereas those in the temporal retina project
ipsilaterally. The nasal region of the LGN receives its afferent input from ganglion
cells in the superior retina (B, C), and the temporal region of the LGN is innervated
by ganglion cells located in inferior retina (A, D). (From Ref. 126.)
65
(2)
Figure 6 (cont.) Part 2. Photomicrographs of cresyl violet-stained coronal sections from the right LGN of a normal rhesus monkey (A) and animals that had the
pressure in one eye elevated for 2.5 (B), 8 (C), and 24 (D) weeks. In all cases,
the nasal region of the nucleus is to the left, and the temporal region is to the right.
Layers 1, 4, and 6 receive retinal input from the normal contralateral eye, whereas
layers 2, 3, and 5 are innervated by the glaucomatous ipsilateral eye. Prolonged
elevation of IOP resulted in a decrease in the size and Nissl substance within neurons receiving input from the glaucomatous eye, resulting in their pale appearance.
Scale bar, 500 m. (From Ref. 126.)
creased more in the magnocellular layers than in the parvocellular layers [125].
Unilateral elevation of IOP severely affects the size, density, and number of neurons in those LGN layers receiving input from the affected eye. One study of
increased IOP for 2.527 weeks shows a greater degenerative effect on magnocellular than parvocellular regions [126]; while another study showed equal loss
of neurons in the magnocellular and parvocellular layers with experimentally
increased IOP for 14 months (Fig. 6) [127]. Signicant loss of CaMKII-a immunoreactivity (indicating blue-on neurons) between and within the principal layers
of the LGN occurs in all ocular hypertensive monkeys with or without signicant
66
Gabelt et al.
(3)
Figure 6 (cont.) Part 3. Low-power microphotographs of coronal sections of the
left LGN from control (left) and glaucomatous (14 months) (right) cynomolgus monkeys, immunostained for parvalbumin. All six layers in the control are strongly immunoreactive for parvalbumin as indicated by numbers. There is overall shrinkage
of the LGN and a decrease in immunoreactivity in parvocellular layers 4 and 6 in
the glaucomatous LGN compared with control. The bar indicates 0.5 mm. (From
Ref. 127.)
optic nerve ber loss [128]. Changes in the distribution of certain neurochemicals
occurs in the visual cortex of glaucomatous primates, suggesting cortical plasticity in recovery from glaucomatous visual damage [129]. Cytochrome oxidase
reactivity in the neurons in the LGN was reduced to the same degree in both the
P- and M-cellular layers with increasing severity of experimental glaucoma [130].
In eyes with IOP increased for 1460 months, ganglion cells and nitrergic
nerve bers in the choroid are signicantly reduced. Whole mount preparations
of the retina stained for NADPH diaphorase revealed a signicant reduction in
positively stained amacrine cells, reduction in diameter of arterioles and changes
in the staining pattern of the retinal vasculature, particularly in the perimacular
region [131]. Vascular and glial changes of the retrolaminar optic nerve was
studied in monkeys with elevated pressures for 1 to 4 years. Independently of
axon loss, the number of capillaries remained constant or decreased only slightly.
Some vessels, especially in the most severely damaged regions, were occluded.
67
The density of glial cells increased. In glaucomatous optic nerves, the density
of alphaB-crystallin and glial brillary acidic protein-positive cells signicantly
increased [132].
Optic nerve head changes have been evaluated in the laser-induced chronic
glaucoma monkey model by several methods. Optic disc cupping occurs symmetrically in early stages of experimental glaucoma but occurs predominantly in the
vertical axis in later stages. Spontaneous reduction of IOP led to a reversal of
cupping, which was signicantly less in later stages of glaucoma [133]. Cupping
in the monkey may have both a compliance component and a true tissue loss
component, analogous to the situation in human infants with congenital glaucoma
[134]. Lowering the IOP and re-examination may be the only way to tell. When
IOP is acutely lowered, there is signicantly less anterior movement of optic
nerve heads with larger deeper cups than those with smaller cups [135].
Glaucomatous nerve heads showed increased labeling for collagen type IV
along the margins of beams in the lamina cribrosa, due to accumulation of basement membranelike materials. Material in the pores of the laminar beams labeled with antibodies to collagen types I, II and IV. These changes were not seen
in optic nerve transected eyes [136]. In glaucomatous nerve heads, there is a
major disruption of the lamina cribrosa beam structure, including a decrease in
collagen density [137,138]. The destruction of collagen beams accompanies an
accumulation and enlargement of collagen-associated proteoglycan laments.
Accumulation of chondroitin sulfate proteoglycans was most evident. Prominent
lamentous heparan sulfate/heparin proteoglycans were also noted in thickened
astrocytic and vascular basal laminae [139].
Elastin is an important component of the ECM of the lamina cribrosa that
provides resiliency and deformability to the tissue. The elastic properties of the
lamina cribrosa are important for buffering the constant uctuations in IOP [140].
Abnormal elastin synthesis by astrocytes of the lamina cribrosa in experimental
glaucoma optic neuropathy was shown to be specic to elevated IOP and not
secondary to axonal loss, as demonstrated by comparison with optic nerve transected eyes [10].
B. Other Monkey Models

1. Optic Nerve Transection
Optic nerve transection (ONT) can be used to distinguish between elevated IOP
induced changes versus those due to the loss of axons that occurs in transection.
Six animals underwent transection of the optic nerve in one eye, preserving the
central vessels as veried by the absence of hemorrhage by indirect ophthalmoscopy at the completion of the transection and again several days later. Briey,
an oculoplastic surgeon performs a lateral orbitotomy [141] using pentobarbital
68
Gabelt et al.
(15 mg/kg I.V. or 35 mg/kg I.M.) or isouorane (1.52% inhalation) anesthesia.

The intraconal space is entered by gentle dissection between the lateral and superior recti muscles, under 2.5-loupe magnication. A malleable retractor is used
to gently retract the globe medially. At all times, pressure on the globe is kept
as light as possible and pressure is released for a few seconds every 2 to 3 mins.
Under visualization with an operating microscope, the optic nerve is exposed and
a sickle knife is used to make a 3 mm linear incision in the dura parallel to the
nerve, as far posteriorly as practical (at least 15 mm posterior to the globe) in
order to avoid damage to the central retinal artery. Dural vessels and use of cautery are avoided. Neurosurgical angled ne scissors are then used to extend the
incision posteriorly several millimeters. The scissors are then inserted within the
dural sheath, and the nerve transected (two cuts each two-thirds through the nerve
or complete transection) under direct visualization [10]. The nerve may be transected within the sheath, or the sheath and the nerve can be transected as one
unit. The ciliary ganglion is used as a marker to verify that we are sufciently
far posterior. The transection is performed approximately 12 mm posterior to
the globe in rhesus monkeys. The retina is then observed by direct and indirect
ophthalmoscopy, to ensure that no central retinal artery occlusion occurred. The
wounds are closed and the animals treated with systemic benzathine and procaine
penicillin (30,000 U/kg, Phoenix Pharmaceutical, Inc., St. Joseph, MO) daily for
5 days and systemic methylprednisolone acetate (Depo-Medrol, 1 mg/kg I.M.
Pharmacia Corp, Pepack, NJ) for 3 weeks, tapering to 0.1 mg/kg over the course
of 710 days. An alternative to the prolonged depomedrol treatment is dexamethasone (1 mg/kg, I.M., Phoenix Pharmaceutical, Inc., St. Joseph, MO) administered for 35 days. Analgesia is provided with buprenorphine HCl (0.1 mg/kg,
I.M., Abbott Labs, Chicago, IL) every 12 h for 3 days, if necessary.
Disc pallor and loss/attenuation of the nerve ber layer may be evident
within one month (Fig. 2) although, in most cases, indirect ophthalmoscopy, slit
lamp biomicroscopic funduscopy, and stereoscopic fundus photography detect
no clear-cut changes in the fundus of eyes approximately 1 month after ONT
compared with their presurgical baseline or with their contralateral control.
Changes in the clinical appearance of the optic disc begin to be visible 5 weeks
after experimental optic nerve trauma [142]. Consistent with ONT, an afferent
pupillary defect develops in the ONT eye. The pupil in the transected eye was
generally larger than in the control eye, and the consensual response to light was
weak in some ONT eyes throughout the 4 weeks. The dilated pupil and absent
or weak consensual response to light may have resulted from damage to the
ciliary ganglion or the parasympathetic efferent bers traveling with the ciliary
nerves during the dissection, so that the iris sphincter muscle received reduced
innervation. No systematic alteration in IOP is measured in ONT eyes, although
transient elevations may occur, presumably related to orbital swelling and pressure on the globe, as can occur after orbitotomy in humans.
69
As mentioned previously, ONT does not elicit responses in astrocytes of

the optic nerve head nor alter the structure of the tissues as seen after experimental
glaucoma [10].
2. Endothelin
Endothelin-1 delivered to the perineural region of the anterior optic nerve via
osmotically driven minipumps has been used to study the effect of chronic vasoconstriction and reduced blood ow to the optic nerve. Optic nerve blood ow
determined by colored microspheres after 7 days for endothelin-1 administration
was signicantly decreased [143]. Chronic ischemia for up to 2 to 6 months
resulted in diffuse loss of axons without a change in the IOP [144]. Confocal
scanning laser ophthalmoscopic topographic analysis 35 months after the onset
of ischemia showed increased cup area, cup volume, and mean cup depth. mERG
after 5 months showed functional changes consistent with inner-retinal damage
[145].
3. Steroids
Early attempts to induce IOP elevation and outow facility reduction by chronic
administration of glucocorticosteroids in a nonhuman primate, analogous to steroid-induced ocular hypertension in the human, were not successful [146]. More
recently, steroid-induced ocular hypertension has been induced for the rst time
in cynomolgus monkeys (Fig. 7) [147]. In this model, monkeys received topical
ocular drops (10 L) of 0.1% dexamethasone 3 times a day for 28 days; 45%
(5 of 11) of monkeys tested developed and maintained ocular hypertension of
greater than 5 mmHg, similar to the frequency in humans [148,149]. Discontinuation of dexamethasone treatment resulted in IOP returning to normal in 2 weeks.
The response in responder monkeys was reproducible but nonresponder monkeys
remained nonresponders. This study identied no statistically signicant evidence
for a link between myocilin mutations and steroid-induced ocular hypertension
in both monkeys and humans.
The use of sustained-release intraocular steroid implants is awaiting testing
in monkeys as another possible mode for inducing ocular hypertension in this
species.
4. Particles/Gels
Intracameral injection of ghost red blood cells causes abrupt IOP elevations of
7090 mmHg lasting 2 to 42 days [150,151]. IOP elevation of 45 mmHg to
70 mmHg for only 2 to 4 days could cause signicant retinal ganglion and axonal
degeneration. Cross-linked polyacrylamide (microgels), an altered form of the
synthetic viscoelastic Orcolon, elevated IOP by 510 mmHg and decreased out-
70
Gabelt et al.
Figure 7 (A) IOP time course of cynomolgus monkeys during rst DEX treatment.
Eleven monkeys were treated with topical glucocorticoid (10 L of 0.1% DEX) three
times a day for 28 days. IOP measurements were recorded every 3 to 4 days during
4 weeks of glucocorticoid treatment and for an additional 2 weeks. An elevation of
IOP in excess of 5 mmHg was observed in ve monkeys that were considered
steroid responders. (B) IOP time course during the second DEX treatment. After
a 6-month washout period, 10 of the 11 monkeys were re-treated with the same
regimen of DEX. Elevated IOP was observed in the same monkeys that had
steroid-induced elevations in IOP during the rst course of glucocorticoids. Filled
circleIOP of steroid responders at each time point; open circlesIOP of the
nonresponding monkeys. Data are mean SD. (From Ref. 147.)
71
ow facility by 4050% for 12 months following exchange of the anterior

chamber with microgel solution [152]. This was due to accumulation of microgels
in the cribriform meshwork and beneath the inner wall of Schlemms canal with
no cellular alterations or inammatory inltrate.
Repeated intracameral injections of 50100 L of sterile 10 m latex microspheres has recently been used to maintain elevated IOP by TM obstruction
for up to 3 years in rhesus monkeys (Fig. 8) [153]. This has the advantage of
producing gradual, chronic elevations of IOP without the need for expensive
Figure 8 Histological analysis showing the initial localization of beads injected

into the anterior chamber to the region of the TM (top) and their intense packing
density following multiple injections (bottom). Beads distributed to the iris had no
adverse effect on its function. (From Ref. 153.)
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Gabelt et al.
ophthalmic equipment, without producing inammation, and without compromising visibility of the optic disc.
5. Enzymes
Injection of alpha chymotrypsin into the posterior chamber rapidly increases IOP
and is also accompanied by iridocyclitis, lens displacement, ciliary body atrophy,
anterior chamber angle deformation, and peripheral anterior synechiae [154
156]. IOP elevation can persist for at least 6 months or longer.
III. CONCLUDING REMARKS

Laser-induced ocular hypertension is currently the most commonly used and most
extensively characterized model for experimental glaucoma in primates. It mimics the optic neuropathy affecting the entire visual pathway, analogous to the
human disease, and also amplies the IOP-lowering effect of drugs. It has thus
provided, and will no doubt continue to provide, useful pathophysiological and
therapeutic insights relevant to the human disease. Other promising primate models including bead injections and steroid administration await more comprehensive characterization to enhance our understanding of the situations in which they
will be most useful.
ACKNOWLEDGMENTS
We thank Drs. Jay McLaren and Richard Brubaker for their contributions to the
aqueous humor formation section and Dr. T. Michael Nork for his contribution
to the photoreceptor section.
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6
Daniel T. Organisciak, R. M. Darrow, and L. S. Barsalou
Wright State University
Dayton, Ohio, U.S.A.
I.
INTRODUCTION
Acute light-induced photoreceptor cell degeneration has been studied for over
35 years as a model of visual cell loss arising from genetic and age-related disease
or light-induced trauma during ocular surgery. The rationale for these studies has
been based largely on morphological similarities between the end stages of disease and the appearance of the retina after experimental retinal degenerations.
While much has been learned, the recent advent of transgenic animal models of
human disease and retinal gene knockouts has resulted in renewed interest in the
light-damage model. In addition to using acute intense light to test the relevance
of genetically modied animals as models of human retinal degenerations, we
have also gained insights into the mechanism of retinal light damage. We now
know that visual cell loss, whether of genetic origin, age-related, or light-induced,
involves programmed cell death. We also know that light exposure induces an
oxidative stress in the retina that may trigger apoptosis, leading to photoreceptor
cell degeneration. Accordingly, genetic predisposition and environmental light
appear to produce synergistic effects on the survival or demise of visual cells.
The great advantage of acute light damage, however, is that if done correctly it
can result in the synchronous involvement of numerous photoreceptors, whereas
genetic or age-related degenerations involve fewer photoreceptors over much
longer periods of time. Retinal light damage, therefore, provides an opportunity
85
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to study cell death during a shortened time course and to determine the order of
events leading to retinal degeneration.
II. GENERAL OVERVIEW

The prevention of light-induced retinal degeneration has been described in numerous studies employing natural or synthetic antioxidants, dietary alterations
of retinol or essential fatty acids, and even manipulation of the light-rearing environment or the start time of light treatment. These various forms of neuroprotection have implications for therapeutic strategies designed to treat or prevent retinal disease. To this end, the following is provided as an experimental guide for
acute light-induced retinal degeneration in animal models. It is intended to maximize experimental outcomes, to reduce variables, and to minimize mistakes
(most of which we have already made). We begin with the selection of animal
models followed by exposure paradigms and expected outcomes. Finally, we
address ocular neuroprotection by looking at endogenous processes in the retina
and exogenous treatments. The interested reader is referred to additional comprehensive reviews or descriptions on mechanisms of retinal light damage and cellular death [18].
III. METHODS/PROCEDURES; ADVANTAGES/

DISADVANTAGES
A.
Animal Selection and Maintenance
1. Species
Most animals will undergo retinal cell loss from intense light, provided that light
exposure conditions are optimized and other precautions are taken. Commonly
used animal models are rodents, which, because of their rod-dominant retinas
and because they are nocturnal, incur retinal damage at light levels only one- to
two-fold greater than ordinary room lighting. In addition, rats or mice are often
selected for transgene expression and gene knockouts, which further increases
their importance. Less frequently used are rabbits. Although their retinas can be
damaged by visible light, the intensity required is much greater than for rats
and most successful studies have employed more energetic blue light [9] or UV
wavelengths. In our hands, we have never been able to induce retinal light damage
in albino rabbits with green light exposures that cause massive visual cell loss
in rats. Diurnal species including, ground squirrels [10], birds [11], miniature
pigs [12], and monkeys [13] have been studied for the effects of UV or visible
light on rod or cone cells, although only monkeys have a foveal region akin to
87
the human retina. Notwithstanding their cost and the need for anesthesia, some
of these species are difcult to breed in captivity and are most often acquired
from the wild. Using captured animals increases experimental variability, and
captivity is potentially much more stressful than for typical laboratory animals.
The intensity of light required to induce retinal damage in diurnal animals is
also higher, often mW or greater, than the W levels used for nocturnal rodents.
Similarly, longer duration light exposure at higher light energy levels can lead to
damage in the inner retinal layers, which compromises interpretations regarding
photoreceptor cell loss.
Other species have not been studied as extensively as mammalian models.
These include sh, amphibians, crustaceans, and insects such as drosophila. However, the zebra-sh model developed by Dowling and associates [14] and albino
and pigmented trout [15] have been used in light-damage studies. Similarly,
transgenic frogs developed by Papermaster [16] and Drosophila mutants offer
additional models for such studies. Although the use of such models is beyond
the scope of this chapter, additional information can be found in Photostasis and
Related Phenomena [17] as well as other sources [1821].
Among the rodent models, light damage typically spares both the cone cells
and the inner retinal layers [2224]. Depending on the intensity of light and the
duration of exposure, the inferior hemisphere of the rat eye is also less susceptible
to damage than the superior hemisphere [25,26]. The same appears to be true
for mice [23,24] and for transgenic rodents [27,28], although c-fos knockout mice
do not exhibit the same pattern of retinal light damage [29]. Whereas genetically
manipulated mice are more widely available and have been used to study specic
proteins that may be involved in retinal light damage, in general, mice are more
difcult to damage than rats. In an earlier study, LaVail et al. [23] demonstrated
a genetic predisposition among mice that leads to retinal light damage following
continuous exposure for 16 weeks. Using young Royal College of Surgeons
(RCS) rats, we reached similar conclusions in animals exposed to light for only
8 h [30]. The reasons for the greater light damage susceptibility of rats are not
clear but may be related to the packing of photoreceptors within the eye and the
relative rhodopsin concentrations of the two species (0.35 vs. 2.0 nmol/eye)
2. Pigmentation
Where practical, albino animals are a better choice than pigmented animals because of their relative light damage susceptibilities. Pigmented animals require
mydriatics and about twofold longer exposure times (at equal light intensity) than
albinos to exhibit the same degree of retinal damage. This is because light is
effectively screened by melanin and only enters the pigmented eye through the
dilated pupil. In albino rats or mice, light enters the eye through the sclera as
well as the pupil and, although reduced in intensity, still penetrates the closed
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eyelids of sleeping animals. Obviously, the choice of mydriatic and its duration
of action, relative to the length of light exposure needs to be considered. Irrespective of this limitation, the mechanism of action of light on photoreceptors is the
same in albino and pigmented animals, and numerous studies have shown that
melanin is unlikely to cause visible-light induced retinal degeneration (see Ref. 3
for references therein).
Our best experience has been with male albino rats, although, in our hands,
female Sprague-Dawley or albino RCS animals are equally susceptible to light
damage. This may not be true, however, for other strains of albino rats, transgenic
rats having rhodopsin mutations, and some mice. To minimize variability, it is
recommended that a single sex of experimental animal be used where possible.
3. Age and Rearing Conditions
Irrespective of the choice of animals to be used, variables associated with lightrearing conditions and age can impact the outcome of acute retinal light damage.
All too often animals obtained from commercial sources are used within several
days of arrival and without consideration for rearing light differences and dark
adaptation. For example, with respect to rodent models, light levels in many
laboratory animal facilities range from over 1000 lux white light at the top of
animal cage racks to less than 120 lux on the bottom shelf. This results in differences in rhodopsin levels among experimental animals and may result in visual
cell loss in the long run. Because complete rod outer segment rhodopsin turnover
in rodents occurs in about 910 days we suggest a minimum of 2 weeks acclimation, at a dened light level, before experimentation. A nice example of the differential effects of light rearing environment on light damage has been published
[31,32]. These authors found that rats reared under higher light intensities are
more resistant to retinal light damage than rats from lower light level environments. However, at the highest light levels, visual cell loss also occurred prior
to intense light treatment.
This variable is even more critical in studies of age-related differences in
light damage susceptibility. Commercial suppliers normally rotate animals from
the top to bottom shelves of the cage racks, but obtaining rats of different ages
at different times can easily lead to differences in rhodopsin and other ocular
proteins which are up- or down-regulated by light and dark rearing conditions
[33]. Nevertheless, animals should be well dark adapted (1216 h), to maximize
rhodopsin content before intense light treatment, and all exposures should be
initiated at the same time of the day.
In our animal facility, cyclic lightreared rats are housed on cage racks
equipped with multiple 7 W night lights (G.E. 7C7, indicator bulbs; Cisco Electric
Co. Columbus, OH) in multiple outlet strips attached to cage racks above each
shelf. By wiring these outlet strips in parallel and with the use of a commercial

Table 1
89
Species Selection for Retinal Light Damage Studies
Animal Type
Nocturnal (rats/mice)
Diurnal (pigs, birds, squirrels, monkeys)

Other models (Fish, amphibians, insects)
Advantages
Notes
Easily damaged by light

(W); albino and pigmented strains;
transgenic/knockouts
Rod/cone cell models of
light damage; some
transgenics available
Transgenics being developed or mutants; known
rod/outer segment regeneration after damage
(some)
Rearing conditions need to

be carefully controlled
Cost, anesthesia, breeding

difcult, UV and mW
levels of light required
Retinal cell types different
from mammalian, not
well studied
dimmer switch and timer, we are able to set light levels uniformly to any desired
illuminance (e.g., 2030 lux) as well as on/off times (e.g., 8 a.m./8 p.m.) for the
entire colony of animals. The 12-h cyclic light period is also adjusted for seasonal
changes in time such as daylight savings time. Overhead lighting in our animal
room has been disconnected. Normally, rats, purchased as weanlings or born in
the facility, are maintained until the age of 60 days (4 rod renewal cycles)
before use. A similar room for dark rearing of rats has an internal, light-protected
door for access and red Plexiglas panels in the overhead light xtures. These
lights are on for less than 30 min per day for routine animal care. Because the
red Plexiglas panels (2423) transmit light greater than 600 nm (Fig. 3), rhodopsin
is not bleached in dark-reared rats during normal care and feeding periods. Table
1 summarizes some of the considerations and conditions discussed in animal
selection and maintenance
B. Acute Light Exposure Paradigms
Based on the foregoing and our experience, the focus of this section will be on
the rat model of light damage. Because retinal light damage exhibits reciprocity
and the bleaching of rhodopsin triggers visual cell loss [25], both intensity of
light and duration of exposure need to be considered along with other factors
that can inuence the extent of photoreceptor cell damage.
1. Light Chamber Design
The relative ease with which rats incur retinal light damage often leads investigators to overlook experimental conditions that can help to reduce variability
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Organisciak et al.
and increase the reliability of their conclusions. One variable is the uniformity
of light during exposure. As intense light is a stress for experimental animals,
they will seek the lowest level of light and attempt to shield their eyes during
exposure. This may be the corners of rectangular cages or plastic exposure chambers. In wire top cages with light from above, animals will often remain under
their water bottles or food supply, if provided on top of the wire cage cover.
Figure 1 depicts the type of light chambers we use to induce retinal degeneration in rats. Our light chambers are made from 1/8 in. thick Plexiglas, molded
to produce a 24 in. long, 6 in. OD cylinder (Dayton Plastics, Dayton, OH). Shown
here is green 2092 Plexiglas [25], but other types of plastics can be used depending on the wavelengths of light desired. The overall advantage of Plexiglas,
or other plastics, is that UV is effectively absorbed by most of these materials
(see Fig. 3). This reduces the potential for conjunctivitis in experimental animals
[25]. The Plexiglas exposure chamber is mounted in a cradle and is surrounded
by 712 in. circular 32W uorescent bulbs (G.E. Cool white FC 12T9-CW).
This provides 360 of relatively uniform light during exposure periods. The
chamber contains an internal wire mesh oor and is tipped 10 to eliminate
wastes. A small 4 in. electric pancake fan mounted at the rear is run at approximately 50% line voltage to reduce heating during light exposure. Our chambers
Figure 1 Plexiglas light exposure chamber for rats.
91
are equipped with on/off timers (EC71D/305 Paragon Electric Corp., Two Rivers,
WI), which can be programmed for different start/stop times or intermittent light
exposures. It is important that the electric fans are on only during light periods. We
use four green chambers with light levels of 12001500 lux (corneal irradiance 170
or 200 W/cm2) measured internally with a light meter (IL 1400A, International
Light Inc., Newburyport, MA). Lower light levels can be achieved by wrapping
the cylinders with commercial screening, which acts like a neutral density lter
(see Fig. 1).
Hyperthermic light exposures are conducted in four vertical 9 in. OD round
chambers, each surrounded by three circular 12 in. uorescent bulbs. Rats are
maintained at approximately 80% relative humidity and increased temperature for
2 h before, as well as during, light exposure (required to raise core body temperature). The cabinet contains heaters set for the desired hyperthermic exposure. The
chamber shown in Figure 2 is from W.K. Noell and was used in his original light
damage study. See Noell et al. [25] and Organisciak et al. [34] for details.
Figure 2 Hyperthermic light exposure chamber.
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Organisciak et al.
Well dark adapted rats are given food and water ad libitum during light
exposure. The animals, two per chamber or one for hyperthermic exposure, are
also unrestrained and unanesthetized during exposure. Typically, light exposure
starts at 9 a.m., although exposures can be conducted at any time provided start
times are consistent. Following light exposure, our rats are maintained in darkness
for as long as 2 weeks before end point measurements of visual cell loss, whereas
many investigators simply return animals to their normal rearing light environment. This does not appear to alter the outcome of most measurements of visual
cell loss.
2. Factors That Inuence the Extent of Light Damage
Aside from the principle of reciprocity, which implies that time and intensity have
an inverse relationship with respect to light damage, many other characteristics
inuence the extent of visual cell lossfor example, the wavelengths of light.
Figure 3A shows the transmission spectra for several types of Plexiglas, or other
plastic materials. Green Plexiglas 2092 and blue 2424 each have an approximately 100 nm band pass, but both bleach rhodopsin with the same efciency,
and bleaching is dependent on light intensity. Figure 3B shows that the energy
for blue and green light is approximately the same at the higher green intensities,
Figure 3 Characteristics of plastics used in light chambers. (A) Transmission

spectra of various plastic materials. (B) Rhodopsin bleaching in rats exposed to
light in chambers composed of green or blue Plexiglas at different light intensities.
93
despite the differences in photometric units (e.g., 1500 lux vs. 170 lux). Accordingly, at similar corneal irradiances, rhodopsin is bleached by about 90% in 5
min. However, in a recent study with 403 nm blue light, retinal was found to be
photoisomerized and rhodopsin photoregenerated [35]. Whatever light lter is
employed, however, it is important to keep in mind that plastic pigments age and
chambers should be replaced periodically (e.g., see old green 2092 [Fig. 3A]).
Likewise, uorescent bulbs also age and should be replaced on a regular
schedule.
Most often, continuous light exposures are used, although intermittent light
and hyperthermic treatment both enhance the extent of light damage in rats (Fig.
4). These light-exposure paradigms have been utilized in an attempt to produce
synchronous visual cell involvement. For the exposure conditions shown in
Figure 4, and with unanesthetized albino rats, we nd that hyperthermia is 18to 24-fold more damaging than continuous exposure under euthermic conditions.
However, see de Lint et al. [36], who measured fundoscopic lesions in hyperthermic-pigmented rats under anesthesia and found only a twofold difference. In our
hands, intermittent light treatment is twofold more damaging than continuous
light exposure of the same intensity and duration [37].
Figure 4 Paradigms of intense light exposure. Weanling rats are maintained in

dim cyclic light or darkness for 40 days before exposure to intense light. When
antioxidants, or drugs, are given (normal injections) these normally precede light
treatment.
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Organisciak et al.
The prior light-rearing conditions of experimental animals, age, and diet

are additional factors that inuence light damage. Irrespective of the type of light
exposure used, dark-reared rats are about two- to threefold more susceptible to
light damage than comparable cyclic light reared animals. Dark-reared rats also
incur RPE cell damage, whereas in cyclic lightreared rats the RPE is less involved. Young cyclic lightreared rats (25 days) are generally quite resistant
to light damage compared with adult animals [38]. Among adult rats we found
a twofold increase in visual cell loss at 12 months versus 2-month-old animals
after 24 h green light exposures. Ironically, dark rearing resulted in no age-related
increase in retinal light damage, but those animals were still more susceptible to
damage than cyclic light reared animals [39]. In rats fed vitamin Adecient
diets, or in those fed essential fatty aciddecient diets, retinal light damage is
also decreased [40,41]. Recently we tested the start time of light exposure as a
variable. Our ndings indicate that light exposure beginning at 1 a.m. causes oneto twofold more damage than light exposure at 9 a.m. [42]. With the exception of
mydriatics, anesthetics such as halothane also reduce light damage susceptibility
in rodents by preventing rhodopsin regeneration [43]. These factors have been
summarized in Table 2.
C.
Outcomes of Retinal Light Damage
Depending on the types of information sought and the measurements used, conclusions regarding the extent and nature of light-induced cell loss can vary.
Among the typical techniques usedhistology, electrophysiology, and biochemistryeach has its limitations, and correlative measurements are recommended.
In this section we point out some of these limitations and other problems associated with interpretations based on the time course of retinal degeneration. Our
Table 2 Some Experimental Factors Which Alter Retinal

Light Damage in Rats
Reduced
light damage
Cyclic light reared

Young (2 mo.)
Euthermic
9 a.m. start time
Continuous light
Vitamin A decient
Decient DHA (22 : 6)
Pigmented (dilated)
Enhanced
light damage
Dark reared
Old (12 mo.)
Hyperthermic
1 a.m. start time
Intermittent light
Normal diet
Normal DHA levels
Albino
Fold
increase
23
2
1824
12
2
12
12
2
95
approach will be a quantitative assessment of visual cell loss arising from the
synchronous involvement of photoreceptors during intense light exposure.
1. Measurements of Visual Cell Loss
Much of our understanding of lights pathological effects on the retina has come
from histology. It is the most widely used approach, providing information on
the types of cells involved and their locations within quadrants of the retina. No
other approach is as good in describing organelle damage within cells during or
after light exposure. From such studies we know that the entire visual cell becomes involved during damage, including the ROS, inner segment mitochondrial
rich region, nucleus, and even the synaptic end plate. Depending on the type of
light and the animal model used RPE damage also occurs, either simultaneously
or shortly after visual cell damage [6,38]. The major drawback is that histology
is descriptive and is often presented as a snapshot that is interpreted as representative damage in the entire tissue. While this may be true, the examples chosen
frequently are those that depict the greatest morphological changes. Accordingly,
histological measurements that assess visual cell loss by morphometric analysis
over the entire eye, including ONL thickness, ROS length, and/or rod cell nuclear
counts, are required. Such approaches require serial sectioning and proper cell
alignment for accurate conclusions regarding ONL volume and ROS length. Obviously this is painstaking and tedious work, requiring considerable expertise.
One of the rst examples of this approach was published by Rapp and Williams
[26] in their detailed analysis of the superior and inferior hemispheres of lightdamaged rat eyes.
Electroretinography is one of the best examples of unbiased functional analysis of photoreceptor survival and loss. Its strength lies in its repetitive use for
following ERG changes in the same animals. By comparing scotopic A- and Bwave amplitudes, latencies, and thresholds, which reect the summed electrical
signals generated primarily by intact photoreceptors and Muller cells, much can
be learned about the extent of visual cell damage and its time course. Photopic
analysis, to determine cone cell responses, provides valuable functional measurements of histologically identiable cell types that survive light damage. This is
particularly useful in cone-dominant animal models because puried cone pigments have never been isolated from the retina. Unfortunately, simple ERG analysis and morphometry of visual cell loss do not always correlate as well as expected. This may be related to RPE cell damage and the subsequent shunting of
current generated in response to light stimuli [3]. Despite this limitation, electroretinography, using Ganzfeld illumination, provides an average summated response from the entire population of synaptically intact photoreceptors remaining
after light damage. In one of the few comprehensive studies comparing ERG
analysis and morphometry in light-damaged rat retinas Sugawara, Sieving, and
96
Organisciak et al.
Bush [44] report a high degree of correlation between the two techniques and
discuss potential drawbacks in electroretinography. The interested reader is referred to this thorough study.
Classical biochemistry offers many opportunities to assess retinal damage
and cell loss because of the variety of measurements that can be made with the
same tissue. As with electroretinography and morphometry, the two eyes of the
same animal can also provide correlative assessments of light damage. This is
based on many studies showing that both eyes have identical photoreceptor cell
numbers and components, and that when intense light exposures are properly
conducted the extent of visual cell loss is the same. Typically, biochemical measurements are average responses determining changes in the entire tissue that
remains following damage or cell loss. Because acute light-induced retinal degeneration induces synchronous cell involvement, much has been learned about the
mechanism(s) of light damage and cell death from these techniques. The relative
ease by which biochemical measurements can be made is another advantage, but
the choice of measurement and the time course of damage can lead to erroneous
conclusions.
Because rhodopsin is essentially contained within the ROS of rod photoreceptors and damaged cellular material is removed by the RPE, it can be used
as an endpoint determination of rod cell survival following light damage. When
compared with the rhodopsin level in similarly dark adapted, but unexposed,
littermate controls, the extent of visual cell loss can reasonably be quantied.
Although this technique works well in normal rats, in RCS rats its use alone
is unsatisfactory. Because phagocytosis is decient in these animals, rhodopsin
accumulates in the partially degraded photoreceptor debris, and estimates of
visual cell loss by DNA measurements differ by as much as 50% from simple
rhodopsin determinations [30].
Through the activation of endonucleases, DNA is degraded in dying photoreceptors, even in the absence of phagocytosis by RPE. However, DNA is also
present in cells of the inner nuclear layers. To quantitate photoreceptor cell DNA
losses, therefore, it is necessary to subtract the DNA content of the inner retina
layers. Based on this approach and using old RCS rat retinas, which have lost
their photoreceptor cells, we determined that about 70% of retinal DNA is contained within the ONL. By using this subtraction technique, reasonable conclusions about visual cell loss and DNA fragmentation in photoreceptors can be
achieved from retinal DNA measurements following light damage.
Similarly, about 50% of retinal protein and lipid is contained within the
photoreceptor cell layer of the adult rat retina. Because extensive light-induced
visual cell loss can result in the loss of up to one half of the retinal protein, this
needs to be controlled for in typical enzyme measurements. For example, the
activity of trans retinol dehydrogenase, which is present in photoreceptors, is
decreased by as much as 25% following light exposure, but the Muller cell en-
97
zyme glutamine synthetase (GS) is unaffected by light [45]. These measurements

need to be reported per retina, because GS activity per milligram of protein would
show about a 50% increase; clearly a different and erroneous conclusion. Therefore, despite the relative ease and advantage of biochemical measurements,
knowledge of the distribution of retinal proteins, enzymes, DNA, or lipid within
the normal retina is essential for quantitative assessments of visual cell loss in
degenerating tissues.
2. Time Course of Damage
The relative order of events leading to retinal damage and degeneration is important, not only from a mechanistic standpoint but also for possible therapeutic
interventions. Time course measurements beginning with the light-mediated
bleach of rhodopsin (the trigger for light damage) and proceeding through the
nal common pathway of cell death can also lead to reliable conclusions regarding endpoint determinations of retinal cell loss. As cellular apoptosis appears to
be a primary cause of retinal degenerations in animal models and in human disease we can also learn much from its measurement. There are, however, potential
problems associated with time course measurements of retinal light damage and
cell loss.
The decision to use one type of measurement or another in light damage
studies rests on the important variable of when the determination is made. In rats
a somewhat normal ERG is recordable for several hours after intense light treatment and rhodopsin regenerates in darkness to about 60% of control over a period
of 6 h to 1 day even though photoreceptors are severely damaged [25,63]. Subsequently, the ERG declines and the level of rhodopsin decreases as photoreceptors
die and degenerate over the next 46 days. Ten to 14 days after intense light,
most of the degenerated photoreceptors have been removed and repair in the
surviving cells is nearly complete. At this time morphometry, ERG, and biochemical measurements more accurately reect total visual cell loss.
Over the same 2-week recovery period, retinal DNA levels decline from
about 90% of control immediately after light exposure to a nal level commensurate with the extent of visual cell loss and the percent of rhodopsin loss. Thus,
although light leads to DNA degradation, often there is little measurable loss
during a typical light exposure period. Conclusions regarding the nature of DNA
degradation also depend on the timing of the measurements and the technique
used. An example of this is shown in Figure 5, which depicts neutral and alkaline
gel electrophoretic analysis of DNA from rat retinas.
Using neutral gels, a pattern of DNA ladders is present in cyclic light
reared rats immediately after 24 h of light exposure. This pattern is not as apparent
in dark-reared rat retinas until 2 days after a 3 h light treatment. In both cases
DNA ladders are not present 4 to 16 days after light treatment. Alkaline gel
98
Organisciak et al.
Figure 5 Gel electrophoresis of rat retinal DNA. (A,B) neutral agarose gel electrophoresis (C,D) alkaline agarose gels. Each lane was loaded with a total of 2 g
DNA from three different rat retinas. (Adapted from Ref. 46.)
electrophoresis, which detects single strand breaks in DNA and larger fragments,
shows a pattern of DNA breakdown that coincides with the apoptotic pattern in
cyclic light rats, but that precedes the same pattern in dark reared rat retinas by
at least 12 h. What does this mean? Simply put, if DNA electrophoresis is conducted 416 days after light exposure, no evidence of the nature of DNA degradation is detectable. Furthermore, by neutral gel analysis, apoptosis could be detected in one group of rats after 12 h in darkness but is barely detectable in the
other group of animals. While this example is for DNA [46], similar time-dependent differences in retinal gene expression and protein or enzyme analysis can
be found. The bottom line is that conclusions based on single time-point determinations of the extent or nature of pathological changes in the retina can vary
considerably depending on when those measurements are made.
D.
Prevention of Retinal Light Damage
Having discussed some variables associated with light-induced retinal degeneration, the goal of this section is experimental design related to its prevention. The
literature is replete with claims that one treatment or another reduces the extent
of visual cell loss from light, but upon further analysis these ndings are often
99
viewed as marginal. That is not to say that extending visual cell life by, say, 5
to 10% is not important. As almost any clinician can attest, there are patient
quality-of-life benets from preserving vision in age-related or genetically based
conditions. Because the mechanisms of retinal cell loss in human disorders and
experimental retinal degenerations bear a striking resemblance, well-designed
interventions in animal models of light damage offer hope for future clinical
trials. With an aim toward improving the efcacy of drug, antioxidant, or dietary
treatments, we discuss some of the problems associated with these in animal
models.
1. Endogenous or Adaptive Processes
The relative susceptibility or resistance of the retina to light damage depends on
a variety of conditions already discussed (see Table 2). These include rearing
conditions and age, both of which contribute to changes in retinal gene expression. Because these changes precede intense light treatment, they are endogenous
in nature and can greatly affect experimental outcomes. Simple differences in
rhodopsin levels, or other visual cell proteins, which are altered by rearing conditions or age, indicate that control measurements should be made before light
damage experiments are done. The same is true for transgenics or retinal gene
knockout models as these can alter in unexpected ways other proteins or processes
in the retina. For example, transgenic rd mice overexpressing Bcl-2 were found
to be resistant to retinal light damage, but also to have lower rhodopsin levels
than controls; a possible reason for the nding [47]. C-fos knockout mice also
have reduced rhodopsin levels and fewer photoreceptor cells than c-fos /
animals [48], although the basic conclusion regarding the importance of this transcription factor in light-induced apoptotic cell death is unchanged.
In addition to providing mechanistic insights into light damage, the use of
genetically modied animal models has implications regarding the progression
of inherited or age-related retinal disease. Early work with RCS rats, which have
a recessive mutation in phagocytosis of ROS tips by RPE, showed that both lightrearing conditions [49] and intense light exposure [50] accelerate the rate of retinal degeneration. In albino mice, LaVail et al. [23] found light damage susceptible and resistant strains, a nding that was conrmed in F1 heterozygotes [24].
In some cases of these spontaneous mutations, retinal light sensitivity led to
studies into the underlying gene defect. However, with prescribed transgene expression or knockout models, questions about specic proteins and the role of
light environment can be addressed more directly. Using transgenic mice expressing a mutated SOD gene, Mittag et al. [51] found that they were more susceptible
to retinal light damage than nontransgenic animals. In mice [27] and in rats [28]
with rhodopsin mutations, light rearing and/or intense exposures were found to
inuence the rates of retinal degeneration. Arrestin knockout mice exhibit re-
100
Organisciak et al.
duced retinal light damage [52] and c-fos knockouts [29] and the absence of
RPE-65, which is required for rhodopsin regeneration [53], prevents light damage
[54]. These studies and others [47,55] have provided useful insights into the
mechanisms of inherited retinal degenerations and retinal light damage.
Using transgenic rats with a P23H rhodopsin mutation or an S-334 truncation, provided by M. LaVail, we compared their relative susceptibilities to retinal
light damage. Figure 6 is an example of results from rats reared in different light
environments and exposed to intense green light.
Overall, P23H line 3 rats were more susceptible than line 2 rats, which
have a slower rate of spontaneous retinal degeneration (LaVail, personal communication), S-334 ter line 4 and line 9 rats were equally damaged by light when
previously reared in dim cyclic light. Why a folding mutation in the amino terminal
region of rhodopsin leads to greater light damage susceptibility than the loss of 15
amino acids from the C-terminal region is unknown at present. However, these
transgenic rats appear to be good models of some forms of human retinitis pigmentosa because the loss of rod photoreceptors is accelerated by intense light [56].
In recent studies we found that normal rats exhibit both increased light
damage susceptibility and resistance to light damage, depending only on the time
of day that light exposure begins [42]. A circadian response was also found to
exist in transgenic P23H and S-334 ter rats [8]. This indicates that endogenous
factors normally expressed in the retina contribute to light damage susceptibility
and that systemic or local signals in the retina may regulate their expression.
A variety of growth factors also provide neuroprotection in rats given intraocular injections prior to light exposure [57]. As these neurotrophic factors
are normally given in microgram concentrations 2 days before a 1-week intense
light treatment, it appears that they elicit changes in the expression of other retinal
proteins that provide protection. In a follow-up study, LaVail and associates
found that -FGF induced a dose-dependent expression of the transcription factors c-fos and c-jun in isolated rat Muller cells. [58]. There are two important
points to be made from studies such as these. The rst is that these factors work
at low concentrations in a dose- and time-dependent manner in rats. The same
may not hold for other rodents, such as mice, which reemphasizes the need for
Cyclic Reared
Normal S-334, line 9 line 4 P23H, line 2 line 3
Dark Reared
Normal S-334, line 4 line 9 P23H, line 2 line 3
Figure 6 Relative light damage susceptibility in different rat models. Results are
based on end point determinations of rhodopsin and DNA in comparison to their
respective unexposed controls. Normal Sprague-Dawley rats are least susceptible
to light damage.
101
careful species selection. The second point is that trauma to the injected eye can
induce a stress response, as shown by partial protection in the eyes of vehicle
injected animals. LaVail et al. [57] controlled for this partial response, further
supporting their important conclusions regarding neurotrophic factors.
2. Exogenous Treatments or Factors
A variety of natural or synthetic compounds have been shown to provide protection against retinal light damage. In fact, a colleague once asked, is there anything that does not protect the retina against light damage?(personal communication, B. Winkler). Quite simply, the answer is yes. The same compounds that
exhibit protection provide no effect when given at the wrong time, at the wrong
concentration, or in the wrong way. There are also a number of variables associated with exogenous agents and neuroprotection in the light damage model. These
include systemic versus local effects, concentration, uptake and half-life or clearance rates, as well as time and intensity relationships during light exposure. In
this section we describe some of these variables in the rat model.
First and foremost the question of efcacy is a function of the extent of
light-induced damage. In our model the goal is to induce 50% photoreceptor cell
loss in untreated or vehicle-injected rats. In doing so, it is possible to reliably
determine whether a drug protects against or enhances retinal light damage by
measuring an increase or decrease in visual cell survival. Too much damage can
overcome a protective effect and too little can mask the effect. In this type of
experiment, the variable most often applied is duration of exposure at a xed
light intensity. With the use of a 50% cell response, it is also possible to compare
the relative efcacy of a variety of drugs or closely related compounds. Using
such an approach we were able to demonstrate that the synthetic antioxidant
dimethylthiourea (DMTU) was more effective in reducing light damage than lascorbic acid, a natural antioxidant present in the retina [59]. It is important to
note that the d-steroisomer of ascorbate, which is an antioxidant but not an enzymatic cofactor in mammals, was as effective as l-ascorbate when given at an
equal concentration [59].
A second variable is tissue uptake and the half-life of administered compounds. For cyclic lightreared rats treated with DMTU, we found complete
protection following light exposures lasting for up to 48 h. By measuring tissue
levels, we found that DMTU has a half-life of about 24 h [60], whereas ascorbic
acid has a much shorter half-life (48 h). DMTU levels in the retina were also
about vefold higher than l-ascorbate 10 min after IP injection. As serum levels
of drugs can also be high, it is important to use perfused animals for accurate retinal
tissue level measurements. We estimate that the rat retina contains about 10 L of
blood. Accordingly, to demonstrate uptake in a tissue such as the retina, if the animal
is not perfused, the concentration of drug should be higher than in blood.
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Organisciak et al.
A surprising number of studies report protection against retinal light damage without measurements of the nature described above. Thus, it is never really
clear whether the administered compound is itself active in preventing the damage or whether it exerts a systemic, perhaps stress, effect leading to protection.
Ideally, an agent that exhibits a primary effect not only will be present in the
retina for a period of time but also will be found at lower levels in the eyes of
light-treated animals than in unexposed controls. The level of ascorbic acid, for
example, can be shown to decrease in the rat retina during light exposure and to
be effective only when given before the start of light treatment [61,62].
To achieve effective levels of drugs in the eye, the route of administration
and the type of compound are also important. We have had good success with
water-soluble antioxidants such as DMTU and ascorbic acid given I.P., I.V., or
I.O. The kinetics of uptake for ascorbate in retina or RPE are similar for I.P. and
I.O. injections although, as expected, the concentrations in the two tissues and
the time courses of their losses were different [62]. The same is not true, however,
for lipid soluble materials such as -carotene or other carotenoids. The reasons
for this are not entirely clear. Using I.P. or oral gavage of these materials in rats,
we have been unsuccessful in demonstrating efcient retinal uptake. It appears
that detergents will be required to solubilize these lipophylic materials for possible tissue uptake and light damage studies.
Finally, a comment about dose response curves. Whether one chooses to
measure tissue uptake or not, or to determine serum levels and clearance rates,
a dose response curve for any drug or compound can be informative and useful in
Figure 7 Dose-response curves for antioxidants used in light damage. Rhodopsin measured 2 weeks after intense light exposure. (A) 8 h () or 3 h () of intermittent light. (B) 24 h () or 12 h () continuous light. (, ) cyclic light and dark
reared rats. (A) (, ) urea; (B) () dehydroascorbic acid; (A) (, ) unexposed
controls S.D. (n 812).
103
studies of neuroprotection. These responses can be different for different animal

models. As shown in Figure 7, the level of antioxidant required for maximal
effectiveness is not the same for rats reared in dim cyclic light or in darkness.
First the level of tissue damage in the two types of rats is different. Second,
the concentration of injected DMTU or ascorbate required to reach a plateau
effect is greater in dark reared rats [60,61]. This is important because working
with lower concentrations will not achieve the same level of protection, compromising the drugs effect. In practice, we use a concentration well above the minimum to achieve protection because this gives a more uniform response among
test subjects. This translates into more reliable results with fewer animals required
for light damage studies.
IV. CONCLUSIONS
In the preceding we have attempted to provide an experimental outline for studies
related to acute retinal light damage and neuroprotection in vivo. By addressing
some problems and pitfalls we hope that future investigators will avoid some of
the common mistakes that we and others have made. While a number of mechanistic interpretations might be derived from such studies, the effects of light on
genetically modied animals, and the prevention of vision loss, has implications
well beyond the use of the models described here. For the present these experimental outcomes will need to be extrapolated to the human condition. However,
with a better understanding of the variables associated with acute light damage,
we may nd simple dietary or other therapeutic approaches to neuroprotection
for those aficted with genetic or age related vision loss.
ACKNOWLEDGMENTS
Supported by NIH grant EY-01959; Ohio Lions Research Foundation; and M.
Petticrew, Springeld, OH.
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to light damage in an arrestin knockout model of Oguchi disease (Stationary night
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Redmond MT, Yu S, Lee E, Bok D, Hamasaki D, Chen N, Goletz P, Ma J-X,
Crouch R, Pfeifer K. Rpe65 is necessary for production of 11-cis-vitamin A in the
retinal visual cycle. Nat Genet 1998; 20: 344351.
Grimm C, Wenzel A, Hafezi F, Yu S, Redmond M. Protection of Rpe65-decient
mice identies rhodopsin as mediator of light-induced retinal degeneration. Nat
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Hobson AH, Donovan M, Humphries MM, Tuohy G, McNally N, Carmody R,
Cotter T, Farrar GJ, Kenna PF, Humphries P. Apoptotic photoreceptor cell death
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7
Retinal Detachment
Ward M. Peterson
Inspire Pharmaceuticals
Durham, North Carolina, U.S.A.
I.
INTRODUCTION
Retinal detachment is an acute pathological endpoint of many ocular conditions

and often results in permanent impairment of central vision if the macula is detached. Models of experimental retinal detachment (RD) have been developed
to understand the disease process and to provide insight into various aspects
of basic retinal biology. For reviews of signicant ndings made in the context
of experimental RD, see Refs. [13]. Although naturally occurring retinal detachments have been observed in cats, dogs, and monkeys, these occur too infrequently to be useful as reliable model systems [48]. To mimic various
aspects of the clinical condition, a number of experimental models of RD have
been developed using surgical and, to a lesser extent, pharmacological means.
On the cellular level, sustained retinal alterations and photoreceptor degeneration are common outcomes of prolonged detachments (3). The signicance of
retinopathic changes in causing protracted or permanent visual dysfunction
in patients suffering from retinal detachment is well appreciated [3,9]. However, investigations of experimental models of RD in the context of either reducing the severity or enhancing the recovery of retinal function are considerably
limited.
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II. CLINICAL RETINAL DETACHMENT

Based on general etiological parameters, retinal detachments are thought of as
belonging to one of three major forms: rhegmatogenous, traction, and serous
[10,11]. In the exudative form of age-related macular degeneration (AMD), choroidal neovascularization can lead to leakage of uid and growth of blood vessels
in the subretinal space, which can produce localized macular separations. Models
of AMD are beyond the scope of this chapter. Photoreceptors receive oxygen
and glucose from the nearby choriocapillaris and even a shallow detachment can
result in photoreceptor hypoxia and ischemia [12,13]. Because of their high metabolic activity, photoreceptors are very susceptible to prolonged hypoxic conditions [14]. Whatever the cause of the RD, failure to achieve retinal reattachment
in a timely manner often leads to irreversible loss of vision in the affected region.
For example, prolonged detachment of the macula results in signicant cone alteration or degeneration in the fovea and invariably leads to permanent loss of visual
acuity, distortion of color vision, and metamorphopsia, even following successful
retinal reattachment [9].
Rhegmatogenous retinal detachment is the most common form of retinal
detachment and occurs as a result of tears or holes in the retina. In the older eye,
the vitreous loses some of its gel-like characteristics and can become liqueed,
and these retinal holes or tears permit liqueed vitreal uid to enter and accumulate in the subretinal space, thus creating and enlarging the detachment. A variety
of surgical techniques (scleral buckle, pneumatic retinopexy, and vitrectomy) are
used to reattach the retina. Retinal reattachment surgery often affords an initial
success rate of 70 to 90% and a nal success rate of higher than 95% following
repeat surgeries [15,16]. Scleral buckle surgery is the most common technique
for treating rhegmatogenous RD and can result in overall reattachment success
rates higher than 90% following a single operation. Thus retinal reattachment
surgery is thought to be largely successful. However, success in RD surgery is
measured primarily, if not exclusively, in terms of achieving retinal reattachment.
Unfortunately, this anatomical success does not parallel recovery in visual function. In a retrospective study, Burton found that of patients treated successfully
with a single operative procedure, 42% achieved better than 20/50 visual acuity.
For cases involving patients with macula-off detachments, only 20% of reattached macula achieved postoperative visual acuities of 20/50 or better [9].
Although decreased postoperative visual acuity can occur as a result of proliferative vitreoretinopathy, cystoid macular edema, or macular pucker, it is generally
thought that degenerative loss of macular cone function is the primary underlying
cause of permanent visual acuity impairment and color distortion following successful reattachment surgery [9,15].
Tractional forces within the vitreous body or along the inner surface of the
retina can exert sufcient mechanical force on the retina to pull it away from the
Retinal Detachment
111
RPE, thereby creating a traction retinal detachment. Traction RD is the second

most common form of retinal detachment and is thought to result in part from
an inappropriate cellular wound healing response along the vitreal-retinal and
retinal-RPE interface. Proliferating, hypertrophic, reactive, or otherwise altered
glial cells (Muller cells and astrocytes), RPE, pericytes, and endothelial cells are
believed to contribute to the formation of the tractional forces [17]. These cells
can proliferate and form a clinically evident membrane structure that contracts
and pulls either perpendicularly (via interactions with vitreous strands) or tangentially to the retinal surface. Traction RD occurs in diabetic retinopathy, penetrating trauma (secondary to internal bleeding), retinopathy of prematurity and proliferative vitreoretinopathy [10,11]. It can also lead to permanent visual loss, and
is generally treated with vitrectomy, which allows the surgeon to remove the
tractional forces. Tractional forces can also contribute to the formation of a retinal
tear by pulling a patch of retina from the RPE, usually creating a horseshoe
tear and increasing the likelihood for the development of rhegmatogenous RD.
Diseases and abnormalities of the choroid or RPE can lead to serous or
exudative retinal detachment [10]. Serous RD occurs without retinal holes or
tears and without apparent tractional forces on the retina, and it is thought in
most cases to originate from choroidal uid leaking into the subretinal space
across a compromised RPE [10,18]. Conditions that can lead to serous detachments include central serous retinopathy, severe hypertension, toxemia of pregnancy, Haradas disease, and various choroidal inammatory disorders. Treatments for serous RD are generally nonsurgical.
Many aspects of rhegmatogenous, traction, and serous RD are studied by
employing an experimental model of non-rhegmatogenous retinal detachment,
which has been used extensively to study photoreceptor degeneration, retinal alterations, and, more recently, to test potential therapeutic approaches for preventing retinopathic changes. This model is described below.
III. EXPERIMENTAL NON-RHEGMATOGENOUS

RETINAL DETACHMENT
A common method for experimentally creating a retinal detachment is to introduce a small needle or micropipette in the anterior globe (usually through the
pars plana), advance the needle or micropipette through the vitreous and into the
retina, and then gently inject uid into the subretinal space (Fig. 1A). The resultant detachment is best described as a non-rhegmatogenous retinal detachment
non-rhegmatogenous because the retinal hole caused by the needle is sufciently
small to prevent shunting of subretinal and vitreal uid [36]. Experimental nonrhegmatogenous RD is sometimes referred to as a subretinal bleb or serous
retinal detachment, although it is important to recognize that this model does
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Figure 1 Diagrammatic representation of techniques used to create experimental

non-rhegmatogenous and rhegmatogenous retinal detachments. (A) The micropipette or small needle (usually 32-G) is inserted through the pars plana, slowly advanced into the vitreous, and into the retina. A small outow is maintained across
the needle tip to prevent obstruction by the vitreal gel, but this is usually done with
very small diameter micropipette tips. After creation of the subretinal bleb and retinal separation, the micropipette or needle is retracted, leaving a hole that either
seals on its own (perhaps through localized blood-clotting) or a hole that is otherwise too small for exchange of subretinal and intravitreal uid. (B) In preparation for
the creation of a retinal hole, the vitreous is collapsed by aspirating and reinjecting
hyaluronidase with the needle that is inserted intravitreally through the pars plana
(top panel). The retinal hole and surrounding detachment are subsequently created
by pressure injection of vitreous against the retina (middle and bottom panels),
resulting in a rhegmatogenous retinal detachment [35].
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113
not formally mimic many aspects of a true serous RD. The model is used to
study many aspects of retina/RPE interactions, including rates of subretinal uid
reabsorption, forces of retinal adhesion, metabolic and pharmacological effects
on the RPE uid pump, and subcellular and cellular effects on the retina and
RPE [1,1923]. Such retinal detachments are also invariably produced when delivering compounds, proteins, and viral vectors (for gene-based therapy) into the
subretinal space [24,25]. More recently, large bullous or complete non-rhegmatogenous retinal detachments are deliberately produced in the clinic as part of
an evolving surgical technique called macular translocation surgery for the treatment of age-related macular degeneration [26]. In this approach, the entire macula
is detached from the underlying diseased RPE and moved to a presumably healthier region of the RPE. Experimental models of non-rhegmatogenous RD have
been produced in rabbit, cat, monkey, and, more recently, rat, ground squirrel,
and pig [23,2733].
A. Preoperative Procedure
Animals undergo general anesthesia in preparation for retinal detachment surgery. A variety of anesthetic combinations have been used for rabbits (singly or
in various combinations): thorazin, pentobarbital, urethane, ketamine, and acepromazine maleate. Cats: combinations of ketamine and sodium pentobarbital;
or ketamine and acepromazine maleate. Monkeys: combinations of ketamine and
xylazine; phencyclidine hydrochloride and sodium pentobarbital; or sodium pentobarbital alone. Rats: a combination of buprenophrine, sodium pentobarbital,
and atropine. Topical anesthesia is administered via retrobulbar injection of xylocaine in rabbits and cats. To visualize the injection and fundus, pupils are dilated
using (singly or in various combinations): phenylephrine, cyclopentolate, tropicamide, and atropine. Some of the pioneering or prototypical models for experiment RD can be found in these references: [23,30,31,34,35].
B. Surgical Procedure: Retinal Detachment
Modications of a basic surgical procedure are used for preparing the eye and
creating a retinal detachment; these approaches often depend on the species and
the investigator. Delineation of the myriad of surgical approaches used will not
be attempted here, but a number of general methodologies are discussed instead.
In rabbits, the superior sclera is penetrated with a needle (usually 22-G) 34 mm
behind the limbus to avoid inserting through the retina, and a glass micropipette
(1550 m tip diameter) or small needle (32-G or smaller) is inserted through
the scleral opening and advanced into the vitreous under the ne control of a
micromanipulator [23,36]. For micropipettes, a small outow of uid can be
maintained across the tip by applying air pressure at 1020 lb/in.2 to prevent
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vitreous gel from obstructing the tip. The tip is advanced until the retina is gently
penetrated and a detachment begins to form. Small and large subretinal blebs
can be made this way.
In cats, a number of modications of a basic surgical technique for inducing
large retinal detachments have been reported [13,30,31]. The procedure begins
with a pre-detachment preparation phase in which an extracapsular lens extraction
is performed through an 180 corneal incision. This is followed by excision of
the posterior capsule and a partial vitrectomy or, alternatively, the posterior capsule can be left intact. The vitrectomy affords better control of the height and
extent of the induced RD. The cornea is sutured closed and allowed to heal for
several weeks before the actual retinal detachment is created. For RD surgery, an
infusion cannula is sewn in place in the cornea (for example, in the inferotemporal
quadrant). A puncture incision is then made with a 20-G needle in an adjacent
quadrant (superotemporal) of the cornea, and the posterior capsule and vitreous
are removed. Fluid-gas exchange is performed following the vitrectomy. Alternatively, the lens and vitreous can be left in place [13]. A glass micropipette with
a at 80100 m tip diameter is mounted on a micromanipulator and then inserted into the 20-G incision. The retinal detachment can then be made in a manner similar to that used in rabbits. Variations of this technique have been adapted
for use in rhesus monkeys [28].
A rat model of retinal detachment has recently been developed and used
for subretinal uid reabsorption studies [33]. Retinal detachments were made in
Long-Evans rats by rst inserting a 26-G guidance needle behind the limbus and
into the vitreous. A 33-G at-tip needle attached to a Hamilton syringe is then
inserted into the barrel of the 26-G guidance needle to create the detachment and
to inject solutions into the vitreous. A specially designed double convex lens for
the rat eye is placed on the cornea to view injections and the fundus. Although
the 26-G needle used to penetrate the rat eye may seem rather large relative to
the size of the eye, this approach does not appear to cause signicant changes
in intraocular pressure or other complications and seems amenable for studying
retinal detachments over a 24-h period.
C.
Surgical Procedure: Retinal Reattachment
In some cases, it is necessary to reproducibly induce retinal reattachment. In cats

and monkeys, a gas/air exchange procedure has been developed to induce retinal
reattachment and theoretically should be applicable for use in other species with
eyes of similar or larger sizes [28,31]. In this approach, animals are rst given
general anesthesia. The eye is then prepared for a mixture of sulfur hexauoride
(SF6) gas and air exchange via an infusion cannula and a drainage cannula at
different quadrants of the globe. In the cat, the infusion cannula is placed in the
inferotemporal quadrant of the cornea approximately 1.5 mm from the limbus.
Retinal Detachment
115
(The infusion cannula can be inserted through a 20-G incision, for example, and
secured with 5.0 Dacron mattress suture.) A second 20-G incision is then made
in the superotemporal quadrant, in which a 20-G blunt beveled tip on a Charles
uted needle is inserted. This allows for a mixture of 50% or 75% SF6 gas and
air to be ushed through the eye until complete exchange is achieved.
D. Solutions
A variety of buffers and Ringers solutions have been used to experimentally
induce retinal detachments. Table 1 shows the effects of some of these buffers
and solutions on the duration of detachments made in a number of species. The
results shown in Table 1 were taken from various published reports and may
represent laboratory-specic ndings. Other factors such as height and extent of
the induced RD are also important factors in affecting the duration of the detachment. Reattachment of experimental non-rhegmatogenous RD usually occurs
over a period of hours to days, assuming that a physiologically based solution
is used to create the detachment. Rabbits and rats appear to spontaneously reattach over a period of hours, and cats and monkeys can take many hours or even
days [1,27,30,36]. For studies of prolonged, controllable, and semi-reproducible
retinal detachments, a useful technique has been developed that enables the researcher to create detachments of xed lengths of time and consists of adding
sodium hyaluronate in the infusion solution; this prevents the retina from reat-
Table 1 Approximate Duration of Non-Rhegmatogenous Retinal Detachments

Following Subretinal Injection of Different Solutions in Various Species
Species
Rhesus monkeys
Rhesus monkeys
Rhesus monkeys
Rhesus monkeys
Cat
Cat
New Zealand rabbits
New Zealand rabbits
Rat
Solutions
Balanced salt saline (BSS)
Glutathione-bicarbonate Ringers solution
Autologous serum
Silicone oil
Balanced salt saline
BSS 0.25% Na hyaluronate
1.5% solution of Na hyaluronate
Ames solution
Modied phosphate buffered
saline
Detachment period
7 days
Complete reabsorption, 27
days
Gradual reabsorption, 3
weeks
No reabsorption
3 days
No reabsorption
No reabsorption, at least up
to 29 days
Few hours
At least 24 hours
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taching [22,30,55]. In cats, for example, retinal detachments of up to 14 months

in duration have been made with this approach [30]. Furthermore, retinal reattachment can be achieved by using the gas/uid exchange technique described above.
In the aforementioned rat model, it was noted that the constancy of the apparent
subretinal bleb size over time depended on the relative osmolarity of the blood
serum, thus suggesting that osmotic differences across the retina and RPE play
a signicant role in determining the rates of subretinal uid reabsorption [33].
The investigators exploited this nding to devise a modied phosphate buffered
saline solution that allowed subretinal blebs to remain relatively constant, at least
for an initial 24-h period. This control over the constancy in bleb size has been
useful, for example, in evaluating pharmaceutical approaches for enhancing subretinal uid reabsorption [33].
E.
Species Considerations
There are signicant anatomical differences between human eyes and the eyes
of various species used for experimental RD studies, and relevant differences
should be taken into account when interpreting the results of animal studies in
the context of human retinal detachments. Rabbit retinas are rod-dominant, lack
a cone-rich macula, and contain extremely limited intrinsic blood vessels that
reside along the nasal-to-temporal myelin wing. The rabbit retina is thought to
receive virtually all of its oxygen from the choriocapillaris. Thus a detachment
would render the entire retina hypoxic, rather than merely the outer retina. The
retinal vascular bed in the cat more closely resembles that of humans, and although the cat retina contains a higher population of cones than the rabbit retina,
it is nevertheless rod-dominant and also lacks a cone-dominant macula. Rhesus
and cynomolgus monkeys contain true cone-dominant macula and would represent ideal animal models for human RD. The retinas of ground squirrels contains
85 to 90% cones, their color vision is dichromatic, and their cone system has
been well characterized electrophysiologically. The vascular bed is extensive,
and so squirrels appear to be an attractive alternative to monkeys for RD research.
Rats also contain an extensive vascular bed, but the retina is rod-dominant
(95%) and lacks a macular region. Finally, the porcine eye, which also does
not contain a true cone-dominant macula, appears to be an attractive model for
studying neurodegeneration and neuroprotection in the context of RD. The size
of the eye, the retinal vasculature system, lens/vitreal volume ratio, and the presence of a cone-enriched macula (or visual streak) give strong credence for studying cone alterations in this species.
F.
Relevance of Model
There were some early concerns about whether the retinal hole created by the
small needle or micropipette was sufciently large to create a true shunt for
Retinal Detachment
117
subretinal and vitreal uid. If so, the model could no longer be thought of as
non-rhegmatogenous and the ability to use the model to estimate RPE uid
absorption, for example, is questionable. To address this concern, Marmor and
colleagues showed that in rabbits the creation of retinal holes with a 1525 m
diameter micropipette had no effect on the rates of subretinal uid reabsorption
[36]. Sealing the hole with cyanoacrylate, mucilage, or an air bubble did not
affect the rate of subretinal uid reabsorption, nor did the creation of multiple
holes in the same bleb. Pederson and MacLellan showed that in rhesus monkeys,
the creation of a subretinal bleb containing a small retinotomy resulted in spontaneous retinal reattachment, whether or not the eye had a vitrectomy [27,34]. In
rats, Mamanishkis and colleagues showed that uorescein injected into the subretinal space did not diffuse into the vitreous through the hole [33]. These results
all provide strong experimental support that small retinal holes do not create a
shunt for subretinal and vitreal uid, hence validating the concept that such retinal
detachments are non-rhegmatogenous in nature.
In humans, detachments enlarge over a period of hours to days, whereas
in the lab, detachments are usually created in less than a minute. Rhegmatogenous RD in an untreated human eye often progresses to total detachment. In the
absence of a signicant retinal hole or tear, experimental non-rhegmatogenous
RD tends to reattach gradually over a period of hours to a few days, and thus
requires additional constituents (such as hyaluronic acid) in the buffer formulations used for subretinal injections to maintain prolonged retinal separation. Because clinical retinal detachments tend to occur in an older eye, the state of the
human vitreous is signicantly different from that of the experimental models.
The vitreal collagen framework collapses in the aging eye, resulting in syneresis,
liquefaction, and pooling of uid within the vitreous gel. Gel liquefaction is
highly correlated with posterior vitreous detachments, which in turn sets up tractional forces on areas of vitreoretinal adhesion [37]. Such traction on areas of
vitreoretinal adhesion may cause retinal tears and subsequent retinal detachment.
Thus, some of the important differences between RD in humans and in animal
models are the origin and context of the diseased condition, particularly the role
of the vitreous. Despite these limitations, a number of important ndings made
in the lab have demonstrable correlative ndings in humans, and have yielded
further insight into the pathology of RD above and beyond what might be expected from clinical or patho-clinical studies alone. Some of these ndings are
described below.
G.
Retinal Changes
For an excellent review of alterations in retina and RPE associated with experimental retinal detachment and reattachment, see Ref. 3. Research in animal models of RD has focused considerably more on rods than cones, and very little of
the work on cones is performed on the cone-dominant macula. Of the animal
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models employed in experimental studies, only certain primate species (including

the rhesus macaques) contain a cone-dominant macula. The retina of the nocturnal owl monkey, which has been used in a series of RD studies, contains a macular region that is primarily made up of rods [38]. Rod and cone outer segments
exhibit a number of well-appreciated differences in terms of their structural and
biochemical interactions with the interphotoreceptor matrix and the RPE apical
membrane [39,40].
Recent studies provide some evidence that rods and cones respond differently to retinal detachment and reattachment. Surviving rods continue to express
high levels of rhodopsin and other cell-specic markers in a detached retina,
whereas cones respond with a rapid reduction in a variety of protein and cellular
markers, such as short wavelength (S)-cone opsin, calbindin D, carbonic anhydrase, peanut agglutinin, and possibly medium to long (M,L)-cone opsin [3,41
43]. Labeling of apoptotic cell death with the TUNEL (TdT-mediated duTPbiotin nick end labeling) technique demonstrated that both rods and cones die
during retinal detachments [42,44,45]. However the signicance of detachmentmediated reductions in measurable expression of cone-specic markers remains
elusive in the context of cone cell death. That is, reductions in the expression
of these markers do not necessarily indicate inexorable death of cones. Further
investigation into the time-course and extent of recovery of these markers needs
to be conducted to appreciate the correlation, if any, between the decrease in
cone-specic cell markers and cone death [42].
There is some experimental and clinical evidence that S-cones are more
susceptible to prolonged or irreversible damage than M,L-cones in both human
and experimental retinal detachment. Distortion in blue-yellow color vision has
been described as a relatively common visual defect following successful retinal
reattachment. Using enzyme histochemistry for carbonic anhydase and immunocytochemical localization of S antigen to differentially identify blue cones and
red/green cones, Nork and colleagues provided evidence for increased loss of
blue cones in human rhegmatogenous RD [43]. Clinical electroretinography
(ERG) has also provided some insight into alterations in cone-mediated function
in patients. Cone ERG results from 19 patients taken prior to and at multiple
time points following successful retinal reattachment surgery provide evidence
that the capacity for S-cone b-wave amplitudes to recover is signicantly reduced
when compared with those of L- and M-cone b-wave amplitudes [46,47]. In another study, multifocal ERG recordings taken from detached and attached areas
of the retina before retinal reattachment surgery were compared with similar recordings taken from the same areas after successful reattachment surgery [48].
These ERG recordings were then compared with corresponding visual eld measures. The authors found that improvement in ERG responses from both areas
were relatively modest and surprisingly did not correlate with the more signicant
improvements in visual eld results. Taken together, these ERG recordings in
human RD point to the utility of conventional full-eld as well as multifocal
Retinal Detachment
119
ERG in extracting information about retinal function, but also suggest that ERG
parameters may not correlate with visual function.
There have been some published reports on experimental RD that have
focused on scotopic and photopic ERG responses during detachment and following reattachment [4852]. Kim and colleagues recorded photopic focal ERG from
small retinal detachments in adult rabbits and showed that b-wave amplitudes
decreased signicantly 30 min following retinal detachment and recovered back
to baseline 3 days later [52]. However, immediately following the detachment,
b-wave implicit times increased and remained signicantly higher than control
eyes even up to 28 days following reattachment. Because reattachment in rabbits
occurs within a few hours following the induction of a small, bullous detachment,
these results indicate that changes in retinal cone function persisted for weeks
following retina/RPE reapposition and that photopic b-wave implicit time may
be a useful parameter to assess loss and recovery of cone function. ERG recordings made from eyes of ground squirrels with RD of varying sizes showed a
clear depression in cone-mediated ERG function in the detachment zone [77].
However, unlike observations made in human RD, no apparent differences in Scone and M-cone ERG amplitudes were observed in squirrel. The reasons for
this discrepancy remain unresolved, but may be a result of species differences
or differences in experimental versus surgical conditions [77].
Although most of the basic and clinical research on retinal detachments
have focused on changes in photoreceptors, signicant alterations in the inner
retina and RPE have also been observed and are believed to play a signicant
role in the aptly named retinopathy of detachment [3]. One of the leading causes
of failure in retinal detachment surgery is the development of proliferative vitreoretinopathy, which is thought to be mediated by de-differentiated, proliferating, and hypertrophic non-neuronal cells, including retinal astrocytes, Muller,
and RPE cells. The proliferative response can lead to eventual contraction of
cells on the vitreal surface of the retina and result in traction retinal detachment.
Understanding the causes of proliferative vitreoretinopathy may provide some
insight into ways of preventing its development after successful reattachment
surgery [15,17].
IV. POTENTIAL THERAPEUTIC OPPORTUNITIES

A variety of experimental parameters have been employed to quantitatively or
semiquantitatively dene the retinopathic effects of experimental retinal detachment and to measure the efcacy of a potential therapeutic approach. A number
of these parameters are listed here:
Immunohistochemical labeling of rhodopsin and cone-opsin
Quantication of cell proliferation with MIB-1 antibody
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Labeling of Muller cells using antibodies directed against glial brillary

acidic protein (GFAP), cellular retinaldehyde binding protein, and glutamine synthetase
Glutamate receptor GluR-2 immunohistochemical labeling
Outer nuclear layer thickness and cell counts
Outer segment length
Quantication of apoptosis using the TUNEL technique
Full-eld and multi-focal ERG
Scotopic and photopic ERG
In the context of experimental RD, it has been only in recent years that
studies have been conducted to investigate new approaches for either reducing
the severity of retinopathic changes or improving retinal recovery following reattachment. Such experimental or neuroprotective therapies aimed at treating retinal changes in the context of RD are not as well dened as those strategies developed in the context of experimental models of light-induced and inherited
photoreceptor degeneration [53]. Retinal detachments and photoreceptor degenerative diseases share a number of similar features, including time-dependent alterations in Muller cells (reactive gliosis and hypertrophy), photoreceptor cells (particularly synaptic changes), and ultimately apoptosis as the mechanism of
photoreceptor cell death [54]. A number of the same survival factors (ciliary
neurotrophic factor, brain-derived neurotrophic factor, pigment epithelium-derived factor, glial-derived neurotrophic factor, basic broblast growth factor, etc.)
that enhance photoreceptor survival and function in the context of experimental
models of photoreceptor degeneration are likely to have a similar effect in experimental RD.
Recent work has shown that brain-derived neurotrophic factor (BDNF) and
glial-derived neurotrophic factor (GDNF) can mitigate the progression of a number of pathological retinal changes associated with experimental retinal detachments in the cat [55,56]. Lewis and colleagues showed that BDNF enhanced the
organization and increased the length of outer segments and signicantly reduced
the proliferative changes of Muller cells while the retina was detached for 7 or
28 days. However, BDNF failed to reduce the overall photoreceptor cell death
in this model. In a separate study, intravitreal GDNF was shown to increase the
mean outer segment lengths in the detached retina when compared with control
detachments [56].
A potentially important difference between models of photoreceptor degenerative diseases and experimental RD in the context of retinal neuroprotection
may be the role of supplemental oxygen. In a pair of recently published studies,
cat retinas were detached for three days, during which some animals were housed
under hyperoxic conditions (70% ambient oxygen) and remaining animals were
housed under normoxic conditions (21% ambient oxygen) [13,57]. Oxygen sup-
Retinal Detachment
121
plementation was shown to promote the survival of photoreceptor cells, enhance

the organizational structures of their outer segments, reduce Muller cell proliferation and hypertrophy, and normalize various aspects of the glutamate signaling
system. These studies elegantly illustrate the importance of oxygen supplementation in decelerating a number of photoreceptor and retinal degenerative processes
in RD. More recently, work in a ground squirrel model demonstrated that hyperoxia also rescued cone photoreceptors in this cone-dominant species, suggesting
that survival of cones with hyperoxia is not necessarily secondary to survival of
rods [58]. These studies on the effects of hyperoxia on photoreceptor survival
provide a strong rationale for testing oxygen supplementation for enhancing visual outcome associated with the treatment of retinal detachments in the clinic.
Two factors play important roles in recovery of central vision following
macula-off detachments: preoperative visual acuity and duration of the detachment [9]. In the absence of signicant degeneration of the fovea, preoperative
visual acuity is correlated with the height of the fovea from the RPE. Therefore,
these clinical observations suggest that the ability to reduce the distance between
the fovea and RPE and to speed retinal reattachment may improve visual outcome
following reattachment. The RPE normally absorbs uid in the subretinal-tochoroidal direction, and this RPE uid pump function can be enhanced in vitro
and in vivo by pharmacological means [1,59,60]. Thus, a pharmacological approach that stimulates the RPE pump could facilitate the removal of extraneous
uid in the subretinal space in RD and may provide a therapeutic approach for
reducing the spread and height of the retinal separation. If sufciently robust,
pharmacological stimulation may reattach the retina without surgery; alternatively, it may be used as a surgical adjunct to minimize the spread of the detachment or facilitate the rate of reattachment. Carbonic anhydrase inhibitors acetazolamide and benzolamide have been shown to enhance subretinal uid
reabsorption in experimental subretinal blebs, presumably by activating the RPE
uid pump, and thereby facilitate retinal reattachment in rabbits [61,62]. However, acetazolamide is poorly tolerated, thus pointing to the need for alternative
pharmacological means for stimulating the RPE pump.
The apical membrane of bovine RPE contains metabotropic P2Y2 receptors
that respond to the natural ligands adenosine 5-triphosphate (ATP) and uridine
5-triphosphate (UTP) by activating membrane transport mechanisms and stimulating net apical-to-basolateral uid absorption in vitro [63]. A synthetic P2Y2
receptor agonist INS37217, when delivered subretinally or intravitreally, has recently been shown to signicantly enhance subretinal uid reabsorption and retinal reattachment following experimental RD in rabbit [64] and rat [33]. Recent
work in a mouse model of experimental RD also showed that INS37217 signicantly enhanced the recovery rate of retinal function (as determined using standard ERG techniques) following retinal reattachment [78]. In the clinic, a pharmacological agent such as INS37217 that limits the spread of retinal detachments
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Peterson
and enhances retinal reattachment may provide a novel strategy for improving
visual outcome following surgery.
V.
MODELS OF RHEGMATOGENOUS, SEROUS,

AND TRACTION RETINAL DETACHMENT
Machemer, Aaberg, and Norton developed an experimental model of rhegmatogenous retinal detachment in the owl monkey [8,29,35]. They dened the conditions that are necessary to produce and sustain long-standing rhegmatogenous
RD, and concluded that alterations of the vitreous structure and creation of a
relatively large retinal hole are required. Their basic technique is described in
Figure 1B. They compared the histological effects of experimental verses naturally occurring retinal detachments in the owl monkey and found many similar
features, including cystoid degeneration of the outer plexiform layer, degeneration of the outer and inner nuclear layers, and appearance of at and multi-nucleated RPE cells [8]. Their model produced cystoid spaces and edema in the inner
retina that developed and enlarged with time, and subsequently reabsorbed and
disappeared following surgical reattachment of the retina [28]. These cystoid
spaces were much larger and prominent than those reported from the experimental models of non-rhegmatogenous RD described by other investigators and do
not appear to be artifacts of histological processing. It is unclear if these ndings
are species dependent, a result of surgical manipulation, or caused by the large
retinal hole, but the observations of cystoid spaces and retinal edema are clinically
relevant because they are found in long-standing human retinal detachments.
Pederson and colleagues published a series of papers in 1982 to 1986 that
examined, among other things, some of the technical differences used in various
approaches for creating rhegmatogenous versus non-rhegmatogenous RD [27,
34,6567]. They were able to produce long term retinal detachments in rhesus
monkeys by inducing a large bullous detachment (1 mL of 20% autologous serum
in Ringers solution injected into the subretinal space), creating a large 23 mm
retinal hole with a hooked tip of a 25-G needle, and following up with a subtotal
vitrectomy. Without the vitrectomy, retinal detachments tended to spontaneously
reattach [27]. In their model of rhegmatogenous RD, they found a transient and
marked decrease in intraocular pressure and an increase in the rate of uorescein
disappearance from the vitreous. Based on these and other ndings, they concluded that there is a posterior-directed ow of uid through the retinal hole and
that the RPE uid pump function is increased.
Many models of traction RD have been developed using different methodologies [2,17,6871]. A reproducible model of traction RD in the cat was created
by rst inducing a serous retinal detachment using the rose bengal method (see
next paragraph), and then injecting dermal broblasts into the vitreous [68]. Trac-
Retinal Detachment
123
tion RD was produced within the rst 2 weeks after broblast injection, with
characteristics of vitreoretinal strands and epiretinal proliferation. Retinal degeneration was observed in this model. The causes of the retinal degeneration may
be attributed to multiple origins, including the associated photodynamic treatment
for inducing the serous detachment, vascular thrombosis, and the prolonged detachment itself. Experimental traction RD was also observed following injection
of autologous broblasts into the vitreous cavity of rabbits, and induction of
perforating ocular injury in rabbits, pigs, and monkeys [6971].
Serous retinal detachment is difcult to model. A reproducible model for
serous RD utilizes intravenous administration of the dye rose bengal, which immediately photosensitizes the choroid and RPE and can be photochemically activated in the eye using a xenon photocoagulator ltered to a central wavelength
of approximately 550 nm [72]. Adjusting the irradiance, duration of light exposure, and the dose of the rose bengal can control the peak and size of the resultant
serous detachment. In the cat, Wilson and colleagues showed that serous retinal
detachments as small as a focal retinal bleb (lasting 1 h) to a massive bullous
detachment comprising the entire retina (lasting 4 months) were induced with
this model. Considerable ischemic degeneration of the detached retina was observed in the region of the photothrombosis at 3 days following photodynamic
injury, but the retina outside this region appeared relatively undamaged. For detachments that lasted for at least 7 days, there was a complete loss of photoreceptor outer segments, and for detachments that lasted at least 14 days, the neural
retina was thin and gliotic with loss of the outer layers. Signicant occlusion of
blood vessels in the retina was also observed after 14 days of detachment. The
need to photosensitize the choroid and induce signicant photoactivated damage
to the retina, RPE, and choroid indicate major disparities between the model
and clinical serous RD. Experimental serous RD has also been created following
occlusion of choroidal circulation with microsphere embolization and in experimental malignant hypertension [7376]. To my knowledge, no systematic studies
of experimental serous, traction, or rhegmatogenous retinal detachments have
been conducted in the context of preventing retinal degeneration.
VI. CONCLUSION
Many experimental models of retinal detachment have been developed in the
past few decades, and a number of them have been used to carefully dene and
understand various aspects of retinal alteration, remodeling, and degeneration.
Quantitative and semiquantitative descriptions of retinal changes in experimental
retinal detachment are important because they provide the investigator with measurable parameters to evaluate neuroprotective and other vision-salvaging strategies. There remains a signicant medical need to enhance visual outcomes fol-
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Peterson
lowing anatomically successful retinal reattachment procedures. Because

macula-off detachments are more frequently seen in the clinic and because loss
of central visual acuity and color distortion are common following successful
macular reattachment surgery, a better understanding of the cellular and electrophysiological effects of experimentally detached macula is needed. In Burtons
study, only 2% of patients operated on within 5 days of macular detachment
were able to achieve postoperative visual acuity of 20/20 [9]. This 2% gure is
illuminating: it tells us that in macula-off detachments it is indeed possible to
restore visual acuity back to normal. The researcher is therefore motivated to
nd ways of utilizing experimental models of retinal detachment to develop new
therapies that enable the capacity of foveal cones to recover fully. However, the
remaining gure of 98% is dauntingit is a measure of the challenges that lay
ahead.
ACKNOWLEDGMENT
I would like to thank Drs. Steve Fisher and Sheldon Miller for their valuable
input and feedback on this chapter.
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8
Retinal Ischemia
Manuel Vidal-Sanz, Mara P. Lafuente, Inmaculada SellesNavarro, Mara E. Rodrguez, Sergio Mayor-Torroglosa, and
Mara P. Villegas-Perez
Universidad de Murcia
Murcia, Spain
I.
INTRODUCTION
In recent years, an increasing amount of experimental work has been directed

toward the search for substances (e.g., trophic factors, pharmaceutical compounds) that could diminish or delay neuronal degeneration secondary to central
nervous system injury or genetic diseases. The concept of neuroprotection as a
possible therapy for degenerative diseases of unknown mechanisms has gained
acceptance among the scientic community. Yet we are still at the stage of gathering bench-laboratory information necessary to understand the neuronal response
to injury, and thus it might take some time before neuroprotection becomes an
option for clinical management.
We will describe some of the methodological approaches used in our laboratory to study the fate of retinal ganglion cells after ischemia and will discuss
the problems, advantages, and disadvantages of these methods. Specically, we
will concentrate on two protocols recently used in our laboratory to induce transient ischemia of the retina. Our aim is to provide the researcher with a detailed
guide on these two experimental models to study the fate of retinal ganglion cells
after ischemic injury and neuroprotection.
129
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Vidal-Sanz et al.
II. METHODS
A.
Animal Care and Anesthesia
We have mainly used adult female Sprague-Dawley rats (200225 g) for our
experiments. The rats were obtained from the breeding colony of Murcia University or from Harlan Interfauna lberica (Barcelona, Spain). Animal care and experimental procedures were done according to institutional guidelines, European
Union regulations, and policies on the use of animals of the association for research and vision in ophthalmology (ARVO). All surgical manipulations were
carried out using general anesthesia.
Different types of anesthesia were used. For retrograde labeling, animals
were administered a mixture of ketamine (75 mg/kg) (Ketolar, Parke-Davis,
S.L. Barcelona, Spain) and xylazine (10 mg/kg) (Rompun, Bayer, S.A.
Barcelona, Spain) in sterile saline. For neuroprotection studies, animals were
anesthetized with intraperitoneal injections of 7% chloral hydrate in saline
(0.42 mg/g body weight), because both ketamine and xylazine have been reported to have neuroprotective effects on ischemia-induced neuronal damage
[1,2].
After experimental manipulations, animals were placed in their cages to
recover from anesthesia and a steroid-antibiotic ointment containing neomycin
and dexamethasone (Fludronef, Iquinosa, Madrid, Spain) was applied over the
ocular surface to prevent corneal desiccation. Rats were fed ad libitum and kept
in cages in temperature-controlled rooms with a 12-h light/dark cycle (light period from 8 a.m. to 8 p.m.). Light intensity in the cages ranged from 8 to 24 lux.
An operating microscope and standard microsurgical instruments were used for
all experimental procedures.
B.
Is the Retina Suitable for Neuroprotection

Studies?
The retina is a highly specialized part of the central nervous system that takes
its position in the front of the head during development. From this location, the
retina looks at the outside world and captures electromagnetic waves within the
visible spectrum; these are transduced into electrical signals that are in turn
processed through the different retinal layers to produce a retinal output. The
retinal ganglion cell (RGC) axons convey this output along the optic nerve toward several relay stations in the midbrain, where it is further analyzed and sent
over to the visual cortex where visual perception occurs. Several characteristics
of the retinocollicular system of the adult rodent make it suitable for in vivo
studies of neuronal survival after injury and administration of neuroprotective
substances.
Retinal Ischemia
131
1. Location
The retina is encapsulated within the eye globe, which is located in the orbit,
and is easily accessible for experimental manipulations. Moreover, the retina is
somewhat isolated from the rest of the CNS, and thus experimental manipulations
of one retina do not usually involve damage to the other retina or other parts of
the brain, and this allows animal survival for short- and long-term studies.
2. Natural Reservoir
The eye globe may act as a reservoir for substances injected into the vitreous.
These substances may be used to treat the retinal cells, or to label selectively the
entire axonal population of RGCs, thus allowing identication of retinal projections into the brain [3].
3. Vascular Supply
In the rat, the ophthalmic artery trifurcates into two posterior ciliary arteries (nasal
and temporal) and one central retinal artery (CRA). The CRA penetrates into the
eye through the inferior and nasal aspect of the optic nerve [47]. Seven or eight
terminal radial branches of the CRA are the main vascular supply to the inner
layers of the retina. The choroidal blood vessels provide nutrients to the outermost
one-third of the retina, and originate from the two posterior ciliary arteries. Arterioles arising from the posterior ciliary arteries, as well as from the CRA, are the
main source of blood ow to the optic nerve head [6]. Because the retina is
encapsulated within the sclera, which is a semi-rigid structure, increments of the
intraocular pressure directly affect retinal blood ow. In addition, the ophthalmic
artery may be identied intraorbitally on its course along the ON sheath before
it trifurcates and is dissected and ligated, thus interrupting retinal and choroidal
blood supply.
4. Optic Nerve Manipulations
The intra-orbital segment of the optic nerve can be readily identied, dissected,
and injured. The ophthalmic artery does not enter the ON, like in most mammals,
but courses within the ON sheath, and this allows experimental manipulations
of the ON without compromising retinal blood supply [8,9].
5. Identication of Retinal Neurons
The great majority of RGCs in the albino rat send their axon to the supercial
layers of the superior colliculi (SCi) [10] and a proportion (35%) of these also
send a collateral to the dorsal lateral geniculate nuclei (dLGNi) [11]. Application
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of retrogradely transported tracers to the main retinorecipient territories (e.g., the

SCi), or to their cut axons in the ON results in the selective labeling of the RGC
population. Because the retina may be examined as a at mount, the entire RGC
population can be observed in one preparation, and this facilitates quantitative
estimations of the RGC population in the normal or experimental retina. In addition to RGCs, the retina comprises ve other different classes of neurons, whose
cell somata and processes are all conned to the retina. The cell somata of these
are organized into three nuclear layers (outer, inner, and RGC layer) with two
intermingled synaptic layers (outer and inner plexiform layers). This layered
structure may be readily examined in retinal cross-sections. Furthermore, specic
classes and subclasses of retinal neurons and cells may be identied using retrograde labeling techniques combined with immunocytochemical techniques [12].
C.
Retrograde Labeling of the RGC Population
It is difcult to distinguish RGCs from the many displaced amacrine cells in the
RGC layer [13] on the basis only of classical anatomical methods [12]. Furthermore, identication of injured RGCs is hindered because these may suffer phenotypic changes, including morphological [14], immunocytochemical [8,12], and
molecular [15,16]. To identify the RGC population we have used retrogradely
transported neuronal tracers applied to the terminals or cut axons of the RGCs.
Identifying and counting RGCs is important when evaluating whether a substance
may have neuroprotective effects.
1. Use of FluoroGold as Retograde RGC Tracer
The RGC population was retrogradely labeled from the superior colliculi (SCi)
with the uorescent tracer FluoroGold (FG) (Fluorochrome Inc., Engelwood, CO)
following protocols that were originally described in 1988 [17]. In brief: The
Midbrain was exposed, the pia mater overlying both SCi was gently removed,
and a small piece of gelatin sponge (Spongostan Film, Ferrosan, Denmark)
soaked in a solution of 3% FG and 10% dimethyl sulfoxide in saline was laid
over the SCi. Because most RGCs in the rat project to the SCi [10], this procedure
results in the labeling of almost the entire RGC population in both retinas [17].
Previous studies from our laboratory [18,19] have shown that following
FG application to the SCi, some RGCs appear labeled as early as 3 days after
dye application, and most RGCs appear labeled by 7 days after dye application.
At this time, the densities of FG-labeled RGCs are similar to those obtained when
other uorescent and nonuorescent retrogradely transported tracers are applied
to the main retinorecipient target regions in the brain [12,17,20]. Furthermore,
FG application to the SCi [19] or the lateral rectus muscle [21] results in the
labeling of the entire population of RGCs or abducens motoneurons, respectively,
Retinal Ischemia
133
for up to 4 weeks after tracer application without leakage or apparent fading.

Longer survival periods, however, were associated with a decrease in the numbers
of labeled cells [19,21], and by 3 months only one-third of the population of
abducens motoneurons was still labeled with FG [21]. At this time point, the
entire population of abducens motoneurons could be labeled again if FG was reinjected into the ipsilateral rectus muscle, suggesting that FG is not toxic to neurons
but disappears slowly from the neurons with increasing survival periods [21].
2. Use of di-ASP as Retrograde RGC Tracer
To identify RGCs in long-term studies we have used two different methods. In
a rst method, we labeled the RGC population with dil, a lipophilic carbocyanine
which persists within the RGC somata for periods of up to 21 months after tracer
application without leakage or fading [17,20]. A second method consists of
applying retrograde tracers to the axons [12,22] or terminals [23] of RGCs,
shortly before animal processing.
Here we describe the use of DiAsp applied intraorbitally to the ocular stump
of the transected ON. The lipophilic dye 4Di-10Asp [DiAsp, D29, N-4-4-4-didecylaminostyryl-N-methylpyridinium iodide, Molecular Probes, Eugene, OR] can
be applied to the RGC target territories or their cut axons to retrogradely label
RGCs, both in control and experimental animals [22,24,25]. In brief: Three days
before sacrice, the left ON is exposed in the orbit, dissected from its surrounding
sheaths, and cut close to its origin in the optic disc [8]. DiAsp is then applied
to the ocular stump of the intraorbitally transected ON. To facilitate dye uptake
by cut axons, crystals of DiAsp were previously dissolved in dimethylformamide
and the solvent allowed to evaporate. Intraorbital transection of the ON induces
RGC death [12,20], but this rst appears between day 4 and 5 after axotomy
[18,26]. Therefore, it is likely that the densities of DiAsp-labeled RGCs 3 days
after dye application would not be affected by axotomy-induced RGC death. However, we cannot exclude the possibility that transient ischemia of the retina prior to
DiAsp application shortens this 45 day period before the onset of cell death.
D. Induction of Retinal Ischemia
A variety of procedures have been described to induce retinal ischemia in laboratory animals. These may be divided into two main groups. One consists of increasing the intraocular pressure (IOP) above systolic levels to interrupt blood
ow within the eye, and the other consists of the ligature of the blood vessels
supplying the retina. Retinal ischemia may be permanent or transient if reperfusion is allowed (for review, see Table 3, Ref. 27). In the following section we
describe the methods used in our laboratory to induce transient ischemia of the
retina by elevation of the IOP [19] or by selective ligature of the ophthalmic
vessels [2832].
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1. General Considerations
Ischemia-induced CNS damage is inuenced by body temperature, which should
be kept constant throughout the duration of the experiment. This is also the case
for experiments aimed at studying ischemia-induced RGC death [33]. In a previous study, in which the room temperature was maintained constant at 23C, the
body temperature of a group of rats anesthetized and subjected to transient retinal
ischemia was monitored with a rectal probe. The body temperature uctuations
in these animals ranged from 35.4C right after initiation of retinal ischemia to
33.8C 2 h later [19]. Because in a large series of similarly prepared experiments
the densities of RGCs surviving different periods of ischemia were very consistent, it is possible that small body temperature uctuations did not interfere with
RGC survival [19]. Furthermore, the temperature within the eye was not monitored; thus, we ignore whether these small changes in body temperature also
inuenced retinal temperature and if this in turn affected RGC survival in those
experiments.
The retinal blood ow may be monitored through the operating microscope.
Inspection of the eye fundus is facilitated by pupil dilation with a topical drop
of 1% tropicamide (Cusi Laboratories, El Masnou, Barcelona, Spain). Corneal
desiccation throughout the experiment is avoided by applying on the cornea a
solution of 2% hydroxypropylmethylcellulose (Gonioftal 4000, Cusi Laboratories, El Masnou, Barcelona, Spain). The placement of a coverslip over the cornea facilitates direct visualization of the eye fundus through the microscope. The
eye fundus of the albino rat shows the central retinal artery dividing into six or
seven radial vessels as well as six or seven radial veins that converge toward the
disc into the central retinal vein.
2. Increase of the Intraocular Pressure Above Systolic
Levels
We induced retinal ischemia in the left eye by increasing the intraocular pressure
(IOP) above systolic arterial levels (see below). In these animals, as well as in
those in which ischemia was induced by selective ligature of the ophthalmic
vessels, the right intact eye served as control with the animals under deep anesthesia, two nylon monolament 6/0 sutures were placed on the superior and inferior
bulbar conjunctiva of the left eye close to the corneo-scleral limbus. These sutures
were pulled tangentially in opposite directions until retinal blood ow was interrupted completely; this was assessed by examination of the eye fundus. The sutures were then tied to a metal frame, specially devised for these studies, to maintain the IOP above systolic levels. Inspection of the eye fundus was required
throughout the duration of the ischemic period to ascertain blood ow arrest (Fig.
1a). Retinal ischemia is characterized by pallor of the iris and intense pallor of
the eye fundus, as well as by blood ow arrest within radial retinal vessels. These
were either empty or showing fragmented columns of arrested red blood cells.
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135
Figure 1 Schematic representations of the methods employed to induce transient

ischemia of the retina by increasing the intraocular pressure above systolic levels
(a) or by selective ligature of the ophthalmic vessels (b). Both procedures induce
choroidal and retinal blood ow interruption. (a) Two sutures were placed on the
superior and inferior bulbar conjunctiva close to the corneo-scleral limbus. These
sutures were pulled tangentially in opposite directions to increase the intraocular
pressure until retinal blood ow was interrupted completely. This was assessed by
examination of the fundus. (b) The optic nerve (ON) is exposed in the orbit and the
dural sheath opened longitudinally. A ne 10/0 suture is placed between the ON and
the sheath and tied around the sheath avoiding damage to the ON. Because the ON
contains the ophthalmic artery, this procedure interrupts retinal blood ow.
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Vidal-Sanz et al.
At the end of ischemia, the sutures were released slowly and this allowed
restoration of the retinal blood ow. Reestablishment of blood ow was characterized by increasing movement of the fragmented columns of red cells, the progressive lling of the radial vessels to their normal pattern, and the pink appearance of the fundus. Reestablishment of blood ow within the retina, if not
spontaneous, may be facilitated with gentle eye massage. Animals in which restoration of the retinal blood ow did not occur, or was associated with retinal or
vitreous hemorrhages, were discarded from the study. Regular ndings at the
termination of the period of ischemia were a moderate edema of conjunctiva and
cornea [19].
3. Selective Ligature of the Ophthalmic Vessels
Under deep anesthesia, the left optic nerve was exposed in the orbit and the ON
sheath was opened longitudinally [8] (Figs. 1b, 2a). A ne 10/0 monolament
suture was carefully introduced between the ON and the sheath, and tied around
the sheath, avoiding damage to the ON (Fig. 2b). Because the ON sheath contains
the ophthalmic artery [6], this procedure interrupts retinal and choroidal blood
ow. The ligature may be released after different ischemic intervals to allow
retinal reperfusion. The eye fundus may be examined directly through the operating microscope to assess retinal blood ow before, during, and after retinal
ischemia (Fig.3) [3032].
E.
Administration of Neuroprotective Substances
Substances to be tested for their neuroprotective effects may be administered

before, during, or after the insult. These may be given systemically or topically
or delivered directly into the vitreous [34]. Topical instillation is a simple procedure that may be done in the alert animal.
For direct administration into the vitreous, the animal is sedated with anesthesia, the pupil is dilated, and topical anesthetic eyedrops are also instilled on
the eye. A 30-G needle is used to produce a penetrating puncture through the
conjunctiva, sclera, choroid, and retina at approximately 12 mm from the corneo-scleral limbus. The needle of 5 L Hamilton microsyringe is then introduced
through this puncture into the vitreous and directed toward the posterior pole to
avoid injury to the lens [34]. Direct injury to the lens results in lens opacity and
may have neuroprotective effects on RGC survival [35,36]. Injection volumes
were not greater than 5 L, and saline was used in most instances as vehicle.
F.
Tissue Processing
After various survival times, animals were given an overdose of anesthetics (ketamine and xylazine) and perfused through the heart rst and briey with 0.9%
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Figure 2 Micrographs taken with the operating microscope illustrating the procedures employed for selective ligature of the ophthalmic vessels. (a) Micrograph
illustrating the posterior part of the eye, the optic nerve head (*) and its surrounding
dural sheath. (b) The dural sheath has been opened longitudinally and a ne suture
is tied around the sheath avoiding damage to the ON (*).
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Vidal-Sanz et al.
Figure 3 Micrographs illustrating
the eye fundus of the retina before
ischemia (a), during ischemia (b), and
shortly after reperfusion (c). These
micrographs were taken through a
video camera connected to the operating microscope. (a) A normal eye
fundus shows typical radial retinal
vessels. (b) During the period of transient ischemia induced by selective
ligature of the ophthalmic vessels,
these appear empty or with fragmented columns of red cells that do
not move. (c) Shortly after release of
the ligature around the ophthalmic
vessels, blood ow is restored within
radial retinal vessels.
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NaCl followed by 4% paraformaldehyde in 0.1 M phosphate buffer. Both eyes

were enucleated and an 8/0 suture was placed in the superior aspect of the conjunctiva near the limbus for orientation purposes. The retina may be examined
in cross-sections or in whole-mounts. In the following section we describe the
methodology employed to obtain attened whole-mounts.
1. Retinal Whole-Mounts
The retinas were dissected out and prepared as attened whole-mounts by making
four radial cuts, the deepest one indicating the superior pole of the retina and the
others indicating the nasal, temporal, and inferior pole of the retina, respectively.
Flattened whole-mounts were transferred to a piece of lter paper and this was
immersed in the same xative for 1 h. The retinas were released from the lter
paper, washed in the buffer solution, and mounted vitreal side up on subbed
slides.
For experiments in which DiAsp was used to retrogradely label RGCs, 0.1
M sodium carbonate buffer (pH 9) was used as mounting media. In animals in
which FG was used as retrograde tracer, mounting media consisted of 50% glycerol in 0.1 M sodium carbonate buffer (pH 9) containing 0.04% of p-phenylenediamine [37].
Retinas were examined under uorescence microscopy (Axiophot, Zeiss,
Oberkochen, Germany) with uorescein (BP 450-490, LP520) and ultraviolet
(BP 365/12, LP 397) lters that allow the observation of the green-yellowish
uorescence of DiAsp and of the white-gold uorescence of FG, respectively. It
is our experience that examination of DiAsp should be done within the rst 24
h after processing the animal because the dye tends to dissipate and fade away,
thus losing its uorescence quality soon after mounting.
2. Photographing and Counting Labeled RGCs
Densities of surviving RGCs in the at mounted retinas were estimated following
previously described methods to determine densities of RGCs within the central
regions of the retina [12,1720,30], where RGCs are the most prevalent [38,39].
In brief, FG-labeled RGCs were counted in a masked fashion from printed
micrographs (400) taken from 12 standard rectangular areas of 0.0864 mm2
of each retina situated, three in every retinal quadrant, at approximately 0.875,
1.925, and 2.975 mm, respectively, from the optic disc. A labeled RGC was
counted if the whole cell or its nucleus was visible within the micrograph. Counts
in the 12 regions were averaged and divided by the area of the picture to obtain
a mean RGC density (cells/mm2) per retina. For groups of similarly treated
retinas, results were reported as mean RGC densities SD (standard deviation). Cell counts were done in a masked fashion from printed micrographs; the
identity of the retinas that led to the micrographs was not known until cell counts
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Vidal-Sanz et al.
from different groups were nished. For statistical analysis, RGC densities were
compared using two non-parametric tests: the one-way ANOVA Kruskal-Wallis
and Mann-Whitney tests, with P values of 0.05 considered statistically signicant.
3. General Appearance of Retinas Labeled with FG
or Di-ASP
In control retinas, only the retinal cells labeled with FG have the typical characteristics of RGCs. These are observed in the RGC layer, but there is a small proportion of displaced RGCs (Dogiel corpuscles) that may be observed in the inner
plexiform or inner nuclear layers. FG-labeled RGCs have the typical punctate
and diffuse FG uorescence, which delineates their soma and occasionally initial
segments of their primary dendrites (Fig. 4a, b). In a recent study, the densities
of FG-labeled RGCs in the control retinas of large series of rats were estimated
following the above-described methods [29,30]. Although there were small variations in the mean densities of FG-labeled RGCs in some control retinas, overall
these were rather consistent [29,30] (Fig. 5). These variations can be attributed
to slight differences in tracer application or in the efciency of the batches of
tracer employed, and also have been observed in previous studies from this laboratory [18,19]. Nevertheless, in the experimental groups, the densities obtained
for the right intact retinas may be used as 100% survival for their contralateral,
left operated retinas.
In the experimental left retinas subjected to various periods of ischemia,
in addition to RGCs, microglial cells also appear intensely labeled with FG. These
cells, which can be easily distinguished from RGCs on the basis of their morphology (cells with bright uorescence in their small soma and multiple ne tortuous
processes), phagocytose the debris of degenerating FG-labeled RGCs and thus
become FG-labeled [24]. FG-labeled microglial cells have been found in a number of studies in which FG was applied to the SCI before retinal injury in the
rat [18,19,36] and goldsh [40].
In control and experimental retinas in which we used DiAsp as retrograde
tracer, the only cells that appeared DiAsp-labeled were RGCs. These showed
typical punctate and diffuse DiAsp uorescence delineating the soma, axon, and,
occasionally, primary and secondary RGC dendrites (Figs. 6, 7). The mean densities of RGCs labeled with di-ASP applied to intraorbital ON stump in a control
group of nine rats (22) was somewhat smaller than the densities of labeled RGCs
when other nonlipophilic tracers were applied to the SC or the ON (1719). This
could be explained by the limited solubility of the dye, which resulted in lower
labeling efciency. Because DiAsp application involves transection of the ON,
the low numbers of DiAsp-labeled RGCs found in our experiments could also
be explained by the axotomy-induced retrograde degeneration of RGCs. However, this is not likely because axotomy-induced RGC death rst appears between
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141
Figure 4 Retinal ganglion cells (RGCs) retrogradely labeled with FluoroGold

(FG) in whole-mounted retinas. FG was applied to the superior colliculi one week
prior to animal processing. (a) The central region of the retina in the superior temporal quadrant of this retina shows FG-labeled RGCs separated by radially oriented blood vessels and axon bundles (218). (b) At higher magnication, FGlabeled RGCs show the typical diffuse and granular uorescence depicting their
somata (436).
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Vidal-Sanz et al.
Figure 5 Micrograph of the whole-mounted right retina in an adult PVG rat 1

week after FluoroGold application to both superior colliculi. FG-labeled RGCs are
evenly distributed throughout the retinal quadrants. The micrograph was prepared
with the aid of a motorized stage on a photomicroscope with a high-resolution camera connected to an image analysis system with an automatic frame grabber device (Image-Pro Plus, V4.1; Media Cybernetics, Silver Spring, MD, USA). The
superior aspect of the retinas is between 1 and 2 oclock orientation. (Scale bar
1 mm.)
4 and 5 days after ON section close to the eye [18,26], and we estimated RGC
densities 3 days after dye application [22]. Moreover, in experiments in which
DiAsp was applied to the SC to retrogradely label RGCs, Thanos [24] reported
densities of DiAsp-labeled RGCs similar to those obtained when we applied DiAsp to the cut ON, indicating that DiAsp has lower labeling efciency than other
neuronal tracers. Finally, the absence of DiAsp-labeled microglial cells in our
experiments further supports that there is no RGC death in the rst 3 days after
ON section.
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Figure 6 Retinal ganglion cells (RGCs) retrogradely labeled with DiAsp applied
to the ocular stump of the intraorbitally transected optic nerve 3 days before animal
processing. The central retina of the superior temporal quadrant shows typical DiAsp labeling of retinal ber bundles as well as the somata and primary dendrites
of RGCs (441).
Figure 7 Retinal ganglion cells (RGCs) retrogradely labeled with DiAsp applied
to the ocular stump of the intraorbitally transected optic nerve 3 days before animal
processing. The central region of the inferior-nasal quadrant shows typical intense
DiAsp labeling of RGC axonal bundles and somata (59).
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Vidal-Sanz et al.
III. DISCUSSION
Several requirements need to be met for the above-mentioned protocols to be
useful. These include, among others (1) reproducibility of the same insult given
to the retina; (2) consistency of the results produced with a given type of insult;
and (3) reliable estimates of the effects of the insult in the survival of the RGC
population and/or other retinal neurons.
A.
Requirements Needed for the Protocols to Be

Useful
1. Reproducible Insult
For an insult to be reproducible, it should be performed equally every time and
should also lead to similar results. Thus, an identical procedure should be performed every time that transient ischemia is given to the retina, whether this is
induced by elevated IOP or by ligature of the ophthalmic vessels. A number of
investigators monitor the level of the IOP reached to induce blood arrest with
the use of intraocular cannulas connected to a manometer system [4146]. In
our experiments the eye fundus was constantly inspected to ensure ow arrest,
but accurate measurements of the IOP were not done. Nevertheless, studies in
which a number of animals were treated with similar periods of ischemia showed
consistent numbers of surviving RGCs [19], suggesting that small uctuations
in the intraocular pressure reached to arrest blood supply did not affect RGC
survival substantially.
Ligature of the ophthalmic vessels seems to be a rather consistent method
to induce blood arrest into the retina [30]. The reproducibility of this method
relies on the ability of the investigator to perform in identical fashion the dissection and ligature of the ophthalmic vessels that run within the ON sheath. Another
variable to be taken into account when performing these studies is the difference
in the period of time (usually a few minutes) from ligature release to full retinal
reperfusion.
2. Consistency of Injury-Induced Cell Loss
The results of a large series of experiments in which transient ischemia of the
rat retina was induced by elevated IOP were highly consistent for the different
groups analyzed after different survival (reperfusion) intervals [19]. Similarly,
consistent results in the amount of RGC loss were observed in another series of
experiments in which transient ischemia of the rat retina was induced by ligature
of the ophthalmic vessels and RGCs were labeled with DiAsp [31] or FG
[29,30,32]. Altogether, these studies indicate that RGC survival after different
periods of transient ischemia and survival intervals is a highly consistent and
predictable nding.
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145
3. Reliable Estimates of Surviving Cells

The studies mentioned above were based on counts of cells retrogradely labeled
with dyes applied to their cut axons or their targets. The efciency of the tracer
employedthat is, the proportion of retinal ganglion cells that becomes retrogradely labeledshould be well established. Another important issue is to be
able to reproduce consistently similar proportions of labeled retinal ganglion cells
every time the dye is applied.
The techniques employed in these experiments sampled similar regions of
the retinas located within the central retina, where higher densities of RGCs are
observed [13], but overlook RGC densities within more peripheral areas of the
retina. While this is a reliable and consistent method to obtain RGC densities, a
more accurate estimate should include counting all the FG-labeled RGCs within
each retina, but this is major job to be done manually. RGC counts with an
automatic image analysis system of a digital image of the retina has been precluded by technical difculties in identifying, single FG-labeled neurons, FGlabeled microglial cells, and FG-labeled cells that are located close together as
it happens for the central regions of the retina (see Fig. 4a,b). Recent advances
in image-analysis may permit in the future more detailed analysis off the RGC
population [23,39], including the identication of different types of RGCs, as
dened by their morphological properties [47].
Screening studies on the effects of neuroprotectants with the above-mentioned techniques rely on the interpretation of labeled RGCs as alive, rescued
neurons. In some instances, neuronal cells were labeled prior to injury with tracers
applied to their main targets in the brain (e.g., Refs. 34,30). In other instances
these cells were labeled after injury with tracers applied to their targets [23], or
their cut axons [31]. While this assumption is generally accepted for many of
these studies [18,19,23,2932,34,35], we cannot be certain that rescued RGCs
retain all their normal physiological properties. Other studies have shown that
retinal cells may undergo functional and metabolic decits before they actually
die [16,48,49]. For example, chronic ligature of both carotid arteries did not induce evident loss of photoreceptors until 9 months later, but functional correlates
indicated that both the a and b waves of the electroretinogram were clearly pathological, as early as 90 days after bilateral carotid occlusion [50]. This highlights
the need for further studies to ascertain functional viability of retinal neurons
surviving ischemic injury [51].
B. Advantages and Disadvantages of These
Methods
1. Increasing IOP Above Systolic Levels
Increasing the IOP above systolic levels with two sutures pulling from both sides
of the eye has the advantage of being a noninvasive method. No puncture is given
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Vidal-Sanz et al.
to the eye and/or the retina, and therefore nonspecic neuroprotective effects
[35] that may originate as a consequence of lens injury [36] are discarded. However, the main disadvantage of this method is that it is tedious and time consuming; constant observation of the eye fundus of the retina to ascertain blood arrest
is required to adjust tension on the sutures when necessary.
The duration of the period of transient ischemia inuences both the amount
and pattern of RGC loss [19]. This fact should be taken into account when designing experiments aimed at investigating possible neuroprotective effects. Furthermore, we ignore whether in addition to blood ow arrest, increased intraocular
pressure also induces compression or axotomy-like damage to retinal axons. If
this were to be the case, an additional axotomy-like insult is given to the retina.
2. Ligature of the Ophthalmic Vessels
Transient ischemia of the retina may be induced with methods that do not increase
the intraocular pressure. Transient ligature of the carotid [48,52] and vertebral
arteries [53] induces retinal ischemia but may also affect the brain. Clamping or
ligating the ophthalmic vessels together with the ON have been used to induce
retinal ischemia [35,5457]. However, these were short-term experiments and, in
addition to retinal ischemia, these procedures also induced ON axotomy. For the
purpose of our studies, aimed at investigating the short- and long-term effects of
retinal ischemia, we have preferred to induce retinal ischemia by the selective ligature of the ophthalmic vessels, avoiding direct mechanical damage to ON bers.
Selective ligature of the ophthalmic vessels was rst used in monkeys by
Hamasaki and Kroll [58] and later applied to rats [2832,51]. Selective ligature
of the ophthalmic vessels induces in the retina pathological ndings that are similar to those found after ischemia induced by elevated IOP, including the loss of
RGCs and the thinning of the IPL, INL, and OPL [59]. This method does not
increase the IOP and, thus, there is no intraocular compression of retinal axons,
avoiding axotomy-like injury to RGC bers. The effects of ligature of the ophthalmic vessels on the ON head were not studied, but it is likely that transient
ligature of the ophthalmic vessels also induces transient ischemia of the ON head.
The main source of blood ow to the ON head in the rat comes from branches
of the posterior ciliary arteries as well as from arterioles arising from the central
retinal artery [6], and thus it is likely that ligature of the ophthalmic vessels affects
blood ow within the ON head.
C.
Further Implications of These Models
Retinal ischemia induced by any of the above-discussed methods induces an early

loss of RGCs [19,31]. The loss of RGCs that appears shortly after retinal ischemia
may be prevented, at least in part, with the use of different neuroprotective agents.
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147
A recent article reviews several studies on RGC neuroprotection after retinal

ischemia [27]. The variety of drugs employed in these experiments may be taken
as an index of the many steps involved in RGC death that may be halted, and
further points to the multiple factors involved in the process of ischemia-induced
RGC death (see Table 7, Ref. 27). The techniques described here do not address
questions related to the specic mechanisms of action of putative neuroprotectants. An efcient neuroprotective drug should diminish injury-induced RGC
death, and this may be accomplished either through a direct effect on injured
RGCs or indirectly by activating other non-RGC neurons or nonneuronal cells
of the retina. Thus, whether the neuroprotective effect of a given drug is directly
mediated through the RGCs themselves, or indirectly mediated through other
cells in the retina, should be tackled with additional methods.
In addition to the early loss of RGCs, retinal ischemia also induces a protracted loss of RGCs [19,31]. The mechanism by which the initial insult triggers
such a long-lasting and prolonged process of RGC death is unknown. Whether
injury triggers the immediate death of a subset of RGCs, and this in turn leads
to secondary RGC death is currently unknown [31]. In this context, it is worth
noticing that ideal neuroprotection should prevent both the early as well as the
secondary slow loss of RGCs that were not primed to die in the rst instance
but that disappear with time [2932].
Therapeutic interventions after CNS ischemia are at present mainly directed
toward the so-called penumbra zone, a region of incomplete ischemia that surrounds the core of the infarcted region, which is most susceptible to reperfusion
damage. The above-discussed models involve complete ischemia of the retina,
and there may be differences in susceptibility of different retinal neurons depending on their location within the retina [55,60]. However, the concept of penumbra zone after ischemia may not be fully reproducible in these models of
retinal ischemia. Moreover, a few minutes of ischemia induce irreversible damage
in the CNS [6163], whereas in the retinal models examined in these studies
(increase of the IOP and SLOV), the transient period of ischemia required to
produce RGC death is substantially longer. For example, 5 min of global cerebral
ischemia induced by bilateral carotid artery occlusion are enough to produce neuronal death of nearly all the CA1 pyramidal neurons within the gerbil hippocampus after 5 min global cerebral ischemia [61,62]. In contrast, periods of 4560
min of pressure-induced retinal ischemia are required to induce RGC death [19].
Transient ischemia of the retina has been used as a basic research model
for glaucoma because ischemia may be involved in the pathogenic mechanism
of this disease. In addition, both ischemia and glaucoma involve progressive loss
of RGCs [19]. However, both the above-discussed methods to induce retinal ischemia elicit a rapid and massive RGC loss, and this is clearly not the case for
chronic neurodegenerative conditions such as glaucoma, where RGC death appears to be a progressive but much slower event. Moreover, retinal ischemia also
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Vidal-Sanz et al.
induces the loss of other non-RGC neurons [59] and this may not be the case
for glaucoma [64].
Ligature of the ophthalmic vessels does not fully resemble the clinical situation of acute interruption of the blood ow either by central artery or vein occlusion. In these diseases, the outer retina is usually not affected, while ligature of
the ophthalmic vessels interrupts blood supply to the retina and choroid, thus
leading to different pathological ndings. Preliminary results indicate that after
ligature of the ophthalmic vessels, not only the RGC layer but the other retinal
layers were severely affected by the initial period of transient ischemia [65].
The ligature of the ophthalmic vessels and the pressure-increased models to
induce retinal ischemia do not fully reproduce human conditions. Nevertheless,
these are useful models in neurobiology research to induce progressive neuronal
degeneration of the RGC population, and are suitable models to examine the consequences of such insults in the different retinal layers after retinal ischemia. The fact
that other retinal layers also degenerate with time after these insults may provide an
opportunity to explore the progressive loss of other non-RGC neurons of the retina,
including the inner and outer nuclear layer neurons. Moreover, these models may
be used as powerful in vivo tools to screen for compounds that may have relevant
neuroprotective effects against ischemia-induced neuronal cell death [2932,65].
IV. CONCLUSIONS
The above described methods to induce retinal ischemia cannot be extrapolated
to clinical conditions in the human eye. Nevertheless, they provide a powerful
tool to examine the fate of retinal neurons after ischemia and to further explore
future therapeutic interventions to lessen the effects of transient ischemia.
ACKNOWLEDGMENTS
This work was supported by research grants from the Regional Government of
Murcia (Fundacion Seneca, PI-92/00540/FS/01), the Spanish Ministry of Science and Technology (BFI2002-03742), The Spanish Ministry of Health (03/13;
FIS PI020407), and the European Union (QLK6-CT-2000-00569 and QLK6-CT2001-00385). The authors thank A. Aviles, M. E. Aguilera, and J. M. Bernal for
technical support.
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9
Drug Delivery
Robert W. Nickells and Cassandra L. Schlamp
I.
INTRODUCTION
The growing concept of being able to protect neurons from damaging stimuli,
or being able to rescue them from actively executing a cell death program, carries
with it the requirement of being able to deliver the appropriate drug or agent to
the target cells. In the eye, this means being able to apply an effective dose of
an agent to the retina and optic nerve. In some cases, it may also mean that the
drug be applied continuously or for a sustained period of time in order to achieve a
therapeutic effect. This chapter describes methods that have been used by various
laboratories to deliver agents, most notably small pharmacological molecules,
successfully. Each method has its advantages and disadvantages, which will be
discussed.
There are four basic modes of application to deliver a drug to the back of the
eye. These are trans-corneal or topical, intravitreal, trans-scleral, and systemic [1].
II. TRANS-CORNEAL APPLICATION OF DRUGS

There is a great deal of interest in the trans-corneal route of drug administration
because it is an easy method of employment for patients and, in the case of
glaucoma, could complement a patients regular use of eyedrops. Also, some of
the ophthalmic drugs currently used to lower intraocular pressure appear to have
a limited neuroprotective effect, making the use of these agents commercially
important.
153
154
A.
Nickells and Schlamp
Application of Drug and Analysis of Effect
The delivery of drugs to the posterior pole of the eye by topical application to
the cornea faces several challenges, including a long diffusion pathway to the
target tissue, corneal impermeability to large molecules, and convectional ow
of intraocular uid in the opposite direction. Protocols designed to examine the
effects of drugs delivered to the cornea must therefore demonstrate penetration
to the posterior pole and biological efcacy. A recent and careful study by Mizuno
et al. [2] to test the effects of nipradilol met these challenges in a study using
male Japanese White rabbits.
B.
Penetration Studies
Wake rabbits were placed in restraining cages. Radiolabeled [3-14C]-nipradilol

(Amersham Pharmacia Biotech) was applied to the lower cul-de-sac of the right
eye twice daily (100 L of a 1 % solution) for 7 days. Care was taken to apply
the agent at the same times each day. Thirty minutes after the nal application,
the rabbits were anesthetized with phenobarbital and the eyes enucleated. The
basic control for these experiments was to apply saline (or appropriate vehicle)
to fellow eyes and to eyes of rabbits that did not receive radiolabeled agent.
Enucleation was performed by the following procedure to minimize contamination of ocular tissues with the radiolabeled nipradilol. An incision was
made along the outer edge of the orbit and the eyelids were clamped together
with a surgical clip. The entire globe, complete with eyelids and surrounding
tissues, was then removed as a pouch. The pouch was then immediately immersed
in hexane and solid carbon dioxide for 2 min and then stored at 15C.
Distribution of the [3-14C]-nipradilol was analyzed by dissecting the tissues
of the eye under semi-frozen conditions, solubilizing them, and counting them
in a scintillation counter. The actual amount of agent that had penetrated different
tissues was calculated from the quench-corrected measurement of radioactivity
and the known specic activity of the radiolabeled compound. Note that quench
corrections are routinely performed automatically by modern scintillation counters and specic activity should be determined as a function of the half-life and
the age of the isotope being used.
Tissues that were analyzed in the Mizuno study included anterior chamber
components (cornea, aqueous humor, iris, and lens) and posterior chamber components (vitreous and retina/choroid). After two applications every day for 7
days, the vast majority of labeled nipradilol was localized to the cornea (1284fold over controls) and iris, with moderate levels being detected in the aqueous
humor. Substantially less nipradilol was detected in the lens, vitreous, and retina/
choroid (threefold over controls), but the levels in the retina were still signicantly higher than in control eyes. Also, the effective concentration of nipradilol
Drug Delivery
155
in this tissue was estimated at 0.12 M, which was in the clinically effective
range of the drug.
C. Efcacy Studies
In many studies, efcacy is measured as the ability to prevent loss of target cells
in the retina. It is important to note, however, that topically applied drugs could
achieve these effects through systemic means (i.e., affecting ocular blood ow)
rather than by directly acting on the target cells. To test the physiological effect
of this dose of nipradilol, Mizuno et al. utilized a bioassay that took advantage
of the antagonist effect of nipradilol on endothelin-1. Nipradilol has a vasodilating
activity, whereas endothelin-1 has a vasoconstricting effect. Fundus photography
was utilized to determine the diameter of the two major retinal arteries at the rim
of the optic nerve head. The change in artery diameter was monitored with successive fundus photographs after an intravitreal injection of 20 L of 5 108 M
endothelin-1. These experiments showed that topical application of nipradilol provided a signicant reduction in the vasoconstriction effects on intravitreally injected endothelin-1 up to 60 min, indicating that sufcient amounts of the topically
applied drug had penetrated to target endothelial cells at the back of the eye.
D. The Route of Trans-Corneal Delivery
to the Back of the Eye
Radiolabeled-drug studies suggest that the route of drugs administered to the
cornea to the back of the eye is periocular, rather than through diffusion across
the cornea and on into the vitreous. The evidence indicating this is the very low
level of accumulation of drug in the vitreous. In addition, nipradilol (molecular
weight of 326.35) is small enough to penetrate through the sclera, where the
permeability constant is inversely proportional to the molecular weight for solutes
between 285 and 70,000 daltons (see Sec. IV). Additionally, studies have shown
that both timolol and betaxolol accumulate in the connective tissue of Tenons
capsule in patients with prolonged usage of these drugs as topical drops [3],
supporting this mode of access to the posterior pole.
III. INTRAVITREAL DELIVERY

Intravitreal application is the most direct method for delivering a compound to
the cells of the retina. It has been used to test recombinant viruses carrying neuroprotective genes [4], recombinant proteins such as growth factors [57], and
small compounds such as upirtine [8].
156
A.
Intravitreal Injections
Injections into the vitreous are relatively straightforward and in most animals can
be accomplished with a suitably small gauge needle. Injections are always made
in anesthetized animals. Rats can be injected with a 2730 G needle with little
difculty. Mouse eyes are more difcult to inject into and require a two-stage
process of rst piercing the conjunctiva and sclera with a 30-G needle followed
by passing a glass micropipette through the hole into the eye [9]. Ideally the
micropipette should be pulled mechanically using Drummond borosillicate glass
capillary tubing (Cat. No. 3-000-210-G/ID 0.704 mm). Injections are easiest using needles with a long tapered point rather than a steep point typical of pipettes
used for single cell injections or electrophysiology. Hand-pulled glass pipettes
can also be used successfully. A total maximum volume of 2 L can be injected
into the mouse eye without damaging the retina. There are reports that up to 10
L injections can be made into the rat eye.
There are several disadvantages to intravitreal injections. The rst is the
transient time of exposure to the drug, since the turnover rate of uids (and hence
clearance of a drug) in the vitreous is quite high [1]. This may necessitate repeated
injections, which are not trivial on small eyes, although, such have been performed successfully in the rat eye for upirtine [8]. Repeated injections also
run the risk of a variety of unwanted side effects, including cataract formation,
endophthalmitis, retinal detachment, and vitreal hemorrhage [l0]. Alternatively,
sustained-release devices have been implanted into the vitreous to allow a more
continuous exposure of small molecules [11], and osmotic mini-pumps have been
used successfully to provide continuous application of larger molecules such as
brain-derived neurotrophic factor [12]. If the neuroprotective agent is a protein,
long-term delivery can also be achieved by introduction of a transgene for the
molecule into surrogate retinal cells by recombinant viral gene therapy (see Chapter 10). A second problem with intravitreal injections is leakage of the injected
uid from the injection site. This is particularly true in the mouse eye if the initial
puncture wound is too large, resulting in a poor seal around the glass pipette. Last,
great care must be taken not to damage anterior chamber structures, including
the lens, with the injection needle. This can signicantly complicate results by
secondarily creating a neuroprotective environment in the eye [13]. Lens damage
is often marked by cataract formation a few days after the injection and affected
animals should be excluded from the data set in experiments employing intravitreal injections. In the mouse, making the initial puncture at a site just posterior
to the limbus and then inserting the micropipette used for the injection at a relatively steep angle can avoid this damage. Care must then be taken to avoid penetrating the retina. It is recommended that initial attempts be made using a tracer
dye to determine the success of the injections. With practice, it is possible to
achieve highly consistent results.
Drug Delivery
157
IV. TRANS-SCLERAL DELIVERY

Trans-scleral delivery of compounds is gaining favor as a method of applying
neuroprotective agents to the retina, because compounds can be delivered to specic regions of the posterior pole without the complications of repeated intravitreal injections or the requirement for applying high concentrations of drug necessary for both trans-corneal and systemic delivery methods. The sclera is an elastic
tissue composed of collagen brils and proteoglycans, which has a highly porous
nature. Several in vitro diffusion studies using isolated pieces of sclera indicate
that the rate of diffusion across this tissue is inversely proportional to the molecular weight of compounds between 285 and 70,000 molecular weight [1,14]). The
molecular radius of molecules also inuences diffusion, such that more globular
compounds diffuse at a higher rate than linear structures of the same molecular
mass [10]. More recent studies indicate that even large biomolecules, such as
immunoglobulins, can penetrate the sclera [15] and retain their biological activity
[16].
Methods of applying compounds to the eye for scleral diffusion fall into
two categories depending on the amount and time course of drug delivery required. For short, one-time applications, drug is injected subconjunctivally as a
single bolus. Diffusion can occur over a time period of minutes, but this rate can
be inuenced by factors such as molecular mass, as indicated above, and by scleral
thickness, which varies from being relatively thin at the equator of a human eye
(averaging 0.39 mm) to relatively thick (1.0 mm) at the optic nerve [17]. Prolonged drug release can be achieved by using a sustained-release method. Like
intravitreal delivery, sustained-release methods range from osmotic pumps to
preparations of drug in slow-release matrices such as cyanoacrylate adhesive. In
addition, Tenons broblasts are readily transduced with recombinant DNA using
adenoviruses [18] or naked plasmids absorbed into a collagen shield [19], methods that can be used to deliver genes encoding proteins small enough to penetrate
the sclera. Because the transduced broblasts have been shown to express a
transgene for several weeks post infection, this method may be used to achieve
a biological alternative to a sustained-release device. Adenovirus infection can
be achieved simply by a sub-conjunctival injection of virus, although this method
often results in a diffuse spread of the infection around the globe (Fig. 1). A more
discrete area of transduction can be achieved by soaking a cellulose sponge with
virus and placing it under a ap of the Tenons capsule and conjunctiva in the
region of interest. Signicant levels of transduction are obtained using as little
as a 5-min exposure. Like any other sustained-release device, the drawback with
the gene transfer method is that it is not permanent and the application of recombinant virus would have to be repeated after between 14 and 30 days, when
transgene expression becomes quiescent and/or the transduced cells are eliminated. Times of persistence may vary depending on the nature of the transgene
158
Figure 1 Gross morphology of an enucleated rabbit eye (OD) showing distribution of a recombinant adenovirus injected subconjunctivally 4 days previously. The
superior part of the eye is indicated with a suture in the cornea and temporal is to
the left of the micrograph. A bolus of 100 L (approximately 1010 virus particles)
of recombinant adenovirus carrying the reporter gene -galactosidase was injected
by introducing a needle under the temporal conjunctiva and working it superiorly
to the point indicated by the arrow. After 4 days, the animal was sacriced and
the eye enucleated, xed briey in paraformaldehyde and then stained for -galactosidase activity. Stained cells are evident along the area of injection and needle
track, while cells in the inferior nasal conjunctiva are not transduced. Histological
analysis of transduced conjunctiva show that Tenons broblasts are readily transduced by adenovirus and therefore may be suitable targets for gene therapy using
secreted small proteins that could diffuse across the sclera.
and the promoter being used to drive expression. In addition, this method must
employ a transgene construct that allows for secretion of the target molecule.
V.
SYSTEMIC DELIVERY
Several systemic routes of drug delivery have been used successfully in ocular
neuroprotection studies, including intraperitoneal or intravenous injections and
oral delivery. Other modes of delivery include subcutaneous and intramuscular
Drug Delivery
159
injections. In general, systemic delivery is the preferred method for protocols

that require repeated applications, although this requires the use of high concentrations of drug, which may have undesired side effects. Detailed descriptions of
systemic delivery methods for different animals can be found in the appropriate
volume of the Laboratory Animal Pocket Reference series. Methods used on rats
and mice are described here.
A. Oral Delivery
One of the most dramatic uses of oral delivery for a neuroprotective agent was
reported recently by Neufeld and colleagues [20]. In this study, the nitric oxide
synthase inhibitor aminoguanidine was introduced into the drinking water of rats,
half of whom had been made ocular hypertensive using the vortex vein occlusion
method. The amount of drug taken in by each animal was determined by measuring the amount of water consumed. In this particular paradigm, the water was
changed 3 times a week, and the rats were kept on the drug for a period of 6
months. Based on the concentration of drug put into the water (2.0 g/L) and the
amount consumed (in mL/day), the authors estimated that the rats consumed
about 60 mg of drug per day.
Although this approach was successfully used in this study, drawbacks are
several-fold. First, it is limited to drugs that are water-soluble. Second, it is important that the drug does not impair the behavior of the animals drinking habits,
primarily by making the water unpalatable. This was a concern in the Neufeld
study, which found that rats consuming aminoguanidine drank an average of
approximately 25% less water than rats drinking untreated water, although this
difference was not statistically signicant based on the variation in the data set.
Third, this method may be prone to overestimating the amount of drug consumed
by not accounting for gravity-induced seepage of water from the drinking bottle.
In a simple experiment testing for gravity ow from standard feeding bottles, it
was estimated that 12 mL of water is lost per day in conditions where the bottles
were undisturbed. It is likely that this loss is greater when animals are agitating
the nozzles during drinking.
More controlled drug studies interested in testing the use of oral delivery
employ the gavage technique [21]. In this method, a drug is taken up as a metered
dose in a syringe, which is then attached to a standard feeding or gavage needle
with a blunt tip. In mice, it is possible to use a 3022 G needle, either straight
or curved depending on the size of the mouse, attached to a tuberculin syringe.
Up to 100 L can be introduced into the stomach of even small mice.
To feed a mouse, it is essential that the animal is rmly restrained with
complete immobilization of the head. The unanesthetized animal is fed by grasping it dorsally and articulating its head down and away from the holder to open
the airway and esophagus. The needle is inserted down the esophagus into the
160
stomach and the uid is administered slowly (Fig. 2). With practice, an experienced technician can feed several hundred animals in a day using this method.
The primary complication with gavage is entering the needle down the trachea
and into the lungs. If this occurs the needle may become obstructed and the
animal may begin to cough or struggle vigorously after the uid is introduced.
Fluid may also come out of the nose. At this point the needle should be withdrawn
immediately. If it appears that uid has been injected into the lungs, the animal
should be euthanized. Another complication is the rupture of the esophagus or
trachea due to excessive force when entering the needle. The resulting tissue
damage can lead to hydrothorax after uid application. The gavage method was
Figure 2
Ref. 21.)
Diagram showing the gavage method for drug delivery in mice. (From
Drug Delivery
161
used successfully to introduce FK506 into rats to test its neuroprotective effects
on retinal ganglion cells after optic nerve crush [22].
The advantages of the gavage method is greater control over the dose and
the timing of application of a target drug. The drawback with gavage feeding is
that it is more labor-intensive for the researcher and disruptive to the animals,
although they will become accustomed to repeated feedings by this method.
B. Intraperitoneal Delivery
Intraperitoneal injections can be a very efcient method of systemic drug delivery
[21]. Injections are made into the caudal left abdominal quadrant to avoid the
cecum in the right. The animal should be held dorsally with the head and tilted
away and down from the holder (Fig. 3). The needle should be introduced with
a quick rm motion to ensure passage through the abdominal musculature. After
Figure 3
21.)
Diagram showing intraperitoneal injections into the mouse. (From Ref.
162
Figure 4 Diagram of subcutaneous injections into the mouse. (From Ref. 21.)
penetration, pull back on the syringe plunger. The presence of any uid is indicative of penetration of an abdominal organ and the needle should be repositioned.
Depending on the compound, injection into an abdominal organ should have no
serious consequences other than altering the rate of absorption of the drug. Up
to 1 mL of uid can be introduced by this method into a mouse, but between
100 and 400 L is typical. This method has been used successfully to test the
neuroprotective effects of brimonidine on retinal ganglion cells [23,24].
An alternative to intraperitoneal injections is subcutaneous administration
of drug (Fig. 4). In general, access of an injected compound to the bloodstream
is less rapid than with intraperitoneal injections, but this is usually not an issue
with experiments that require prolonged drug exposure. In addition, subcutaneous
injections are less likely to damage the animal, and therefore may be more desirable for conditions that require repeated dosing.
C.
Intravenous Delivery
Drugs introduced intravenously (I.V.) can reach the retina very rapidly. In humans, for example, uorescein injected into a vein of the arm reaches the retinal
arteriole system in 1215 s and completely lls the vasculature of the retina and
choroid within 25 s in a healthy person. The best veins for delivering drugs vary
with the animal used. The most accessible site for an I.V. injection in the mouse
and rat is in one of the lateral tail veins, which are easily found although quite
small [21]. Injections are typically carried out by placing the mouse in a restraining device with a hole that allows the tail to stick out (Fig. 5). Warming
the tail will dilate the vein for better detection. Injections are made with a 27-G
Drug Delivery
163
Figure 5 Diagram of intravenous injection into a mouse through the lateral tail
vein. The mouse is constrained in a chamber with the tail extending through a hole.
(From Ref. 21.)
needle (or smaller), which is inserted into the vein and extended 12 mm. A lack
of resistance to the injected material is indicative of successful entry into the
vessel. In some cases, it may be possible to see blanching of the vein as the
injected uid lls it. The major drawback with this method of delivery is the
inability to successfully perform repeated injections on some animals (such as
mice) because of scarring of the veins.
ACKNOWLEDGMENTS
This work was supported in part by NIH grant R29 EY 12223, a grant from
the American Health Assistance Foundation, and by an unrestricted grant from
Research to Prevent Blindness to the Department of Ophthalmology at the University of Wisconsin. The authors are grateful for the helpful discussions of Drs.
Alyson Jarvis, T. Michael Nork, and Barbara Faha, and the assistance of Mr.
Lou Kohl.
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10
Przemyslaw Sapieha and Adriana Di Polo
University of Montreal
I.
INTRODUCTION
Gene transfer technology is gaining ground as a tool to investigate and promote

neuroprotection in the retina. In the past few years, a number of studies have
established proof-of-principle for the efcacy of gene delivery using viral vectors
to enhance neuronal survival in animal models of retinal diseases. Several factors
have contributed to the progress in this area, such as the elucidation of the genetic
basis of inherited retinal diseases, the availability of natural, experimental, or
transgenic animal models, and the development of recombinant viral vectors suitable for in vivo gene delivery.
The design of appropriate neuroprotective strategies is the rst step in tackling the complex problem of neuroprotection in the retina. A sensible strategy
should consider several factors, including (1) the cell type affected; (2) the mechanism and cause of death; (3) the appropriate target cell for gene transfer; (4) the
time-course of death; (5) the developmental stage of the experimental animal at
the moment of therapy; (6) the optimal viral vector for gene delivery; (7) the
time-course of vector-mediated transgene expression; and (8) the available animal
models. There may be as many neuroprotective schemes as there are retinal diseases or injury models. The goal of this chapter is to provide the reader with an
up-to-date account of current methods and tools for viral gene transfer that may
serve as a guide to investigate neuroprotection in the retina.
167
168
Sapieha and Di Polo
II. NEUROPROTECTIVE STRATEGIES

A variety of gene delivery strategies have been explored to promote neuronal survival in the retina following injury or disease. A gene transfer protocol that leads
to the complete and permanent rescue of degenerating retinal neurons is yet to be
established. However, the following strategies successfully enhanced cell survival
by delaying, in some cases considerably, the time-course of neurodegeneration.
A.
Gene Supplementation
Many retinal disorders are caused by genetic defects that result in a lack of one
or more essential gene products. Delivery of a healthy copy of the mutated gene
to the affected cell may restore loss-of-function decits and lead to neuroprotection. This approach has been tested in the retinal degeneration (rd) mouse, a
model of autosomal recessive human retinitis pigmentosa (RP). Defects in the
rod specic cGMP phosphodiesterase -subunit (-PDE) underlie irreversible
and rapid photoreceptor loss in this model [1,2]. Transient rescue of the rd phenotype was achieved following delivery of the wild-type -PDE cDNA into the
retina using several viral vectors; adenovirus (Ad) [3] encapsidated Ad (gutted
vector) [4], adeno-associated virus (AAV) [5], and lentivirus (LV) [6]. Furthermore, Ali et al. demonstrated that supplementation of the peripherin-2 gene using
AAV vectors resulted in the preservation of outer segment ultrastructure and
function for up to 10 months in the rds mouse [7], a model for autosomal dominant RP and macular dystrophy.
B.
Ribozyme Therapy
Retinal diseases caused by dominantly inherited mutations may result in the production of abnormal gene products that affect cellular trafcking, metabolism,
and function. Cell death due to accumulation of these toxic products can be minimized by delivery of ribozymes, small RNA molecules that can be designed to
cleave mutant RNA transcripts while leaving the wild-type mRNAs intact. Lewin
et al. demonstrated morphological and functional protection of photoreceptors
following AAV-delivery of ribozymes in a mutant rhodopsin transgenic rat model
of autosomal dominant RP [8]. Long-term studies on the effectiveness of this
form of therapy indicated that the progression of photoreceptor loss was considerably delayed for up to 8 months of age [9].
C.
Neurotrophic Factor Therapy
The main limitation of the gene correction therapies mentioned above is that the
genetic basis of prevalent forms of retinal disorders, such as glaucoma or age-
169
related macular degeneration, remains unknown. In addition, retinal diseases that

arise from mutations in several genes (polygenic diseases) or a combination of
environmental and genetic factors represent a challenge in the design of potential
gene therapies. Alternative therapies would involve the use of neurotrophic factors, with a broad spectrum of action, capable of providing a more generic form
of neuroprotection. Neurotrophic factors bind to specic cell surface receptors
triggering intracellular signaling pathways that lead to neuronal survival [10
12]. Because most neurotrophic factors are secreted and diffuse well within the
retina, this type of approach allows one to select either the affected cell or its
supporting cells as targets for gene transfer. For example, delivery of the brainderived neurotrophic factor (BDNF) gene to Muller glial cells using an Ad vector
resulted in temporary protection of retinal ganglion cells in an optic nerve axotomy rat model [13]. Ad-mediated delivery of ciliary neurotrophic factor (CNTF)
has been shown to slow photoreceptor loss in the rd and rds mouse [14,15].
Basic broblast growth factor (bFGF) delivered by Ad vectors delayed retinal
degeneration in the Royal College of Surgeons (RCS) rat [16]. More recently,
AAV-mediated delivery of bFGF delayed photoreceptor cell death in transgenic
rats carrying a mutant rhodopsin [17] and retinal ganglion cell loss after axotomy
[18].
D. Inhibition of Apoptosis
Apoptosis or programmed cell death appears to be a common mechanism of
neuronal loss in the injured or degenerating retina. Retinal ganglion cells have
been shown to die by apoptosis after optic nerve axotomy and in experimental
glaucoma [1921]. Photoreceptors also die by apoptosis in several inherited
mouse models of RP [22,23], the RCS rat [24], light-induced retinal damage
[25] and experimental retinal detachment in the rat [26]. The identication of
intracellular components of the cell death program (e.g., bcl-2 family members
and caspases) has motivated experimental strategies involving transfer of antiapoptotic genes. For example, gene delivery of bcl-2 using Ad resulted in moderate protection of photoreceptors in the rd mouse model [27]. Recently, Ad-mediated gene transfer of the X-linked inhibitor of apoptosis protein (XIAP) resulted
in partial protection of retinal ganglion cells following axotomy [28] and high
intraocular pressure [29].
III. SELECTION OF A GENE DELIVERY SYSTEM

Genes can be introduced into cells via nonviral and viral vectors. Nonviral methods include liposome-mediated DNA transfer, DNA carried on ballistic metal
particles (gene gun), and micro-injection techniques [30]. These gene transfer
Photoreceptors
Retinal ganglion cells
RPE
RPE
Photoreceptors
Adenoassociated
virus
(AAV)
Lentivirus
(LV)
Efcient
Can infect
stem cells
Low
Requires
helper virus
Efcient
Infection
in vitro
Peak: N/D
Duration: 12
weeks (longest time examined)
Peak: 57
days
Duration:
13 weeks
Several
months with
immunosuppression
Plateau: 24
weeks
Duration:
3 months to
1 year
Time-course
retinal
transgene
expression
wtAAV:
Chromosomal
integration
rAAV:
chromosomal
integration
in vitro
N/D in vivo
wt LV:
Chromosomal
integration
rLV: Chromosomal integration
wt Ad: Episomal
rAd: Episomal
Localization
of transferred
DNA
P3/P4 Biosafety Level

Facility
P1/P2 Biosafety Level

Facility
P2 Biosafety
Level Facility
Biosafety
(NIH
guidelines)
1010 1011 pfu/

mL
1012 1014 pp/

mL
1010 1012 ip/
mL
108 109 iu/mL

Packaging cell
lines: 106
iu/mL
4.5 kb
10 kb
Titer stocks
8 kb
Cloning
capacity
Abbreviations: RPE: retinal pigment epithelium; r: recombinant; wt: wild-type; pfu: plaque forming unit; pp: physical particles; ip: infectious particles; iu:
infectious units; N/D: not determined.
Muller cells
RPE
Photoreceptors
(early development)
Adenovirus
(Ad)
Main target
retinal cells
in vivo
Viral Vectors Widely Used in Retinal Neuroprotection Research
Viral vector
Table 1
170
Sapieha and Di Polo
171
methods often yield low DNA transduction rates that result in limited transgene
expression in vivo. In contrast, viral vectors have the ability to efciently infect
proliferating and post-mitotic cells and support adequate transgene expression
levels. Improvements in the design of these vectors and the methods to increase
viral titers and purity have contributed to their popularity. What makes a viral
system suitable for therapeutic use? Let us imagine an ideal vector with the following characteristics: (1) it can transduce a large number of target cells; (2) it
mediates gene expression specically in the target cells; (3) it allows stable expression of the delivered gene product at adequate levels; (4) it is safe, entailing
no toxic or immunogenic response in the target tissue; (5) it has no limitation in
the size of DNA that it can accommodate; and (6) it is easy to produce in large
volumes of high purity, high titer stocks. Although such a perfect vector does
not yet exist, there are a number of useful viral vectors available for gene transfer
research. Here we will focus on Ad, AAV, and LV vectors, the most widely used
vectors in retinal neuroprotection research (Table 1). The following sections are
not intended to endorse any vector in particular but to highlight their advantages
and disadvantages for different gene transfer applications.
IV. ADENOVIRUS (Ad)

Ad vectors are an efcient tool to study the effect of in vitro and in vivo gene
expression on neuroprotection. The major advantages of this system are that Ad
can efciently infect post-mitotic cells and that it can be easily grown to high
titers. Ad contains a linear double-stranded DNA genome of approximately 36
kilobases (kb) encapsidated in an icosahedral protein shell. Immediate early genes
(E1, E2, E3, and E4) orchestrate viral gene transcription and suppression of the
host immune response, whereas late genes are necessary for viral assembly [31].
Most recombinant Ad belong to the group C Ad type 2 or 5. Ad vectors were
initially generated with deletions of the early region 1 (E1), which contains
genes required for virus replication [32], rendering vector replication defective
and more suitable for gene transfer into mammalian cells. A major disadvantage
of these early Ad vectors is the strong cytotoxic and immune response elicited
upon infection of the host cells [33]. Recent versions of Ad vectors have been
produced in which the entire viral genome, except for the terminal repeat regions
required for viral assembly, has been replaced by exogenous gene sequences.
These so-called gutless vectors exhibit considerably reduced immune response
[34,35], but can only be produced in the presence of a helper virus that provides
all the proteins required for viral replication [36]. Because it is often difcult to
fully separate the gutted vector from the helper virus, we will focus here on
novel methods to produce recombinant Ad vectors without the need for helper
virus (see below).
172
A.
Sapieha and Di Polo
Ad Tropism in the Retina
Although Ad vectors show efcient transduction of a wide variety of cell types

in vitro, the cellular tropism for viral infection in vivo appears to be more complex. Our studies using Ad vectors injected into the vitreous chamber of adult
rat eyes demonstrated that Muller cells, the predominant glial cell in the retina,
is the main target for Ad infection in vivo [13]. This approach has proved to be
useful for delivery of genes encoding diffusible neurotrophins to promote neuroprotection of axotomized retinal ganglion cells. Ad vectors have also been shown
to efciently transduce the retinal pigment epithelium (RPE) following subretinal
injections [37,38]. Together, these studies indicate that nonneuronal cells in the
adult retina are the preferred cellular targets for recombinant Ad. Nevertheless,
some experimental conditions support limited Ad transduction of retinal neurons.
For example, intraocular administration of Ad in animals at early developmental
stages may result in modest infection of photoreceptors [3]. In addition, introduction of Ad to the brain (e.g., superior colliculi) or to the transected optic nerve
stump results in retrograde transport of viral particles that mediate gene expression in retinal ganglion cells [28,39]. Because all these studies involved
transgenes directed by the ubiquitous cytomegalovirus (CMV) promoter, it is
likely that the presence of cellular receptors for Ad in nonneuronal retinal cell
types mediates the observed viral tropism in vivo. The coxsackievirus and adenovirus receptor (CAR) protein involved in Ad attachment and infection has been
identied [40]. In addition, integrins v3 and b5 participate in Ad internalization [41]. The specic cellular localization of these receptors in the retina remains
to be dened.
B.
Immunological Response to Ad and Transgene

Expression
Most studies have shown robust Ad-mediated transgene expression detectable

soon after vector administration, but this expression is transient and decreases
within a few weeks. This has been attributed mainly to a host immune reaction
to viral and transgene products in which the infected cells are targeted for rapid
T-cell mediated clearance [42,43]. This is supported by the observation that Admediated gene expression is prolonged in immunodecient animals [42]. It is
possible that other factors such as promoter silencing or loss of viral DNA, since
the Ad genome remains episomal, contribute to this limited expression [44]. As
such, Ad represents an attractive system in gene therapy applications that require
the expression of a gene product for a limited amount of time. For applications
that require sustained transgene expression, several approaches have been explored. Repeated administration of Ad to enhance transgene expression has
proved to be inefcient due to the production of neutralizing antiviral antibodies
173
[45,46]. Alternative strategies have been implemented to by-pass the natural immune response. Daily administration of immune-suppressant drugs, delivered by
subcutaneous injections [13] or osmotic minipumps, can effectively sustain
transgene expression in the retina for several months. Similarly, coadministration
of modulators of the immune response, such as CTLA4-Ig, prolong Ad-mediated
gene expression [47].
The ocular immune response elicited by Ad has been shown to depend on
the route of administration of the vector. It is now clear that injection of Ad
vectors into the vitreous space leads to a stronger immunological reaction than
subretinal injection [3,42]. This nding highlights important differences in the
ocular immune privilege response between the intravitreal and subretinal compartments following Ad injection. It is interesting that, the immune response triggered by intravitreal administration of Ad vectors has been shown to promote a
moderate degree of neuroprotection [13,43]. This has been partially attributed to
the production of cytokines and neurotrophic factors by activated T cells [48].
C. Preparation of Ad Vectors
E1 or E1/E3 deleted Ad vectors are widely used in most gene transfer applications. Most methods to generate such vectors rely on standard molecular biology
and tissue culture techniques. Traditional protocols for the production of recombinant Ad involve homologous recombination of two pieces of DNA: (1) a plasmid
carrying the gene of interest, usually replacing the E1 genes, and the 5 end of
the Ad genome, and (2) the 3 end of the Ad genome. Both DNA constructs are
cotransfected into low passage 293 cells, a human embryonic kidney cell line
engineered to express the E1 gene products in trans to allow replication and
propagation of Ad [49]. Recombination events result in the production of functional viral particles that have incorporated the gene of interest into their genome.
Single viral clones are identied as plaques using standard overlay techniques
and are further characterized by polymerase chain reaction (PCR) or dot-blot
analysis of viral DNA. Viral clones containing the gene of interest are then propagated in 293 cells. The purication of Ad preparations is routinely done by cesium
chloride gradient ultracentrifugation and the viral titer is established using standard plaque titration assays. A detailed protocol to prepare Ad using this method
has been described elsewhere [50]. Commercial kits for the production of Ad
vector using this technique are available (Qbiogene, Carlsbad, CA).
This approach has proved to be useful but time-consuming due to the low
efciency of homologous recombination in mammalian cells and the need for
repeated rounds of plaque purication to eliminate contamination with wild-type
virus. A modication of this approach has been developed where Ad is reconstituted in yeast [51] or bacteria [52,53], which are endowed with a more efcient
recombination machinery and are easier to manipulate than mammalian cells. In
174
Sapieha and Di Polo
addition, the recombinant viral DNA is isolated from single clones, allowing one
to generate homogeneous Ad preparations, free of contaminating wild-type virus,
following transfection into 293 cells.
Recently, a novel method that does not require homologous recombination
has been developed to generate E1/E3- or E1/E4-deleted recombinant Ad by in
vitro ligation [54,55]. First, the gene of interest is inserted into a shuttle plasmid
to generate an expression cassette. Then, this cassette is excised and ligated into
another vector containing the complete Ad genome with E1/E3 or E1/E4 deletions. The resulting recombinant Ad DNA is grown and puried following transformation into bacteria. This DNA is then used to transfect 293 cells where viral
particles carrying the desired expression cassette are propagated. Because transfection is done with DNA from a single clone, there is no need to screen plaques
following transfection. Commercial kits to generate recombinant Ad by this
method are available (Adeno-X, Clontech Laboratories, Palo Alto, CA). One
drawback of this technique is the need to transform bacteria and transfect 293
cells with large plasmids, which may result in poor yields. Alternative methods
using cosmid technology have been designed to select clones containing full-size
genomes while excluding small and incomplete DNAs that are often produced
following transformation of bacteria with large plasmids [56].
V.
ADENO-ASSOCIATED VIRUS (AAV)
AAV is a member of the parvoviridae family initially identied as a contaminant

of Ad stocks. AAV requires a helper virus (e.g., Ad or herpes simplex virus) for
replication. The wild-type virus houses a single-stranded genome of 4680 base
pairs (bp) containing two genes, rep and cap, that encode proteins involved in
replication and encapsidation, respectively. The AAV genome is anked by two
identical 145-bp inverted terminal repeats (ITRs), which are essential for packaging, replication, or integration. Recombinant AAV vectors derived from human
parvovirus AAV-2 have been produced by substituting all viral sequences, except
for the ITRs, for a transgene of interest [57,58]. However, packaging of functional
AAV particles requires the presence of the rep and cap proteins typically provided in trans. The recombinant AAV system has several advantages for in vivo
gene transfer research: (1) it is not pathogenic and has not been implicated in
the etiology of any known human disease [59]; (2) mediates long-term transgene
expression that persists for several months in vivo [60,61]; (3) the absence of
viral sequences results in minimal immune response or cytotoxicity in the target
tissues [62,63]; and (4) it can efciently infect post-mitotic cells in vivo [64].
In the absence of helper virus, wild-type AAV can integrate at a specic
site on the q arm of chromosome 19 to establish latent infection [65]. However,
the lack of rep proteins has been shown to compromise integration specicity
175
leading to random insertion of recombinant AAV [66,67]. Although viral integration into the genome may contribute to the stability of AAV-mediated transgene
expression, a careful evaluation of the risks associated with insertional mutagenesis is required before implementing AAA-based therapies. A disadvantage of
AAV vectors has been the size constraint for packaging genes larger than 4.7
kb. Although methods have been developed to increase the size of delivered
transgenes by trans-splicing of two independent vectors coadministered to the
same tissue [68], this remains a limitation of the AAV system. The laborious
work needed to produce AAV vector stocks has often been regarded as a disadvantage of this vector system; however, recent availability of reagents and improvements in the protocols, described below, have greatly facilitated the preparation of high-titer and pure AAV stocks.
A. AAV Tropism in the Retina
AAV-mediated gene expression can be restricted to photoreceptor cells when
under the control of a well-characterized murine rhodopsin promoter sequence
[69]. More recently, retinal ganglion cells have been identied as the primary
targets for AAV infection in the inner retina following intravitreal injection of
viral vectors [70]. Thus, subretinal injection of AAV vectors results mainly in
gene transfer to photoreceptors and RPE cells, whereas intravitreal injection
allows infection of cells in the ganglion cell layer. Unlike Ad, AAV appears to
have a preferential tropism for retinal neurons rather than glial cells. This is consistent with studies in the brain showing AAV transduction of subsets of neurons
rather than astrocytes, oligodendrocytes, or microglia [71,72]. It is interesting that
genetic modication of capsid proteins has been shown to allow AAV targeting of
cells normally resistant to infection [73].
B. Time Course of AAV-Mediated Transgene
Expression
Recombinant AAV vectors have a slow onset of detectable transgene expression
in the retina, which typically reaches a plateau between 1 and 8 weeks following
administration of the vector depending on the animal species [74] In rodents,
peak expression is normally found 3 to 6 weeks postinjection of the vector
[60,70,75,76]. Similar time-dependent increases in AAV-mediated expression
have been observed in brain [77], muscle [78], and liver [79]. The mechanism
for this in vivo delay to reach peak expression levels in the retina has not been
determined, but may be related to the requirement of the single-stranded AAV
genome to be converted to a double-stranded form for the transgene to be expressed [80,81]. This feature of AAV-mediated gene expression should be taken
into consideration when designing neuroprotective strategies. Experimental para-
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Sapieha and Di Polo
digms with early onset and fast retinal neurodegeneration may be less amenable
to AAV-based therapies. Interestingly, AAV mediates long-term transgene expression that can persist in the retina for at least one year after vector administration [60,61].
C.
Preparation of AAV Vectors
Because AAV depends on the presence of certain Ad helper genes to replicate

and mount an effective infection, traditional methods have relied on the use of
Ad for packaging recombinant AAV. These protocols involve cotransfection of
two constructs, a vector plasmid carrying the transgene expression cassette, and
a helper plasmid that provides the rep and cap genes, into cells infected with
helper Ad. Although separation of the resulting recombinant AAV from Ad has
been routinely performed on cesium chloride (CsCl) gradients, via column chromatography, or by heat-inactivation, Ad remains a frequent contaminant of these
preparations. The presence of helper virus can result in unwanted cellular host
immune response [82], confounding the interpretation of the results following in
vivo gene transfer of contaminated AAV stocks. These concerns were addressed
by the development of constructs that provide the essential helper genes in a
plasmid, but lack the structural and replication Ad genes [63,8385). For example, the AAV rep and cap genes and the Ad helper genes have been introduced
in a single helper plasmid, pDG [83], eliminating the need for Ad infection.
In addition, the use of helper plasmids has been shown to improve the titer of
recombinant AAV stocks possible due to strong cap gene expression that enhances virus encapsidation [86].
Current methods of AAV preparation in most laboratories are based on
cotransfection of low passage 293 cells with a vector plasmid containing the
transgene of interest anked by the ITRs and a helper plasmid. Once the AAV
particles are assembled within the host cells, the virus is extracted by freezing and
thawing the cells and subsequently puried. Traditional AAV vector purication
protocols that involve precipitation of the virus with ammonium sulfate followed
by repeated rounds of CsCl density gradient centrifugation lead to poor viral
recovery and low infectivity. A new purication strategy based on density gradient purication using iodixanol has been reported [87]. Iodixanol (Nycomed
Pharma, Roskilde, Denmark) is a nonionic iodinated density gradient media,
which, unlike CsCl gradients, does not promote aggregation of AAV and cell
proteins that normally make the purication of AAV particles difcult. The fraction containing the AAV is further puried on a heparin/agarose column. This
step relies on the high afnity of this virus for heparan sulfate proteoglycan [88],
its main cellular receptor for attachment and internalization [89]. The eluted fraction containing the virus is then concentrated and its titer is estimated. A detailed
protocol describing this procedure has been described elsewhere [87].
177
In the absence of helper virus, AAV cannot mount a productive (lytic)

infection. Exposure of cells to AAV does not result in the formation of plaques
as with Ad. Consequently, indirect methods are used to estimate how much virus
is present in a given stock. Because not all virion particles are necessarily capable
of successful infection, virus quality is often assessed as the ratio of physical
particles to infectious particles in a given stock. A quantitative competitive (QC)PCR method is currently used to estimate total viral particles or copies of recombinant AAV genomes [90]. DNA dot blot analysis can also be used to estimate
total viral particles [91], but it is more time-consuming and unreliable than PCRbased methods. Infectious particles are determined by a infectious center assay
(ICA) using the C12 cell line [92] that contains integrated wild-type AAV rep
and cap genes. ICA involves infection of C12 cells with the AAV preparation
followed by infection with Ad. The recombinant AAV genome is amplied in
those cells successfully infected by AAV virions. Subsequently, the number of
cells expressing detectable amounts of recombinant AAV DNA are quantied
using radiolabeled probes [87].
Methods for manufacturing high-titer AAV stocks based on transient transfection of 293 cells in the absence of infectious helper virus have proved particularly useful for preclinical studies that require rapid testing of therapeutic genes
in a variety of animal models. While this method provides versatility, it is laborious and expensive to scale up due to the large amounts of DNA needed for
transfection and the low efciency of cotransfection protocols. As AAV vectors
move toward the clinical arena, improved methods to produce large quantities
of pure, high-titer stocks are required. Packaging cell lines engineered to contain
the AAV rep and cap genes have been particularly useful [92,93]. The transgene
of interest can be delivered to packaging cells via a recombinant Ad to generate
recombinant AAV stocks in the presence of helper Ad [94,95]. Alternatively, the
rep and cap genes have been delivered via a recombinant herpesvirus to cells
that carry a stably integrated AAV provirus with the transgene of choice [96].
These strategies are promising for scaling up AAV stocks, but they inevitably
result in contamination with helper viruses and require thorough purication prior
to any clinical application.
VI. LENTIVIRUS
Lentivirus (LV), a genus of retroviruses, consists of two identical single-stranded
RNA molecules and enzymes required for replication within a viral protein core.
Following virus internalization, the viral RNA is reverse-transcribed into doublestranded DNA and transported to the cell nucleus [97]. Viral DNA is then permanently integrated into the host genome to become a provirus. The retrovirus genome contains gag, pol, and env genes anked by long-terminal repeats (LTRs).
178
Sapieha and Di Polo
These genes encode proteins essential for replication, encapsidation, internalization, and reverse transcription. Replication-decient recombinant retroviral vectors have been generated by substituting all viral genes for a transgene of interest
with the exception of the cis-acting sequences required for vector propagation,
such as the reverse transcription initiation site and the packaging site [98]. Functional recombinant retrovirus particles can be generated in culture when the gag,
pol, and env gene products are provided in trans.
Most retroviral vectors can only transfer genes into cells that are actively
proliferating [99]. Thus, their use in neuroprotective strategies that typically involve gene transfer into fully differentiated cells is rather limited. An exception
to this rule are LVs, such as the human immunodeciency virus (HIV), which can
efciently infect nonmitotic cells [100]. This ability relies on nuclear localization
signals in the preintegration complex that allow entry into the nucleus without
the need for nuclear membrane fragmentation [99]. Other advantages of the LV
system are its relatively large cloning capacity, close to 10 kb; its ability to mediate high levels of transgene expression in vivo; and the lack of immune response
in the target tissues [101,102]. Recently, recombinant LV was shown to efciently infect hematopoietic stem cells, extending its potential use as a therapeutic
vector [103].
The main concern with LV vector systems is the risk of generating replication competent recombinant (RCR) virus during the production of viral stocks.
Because HIV is a human pathogen, considerable work has been done to increase
biosafety of LV production systems. The latest methods to generate safer HIVbased vector systems are described below. Other concerns include low vector
titers and the risks associated with insertional mutagenesis as the vector integrates
into the host genome. The issue of transgene silencing still requires further investigation. The lack of immune response associated with LV recombinant infection
and its ability to stably integrate into host DNA are promising features for persistent gene expression. A systematic study of the time course of expression mediated by LV vectors in the retina should resolve this issue.
A.
Cellular Tropism of LV Vectors
LV-mediated gene transfer and expression in the retina in vivo was rst characterized by subretinal injection of a vector carrying the green uorescent protein
(GFP) gene [104]. Using the ubiquitous CMV promoter, the main cellular targets
of LV vectors were shown to be photoreceptors and RPE cells with some bipolar
and Muller cells. When a rhodopsin-specic promoter was used, transgene expression was restricted to the photoreceptor layer [6,103]. The infection pattern
of LV vectors following intravitreal injection remains to be characterized.
An attractive feature of HIV-based vectors is their ability to efciently
infect cells in vitro. For example, RPE primary cultures have been transduced
179
with LV to express GFP. Transgene expression persisted in the infected RPE

cells following their transplantation into the subretinal space of a host [105].
HIV-based vectors have been shown to efciently transduce human CD34 hematopoietic stem cells that were capable of long-term engraftment of nonobese
diabetic/severe combined immunodecient (NOD/SCID) mice [103]. Recently,
neural stem cells have been isolated from the RPE in the ciliary margin of the
adult eye [106]. These cells can express retinal specic markers when they are
differentiated in vitro. Thus, ex vivo transduction of stem cells using HIV-based
vectors followed by retinal transplantation may be useful in the design of neuroprotective strategies.
B. Preparation of LV Vectors
LV has a more complex genome and, consequently, a more complicated replication cycle than other retroviruses. In addition to the three structural gag, pol, and
env genes, LV has additional regulatory genes: vif, vpr, vpu, tat, rev, and nef
genes [107]. Traditional methods to generate recombinant LV vectors involved
a vector plasmid with all viral genes deleted, containing only the transgene of
interest anked by the essential cis-acting elements. The viral proteins required
for virus propagation were provided in trans by cotransfection of a helper plasmid
into 293 cells. This procedure resulted in low titer preparations and in high risk
for generating RCR virus. This is a major concern because the considerable overlap that exists between cis-acting sequences in HIV vectors and helper constructs
increases the chance of homologous recombination leading to the production of
RCR viral particles.
A better understanding of the HIV replication cycle has led to new advances
in the eld of LV vector packaging. The latest generation of LV vectors have
only three of the nine HIV-1 genes: gag, which encodes the main structural proteins; pol, responsible for the production of viral replication proteins and rev,
encoding a regulator required for gag and pol gene expression. The HIV envelope
has been substituted by other viral envelope proteins such as the vesicular stomatitis virus G-protein (VSV-G) due to its high stability and broad tropism [108].
Methods to generate recombinant LV particles from three or four separate transcriptional units containing gag/pol, rev, VSV-G, and the transgene of interest
following transfection into 293 cells have been recently described [101,109]. By
distributing the required sequences in multiple plasmids, the risk of creation of
RCR virions was minimized. In addition, the overlap between vector and helper
sequences was markedly reduced.
The safety level for production of LV has been further improved by the
development of self-inactivating (SIN) HIV-1-derived vectors. Two independent
research groups have reported HIV-based vectors in which the regulatory sequences (promoter and enhancer elements) contained in the 3 LTR have been
180
Sapieha and Di Polo
deleted [110,111]. By eliminating these cis-acting elements, the virus loses its
capacity for gene expression directed by both LTRs, resulting in inactivation of
the integrated virus in the infected cells. In addition, the expression of the
transgene of interest can only be controlled by an internal heterologous promoter.
A stable, high-output packaging cell line to produce this latest generation of LV
vectors has been recently reported [112]. These results represent the latest advancement in the efforts to render HIV-based vectors safe for clinical use. Although these novel vectors greatly minimize the risk of RCR virus infection to
users during manipulation and preparation, the biosafety of LV vectors in vivo
has yet to be demonstrated.
VII. SUMMARY
Several independent research laboratories have recently established proof-ofprinciple for the efcacy of gene therapy for neuroprotection in animal models
of retinal injury or disease. Three viral systems, Ad, AAV, and LV, have been
used for most gene delivery applications to the retina both in vitro and in vivo.
The pros and cons inherent in each of these vectors need to be carefully weighed
when designing a gene transfer strategy. Recent progress in the methods to create
these recombinant viruses has greatly increased the ability of researchers to generate high-titer and pure stocks while reducing production times. In addition, the
likelihood of adverse reactions in the host tissues and the risk of RCR virus
generation have been minimized. As these viral vectors progress toward preclinical and potential clinical human trials, highly stringent standards for their
efcacy, biosafety, ease for scaling up production and purity will be expected.
Future in vivo characterization of these viral vectors will be essential to fully
assess if these requirements are met for clinical applications.
ACKNOWLEDGMENTS
Supported by the Canadian Institutes of Health Research (CIHR) and the Foundation Fighting Blindness. A.D.P. is a scholar of Fonds de la Recherche en Sante
du Quebec.
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11
Christine A. Curcio and Kenneth R. Sloan
University of Alabama at Birmingham
Birmingham, Alabama, U.S.A.
I.
INTRODUCTION
A necessary milestone in establishing the safety and efcacy of new neuroprotective interventions is histological evaluation of the retina in treatment and control
groups. It is likely that any treatment will be quantitative in its effectthat is,
that a disease process will be slowed rather than completely halted. This means
that the evaluation will have be quantitative rather than qualitative in nature and
will have to be designed to detect small but potentially signicant effects. The
purpose of this chapter is to familiarize the reader with methods of counting cells
in retinal whole mounts and sections. Examples will come from our work on
photoreceptors and ganglion cells in human retina and the effects of aging and
degeneration on these cell populations [16]. It should be emphasized that counting cell bodies is only the beginning of a complete evaluation of neural retina
following treatments, because even modest loss could be accompanied by functionally signicant changes at the subcellular or synaptic level. This chapter does
not address the methods required for such studies.
II. WHAT ARE YOU MEASURING? TOTAL CELLS

VERSUS TOPOGRAPHY
The total number of cells of a given type within the retina is a single number
that is easy to understand and easy to analyze using straightforward statistical
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techniques for assessing between-group differences (e.g., t-tests or analysis of

variance). The total number of cells in the retina is determined by counting cells
in small samples and dividing the counts in each sample by the area of the sample
to achieve density (number of cells per square millimeter of retinal surface, or
planimetric density). The density is multiplied by total retinal area to achieve
total number. Samples must be representative of the whole tissue, and their locations must be selected with some knowledge of the variations in cell density
expected with retinal position. The topography of cell loss (in a disease) or reduction of cell loss (for a treatment) can be analyzed further to identify regionally
specic effects.
Our studies of aging human macula involved both determining the total
number of cells and identifying changes in the topography of cell density [3,4].
Figure 1 shows that the total number of cones in the central 0.8-mm diameter
of macula was remarkably constant over adulthood (31,200 3,100 cones). The
use of total numbers enabled us to avoid the high between-individual variability
that is present in the peak density of foveal cones, in the very center of the fovea.
In the same eyes the total number of rods within the central 8-mm diameter
decreased 30%. We used this approach to demonstrate that the loss of rods in
aging human retina was selective for the parafovea, the details of which were
then elucidated with maps of the differences between young and old eyes. The
specics of determining total cell number and analyzing retinal topography will
be explained further below.
Figure 1 Number of cones in fovea and rods in human macula. (A) Cone density
was integrated over a 0.8 mm diameter circle centered on the foveal center. Solid
circles, donor eyes. Open circles, normal eyes surgically removed from patients
with craniofacial tumors. (B) Rod density was integrated over a 8-mm-diameter
circle centered on the foveal center in donor eyes. (From Ref. 4.)
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III. WHOLE MOUNTS VERSUS SECTIONS: WHICH

SHOULD YOU USE?
Morphological assessment typically involves histological sections prepared using
conventional techniques such as parafn embedding, cryosectioning, or epoxy
embedding for electron microscopy. However, unlike most tissues, the retina can
be studied as a whole mount, offering distinct advantages for quantitative studies.
In a retinal whole mount, topography is preserved, with major landmarks still in
place. With the exceptions noted below (Sec. VIII), the total population of a
homogeneous cell type occupies layers that are only one to two cells deep, permitting its visualization within a small range of focus. Cells in individual layers can
be visualized using type-specic antibodies or by distinctive uorescence patterns
achieved with nucleic acid dyes such as DAPI or ethidium homodimer (Molecular
Probes). Shrinkage can be negligible in whole mounts except around cut edges.
Large samples are easily attainable, so that the study of minority cell populations
and the detection of small effects are possible. High-contrast images of specic
cell types that are achievable in a limited focal plane should be the goal, as these
images may be conducive to either automated counting or counting that can be
done by relatively low-level personnel.
Photoreceptors present a challenge to whole-mount studies, because their
extreme compartmentalization is reected in the composition of the retinal layers
viewed en face.
1. Inner segments. In human retina, we counted photoreceptors at the
level of the inner segments, the cross-sections of which form a mosaic in a single
focal plane. Cone inner segments are distinguishable from rods by their threefold
larger diameter and their more highly refractile properties in most parts of human
retina. However, in retinal degenerations, the morphology of photoreceptors
changes as outer segments are lost and the inner segment shortens and broadens.
Therefore, in our study of eyes with age-related maculopathy [5], we validated
counts of rods and cones made on the basis of size differences of inner segments
using carbonic anhydrase histochemistry [7], which stains cone nuclei even in
degenerated retina. In other species, the difference between rod and cone inner
segments is not as prominent as it is in human retina (e.g., rabbit [8]) or inner
segments may form tiers rather than a mosaic (e.g., pig [9]). In these species,
examination of multiple focal planes is required to distinguish between rod and
cone inner segments.
2. Nuclei. Cone nuclei occupy a single layer in the outer nuclear layer
of most mammalian retinas, but rod nuclei form multiple rows that require many
focal planes for analysis.
3. Outer segment. The rst widely used photoreceptor-specic antibodies
were directed against proteins associated with transduction in the outer segment.
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Other investigators have used lectins that bind cone and rod sheaths. The validity
of these markers is beyond the scope of this chapter. However, it is worth mentioning that outer segments are fragile and could be easily lost in the preparation
of a whole mount. Therefore, with outer segmentspecic markers one should
verify that the vast majority of inner segments have an attached outer segment,
which we did when counting short-wavelengthsensitive cones in human retina.
Some cone markers like calbindin stain cones in their entirety [l0].
The major challenge to accurate counts of ganglion cells is the substantial
minority population of displaced amacrine cells, which may overlap in size and
morphology with ganglion cells. The proportion of ganglion cell layer neurons
that are amacrine cells varies among species and within a species, with retinal
region [11]. The sine qua non of a retinal ganglion cell is the presence of a
projection axon to the brain. Some markers (e.g., BRN3A and BRN3B [12]) have
been used in conjunction with injections of a retrograde tracer in the brain to
identify ganglion cells. However, tracer injections only reach some of retinorecipient targets in the brain, and within a given target, not all ganglion cell terminals
may take up and transport tracer. Therefore, the gold standard for validating retinal ganglion cell counts is comparison to optic nerve axon counts in the same
eye [13]. If the total number of ganglion cells is the only desired outcome variable
(and not overall topography, or counts of a particular subtypes), one should count
optic nerve axons rather than ganglion cell bodies. It is possible to obtain unbiased
counts of optic nerve axons using high-throughput morphometric techniques developed for neurogenetic studies by Williams and colleagues [14]. These authors
determined total axon number from the total cross-sectional area of the nerve
and the number of axons within unbiased counting frames on electron micrographs that were systematically placed within the nerve. Axons were counted
directly on the negatives.
Despite the many advantages of retinal whole mounts for quantication,
sections will continue to play an important role in assessing the retina in neuroprotection studies. Stained sections currently afford greater detail than wholemounts and are amenable to multiple marker studies in single animals. Epoxyembedded tissues can be used for electron as well as light microscopy. There is
a wide range of routine, special, and advanced stains and high-throughput processing technologies designed for parafn sections, the mainstay of clinical
pathology laboratories. Finally, it should be stressed that a complete census of
retinal cells currently requires a combination of techniques. In a study that
has provided the most complete enumeration of the neurons in the C57B1/6
mouse retina, photoreceptors were counted in wholes mounts, interneurons and
Muller cells were counted in vertically oriented ultrathin sections examined
by electron microscopy and optical sections examined by confocal microscopy,
and ganglion cells were counted in electron micrographs of optic nerve sections
[13].
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IV. VISUALIZING TISSUE FOR COUNTS

The investigator has several choices for imaging retinal tissue for cell counts.
These include making counts directly from a microscope using an ocular reticule
to delimit counting elds, making counts from a video image generated by a
camera mounted on a microscope, producing photographs that can be printed or
scanned for off-line analysis, and capturing digitized images for off-line analysis.
In our studies of photoreceptors and ganglion cell layer neurons in human retina,
we viewed tissue with a computer-video-microscope system that included a 60
1.4 N.A. oil-immersion objective, Nomarski differential interference contrast
optics, a digitizing tablet, and a computer-controlled stepper motor stage.
We viewed the tissue on a video monitor that provided 20003000 nal magnication, depending on the objective used. The video image was combined
with custom graphical overlays that were manipulated using a digitizing tablet
to mark counted cells. This arrangement allowed us to optimize the focus for
counting for photoreceptor inner segments and to count while focusing through
the thickness of the human macular ganglion cells layer. A computer controlled
the movements of the stage so that the retinal sheet could be sampled systematically in a manner that substantially eliminated the operator error inherent with
manual stage control. Many of these functions, which required custom software
in our studies, are now available in commercial stand-alone packages (Image
Lab, Metamorph) or are bundled with image processing software available for
confocal microscopy.
V.
SAMPLING CONSIDERATIONS
The past 15 years have seen signicant advances in stereology, the branch of
applied mathematics that seeks to deduce unbiased information about threedimensional tissue structure from two-dimensional sections. These methods eliminate bias (i.e., systematic over- or under-counts) and have practical implications
for investigators in neuroprotection research. A thorough discussion of the mathematical foundations of spatial sampling is beyond the scope of this chapter.
A. Sampling Windows and Exclusion Rules
A counting window is an articial boundary on the visible part of the tissue in
which counts are made. In order to ensure statistical independence of samples,
cells should be countable in only one window. The science of counting windows
developed for sections through three-dimensional tissues (see Sec. VII) also apply
to two-dimensional retinal whole mounts. For instructional purposes, it is useful
to imagine a retinal whole mount that is completely covered by contiguous count-
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Curcio and Sloan
Figure 2 Unbiased counting frames. A restricted eld of view of tissue, delimited

by thin dashed lines, contains proles of cells cut in cross-section. In addition to
the proles completely within the frame, one counts all proles partially within the
frame as long as they do not touch or cross the solid exclusion lines. Thus, only
the hatched cells are counted as in. (From Ref. 15.)
ing windows, like wallpaper. In this example, many cells fall on boundaries between adjacent windows, but they can be considered in only one window in
order to avoid overcounts, and thus rules for determining what is in are required. In the unbiased counting window shown in Figure 2 [15], one counts all
the proles completely inside the frame provided that they do not in any way
touch or intersect the full-drawn exclusion edges or their extensions. In our studies, cells that crossed the left and bottom borders of the counting window were
considered in, and cells that crossed the right and top borders of the counting
window were considered out. These rules apply regardless of whether counting
windows are contiguous or widely spaced. The size of the counting window in
tissue dimensions is determined by calibrating the optical and video components
of the system with a calibrated micrometer slide.
B.
Systematic Random Sampling for Determining

Total Cell Number
For statistically meaningful counts, the sites where counts are made must be
chosen independently of their content [15]. That is, sites cannot be chosen because they have features of interest. The choice of sampling pattern across the
retina is dictated by whether totals or topography is the outcome variable of
interest.
195
If the total number of cells is desired, then the method used in a recent
study of cone survival in rd/rd mouse retina is appropriate [16]. These investigators randomly positioned a regular grid over the tissue, and they counted cells
in windows located at 50 evenly spaced sites within the grid. In this approach,
the regional specializations containing higher density of cells are sampled in proportion to their retinal area. A regional specialization such as a fovea or area
centralis is small relative to the entire retina and contributes little to the total
number of cells. In human retina, the small and densely packed foveal cones
constitute only 0.7% of the total number of cones (32,000 vs. 4.6 million), because they occupy only a tiny area. It would therefore be possible to skip the
entire fovea in a systematic sampling scheme and not appreciably change the
total number of cones. However, topographic information is clearly lost by such
an approach.
C. Systematic Sampling for Visualizing

Topography
If analysis of retinal topography is planned, then the sampling scheme must take
into account the presence of regional specializations. An efcient sampling
scheme is one that balances the competing goals of avoiding artifacts caused by
interpolating densities between widely spaced points (undersampling) and to
avoid collecting more data than necessary to achieve a density estimate with
tolerable error (oversampling). Cell densities in primate retina are approximately
radially symmetric around the fovea and change most rapidly near the fovea. In
our topography studies we counted photoreceptors and/or ganglion cells at 100
120 locations that were closely spaced in the fovea and less closely spaced away
from the fovea. We used a spiral pattern that evenly tesselated the retinal surface
(Fig. 3).
D. How Much Is Enough?

It is possible to obtain smooth contour maps, reective of low variance data, with
remarkably few samples. In our studies of aging and degeneration, we counted
cells within a 12-mm-diameter area centered on the fovea. We counted photoreceptors in a single 39-m-square counting window and ganglion cell layer neurons
in adjacent 39-m-square windows until a total of 15 cells was obtained. Approximately 1385 cones (0.17% of the total within central 12 mm diameter), and 1815
ganglion cell layer neurons (0.3% of the total) were counted in these studies. We
found empirically that this sample size was adequate to create a smooth map (see
Sec. VI.B). In a recent study of mouse retina [16], 2.85% of the total number of
counts were sampled. The validity of the sample size in this case was determined
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Curcio and Sloan
Figure 3 Sampling points used to produce smooth maps of cell density in human
retina. (A) Cells were counted at each point. Center of spiral pattern is the point
of highest cone density in the fovea. (B) Central portion of A, showing a denser
grid of sampling points in the fovea. (From Ref. 17.)
by showing that counting twice or three times as many cells did not change the
outcome. It is possible that even fewer cells could have been counted.
VI. METHODS FOR ANALYZING TOPOGRAPHIC

CHANGES
A.
Retinal Model
The retina, being part of sphere, must be cut to be attened. This is not a problem
for generating counts of total numbers [16]. On the other hand, the cut edges may
interfere with topographic analysis. For our topography studies, we developed a
method for reconstructing the human retina by aligning the cut edges of a threepiece whole mount using major retinal vessels as landmarks [17]. Our studies of
aging and degeneration used only the macula, and reconstruction of the entire
retina was not necessary. In both cases (whole retina or macula), we found it
convenient to use a digital model consisting of locations on the retinal sphere
that were indexed by spherical coordinates and an associated cell density [17].
The spherical coordinates were referenced by the center of highest cone density in
the foveal center, and the directions nasal [l80] and temporal [0] referred to the
appropriate sides of a line passing through the foveal center and perpendicular to
a line through the fovea and optic disc center of a standard left eye. This coordinate
system allowed us to combine maps from eyes of different individuals. Note that
197
our coordinate system was ideally suited for human retina, because the fovea, the
point of highest cone density, and the optic disc were readily identied internal
landmarks. However, a coordinate system for a species without grossly visible retinal landmarks should be done in reference to eye position in the head, a process
that begins with marking directions on the eyes when they are still in the skull.
Data points in our retinal model were connected into a mesh of triangular
patches which closely approximated a sphere. A value at any point within a patch
could be determined by calculating a weighted average of the values at its three
vertices, yielding an acceptable approximation to the value found in the tissue
itself at that location. Computer graphic display techniques were used to produce
a variety of images. The values associated with vertices of the triangular patches
were linearly interpolated across the patch. Video look-up tables were manipulated to produce false-colored smooth surfaces, terraced surfaces in false color
(Fig. 6, Ref. 4) and gray scale (Fig. 4), and traditional black-and-white contour
maps [18]. Plots of average cells per square millimeter as a function of eccentricity along selected meridians and maps of average density were created by resampling models of individual eyes at a set of standard locations, in which retinal
directions were preserved. The total number of cells in selected regions was calculated by multiplying the mean density of each triangular patch on the model by
its area on the spherical surface of the retina.
B. Graphical Methods
A map is the most direct way to inspect retinal topography. We displayed our
data in the polar azimuthal equidistant projection, familiar from human perimetry,
which preserves radial distances and distorts circumferential distances. In addition to maps of individual eyes we created maps of mean cell density in a group
of eyes by resampling data from individual eyes at standard locations that reected the weighting of the original sample pointsthat is, with more locations
near the fovca (Fig. 5A,B) [4].
A difference map is a comprehensive and effective way of seeing overall
differences in retinal topography. We created maps of the difference between
groups using a measure of local variability, so that a difference in a location
where variability was high would be displayed less prominently than a difference
in a location where variability was low. In our study of aging human macula [4],
cell density in an older age group (test population) was compared to a younger
age group (reference population). We computed the difference between log (density) at each standard location in all possible pairs of test and reference eyes.
The mean of the pairwise differences at each location was used to create a colorcoded map. Figure 4C shows a gray-scale version of the difference map, and a
color version is shown in Figure 6G of Ref. 4. This approach was used to demonstrate the exquisite localization of age-related rod loss in the human macula.
Figure 4 Rod topography in young and elderly eyes. (A) Donor eyes, 2737 yr. (B) Donor eyes, 8293 yr. (C) Differences between eyes in A and B. Maps are displayed as fundus view of left eyes. Black oval denotes the optic disc.
Rings of isoeccentricity are at 2 mm intervals. Gray scale in C indicates rods per square millimeter in panels A and B
and the mean difference in density between older and younger eyes in panel C, as follows. As applied to panels A and
B, the gray scale indicates 16 discrete bands of 12,500 rods/mm2 between 0 (black) and 200,000 (white). Gray scale
applied to panel C indicates 16 bands of 0.02 log units between 0.16 (black, or 31% lower) and 0.16 (white, or 44%
higher). In panel C, light shades indicate locations where the older eyes have higher mean density than the younger
eyes, and dark shades indicate where they have lower mean density. There is an annulus of deep rod loss in the parafovea. (From Ref. 4.)
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Curcio and Sloan
199
Figure 5 Rod and cone loss in exudative age-related macular degeneration. (A)
Sampling pattern for photoreceptor counts in eyes with exudative age-related maculopathy (ARM). The small oval indicates the optic disc. The large irregular pattern
indicates an area of almost complete photoreceptor loss overlying brovascular
scars and atrophy of the retinal pigment epithelium (RPE). To quantify rod and
cone loss, counts were made at 0.1 mm intervals along 48 arbitrarily placed meridians (hatched lines). Counts were also made at comparable locations in 34
age-matched control eyes. (B) Rods but not cones are lost around the margins of
brovascular scar and RPE atrophy in an eye with exudative ARM. (Dark circles,
rods; gray circles, cones.) (From Ref. 5.)
Figure 6 Disector. A block of retinal tissue (A) containing particles ae is cut into
serial sections (B). Hatched proles in BD are cells that are transected by the
sectioning plane. A pair of sections, distance t apart, is drawn from the stack. The
top (reference) plane contains an unbiased counting frame of area A (C), and
the lower (look-up) section does not (D). The volume of the disector is A t. Only
cell b is counted as in, because it falls within the counting frame in the reference
section (C), and it is not present in the look-up section (D). This diagram shows
a physical disector (i.e., identifying cells in separate sections) An optical disector
involves counting cells identied in the top and bottom focal planes of the same
thick section. (From Ref. 23.)
200
C.
Curcio and Sloan
Specialized Methods for Regional

Photoreceptor Loss
Figure 5 shows a different systematic sampling strategy employed to assess the

relative rate of cone and rod degeneration surrounding areas of nearly complete
photoreceptor loss vitread to brovascular scars and RPE atrophy in eyes with
late exudative age-related maculopathy [5]. Here we counted photoreceptors at
0.1 mm intervals along arbitrarily chosen tracks that crossed the boundary between intact and degenerated photoreceptor mosaic. Photoreceptor degeneration
was expressed as loss relative to controls as a function of distance from the margin
of the intact photoreceptor mosaic. Counts were compared to those in matched
locations in control eyes using the same measure described in the previous section
for maps. In the gure, the dashed line indicates the variability in cell density
among control eyes, calculated as follows. At each retinal location, we computed
the differences between log(density) for each pairwise comparison of one randomly chosen control eye and the other controls. This computation, done for both
cones and rods, established 95% condence intervals for differences among the
controls. Results were similar regardless of which control was chosen. Then we
computed the differences in log(density) for pairwise comparisons of the ARM
eye and the controls. Directional differences between ARM eyes and controls
that fell below the lower condence limit for controls were considered signicant
loss. We then reported the percentage of counting sites with signicant loss and
the percentage of sites with loss where either rod or cone loss predominated.
VII. COUNTS IN SECTIONS

It is possible to count cells in sections through the full thickness of the retina,
(i.e., in the plane orthogonal to a retinal whole mount). Two different approaches
have been employed for counting retinal cells in sections, depending the layer
of interest. In both cases, one must carefully attend to the location of samples
in order to ensure that counts are taken from comparable locations (see Sec. VI
A).
In the mammalian retina, photoreceptor nuclei form a thick layer in the outer
nuclear layer (ONL), typically with a single row of cones external to multiple rows
of rods. Many studies of retinal degeneration and treatments in animal models
have quantied photoreceptors as either rows of ONL nuclei or thickness of ONL
layer [19]. Use of ONL thickness compares favorably with counts of photoreceptor nuclei and can be done quickly [20]. This approach is suitable for quantifying
the more numerous rods but is less suitable for quantifying the less numerous
cones, because the sample of cones is very small in single sections [21]. Note
that counts of cells along the length of a retinal section (typically expressed as
201
cells/100 m) assumes that the counted particles (usually nuclei) do not differ in
size between treatment and control groups, which may not be true for degenerating
photoreceptors. Counts along single slices also assume the overall size of the
retina does not differ between groups. It is possible to section the retina serially
and determine a total number of cells using a subsample of sections in order to
obtain a sample size comparable to that achievable in a whole-mount. Such an
approach is feasible for very small eyes, such as embryonic tissues.
The current standard for cell counting in sections is the disector, an unbiased counting method that uses counts obtained in parallel planes separated by
a known distance in the tissue (Fig. 6). The rule is to count only those particles
that appear within an unbiased counting frame in one plane (the reference plane)
but not in the matched plane (the look-up plane). The number of nuclei (conventionally called Q ) is contained within the volume of the disector. The volume
of the disector is equal to the area of the counting frame, multiplied by the distance between planes. If the specimen has been sampled systematically by multiple disectors, the number per volume is calculated by summing the area and Q
over all disectors. Reference and look-up planes are separated by a distance that
is one-quarter to one-third that of the particles being counted. There should be
only one counted particle per cell, and the fewest assumptions are required for
smooth convex objects, so cell nuclei are the typical counting particle. In order
to count nuclei that are 8 m in diameter, planes that are 23 m apart are
required.
An efcient way to implement a disector is to track through successive
focal planes in a thick slice of tissue, a method known as the optical disector. It
is possible to use a high numerical aperture objective to identify the reference
and look-up planes a relatively thick section (25 m). Confocal microscopy
can also be used to optically section tissue for counting of uorescent cells and
will likely be used more frequently for this purpose as these instruments become
more widely available. The alternative to an optical disector is the physical disector, which involves either semi-thin or ultrathin sections prepared for transmission conventional electron microscopy. In order to identify the same cells in the
reference and look-up planes, the sections are aligned by reference to local ducial landmarks such as blood vessels. A physical disector is difcult to apply to
the ONL of the retina. The rod nuclei are morphologically homogeneous and
form a paracrystalline array like ball bearings in a box, and there are no nearby
blood vessels. Thus, the reference and look-up planes can be aligned spuriously,
introducing inaccuracies into the number of cells missing from the look-up plane.
However, in the inner nuclear and ganglion cell layers, the presence of capillaries
and multiple, distinctive cell populations together facilitate the chore of aligning
sections. Disector methods have been successfully used for determining cell densities in these layers [13], and they are the method of choice in contemporary
morphometric studies of brain [22].
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Curcio and Sloan
VIII. CHALLENGES OF THE MACULA

For either whole-mounts or sections, the primate macula presents unique challenges for cell counting, due to its extreme thickness and the presence of the
fovea. In the macula of human and monkey, ganglion cells are up to six cells
deep. In whole mounts, conventional Nissl stains that involve dehydration of
tissue reduce thickness by factors up to two-thirds [2]. We used unstained tissue
that was cleared with dimethyl sulfoxide rather than ethanol and xylene and
viewed with differential interference contrast optics [2,3,6]. We continuously focused through the tissue while counting nucleoli of ganglion cell layer neurons.
Studies using uorescent markers for inner retinal cells would require multiple
image planes. On the photoreceptor side of the fovea, the external fovea is an
inward dip, where photoreceptor inner segments do not form a single plane.
Therefore, macular photoreceptors, like the ganglion cells, also require continuous adjustment of focus to nd the optimal plane for counting.
Another challenge to cell counting in the macula is the sharp changes in
cell density near the foveal center. The cone distribution has a very small (100
m or less) area of very high density in the foveal center that drops by 90%
within 1 mm. Therefore, counts in this area require precisely specied sample
locations. Poor control of location can insert noise into cell counts and make
effects difcult to detect. In our whole-mount studies, we set the point of highest
cone density as the foveal center and used a computer-controlled stepper-stage
on the microscope, so that position relative to the foveal center could be specied
accurately. In studies requiring sections, we sectioned serially into the fovea so
that the center could be identied by the absence of ganglion cell and inner nuclear layers, and the absence of Henle bers cut in cross-section. Other challenges
to studies using the macula include long incubation times required for complete
penetration of the retina by immunoreagents, the absence of certain well-established markers in the foveal region [l0], and the predilection for postmortem
swelling in inner retina that can impede the identication of the foveal center.
ACKNOWLEDGMENTS
Supported by NIH grant EY06109 and unrestricted funds from Research to Prevent Blindness, Inc., to the Department of Ophthalmology, University of Alabama School of Medicine.
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Jeon C-J, Strettoi E, Masland RH. The major cell populations of the mouse retina.
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Williams RW, Strom RC, Rice DS, Goldowitz D. Genetic and environmental control of variation in retinal ganglion cell number in mice. J Neurosci 1996; 16:7193
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12
Ex Vivo and Whole-Mount Retinal
Preparations
Arthur J. Weber
Michigan State University
East Lansing, Michigan, U.S.A.
I.
INTRODUCTION
Despite its relatively small size, the brain is an enormously complex organ that
poses many challenges to those trying to understand its organization, function,
and dysfunction. Fortunately, however, the brain comprises a number of more
simple and well-dened systems, and many of these have proved to be good
models for studying complex brain mechanisms. The visual system is one area
of the brain that has received considerable attention over the years, in part because
its functional integrity can be assessed easily using light stimulation, but also
because it is naturally divided between the eye and the rest of the brain. In addition, several primary components of the central visual pathway, including the
eye, optic nerve, and visual cortex, are readily accessible for experimental manipulation and/or therapeutic intervention.
II. THE RETINA AS A MODEL FOR STUDYING

NEUROPROTECTION
Over the past several years considerable research has focused on the structure
and function of the normal retina [15] and following injury to either the optic
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nerve [613] or visual cortex [1419]. Because it is a relatively isolated organ,

and because the anatomy, physiology, and pharmacology of many of its neurons
are well dened, the retina provides a good model for studying degeneration and
neuroprotection within the central nervous system. In addition, the retina can be
studied in vivo, or removed from the eye and examined for an extended period
of time as an isolated tissue preparation [2025]. Because removal from the eye
does not disrupt its cellular organization, neurons within the isolated retina retain
their normal spatial and connective relations (synaptic and gap junctional), and
their responses to light stimulation. This is in contrast to other isolated brain
preparations where tissue acquisition commonly results in a signicant disruption
of afferent and efferent connections, neurons at the tissue margins are damaged
during isolation, and the functional integrity of the isolated preparation can be
assessed only by means of electrical stimulation. The maintenance of normal
cellular relations within the isolated retina also is important with respect to neuroprotection, where survival mechanisms need not involve only direct interactions
between the drug and the injured neurons.
The cellular organization of the vertebrate retina, which is conserved across
most species, also makes it an ideal tissue for use in degeneration and neuroprotection studies. Although the retina contains several million neurons, these are
arranged into three distinct layers of nerve cells, separated by two layers of synaptic connections. And while many subclasses of retinal neurons have been described, it is generally agreed that most retinas contain only ve major classes
of neurons. From outside to inside, these include the photoreceptors, horizontal,
bipolar, and amacrine cells, and the ganglion cells [1]. Of these different cell
types, perhaps the best studied are the ganglion cells. These neurons represent
the nal stage of visual processing within the retina and their axons form the
optic nerve, a primary site of injury in many optic neuropathies. In addition,
ganglion cells and their axons give rise to the different functional streams that
compose the central visual pathway [2,5] Finally, because they are relatively
large and form a distinct layer near the inner retinal surface, ganglion cells are
more accessible, both in vivo and in the isolated retina than most other neurons
[1].
III. GANGLION CELL CLASSIFICATIONS

AND METHODS OF STUDYING
To date, at least thirty morphologically distinct types of ganglion cells have been
reported in the vertebrate retina [2628]. Despite this relatively large number,
however, our current understanding of ganglion cell structure and function is
based almost exclusively on a select group of neurons from the cat and primate
retinae. In the cat, the three major anatomical classes of ganglion cells are the
207
alpha, beta, and gamma cells, and there is direct intracellular evidence that these
correspond with the Y-, X- and W-cells described physiologically [2,2931]. Of
these different classes of ganglion cells, the alpha and beta cells have been described most completely. In brief, alpha cells represent about 5% of the ganglion
cells in the cat retina. They have the largest cell bodies (2535 m diameters),
and large, radially oriented, dendritic arbors that commonly originate from ve
or six primary dendrites and display a regular pattern of branching. Beta-cells
comprise about 55% of the ganglion cells in the cat retina. They have mediumsized somata (1820 m diameters), and their small to medium-sized dendritic
trees often originate from a single primary dendrite that then gives rise to a compact, bushy dendritic arbor. Functionally, Y-cells have high contrast gain, large
receptive elds, fast conducting axons, and are considered to be involved primarily with object detection. By contrast, X-cells have small receptive elds, are of
highest density in central retina, and are considered to be involved primarily with
the analysis of ne detail.
The primate retina also contains three major classes of ganglion cells.
Anatomically, these are the parasol, midget, and small-eld bistratied cells
[4,5,20,22,25,3236]. Parasol cells represent approximately 10% of the ganglion
cells in the primate retina. At all retinal eccentricities, the somata and dendritic
elds of these neurons are among the largest in the ganglion cell layer. Similar
to the alpha cells of the cat retina, the dendritic arbors of primate parasol cells
commonly originate from three or four large primary processes that branch regularly and form a radially symmetric arbor. Functionally, parasol cells have large
receptive elds with rapidly conducting axons, and they respond best to achromatic stimuli of high temporal and low spatial frequency [3,5,3740]. Combined
with their relatively uniform distribution acrosss the retina, these neurons also
are considered to be involved primarily with object detection. Midget ganglion
cells represent about 80% of the ganglion cells in the primate retina. They have
medium-sized somata and their small to medium-sized dendritic trees often originate from a single dendrite that then gives rise to a compact, bushy dendritic
arbor. Midget ganglion cells have small receptive elds and respond best to chromatic stimuli (primarily redgreen) of high spatial and low temporal frequency
[3,5,37-40]. Based on these characteristics, and their high density and one-toone synaptic arrangements with midget bipolar cells within the fovea, these neurons are considered to subserve ne spatial discrimination. Small-eld bistratied
cells represent about 58% of the ganglion cells in the primate retina. These
neurons have somata and dendritic elds that are similar in size to those of surrounding parasol cells, and they respond best to short-wavelength (blue) stimuli
[4,5,36].
For more than 100 years, anatomists have been interested in the structure
and classication of neurons in different regions of the brain, including the retina.
Some of the earliest anatomical work in the retina was conducted by Tartuferi
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and Ramon y Cajal using modications of the osmium-dichromate-silver staining

method of Golgi, and by Dogiel, using the methylene blue staining routine of
Ehrlich [41]. More recent studies aimed at dening the different morphological
classes of ganglion cells in the vertebrate retina also have relied on these early
techniques. In addition, neurobrillar stains have been used to study the morphologies of ganglion cells in the normal retina [42], and following damage to the
optic nerve [8]. A number of different retrograde tracing methods also have been
used to examine ganglion cell morphology. Some of these have involved direct
application of various dyes and neuronal tracers to either the intact retina or the
severed optic nerve [4346]; others have used micro-injections of tracers into
specic retinal target structures in order to dene the central projection patterns
of specic ganglion cell types [4749]. The major advantage of these staining
techniques over the more traditional Nissl-staining methods [50] is that they provide detailed information about not only the cell soma but also the dendritic arbor.
This enhances the accuracy of ganglion cell classication and provides a ner
level of detail for determining injury- and/or treatment-related changes in ganglion cell morphology.
Surprisingly, to date few of the techniques described above have been applied to studies of retinal ganglion cell degeneration or neuroprotection. Most
likely this is because of their capriciousness. Although silver staining methods
reveal exquisite morphological detail, they often stain only select subsets of neurons. In addition, the pattern of staining can be highly variable across retinas, or
even within the same retina. Various approaches using retrograde tracers also
have been hampered by the fact that they often label only small populations of
ganglion cells, the labeling is inconsistent and nonuniform across and within
retinas, and the tracer often fails to label ne distal dendrites. Furthermore, in
those cases where the insult occurs at the level of the optic nerve, the potential
for labeling ganglion cells from a central target are greatly diminished or eliminated. One approach that has been used with relatively good success to study
retinal degeneration and neuroprotection in the rat involves placing a piece of
gelatin sponge soaked with either Fluoro-Gold (Fluorochrome, Englewood, CO)
or the carbocyanine dye DiI (Molecular Probes, Eugene, OR) over the superior
colliculus and visual thalamus in vivo [11,51]. Because both of these retrograde
tracers are capable of labeling ganglion cells long term, it is possible to prelabel
ganglion cells, induce the retinal injury, and still provide an adequate survival
period for assessing the treatment strategy. One limitation to this approach, however, is that it is most practical only in small vertebrates, such as the rat, where
the central target nuclei can be easily accessed and covered with the tracer-soaked
sponge. In addition, the tracers label primarily the cell soma, and the fate of tracer
released from degenerating ganglion cells is always a concern. Finally, recent
evidence indicates that, following long-term survival periods (6 wk), Fluoro-
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Gold migrates not only from ganglion cells but from the retina in general (Bal
Chauhan, personal communication).
IV. THE ISOLATED RETINA PREPARATION

In our analyses of retinal ganglion cells from cats with neonatal visual cortex
damage [19] and monkeys with experimental glaucoma [25], we sought to avoid
the limitations of the silver impregnation and retrograde labeling techniques by
combining an isolated retina preparation with intracellular staining techniques.
This approach provided several advantages. First, it allowed us to visualize and
target single neurons. This was especially important in the cats with neonatal
visual cortex damage, where there was a signicant reduction in the number of
surviving ganglion cells. Second, using the isolated retina preparation we were
able to examine ganglion cells from matched areas of each retina, thereby reducing sample variability. And nally, like the silver staining methods, the intracellular approach allowed us to label and compare qualitatively and quantitatively
morphological features associated not only with the cell soma but also the dendritic tree and intraretinal segment of the axon.
Similar procedures are used to isolate and maintain our cat and primate
retinas. These are outlined diagrammatically in Figure 1 and described in more
detail below. Following the desired survival period, each animal receives an overdose of pentobarbital sodium. The eyes then are removed and placed into the
same solution that will be used to sustain them during the course of the experiment. We have used both Ames media [2022,46] and the articial cerebral spinal uid (aCSF) described by Saito [29] with equal success. If the eyes must be
transported, we have found it best to either leave them intact, or design a small
chamber for providing oxygenation during transport. The anterior segment of
each eye (from the ora serrata forward) is removed using a scalpel blade and pair
of small scissors, and the resulting posterior eyecup is placed into a beaker of
aCSF (pH 7.4). The beaker contains a stainless steel basket that holds the tissue
off the bottom, and the aCSF is oxygenated with 95% O2 and 5% CO2 at room
temperature using a glass gas dispersion tube with a fritted ending. Separation
of the anterior and posterior segments is performed in a petri dish over gauze
that has been moistened lightly with aCSF to prevent the tissue from sticking.
Good separation, with complete removal of the vitreous, seems to work best if
the initial incision is made 23 mm posterior to the ora serrata, and the two
segments then are folded back away from each other using two pair of forceps
with serrated tips. The goal is to have the vitreous remain rmly attached to the
anterior segment and removed from the posterior segment by peeling it away
from the retinal surface; pulling the vitreous perpendicular to the surface can
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Figure 1 Schematic diagram outlining the steps used in preparing the isolated,
living retina preparation. The posterior eyecup is stored and dissected in oxygenated articial cerebrospinal uid (aCSF) prior to being placed, ganglion cell layer
up, into the recording/injection chamber. This chamber, which also receives oxygenated aCSF, ts onto the stage of an upright microscope. Single neurons in the
isolated retina are viewed and targeted for intracellular analysis and injection by
prestaining with the vital dye Acridine Orange. A high-resolution video monitor is
used to provide visual stimulation of the retina, and an intracellular ampler is used
to obtain biophysical measurements.
result in mechanical damage to the retina. If vitreous remains attached to the

posterior eyecup, it often can be removed prior to placing the retina into the
recording and injection chamber using the reverse side of a pair of curved forceps
and grasping the vitreous in an area of the retina that will not be examined.
Treating the retina briey with a low concentration (0.1%) of collagenase (Type
II, C-6885, Sigma Chemical, St. Louis, MO) also has been used [20]. Again, it
is important to try to peel the vitreous from the retina by detaching it from the
margin of the eyecup and drawing it horizontally across the surface of the retina.
A clean retinal surface is imperative for good oxygenation of the tissue, clean
microelectrode penetrations and recordings of ganglion cells, and successful postinjection processing.
Once a posterior eyecup with a clean retinal surface is obtained, one can
either separate the retina from the choroid and sclera using a ne (# 00) artists
211
brush, remove only the sclera using forceps and scissors, or leave the entire eyecup intact. Complete isolation of the retina is suitable for intracellular staining
studies, but not those involving electrophysiological recordings, where dissection-related damage to the photoreceptor and pigment epithelia layers could affect
light responsiveness. For these studies it is best to either remove only the sclera
or leave the eyecup intact. Final preparation of the retina for placement into the
injection/recording chamber is performed in a petri dish containing aCSF. The
eyecup is placed on a submersed, perforated, stainless steel platform, and a bubble
stone is used to maintain oxygenation. Regardless of which type of isolated retina
preparation will be used, three to four radial cuts, made away from the area of
interest, are used to atten the tissue; the optic disc, fovea, and retinal blood
vessels serve as references for positioning these incisions. The retina then is
placed, ganglion cell layer up, into the injection/recording chamber, which has
been lled with oxygenated aCSF. While many different types of chambers are
available commercially, we prefer one of our own design. The chamber consists
of a rectangular piece of Plexiglas that has a circular well in the center. A pair
of concentric Plexiglas rings t into the well. The outer ring contains strands of
nylon stocking that form a platform and hold the tissue above the chamber bottom. The inner ring also contains strands of nylon stocking, and when press t
into the outer ring, forms a sandwich that holds the tissue securely in place.
The chamber then is mounted onto the stage of an upright microscope (Nikon
Optiphot-2) equipped with epiuorescence. The tissue is perfused with warm
(36C), oxygenated aCSF using a gravity ow system. An aspirator bottle containing the aCSF is placed approximately 24 in. above the tissue preparation, and
the solution is oxygenated with 95% O2 /5% CO2 using a glass gas dispersion
tube. The aCSF then passes through a ow meter and water jacket where the
ow rate is adjusted to 46 cc/min and the solution is warmed just prior to
delivery into the tissue chamber. The temperature of the water jacket, which
consists of a series of stainless steel tubes traversing a water-lled compartment,
is adjusted using a temperature-controlled circulating pump (Isotemp 2100,
Fisher Scientic). The aCSF is warmed by making ve passes through the Plexiglas water jacket before being dispensed to the tissue chamber. The solution ows
over the tissue, and then is drawn off from a separate well using a vacuum pump
and sidearm ask. Not drawing the aCSF directly from the injection/recording
chamber prevents uctuations in the chamber uid level. The stainless steel needle used to draw off the uid is moveable, thereby allowing one to adjust the
depth of the aCSF in the chamber. This is of particular importance for the physiological recordings, where uid depth affects the capacitance of the electrode, and
therefore the quality of the electrical signals recorded.
In all cases, the retinal dissections are performed in dim light, and the retinas are allowed 30 min in compete darkness in the injection/recording chamber
before being studied. Single ganglion cells are viewed under epiuorescence us-
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ing a 40 water immersion objective (Nikon, N.A. 0.55) with a working distance
of about 1.6 mm. In order to aid visualization, the retinas are treated periodically
with two to three drops of the vital dye Acridine Orange (A-4912, Sigma, 1mM
in aCSF, see Fig. 1). This often is not necessary when working with a completely
isolated cat retina, where individual ganglion cells can be viewed under standard
illumination using either differential contrast optics, or by simply misadjusting
the microscope condenser. However, because of the increased number of ganglion cell axons, it is difcult to view individual cells in the primate retina without
Acridine prestaining. When ganglion cells are viewed using epiuorescence, a
neutral density lter (ND4) is used to reduce the intensity of the mercury vapor
light reaching the tissue, and cells are viewed only long enough to either align
the electrode with the selected cell (physiology studies), or quickly penetrate the
cell (anatomical studies).
V.
ELECTRODE PREPARATION AND

INTRACELLULAR STAINING
Intracellular injections and/or recordings are made using glass microelectrodes

and a four-axis hydraulic micromanipulator (Narishighe, Japan). The glass micropipettes are pulled on a Flaming-Brown P-87 horizontal micropipette puller (Sutter Instruments, Novato, CA). For anatomical studies, we have found that a 3%
solution of the uorescent dye Lucifer Yellow CH (Sigma, L-0259) in 0.1 M
LiCl (pH 7.6) works well for labeling both cat and primate ganglion cells. For
combined electrophysiological and anatomical studies, we ll the pipettes with
a solution containing 2% Neurobiotin (Vector Labs, Burlingame, CA) and 0.05%
pyranine (Aldrich, Milwaukee, WI) in 1 M potassium acetate buffer (pH 7.6);
Lucifer Yellow CH precipitates in 1 M potassium acetate, and therefore we do
not use it with this buffer. However, it may remain soluble in 200 mM acetate
buffer. Because both uorescent dyes have excitation and emission wavelengths
similar to Acridine Orange, it is possible to view the electrode tip and the
prestained ganglion cells using the same epiuorescence lter combination (B3A/DM505, Nikon). This makes it easier to target single ganglion cells and results in a more stable preparation, because lter cubes do not need to be exchanged during cell penetrations. The electrodes are beveled from an initial resistance of 80100 M to a nal resistance of about 3545 M using a K.T. Brown
type beveler (BV-10, Sutter Instruments). For intracellular injection with Lucifer
Yellow CH, the electrode is positioned adjacent to the cell soma and advanced
slowly along its axis until it is seen to penetrate the cell soma. Successful penetration of the cell membrane is recognized by the sudden lling of the soma by the
small amount of dye that leaks from the electrode tip. Complete lling of the
soma, dendritic tree, and intraretinal axon then is achieved by passing 15 nA of
213
negative current through the electrode. Typically-2 min is required to ll parasol,

midget, and beta ganglion cells, whereas 23 min is needed for the larger alpha
cells. Intracellular penetration and recording using the potassium acetate-Neurobiotin electrodes is accomplished using a similar strategy; however, here the negative capacitance circuitry of the intracellular amplier (AxoClamp 2B, Axon
Instruments, Union City, CA) is used to gently ring the electrode tip and aid
with penetration of the cell membrane. Successful penetration is indicated by a
negative deection of about 5060 mV in the recorded electrode potential, and
large intracellular spikes. Injection of Neurobiotin is achieved using positive current pulses (12 nA) of 100200 ms duration, delivered for 35 min.
Regardless of the injection procedure, it is imperative that optimal electrode
conguration and injection parameters be established for the intracellular dyes
and buffers being used, and the cells being studied. Nonoptimal conditions can
result in morphological artifacts that could be misinterpreted as degenerative
changes. In addition, because it is not uncommon for a bond to develop between
the cell membrane and electrode, quick withdrawl of the electrode is needed in
order to prevent the cell soma from being damaged mechanically or excised from
the tissue.
Upon completion of the injections and/or recordings, the retina is removed
from the chamber and drop-xed in a solution containing 4% paraformaldehyde
in 0.1 M phosphate buffer. Retinas containing Lucifer Yellow CH-lled ganglion
cells then are washed with buffer, mounted onto subbed slides, and coverslipped
using DePeX (BDH Laboratory, Poole, England), a non-autouorescing mounting medium. Neurobiotin-labeled retinas are washed with 0.01 M phosphate buffered saline, dissected from the sclera and pigment epithelium using a ne artists
brush (# 00), and processed using Vector Labs Vectastain ABC Elite (PK-6100)
and DAB substrate (SK-4100) kits. These retinas also are then mounted and
coverslipped using DePeX.
VI. RETINAL SAMPLING, MAPPING,

AND ANALYSIS
Although the intracellular technique provides detailed lling of single ganglion
cells, it does not permit large numbers of neurons to be sampled across the entire
retina. Therefore, one typically needs to select a specic area of the retina for
study. This also is necessary because ganglion cell size is not constant, but increases with increased retinal eccentricity. Thus, for accurate morphological comparisons to be made, it is necessary to match ganglion cell samples for retinal
location. Fortunately, the ability to visualize specic retinal landmarks, such as
the optic disc, fovea, area centralis, and retinal blood vessel pattern, in the isolated
retina make it possible to coordinate cell sampling across retinae.
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In order that qualitative and quantitative comparisons be made for retinal

ganglion cells of like retinal eccentricity, the position of each injected cell relative
to the location of the optic disc and either fovea (primate) or area centralis (cat)
is mapped in the xed and mounted retina using a microscope stage digitizing
system (AccuStage, Shoreview, MN). Because this system employs a set of mechanical stage encoders, accurate reconstruction of the retinal map and cell locations is not inuenced by changes in microscope objective magnication. In the
case of the uorescent-labeled neurons, this map also serves as a guide during
the capture of individual cell images using the confocal microscope. The application of confocal microscopy greatly enhances ones ability to acquire and analyze
ganglion cell morphological data efciently; complete high-resolution cell reconstructions can be achieved in minutes, instead of hours, as needed using more
conventional techniques. In addition, because the images are captured as a set
of digital optical slices, it is possible not only to view each cell in three-dimensions from any perspective but also to compress the optical slices into a single
image that includes the soma, dendritic tree, and intraretinal axon (Fig. 2). This
feature is very useful given the highly three-dimensional structure of ganglion
Figure 2 Samples of parasol and midget ganglion cells from glaucomatous eyes
that were injected intracellularly with the uorescent dye Lucifer Yellow CH and
captured using confocal microscopy. Note that the earliest changes are associated
with the dendritic processes. (Adapted from Ref. 25.)
215
cells, and one that is not readily available using standard video image capture
and analysis systems. Aside from the ease of capture and the ability to freely
manipulate cell images, a third advantage of being able to apply confocal microscopy is that most quantitative measurements can be made directly from the digital
images using the systems own morphological analysis software. This enhances
the efciency of the analysis and reduces the potential for magnication-based
errors. However, since most confocal image formats now are compatible with
current image analysis packages, cell images can be downloaded and analyzed
ofine to reduce cost and increase exibility. For our purposes, we combined
intracellular staining and both video-enhanced and confocal microscopy to compare ganglion cell morphologies following either neonatal damage to visual cortex in the cat [19] or experimental glaucoma in primates [25]. Qualitative and
quantitative ganglion cell comparisons included the assessment of differences in
dendritic eld organization, cell body size, dendritic eld area, and axon diameter
for ganglion cells of specic classes. In the cat studies, we found that a selective
loss of beta ganglion cells due to neonatal damage to visual cortex affects only
the development of surviving beta, and not alpha, ganglion cells. This suggests
that the dendritic elds of ganglion cells in the vertebrate retina achieve their
nal morphologies based on competitive interactions among ganglion cells of the
same, and not different, classes. With respect to the primate studies, application of
the intracellular staining technique allowed us to demonstrate for the rst time
that the earliest signs of ganglion cell degeneration in glaucoma occur at the level
of the distal dendritic tree, and that the pattern of degeneration is similar for both
midget and parasol type cells. In brief, the degenerative changes include a thinning of the proximal and distal dendrites, abrupt reductions in dendritic process
diameter at branch points, and a general decrease in the complexity of the dendritic tree (Fig. 2). These ndings have at least three signicant implications.
First, they indicate that the onset of glaucoma-related ganglion cell death occurs
earlier than previously thought based on Nissl-stained estimates of ganglion cell
loss alone. Second, because ganglion cells receive all of their input from more
distal retinal elements via their dendrites, it is reasonable to assume that early
decits in ganglion cell function also occur; this is the focus of our structurefunction studies. And nally, since changes at the level of the cell soma occur
later, they suggest a window of opportunity for effective neuroprotection intervention.
Because our structure-function studies employ the use of a nonuorescent
dye, the ganglion cells for this work are analyzed using the Neurolucida/
NeuroExplorer cell reconstruction and analysis system (MicroBrightField, Inc.,
Colchester, VT). This system incorporates a motorized stage and video-based
(Hamamatsu C-5985 chilled CCD camera) image analysis software. Individual
cells that have been lled with Neurobiotin are reconstructed three-dimensionally
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from a live video image by tracing and recording the spatial relations of each
cell component (e.g., soma, dendrite, dendritic branch point, spine). Once the
data have been entered, it is possible to rotate the cell image in any plane to view
the vertical and horizontal dimensions of the cell. It also is possible to apply a
number of standard analysis routines, including Sholl and polar analyses of the
cells dendritic tree. These provide valuable information concerning the
branching pattern and complexity of the dendritic arbor, and whether it shows
any asymmetry (Fig. 3). In addition, it is possible to compare differences in dendritic spine content, number of dendritic nodes, number and type (primary, secondary, etc.) of dendritic branches and branch length, surface area of the soma
and dendritic eld, and dendritic eld volume. All of these features are important
because the structural integrity of the soma and dendritic eld inuences the
spatial and temporal response properties of ganglion cells. Although the current
focus is to correlate degenerative changes in ganglion cell morphology with
changes in ganglion cell function, these techniques also can be used to establish
whether different neuroprotective treatment strategies result in the recovery of
normal ganglion cell structure-function following retinal injury.
Figure 3 Summary of the anatomical and physiological data that can be obtained
using the isolated retina preparation. Both biophysical and visual stimulation information are collected, and these data then are compared with the ganglion cells
structural integrity.
217
VII. USE OF THE ISOLATED RETINA

PREPARATION FOR BIOPHYSICAL
AND VISUAL STIMULATION STUDIES
As noted previously, one of the main advantages of working with the isolated
retina preparation is that its neural circuitry is preserved. Thus one can analyze
not only the morphology, but also the intrinsic and visual response properties of
single neurons (Fig. 3). Biophysical analyses, which evaluate membrane integrity,
include measurements of resting membrane potential, membrane time constants,
threshold levels for activation, and rate of ring in response to intracellularly
applied steps of depolarizing current. In our setup, the stimuli used for these
tests are presented, and the cellular responses collected and analyzed, using a
commercial stimulus presentation and data acquisition and analysis package
(pClamp6, Axon Instruments). Visual stimulation of the isolated retina can be
achieved by presenting light stimuli (ashing or patterned) via either the camera
port or the condenser of the microscope. Because we do not remove the sclera
from the retina preparation, our system involves positioning a high-resolution
XYZ video monitor (Tektronix 608) over the camera port of the microscope (Fig.
1). The video monitor is aligned with the center of the camera port, and the cell
to be studied is positioned in the center of the microscope eld. Visual patterns
consisting of drifting and counter-phased light and dark bars are projected onto
the isolated retina through the 40 objective. The various patterns are generated
and presented in random order by computer control of a Picasso CRT Image
Synthesizer (Innisfree, Fenstanton, Cambridgeshire, England). Sinusoidal-modulated color diodes also are used to examine the response properties of primate
ganglion cells.
VIII. USE OF THE WHOLE-MOUNTED RETINA FOR

STUDYING NEUROPROTECTION
As a rst assessment of neuronal degeneration or survival following a particular
manipulation or treatment strategy, it often is useful to start by examining a large
population of neurons over a wide region of the tissue being studied. Since this
is not possible using the isolated retina-intracellular technique, we used Nisslstained retinal whole mounts in our initial studies of retinal ganglion cell survival
following optic nerve injury and brain-derived neurotrophic factor (BDNF: Regeneron Pharmaceuticals, Tarrytown, NY) treatment in the cat [13]. The retinal
whole mount is a unique tissue preparation, and it is ideally suited for studying
changes in ganglion cell size, number, and density. Cats too, offer several advantages for use in neuroprotection studies. First, as noted previously, the morpholo-
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gies and central projection patterns of cat ganglion cells are comparable to those
of the primate. Second, the cat eye, and particularly the vitreal chamber, is similar
in size to that of the primate. This provides for a more direct comparison between
drug application and neuronal survival than is possible following the treatment
of different sized eyes, where dose and diffusion differences might be important
limiting factors. Finally, it is relatively easy to achieve uniform Nissl staining of
ganglion cells in the cat retina (Fig. 4). This is not always the case in the primate,
where the higher number of ganglion cells, thicker nerve ber layer, and often
more difcult to remove vitreous can result in one having to stain for a specic
area of interest.
Figure 4 Schematic and photomicrograph showing the approximate region of the

cat retina used to study changes in ganglion cells size, number, and density following optic nerve crush and treatment with BDNF. Note the clarity of ganglion cell
staining (Nissl) in the cat retina and the uniformity of cell size at the retinal eccentricity used (dotted line). OD: optic disc; AC: area centralis. (Adapted from Ref.
13.)
219
A central issue in any study that involves the measurement of ganglion cell
numbers is cell identication. In the cat, as with most vertebrate retinas the ganglion cell layer also contains a signicant number of amacrine cells [5254].
While most ganglion cells in the rat can be identied using the sponge-based
retrograde labeling method described previously, this approach is not readily applicable in the cat due to the greater depth and spatial separation of the colliculus
and visual thalamus. Nevertheless, in the cat one can distinguish most ganglion
cells and displaced amacrine cells based on soma size and cytoplasmic differences; the amacrine cells are among the smallest neurons in the ganglion cell
layer, they show a low ratio of cytoplasmic-to-nuclear volume, and they often
contain a prominent basophilic nuclear fold [52].
In our analysis of the neuroprotective effects of BDNF in cats following
optic nerve injury, we selected for quantitative analysis a region of the retina
that occupied 1.74 mm2 and was located 3.0 mm above and 1.5 mm temporal to
the area centralis. This region was chosen because of the relatively constant size
and density of ganglion cells in this area of the cat retina (Fig. 4)[26]. Welldened retinal landmarks such as the optic disc, area centralis, and retinal blood
vessel pattern were used, along with the microscope stage digitizer, to properly
orient each retina on the microscope stage, and to standardize the starting point
and stage movements for cell sampling. From the starting point, 42 digital images
(41,000 m2 /image) then were obtained systematically using the Hamamatsu
high-resolution video camera and a 40 objective. The retinal images were collected as three dorsal-ventral passes composed of 14 images each. Double counting was avoided by separating each sample column horizontally by 500 m (using
the Digitizer readout), and vertically by using each previous image as a reference
for the next. Cell size, density, and number were determined directly from the
digital images using an image analysis and measurement software package (Image Pro Plus, Media Cybernetics).
By standardizing the measurements and using a region of the retina that
could be identied reliably and sampled systematically across retinas, we were
able to demonstrate the neuroprotective ability of BDNF in an eye comparable
in size to that of the primate. Further, by testing different amounts of the drug,
we were able to demonstrate not only that 30 g of BDNF is needed to obtain
maximum ganglion cell survival in the cat, but also that increasing the drug dose
results in a decrease, rather than increase, in the level of neuroprotection (Fig.
5). Because the sample areas were standardized for each retina, changes in cell
density mirrored the changes in cell number. Cell size measurements suggested
a complex response among the different classes of ganglion cells. While 30 g
of BDNF retained the largest number of ganglion cells, 90 g minimized the loss
of medium-sized neurons and retained normal proportions of large, medium, and
small ganglion cells.
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Figure 5 Photomicrographs and graph demonstrating changes in ganglion cell

number following optic nerve crush and either no treatment or treatment with different levels of BDNF. Treatment with 30 g of BDNF resulted in the greatest cell
survival, primarily by rescuing the medium-sized beta cells. Atrophic ganglion cells
in the untreated retina are recognized by their irregular shape and clumped chromatin (arrows). (Adapted from Ref. 13.)
IX. SUMMARY
In this chapter I have attempted to highlight some of the advantages of using the
retina as a model for studying neuronal degeneration and neuroprotection. I also
have tried, without going into detail, to make the reader aware of some of the
more traditional approaches that have been used to study retinal ganglion cell
morphology over the years. Although many of these might be considered outdated, there remain areas where such traditional methods could be applied to
current studies of retinal degeneration and neuroprotection. Our use of the isolated retina and intracellular staining method, along with the Nissl-stained retinal
whole mount, reects both ends of the spectrum. The highly traditional Nisslstained approach continues to serve as a quick and reliable means for making a
221
rst assessment of neuronal survival based on the evaluation of a large number of

cells. The intracellular approach provides a more detailed analysis of the cellular
changes that occur at the single cell level. It is hoped that the procedures described
here will provide a foundation for others also seeking ways to enhance their
understanding of the injured retina and its responses to different neuroprotection
treatment strategies.
ACKNOWLEDGMENTS
Supported by NIH/NEI grant EY11159 and grants from the Michigan State University Foundation, The Glaucoma Foundation, Alcon Laboratories, and The
American Health Assistance Foundation.
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13
Detection of Single-Cell Apoptosis
William G. Tatton, Ruth M. E. Chalmers-Redman,
and Nadine A. Tatton
Mount Sinai School of Medicine
New York, New York, U.S.A.
I.
INTRODUCTION
Our initial denitions of the apoptotic process were based on a set of morphological changes that occurred in the cell nucleus and cytoplasm. As rst described
by Kerr using electron microscopy [1] the earliest visible change in the apoptotic
cell was the aggregation of chromatin into compact masses along the nuclear
membrane. Eventually, more and more compact granular masses appeared and
lled the nucleus, combined with a gradual reduction in nuclear volume. At the
same time the cytoplasm displayed progressive condensation, but with preservation of organelles. Apoptotic bodies consisting of discrete spherical or ovoid
fragments containing highly condensed chromatin were then phagocytosed and
lysed by nearby cells. Initially these changes were thought to encompass the
entire apoptotic process, but now it is clear that apoptotic bodies are part of the
nal degradative phase of apoptosis (Fig. 1).
Interestingly, the appearance of these morphological changes is still used
as a standard by which to determine whether cells have died via apoptosis. Now,
however, we recognize that it is probably of greater importance to determine
what apoptotic signaling pathway the cell has followed because knowledge of the
pathway will ultimately provide us with information necessary to design agents to
selectively block apoptosis. It is worth noting that not all apoptotic pathways
require the involvement of the mitochondria.
225
226
Tatton et al.
Figure 1 Schematic illustrating the three phases of apoptosis: the initiation

phase, decisional phase, and degradative phase. Four hypothetical initiating insults are shown: three that involve a mitochondrial decision phase and are mitochondrially dependent and a fourth that is mitochondrially independent. The multiple initiation and decision phase signaling pathways converge onto common
events in the degradation phase. The diagram is meant to emphasize the role of
mitochondrial decisional processes in some forms of apoptosis, including the role
of BAD, BAX, and BCL-2-like proteins in the mitochondrial decisional process.
(From Ref. 39.) The diagram is not meant to fully illustrate all apoptosis signaling
pathways.
II. APOPTOTIC DEGRADATION FACTORS

RELEASED BY DECREASED MITOCHONDRIAL
MEMBRANE PERMEABILITY
At least two factors that signal for apoptotic degradation and that are released
from mitochondria have been identied: cytochrome c (CytC) and apoptosis initiation factor (AIF), a 50 kD avoprotein. Released CytC interacts with apoptosis
protease activating factor-1 (Apaf-1) [2], dATP/ATP and procaspase 9 to form
a complex known as the apoptosome [3], in which procaspase 9 is converted to
caspase 9 (see Fig. 1). Caspase 9 then converts procaspase 3 to activated caspase
3 along with activating caspase 7. The apoptosome seems to function as a multicaspase activating complex (see Ref. 4). Caspase 3 in turn activates DNA frag-
Detecting Single-Cell Apoptosis
227
mentation factor [5], an endonuclease activator which enables DNA cleavage and
acinus which enables chromatin condensation [6]. The second factor released
from mitochondria, AIF, induces DNA loss, peripheral chromatin condensation,
and digestion of chromatin into 50 kbp-fragments [7]. The caspase-3 inhibitor
ACE-DVAD-FMC inhibits chromatin condensation induced by AIF. The proteases that occur in the early stages of the apoptotic caspase-activation cascade,
pro-caspase 2 and 9, have been found to be concentrated in the intermembranous
space of mitochondria [8].
III. METHODS OF DETECTING SINGLE-CELL

APOPTOSIS
We have developed a number of protocols to examine apoptosis in situ, with
particular interest in human postmortem tissue. These include a uorescent double-labeling method to simultaneously visualize DNA fragmentation and apoptotic chromatin condensation [9] and several microwave-based antigen retrieval
methods for immunocytochemistry of apoptosis-related proteins on parafn sections. We have also modied these protocols for use on xed, frozen sections and
on cultured cells when examining apoptotic changes in different model systems.
Gavrieli rst published a method of in situ end labeling [10] that allowed the
visualization of DNA fragmentation in tissue sections. Popularly known as the
TUNEL method, or ISEL (in situ end labeling), this was a breakthrough in
the examination of apoptotic cell death in tissue sections and in cultured cells.
Unfortunately, ISEL when used alone, cannot provide unequivocal identication of an apoptotic nucleus. Endonucleases that digest DNA can create single- and/or double-strand breaks reected as high molecular weight (50300 kbp)
DNA fragments on gel electrophoresis [11]. Often the DNA digestion continues,
with the ultimate production of low molecular weight oligonucleosome-sized
fragments [12,13]. Single-strand DNA breaks likely accumulate in the linker regions between oligonucleosomes prior to the double-strand DNA breaks which
create a ladder pattern when observed by gel electrophoresis [14]. The ISEL
method was considered to be selective in detecting these double-stranded breaks
between oligonucleosomes.
However, the terminal transferase enzyme (TdT) commonly used for ISEL
is generally supplied with a protocol that requires increased cobalt chloride (2.5
mM) in the reaction mix, allowing TdT to label double-strand breaks (protruding,
blunt, and recessed ends) as well as single-strand DNA breaks [15,16]. It is important to note that cells dying by necrosis have also been reported to contain singleand/or double-strand breaks, and thus the ISEL method may not always distinguish between the two modes of cell death [1719]. A second, independent
method is therefore required to conrm apoptosis where there is demonstrable
228
Tatton et al.
DNA fragmentation. The cyanine dye YOYO-1 (Molecular Probes) binds to

DNA (and RNA) and allows the visualization of apoptotic chromatin condensationan event that is independent of DNA fragmentation. Use of confocal laser
microscopy has offered us the additional advantage of high resolution of these
nuclear labels (see Refs. 9,20,21).
IV. ISEL/YOYO FOR HUMAN BRAIN POSTMORTEM
PARAFFIN SECTIONS
Standard, 5-m-thick parafn sections are stored at 4C after sectioning. Slides
are run through a standard series of baths to remove all parafn and rehydrate
the sections.
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
Xylene 115 min.

Xylene 210 min.
100% Ethanol 110 min.
100% Ethanol 210 min.
50% Ethanol10 min.
dH2O10 min.
Antigen Unmasking Solution (Vector Labs). Dilute 3.77 mL of this
stock solution in 400 mL distilled H2O in a glass container (a Pyrex
measuring cup is good). Add eight slides in a plastic rack. Microwave
slides for 3.5 4 min, power setting 7nal temperature reached is
approximately 60C. Temperature should not be above 62C in order
to avoid producing DNA degeneration due to excessive heating (i.e.,
65C or more) and nonspecic labeling. Let slides cool, submerged
in the buffer for 30 min on the bench.
Important: Only eight slides should be done at one time. We have
found that if there are more slides in the rack, the heating is uneven
across all slides, resulting in unreliable labeling. Power settings and
time vary with each microwave to achieve a nal temperature of
60C (we use an 850 W Kenmore with a turntable).
3% H2O2 /Methanol, room temperature, l0 min. PBS rinse3 min.
RNAseA digest (100 g/mL in 2 SSC) at 37C for 25 min). Digest
time should be decreased for retinal sections and for embryonic tissue.
The RNAse digest decreases background since terminal transferase
(TdT) can tail from single-strand RNA.
2 SSC rinse, 5 min.
Proteinase K digest (20 g/mL in T.E. pH 8) at 37C for 35 min.
Too long a digest time will increase nonspecic labeling. We do a
very brief digest for embryonic tissue (or retina), always less than 3
min.
229
12. 0.1 M glycine/0.1 M Tris pH 7.2, ice-cold, 1015 min. Stops the
enzyme digest and also helps reduce nonspecic background.
13. Equilibration buffer (Intergen), room temperature, 2030 min. Blot
off excess before adding reaction mix.
14. TdT/BODIPY-dUTP reaction mix, at 37C for 60 min. Thirty minutes provides inadequate and unreliable labeling; 90 min does not
provide any appreciable increase in signal to noise ratio. Cover each
tissue section with a paralm coverslip (allows the spread of a small
volume of labeling reaction mix evenly over section). Can use 12 L
on a mouse brain cross-section, 20 L on most human SNc sections
(unless it is really big, then 25 L).
15. 2 SSC stop bath, 3 10 min, prewarmed to 37C. Slides are washed
in 400 mL volumes of 2 SSC to effectively remove nonspecically
bound BODIPY-dUTP. It is important to use a large volume to remove excess label and that the temperature be 37C.
16. PBS, 2 5 min. Important to rinse all 2 SSC away to get good
YOYO staining.
17. YOYO (1 :500 in PBS), 30 min, dark, RT.
18. PBS rinses, 5.
19. Use Aquamount (Gurr) or GelMount (Biomeda) for coverslipsboth
are water soluble and dry quickly. Let slides dry in fume hood overnight, then store in fridge. We have found that the best color/brightness will be in the rst 72 h (apoptotic nuclei appear orange if you
have a lter that lets you do simultaneous red/green imaging). After
72 h, the uorescence is a little less bright, but still good for at least
another 34 months.
In order to get consistent labeling it is important that all concentrations of
enzymes and their buffers are accurate and are applied at the stated temperature.
This is also essential for the 2 SSC stop baths that follow the labeling reaction.
We prewarm all digestion enzymes before applying to the slides. The TdT reaction mix is made up in the sequence given below just before use. The TdT enzyme
is stored at 20C and should be kept in a freezer block or on ice when in use.
We use less enzyme and dUTP in our labeling reaction than is given in the product
spec sheet. We have tested a broad range and found that 1.4 L of dUTP and
1.4 L of TdT per 100 L reaction volume gave as good or better labeling on
tissue sections than the volumes recommended in the manufacturers spec sheet.
TdT Reaction Mix

20 L
5 TdT buffer
10 L
CoCl2
67.2L
dH2O
230
Tatton et al.
1.4 L
1.4 L
100 L
BODIPY-TR-14-dUTP (Molecular Probes)

TdT (Boehringer Mannheim/Rocher)
We tested different manufacturers TdT samples; Boehringer/Roche was the most

reliable and gave the best labeling. The sample includes the enzyme, reaction
buffer, and CoCl2.
Tdt: Boehringer/Roche, cat. # 220 582
BODIPY-TR-dUTP: Molecular Probes, cat. # C7618
YOYO-1: molecular probes, cat # Y3601
Equilibration Buffer: Intergen, cat # S7106
Other useful things to control for successful labeling:
1. A short postmortem interval for all pathology cases (ours were less
than 6 h on average). You will notice in many European publications the postmortem intervals are 20 h or greaterthis cannot produce reliable ISEL because
many nuclei will label because of extensive DNA degradation that occurs postmortem. Try to get less than a 12 h postmortem interval if you can.
2. The tissue should be xed in buffered formalin and kept refrigerated
to provide good DNA preservation for ISEL. Note: We saw no difference in
labeling with tissue kept in xative for 6 months, 2 years, or 3 weeks when we
initially tested human brain cryosections.
3. Proteinase K digestion is criticaltoo long and your background signal is increased (and it is impossible to get rid of), too short and you dont get
good access to the DNA in the nucleus. Always do a test run on a few slides,
trying different times. Embryonic tissue is more fragile, needs maybe about half
the digest time of adult brain. With some fragile tissues, we have found that a
brief exposure to Neuropore (Trevigen) offers good permeabilization. Again the
time of exposure must be tested (i.e., 515 min).
V.
IMMUNOCYTOCHEMISTRY FOR APOPTOSISRELATED PROTEINSHUMAN POSTMORTEM

PARAFFIN SECTIONS
A.
GAPDH (Monoclonal AntibodyChemicon)
We and others found that GAPDH (glyceraldehyde 3-phosphate dehydrogenase)

plays a role in the apoptotic pathway of some cells. In our partially neuronally
differentiated PC12 cells that enter apoptosis after serum and NGF withdrawal,
we found that there is an increase in cytoplasmic GAPDH as well as nuclear
accumulation of this protein. Blocking GAPDH nuclear accumulation, either by
antisense oligonucleotides [22] or with deprenyl-related propargylamines [23],
231
reduced the incidence of apoptotic nuclei in vitro. It is not yet clear what role
GAPDH plays in the nucleus of cells entering apoptosis. We do know that nuclear
GAPDH accumulation occurs before the nuclear degradative changes. This protocol works well for adult brain tissue, but we have noted that we get better results
if sections are stored cold, and reacted soon after being cut.
Dewax sections according to standard protocol (see ISEL/YOYO for details).
1. Antigen Unmasking Solution (Vector Labs). 400 mL in Pyrex measuring cup. Heat on High power for about 5 min to bring to boil. Add
slides in plastic rack and heat at Power setting 8 for 4060 s to reach
boiling, the let slides boil for 1 min only. Let slides cool in buffer
for 3040 min on benchtop.
2. PBS rinse, 1 5 min.
3. Methanol, 20C, 10 min. Rinse with PBS.
4. RNAse A digest, 100 g/mL in 2 SSC, prewarmed to 37C, for 3
min only! This step allows antibody access to GAPDH, which is often
found in association with RNA in the cell.
5. Ice-cold 0.1 M glycine/0.1 M Tris buffer (pH 7.2) for 5 min to stop
enzyme digest and decrease background.
6. PBS rinse, room temperature, 5 min.
7. 10% Normal Goat Serum/0.2% Tween 20, 15 min to block.
8. Primary Antibody (1: 200) in 1% NGS/0.2% Tween 20/0.1 M
PBS overnight, 4C. Individual sections are covered with a paralm
coverslip.
9. PBS rinses, room temperature, 4.
10. Secondary Antibody, goat anti-mouse IgGAlexa 594 (1:200), in 1%
NGS/0.2% Tween 20/0.1 M PBS, 1 h 37C.
11. PBS rinse, 3.
12. Coverslip with Gelmount (Biomeda) or Aquamount (Gurr) as described above.
1. Human Retinal Sections
We have found that the microwave heat treatment can be too harsh (as detailed
for GAPDH) and have often substituted a brief (about 10 min, room temperature)
Neuropore permeabilization step instead or no permeabilization step depending
on the quality of the case samples. Care trust be taken not to overexpose retinal
sections to this agent.
We also nd that there is a high level of nonspecic, dull background autouorescence in human adult (aged) retina largely in the red range that we do
not see in the brain/spinal cord sections. This is distinct from the bright autouorescent granules that accumulate in retinal ganglion cell neurons. For this reason,
we do not usually do more than one antibody on human postmortem sections
232
Tatton et al.
and often use DAPI as a nuclear counterstain (bright blue) or YOYO (bright
green) to visualize the chromatin pattern. Confocal imaging allows the section
to be viewed in the far red range and thus distinguish nonspecic autouorescent
signal from antibody-specic signal (see Ref. 20, for details). Note that without
an RNase A digest, YOYO will also stain cytoplasmic RNA.
2. Fluorescent Secondary Antibody Conjugates
We routinely use the Alexa dyes from molecular probes. They come in a broad
range of excitation/emission wavelengths, the colors are bright, intense and hold
up extremely well to confocal laser scanning. As a general rule, we have found
that YOYO staining should be done after the secondary antibody step.
B.
Caspase 3-Active Fragment
Although there are a few antibodies now available to the activated fragment of
caspase 3, we have found that we get excellent, reproducible results with the
New England BioLabs product. Control protein samples are also provided in case
you want to use this product for immunoblots. We have also used an antibody
against caspase 3 (recognizes both pro-caspase 3 and activated caspase 3) from
Pharmingen, which is also very reliable (see Ref. 20). For use on human brain
sections, we still use a microwave antigen retrieval method (as described for
GAPDH) but do not use any subsequent enzyme digests. The protocol described
below has been used on parafn sections of human retina.
Dewax according to the standard protocol.
1. Rinse sections in PBS for 5 min, room temperature.
2. Block 10% NGS/02.% Tween 20/0.1 M PBS, 1 h RT. Alternately,
substitute a 10 min permeabilization step with Neuropore followed by
20 min blocking step with 10% NGS/0.1 M PBS. Blot off excess.
3. Primary antibody (rabbit polyclonal) 1:100 in 1% NGS/0.2% Tween/
PBS, overnight at 4C. Sections are covered with a paralm coverslip.
5. Secondary antibodyAlexa 594 goat anti-rabbit 1: 200 in 1% NGS/
0.2% Tween/PBS, 40 min, 37C.
7. YOYO (1:500) in PBS, 30 min, RT, in the dark.
8. PBS rinses, 3 5 min.
9. Coverslip with Gelmount or Aquamount.
C.
Caspase 3 or BaxRapid Method for

Brighteld Microscopy
This is an alternate protocol developed for human brain parafn sections and is
an adaptation of the Biomeda Autoprobe kit, which gives an intense one-step
233
labeling for brighteld viewing. Unlike the Autoprobe original protocol, we have
extended some of the times because of antigen accessibility in sections from
old formalin-xed blocks (as opposed to freshly perfused tissue). We have also
increased the primary antibody incubation to overnight at 4C. Note that the
pepsin reagent digest times would likely need some changes in incubation times
if used for human retinal sections.
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
Xylene 115 min.

Xylene 210 min.
100% Ethanol10 min.
100% Ethanol10 min.
Endoblocker (1 mL in 5 mL 100% ethanol), 5 min, room temperature.
95% Ethanol3 min.
50% Ethanol3 min.
d H2O5 min.
Pepsin reagent 12 min at 37C (prewarmed).
1 AutoBuffer (AB), three quick rinses.
Antigen Unmasking Solution (Vector labs) 3.77 mL stock AUS in
400 mL dist. H2O. Bring solution to boiling (in glass Pyrex measuring
cup (4-cup size), at high power, about 5 min. Add 810 slides in
plastic rack to solution, bring to boil in approximately 30 s (power
setting 8) and then let boil for 1 min only. Let slides sit in buffer on
the benchtop for 3040 min.
1 AB 5 min RT
Tissue conditioner (Biomeda), 1 drop/mL AB, 10 min, room temperature.
Primary antibodyCaspase 3 (1 :500, Pharmingen) or Bax (Santa
Cruz) (1: 200) in primary antibody diluting buffer (Biomeda) O/N
4C.
AB rinse, 2 5 min.
Secondary antibody, universal antibody (Biomeda kit), 40 min, 37C
AB rinse, 2 .
Peroxidase reagent, 37C, 30 min.
AB rinse, 1.
Working chromogen solution, 15 min.
Dist H2O, stop bath, 2 5 min.
Coverlip with Gelmount or Aquamount (Gurr).
Useful information. You can likely nd a different antigen retrieval method for
every day of the month when you search the literature. We found that it helps
to start with the mildest approach possible (i.e., use an unmasking solution at
room temperature rst) and then gear up to more aggressive heat treatments and
then possibly choose to combine heat treatment with some type of enzymatic
digest. We have found that we will vary our retrieval protocols according to
234
Tatton et al.
whether we are working with adult tissue or embryonic tissue (gentler methods),
brain or retina (gentler methods). But most important is to nd out what your antigen
of interest does in the cell. Is it found in a membrane, is it bound to RNA, is it found
in vesicles, in the nucleus? All of these will help determine what you need to do to
make it accessible, often with a limited number of trial runs.
VI. METHODS FOR EXAMINING APOPTOTIC
CHANGES IN CULTURED CELLS
Many of our detection methods were rst developed for in vitro models of
apoptosis. These include ISEL/YOYO, measurement of mitochondrial membrane
potential, immunocytochemistry for apoptotic proteins, DNA-binding dyes to
visualize chromatin condensation, and so on. We generally use YOYO-1 for looking at chromatin condensation for tissue sections or cultured cells. This choice
has really been determined by the capabilities of our krypton-argon laser. The
Hoechst dye (bisbenziamide) and DAPI are also both excellent dyes for examining nuclear DNA and will not stain cytoplasmic RNA. Both are bright blue and
require UV excitation, are long-lasting and slow to quench (see molecular
probes). Either can be applied to tissue sections following antibody incubations
in place of YOYO-1.
A.
Hoechst Dye or YOYO-1 for Chromatin Condensation

For monolayer cell culture:
1.
2.
3.
4.
5.
6.
B.
Wash cells 3 with PBS to remove serum/media.

Fix cells with 4% paraformaldehyde, 30 min on ice.
Rinse well with PBS, 3.
Add Hoechst dye (5 g/mL PBS), 2030 min at room temperature,
in the dark. Or use YOYO-1 (1 :1000 in PBS) for 2030 min at room
temperature, in the dark.
Wash 5 PBS.
Mount in Aquamount (Gurr). We have found that Gelmount is not a
good choice for cultured cells stained with YOYO-1. It appears to
bleed out of the cells after 24 h; therefore, we recommend Aquamount for mounting cell culture coverslips.
ISEL/YOYO for Monolayer Cell Cultures

1. Wash cells 3 with PBS to remove serum/media.
2. Fix cells with 4% paraformaldehyde, 30 min, on ice. With neuronal
cultures we nd it best to keep cells in xative in the fridge overnight
after the initial 30 min on ice, then rinse with PBS the next day.
235
3. Rinse well with PBS, 3.

4. Dip in 100% methanol, 12 min, room temperature. It is important
to keep this step short as apoptotic nuclei can dissociate from the
remaining cell soma in cultured cells.
5. PBS rinse, 1, 5 min.
6. RNAseA digest (100 /mL in 2 SSC), 10 min, 37C
7. Rinse, 2 SSC, 2 5 min.
8. 0.1 M glycine/0.1 M Tris, pH 7.2, 20 min RT. Note that the proteinase
K digest is omitted (we found that it really served to detach cells from
the coverslip rather than improve ISEL signal).
9. Equilibration buffer (Intergen), 1530 min, room temperature.
10. TdT/BODIPY dUTP-red/reaction mix, 60 min, 37C.
11. Rinse, 2 SSC (prewarmed), 3 10 min, 37C.
13. YOYO-1 (1 :1000 in PBS), 2030 min, room temperature, in the dark.
14. PBS rinses, 5.
15. Aquamount coverslips, let dry in fume hood, then store in fridge.
We grow cells on 22-mm square coverslips for ISEL/YOYO or 12 mm
round coverslips. The 22-mm coverslips are kept in 35-mm petri dishes and are
easy to ush with different solutions. A paralm coverslip can easily be applied
to these in order to use a minimal volume of TdT reaction mix (3040 L). If
using the round coverslip, we have found it easiest to transfer them to a ceramic
well plate rather than keep them in a 24-well plastic tray. We invert them in
each well, adding reagents under the coverslip (it will save on TdT reaction mix
volumeuse about 70 L).
VII. MITOCHONDRIAL MEMBRANE POTENTIAL AS

A MEASURE OF APOPTOTIC CHANGE
There is an increasing body of evidence that mitochondria play a critical decisional role in number of forms of apoptosis including those initiated in neurons
by withdrawal of trophic factors, hypoxia/ischemia, and glutamate excitotoxicity.
Using partially differentiated PC 12 cells, apoptosis is induced by removal of
NGF and serum. We have found that there is a signicant decline in mitochondrial
membrane potential after trophic withdrawal, and that this precedes the appearance of nuclear apoptotic degradation.
We have used a xable mitochondrial potentiometric dye, chloromethyltetramethylrosamine (CMTMR or Mitotracker orange) to measure M. Recently, the use of CMTMR and related dyes has been questioned (see Ref. 24).
The mitochondrial uptake of cationic dyes used to estimate M depends on
the difference between the plasma membrane potential and M , and on the
236
Tatton et al.
time of incubation [25]. We [2630] and others [3134] have used rhodamine
derivatives, with a chloromethyl moiety that binds to matrix thiols for this purpose. These derivatives offer the advantage that xatives bind them to matrix
proteins allowing immunocytochemistry to be subsequently carried out in the
same cells. The purported disadvantage in using the xable dyes has been thought
to result from the dyes not being Nernstian in their outward movement from
mitochondria (i.e., if M decreases during the incubation time, the dyes will
not leave mitochondria in proportion to the extent of the decreases). Accordingly,
the xable dyes were proposed to offer less sensitive measures of M [24].
Furthermore, the binding of the xable dyes to thiols has led to the consideration
of the possibility that reactive oxygen species (ROS) levels rather than M
might determine their in situ uorescence [35] and that the dyes might damage
mitochondria and open the PTP, dissipating M [36,37]. We have compared
the sensitivity of the xable dye CMTMR to other well-established mitochondrial
potentiometric dyes and found that if cells are incubated with CMTMR for short
periods before xation (i.e., l530 min), they offer sensitive measures of M
that are in agreement with those provided by other dyes such as JC-1. Ideally,
M should be measured using more than one dye in order to conrm that observed changes in uorescence can be reproduced. However other potentiometric
dyes such as TMRM and JC-1 (molecular probes) require the capability of live
cell imaging which may not be possible for many laboratories. For this reason
we have only supplied a protocol for CMTMR.
CMTMR (Mitotracker Orange) enters mitochondria proportionally to the
potential difference between the cytoplasmic compartment and mitochondrial
matrix. After entering mitochondria, the chloromethyl groups of CMTMR react
with thiols on proteins and peptides to form aldehyde-xable conjugates and
remain sequestered in the mitochondria after permeabilization and xation
[33,38]. The CMTMR mitochondrial uorescence represents the highest level of
potential difference between the mitochondrion and adjacent cytoplasmic compartment (or the nuclear compartment) of the living cell during the period of dye
exposure prior to xation. CMTMR uorescence intensity as an estimate of M
is measured and plotted as a frequency distribution (see Ref. 30 for details).
A.
Mitotracker (Orange) plus YOYO-1

20 50 g vials; MW 427.37
Add 850 L sterile DMSO (N.B. the full 850 L will not t in vial
from Molecular Probes); gives a stock solution of 0.1 mM.
Store as 25 L aliquots at 20C, protect from light and moisture.
1. Dilute mitotracker stock 1:1000 in prewarmed media.
2. Aspirate media from cells and add mitotracker-media for 15 min, return cells to 37C, CO2 incubator.
237
3.
4.
5.
6.
7.
8.
Rinse 3 in HBSS.
Fix cells with 4% paraformaldehyde, for 2030 min on ice.
Rinse 23 in PBS.
Expose cells for 12 min to methanol, room temperature.
Rinse cells with PBS, 2.
Incubate cells with YOYO-1 (1:1000 in PBS) for 30 min, room temperature.
9. Rinse cells in PBS, 5.
10. Mount coverslips with Aquamount. Once dry, store slides at 4C.
B. Mitotracker, Bax (or GAPDH), and YOYO-1
Note that for cells that have been incubated with CMTMR and xed, no detergent
should be used in later steps (such as Triton or Tween) because it will result in
CMTMR bleeding out of the mitochondria. CMTMR-treated cells may be stored
in PBS at 4C for at least 1 week, if necessary. In the following protocol we
have used Bax antibody (Santa Cruz, N-20 fragment), Bcl-2 (Santa Cruz), or
GAPDH (Chemicon), but virtually any antibody may be used that does not require a detergent permeabilization to access its antigen. Note that we have also
exposed CMTMR-treated cells to a cold ethanol postx without any problems,
and we have also used a diluted (1:3) solution of Neuropore for 5 min when a
permeabilization step was required with no damage to CMTMR.
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
Rinse cell-coverslips with PBS, 1 5 min.

Expose briey to methanol, 12 min, room temperature.
Rinse in PBS, 2 5 min.
Block with 10% NGS/PBS for 20 min, room temperature.
Primary antibody Bax or Bcl-2 (1:200) or GAPDH (1 :500) in 1%
NGS/PBS, overnight, 4C.
Rinse cells with PBS, 2 5 min.
Secondary antibody, goat anti-mouse or goat anti-rabbit Cy5-IgG (or
Alexa 690), 1:200 in 1% NGS/PBS for 1 h, room temperature (or
40 min, 37C).
Rinse cells with PBS, 2 5 min.
YOYO-1 (1 :1000 in PBS) for 2030 min, room temperature.
Rinse cells with PBS, 5.
Mount coverslips with Aquamount, let air dry, and then store at 4C,
in the dark.
This protocol allows for triple labeling of the cell. Note that to visualize Cy5,
laser excitation of the uorophore will be necessary. CMTMR emits in a range
similar to Texas Red or TRITC; therefore, appropriate secondary antibody conjugates must be chosen that do not overlap with CMTMR or with YOYO-1. Alter-
238
Tatton et al.
nately, a double-label may be employedCMTMR with YOYO-1 or DAPI,

CMTMR with any other antibody using an Alexa 488 -IgG conjugate (if no confocal is available). Note that GAPDH is used at 1:500 dilution on cultured cells,
but at 1: 200 on tissue sections.
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duces acute promyelocytic leukemia cell apoptosis via a hydrogen peroxide-dependent pathway. Blood 1999; 94:21022111.
Sugrue MM, Wang Y, Rideout H, Chalmers-Redman RM, Tatton WG. Reduced
mitochondrial membrane potential and altered responsiveness of a mitochondrial
membrane megachannel in p53-induced senescence. Biochem Biophys Res Commun 1999; 261:123130.
Wadia JS, Chalmers-Redman RME, Ju WJH, Carlile GW, Phillips JL, Fraser AD,
Tatton WG. Mitochondrial membrane potential and nuclear chages in apoptosis
caused by serum and nerve growth factor withrawal: time course and modication
by (-)-deprenyl. J Neurosci 1998; 18:932947.
Gilmore K, Wilson M. The use of chloromethyl-X-rosamine (Mitotracker red) to
measure loss of mitochondrial membrane potential in apoptotic cell is incompatible
with cell xation. Cytometry 1999; 36:3558.
Macho A, Decaudin D, Castedo M, Hirsch T, Susin SA, Zamzami N, Kroemer G.
Chloromethyl-X-Rosamine is an aldehyde-xable potential-sensitive uorochrome
for the detection of early apoptosis [see comments]. Cytometry 1996; 25:333340.
Poot M, Gibson LL, Singer VL. Detection of apoptosis in live cells by MitoTracker
red CMXRos and SYTO dye ow cytometry. Cytomery 1997; 27:358364.
Poot M, Pierce RC. Detection of apoptosis and changes in mitochondrial membrane
potential with chloromethyl-X-rosamine. Cytomery 1999; 36:359360.
Ferlini C, Scambia G, Fattorossi A. Is chloromethyl-X-rosamine useful in measuring
mitochondrial transmembrane potential? Cytometry 1998; 31:7475.
Minamikawa T, Sriratana A, Williams DA, Browser DN, Hill JS, Nagley P. Chloromethyl-X-rosamine (Mito Tracker Red) photosentises mitochondria and induces
apoptosis in intract human cell. J Cell Sci 1999; 112:24192430.
Scorrano L, Petronilli V, Colonna R, Di Lisa F, Bernardi P. Chloromethyltetramethylrosamine (Mitotracker Orange) induces the mitochondrial permeability transition
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Calarco PG. Polarization of mitochondria in the unfertilized mouse oocyte. Dev
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Tatton WG, Olanow CW. Apoptosis in neurodegenerative diseases: the role of mitochondria. Biochim Biophys Acta 1999; 1410:195213.
14
Imaging of Retinal Ganglion Cells
Joshua P. Vrabec and Leonard A. Levin
I.
INTRODUCTION
In recent years, the study of neuronal apoptosis has expanded into the eld of
digital technology. Many aspects of neuronal physiology, such as ion ux, generation of reactive oxygen species, and changes in membrane potential can now
be observed in real time. As a result, digital imaging of individual neurons labeled
with condition-dependent uorescent dyes will most certainly help dene the
temporal relationships of the many interlinked apoptotic pathways.
Neuronal apoptosis involves the generation of reactive oxygen species
(ROS) and a loss of mitochondrial membrane potential (M) via the opening
of the mitochondrial permeability transition pore (MPTP) [1,2]. Although the
precise relationships of these events to the grand scheme of the apoptotic cascade
are not clear, they likely are key events in the initiation of apoptosis. Therefore,
blocking either ROS release or MPTP opening may be attractive pharmacological
targets for neurodegenerative diseases in the future.
In order to better elucidate these events in real time, we describe our method
for digitally imaging rat retinal ganglion cells (RGCs) using uorescent light
microscopy. Methods for detecting release of superoxide anion and for detecting
changes in the mitochondrial membrane potential are outlined below. Similar
methods can be used for imaging other reactive oxygen species, calcium levels,
pH, and other intracellular processes [36].
241
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Vrabec and Levin
II. RETROGRADE LABELING OF RETINAL

GANGLION CELLS
In order to differentiate retinal ganglion cells from other retinal neurons in mixed
culture, we take advantage of the unique anatomical connections between the
brain and the cell bodies of RGCs. When an appropriate dye is injected into the
superior colliculus of an anesthetized rat, the dye will ow via retrograde axonal
transport in the axons of RGCs back to the cell bodies located in the inner retina [7]. Take care to select a uorescent probe that will work in this manner.
DiI-C18, DAPI, and several other dyes are available, but it is important that the
dye chosen have an excitation/emission spectrum that will not interfere with
the experimental probe. For example, if a red and green emitting dye such as
JC-1 will be used for imaging M , then you should select a blue-violet dye
(e.g., DAPI) for the retrograde labeling of RGCs. In the methods described below
for RGC imaging of both M and superoxide anion, DAPI was used for retrograde labeling.
III. RETINAL GANGLION CELL CULTURE

Long-Evans rat pups, age P3, are injected with either DiI-C18 (Molecular Probes)
or DAPI (Molecular Probes) into each superior colliculus for the retrograde labeling of retinal ganglion cells [8]. After waiting for at least 2 days, pups are sacriced by CO2 asphyxiation and their retinas are dissected. The retinas are then
dissociated according to previous methods [8], and plated on a poly-L-lysinetreated eight-well chambered cover glass for 2 h 37C, 5% CO2, 80% humidity.
After the incubation period is complete, follow the specic JC-1 or HEt staining
protocols for imaging.
Cultures are incubated for 2 h for two reasons. First, the dissociated cell
mix that is added to the well needs time to become attached to the poly-L-lysine
coated surface of the wells. Second, some of the cells in primary neuronal cultures
such as these begin to undergo stress- or axotomy-related apoptosis, which can
be as much as 50% by 24 h [8,9]. Imaging soon after culture reduces the possibility that the cell being imaged will be in the process of dying from axotomy,
although this obviously cannot be excluded. Longer-term cultures are more likely
to have signicant amounts of apoptosis.
IV. SETTING UP FOR IMAGING

A.
Choice of Dyes
JC-1 (Molecular Probes) is a dual-emission uorescent probe that associates with

the mitochondrial inner membrane. The dye exists as two forms (monomer and
Imaging of RGCs
243
J-aggregates) depending on the membrane potential of the inner mitochondrial

membrane with which the dye is associated. The monomer associates with the
inner membrane regardless of membrane potential, but J-aggregates will only
form if an adequate membrane potential is present. While both forms of JC-1 have
identical excitation spectra, each form has its own unique emission spectrum. The
monomer form emits at 535 nm and the J-aggregates have a peak emission at
580 nm. With the proper imaging equipment, these unique characteristics allow
one to measure the emission intensity of each form of JC-1 and determine the
membrane status of mitochondria in intact RGCs. Specically, one can measure
the ratio of 580/535 nm emission over time to detect changes in M brought
on by specic pharmacologic treatments.
Dihydroethidium (HEt) is a dye that has a peak emission around 580590
nm when converted to its active form by superoxide anion. When used in this
method, it can be used to detect the superoxide burst occurring during apoptosis
[10,11].
B. Equipment Needed
Listed below is the equipment necessary for imaging over short time intervals,
and also the additional devices needed if one is to perform imaging experiments
that will last several hours (microscope stage incubator, pH control, etc.). While
each device can be purchased separately, there are some companies that will
provide most, if not all, the necessary imaging equipment in a bundled package.
Equipment necessary for imaging:
1. Inverted uorescent microscope (Zeiss Axiovert S100 or a similar
model) tted with appropriate lters (discussed below) and 100 oilimmersion objective
2. Sutter Instruments Lambda 10-2 lter wheel (or a similar model) with
lters. To image with JC-1, a dual-emission probe, the lter wheel
should be tted along the emission light path (i.e., between the microscope and digital camera).
3. Dye-specic lter sets (obtain from Chroma or Omega Optical)
a. RGC identication
DAPI: Excitation: 330 nm
Dichroic: 400 nm
Emission: 450 nm
b. M measurement
JC-1: Excitation: 480 nm
Dichroic: 505 nm
Emission: 535 nm and 580 nm lters (Lambda 10-2 lter
wheel)
c. Superoxide anion measurement
244
Vrabec and Levin
HEt: Excitation: 515 nm

Dichroic: 565 nm
Emission: 580 nm long pass
4.
5.
6.
7.
8.
1.0 log (10%) neutral density lter. This should be tted in the path
of the microscopes light source, and will cut down on the fading of
JC-1.
Photometrics SenSys digital camera
UniBlitz shutter driver and shutter (important component for reducing
the amount of dye fading)
Pentium III computer or better, loaded with MetaFluor 4.0 software
(Universal Imaging Corporation), or later version
Lab-Tek II chambered cover glass for cell culture (0.20.5 mL working volume, 0.7 cm2 growth area) (Fisher Scientic)
Optional accessories for long-term studies (all but the CO2 tank and regulator
are obtained from Harvard Apparatus):
CSMI microscope plate incubator (#65-0101)
TC-202A temperature controller w/ thermistor (#65-0045)
pH controller (#70-2116)
Watson Marlow 205CA perfusion pump (#72-0501)
Perfusion tubing (#72-0571)
CO2 tank with regulator
V.
MEASURING MITOCHONDRIAL MEMBRANE

POTENTIAL OR SUPEROXIDE ANION IN
CULTURED RETINAL GANGLION CELLS
A.
Dye Treatment
Due to the dyes extreme sensitivity to light, all procedures involving JC-1 or
HEt should be carried out in the dark. Prepare a 4 mM (JC-1) or 3.2 mM (HEt)
stock solution in dimethyl sulfoxide (DMSO). Aliquots should be stored at
20C and protected from light.
I.
Staining protocol for JC-1

1. Thaw out the 4 mM stock solution of JC-1 in the 37C water bath.
2. Add 7 mL of Neurobasal medium to a 15 mL Falcon tube, and place
in a 37C water bath.
3. Once thawed, add 5.37 L of the 4 mM stock to 7 mL of Neurobasal
medium in a sterile incubator. Important: Do not add the JC-1 directly
to the media. Tilt the tube containing the media at a 45 angle, then
Imaging of RGCs
4.
5.
6.
7.
8.
9.
10.
11.
245
add the JC-1 to the inside lip of the tube, taking care to keep the
JC-1 away from the media. Then, carefully close the tube, and IMMEDIATELY vortex the solution.
Now that the 2-h incubation is complete, remove the plate from the
incubator.
Using a sterile glass pipette, carefully aspirate the culture media from
a well (touching only the side of the well), and immediately add 400
L of the JC-1 treating solution (prepared above).
Repeat with the other wells on the plate.
Place the plate in the 37 incubator for 15 min.
While cells are in the incubator, add 200 L of 1 M HEPES buffer
to 9.8 mL of HBSS and vortex to make a 20 mM HEPES washing
solution. If buffering is not desirable, use HBSS alone.
After the 15 min incubation is complete, aspirate a well, and immediately add 400 L of the HEPES/HBSS wash to the well. Repeat with
the other wells on the plate.
Wash cells a second time: Aspirate a well, and this time add 350 L
of HEPES/HBSS solution to the well. Repeat with the other wells.
Place plate on microscope stage and prepare for imaging.
2. Staining protocol for HEt

HEt begins a slow conversion to ethidium after it contacts cell media. Therefore,
it is only loaded into cells just before imaging is to commence.
1. All procedures done in darkness/minimal light.
2. Thaw HEt aliquot (3.2 mM in DMSO).
3. Make a 1000-fold dilution with the imaging media (clear HBSS and
chemical treatment) as the diluent.
4. Place the 3.2 M HEt solution in 37 water bath 15 min prior to cell
culture incubation completion.
5. Remove plate of cells from incubator after 2 h incubation.
6. Aspirate the media from each well, adding 350 L of 3.2 M HEt
solution immediately after each aspiration.
7. Incubate plate at 37 for 10 min.
8. Place plate on microscope stage and prepare for imaging.
B. Imaging of Mitochondrial Membrane Potential
in Retinal Ganglion Cells
Due to differences in the number and size of mitochondria in cells, the absolute
amount of JC-1 uorescence emitted will vary. As a consequence, for a population of cells in a well, the changes in M after an appropriate stimulus will also
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Vrabec and Levin
vary substantially. Therefore, one cannot simply measure the average uorescence from a well if the purpose is to measure the timing of acute changes in
M . Instead, individual cells should be observed for these types of experiments,
and this can be achieved by selecting specic regions for measurement using the
MetaFluor software. An advantage of measuring M in this way is that small
changes in M due to pharmacological treatments can accurately be detected.
However, the number of RGCs in a single eld of a mixed retinal culture under
a 100 objective is low, and does not allow for the measurement of many cells
simultaneously. A section below describing data analysis discusses how to combine experiments to obtain signicance.
During imaging, the digital camera, lter wheel, and microscope shutter
are all under the control of the imaging software program (MetaFluor), which
integrates all of their functions. This allows the experimenter to take a relatively
passive role during the actual acquisitions of images. Before beginning any experiment, the program must be set up properly. Variables such as acquisition number,
time between acquisitions, camera settings, optical lters to use, and so on must
be decided on beforehand. The advantage is that MetaFluor does the rest after a
simple mouse click starts the imaging process. Remember that for measuring
M , two separate emission lters are needed (535 nm areas of low M ,
580 nm areas of high M ). Thus, a typical imaging cycle for JC-1 will involve
taking one picture at 535 nm, followed by a picture at 580 nm. MetaFluor can
then automatically calculate a ratio of the 580 nm and 535 nm uorescent intensities (580/535 nm). The data can be transferred while imaging to Microsoft Excel,
for further analysis after the experiment. A representative from the software company is an excellent resource for describing the complete capabilities of the software package.
The experiments measuring M start by obtaining images once per minute
for 5 min and calculating the ratio of 580/535 nm. After this initial baseline
uorescence has been determined, a chemical treatment is added to the well.
Then, additional images are acquired once per minute for 20 min, up to 12 h
(long-term imaging equipment needed, see above.) Chemical treatments are selected for their ability (proven or proposed) to alter M in some fashion. An
excellent control is 10 M valinomycin, a K ionophore for the mitochondrial
inner membrane, which causes collapse of the M within minutes.
1. Imaging Protocol
1.
2.
Using an appropriate lter set for DAPI, locate an RGC in the well.
Switch to the 100 oil-immersion lens, and refocus. The nucleus
should be stained a bright, vivid blue-violet color. Check that the cell
appears intact and living.
Note: Other light purplecolored cells may be found in the well, but
Imaging of RGCs
3.
4.
5.
6.
247
these are not retinal ganglion cells. Some DAPI-stained RGCs die and
lyse during the incubation period, releasing small amounts of DAPI
that may be taken up by nearby cells (bipolar cells, macrophages, etc.).
These cells that have secondarily taken up DAPI appear as very faint
purple cells scattered throughout the plate. If one concentrates on locating only cells that have their nucleus stained (not the entire cytoplasm),
and that staining is an intense purple (not faint), then there should be
no confusing the RGCs from other retinal cells in this system.
Change the lter block to the position for JC-1 excitation. The cell
should have bright orange spots or streaks (these are the stained mitochondria) against a bright blue background. If the cell appears entirely
green, it is probably dead as a result of the dissociation procedure, and
should not be studied. Focus on the cell, and again check that it is
centered in the eld of view. Manually close the shutter and divert the
microscope light to the lter wheel/digital camera apparatus
Using the imaging software, complete one cycle of imaging. The digital camera will acquire one image of the uorescent intensity at both
535 nm and 580 nm.
Using the imaging software, encircle a region of the just-imaged cell
for measurement.
Start the experiment, which will continue to image the same cell to
the specications of the program setting while making uorescent measurement.
2. Imaging Protocol for Dihydroethidium (HEt)

This procedure is essentially identical to that used for imaging M with JC-1.
3. Common Problems with Obtaining an Image
The light path in the microscope was not properly diverted to the lter
wheel/digital camera apparatus.
The shutter did not open. Light should pass through the plate during
the image acquisitionif it did not, then the shutter did not open properly. This usually can be xed by turning the power for the shutter
controller off, then back on after a few seconds. Or, try hitting the Reset
button on the shutter controller a few times, then try imaging again.
The proper lters are not in the lter block and/or lter wheel.
The cell moved out of the cameras eld of view. Oil microscopy using
a 100 objective can be tricky. Any slight jarring of the microscope
apparatus or vibration of the table it is placed on may cause the cell to
move. Addition of chemical treatments by pipetting into the well while
248
Vrabec and Levin
imaging may also cause problems. Make sure the microscope is on a

very stable, heavy table. The poly-L-lysine solution used to adhere the
cells should be in good condition. If necessary, addition of agents by
perfusion pump may be necessary.
REFERENCES
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
Zamzami N, Susin SA, Marchetti P, Hirsch T, Gomez-Monterrey I, Castedo M, et

al. Mitochondrial control of nuclear apoptosis. J Exp Med 1996; 183(4):15331544.
Marchetti P, Castedo M, Susin SA, Zamzami N, Hirsch T, Macho A, et al. Mitochondrial permeability transition is a central coordinating event of apoptosis. J Exp
Med 1996; 184(3):11551160.
Zochowski M, Wachowiak M, Falk CX, Cohen LB, Lam YW, Antic S, et al. Imaging membrane potential with voltage-sensitive dyes. Biol Bull 2000; 198(1):1
21.
Frank J, Biesalski HK, Dominici S, Pompella A. The visualization of oxidant stress
in tissues and isolated cells. Histol Histopathol 2000; 15(1):173184.
Takahashi A, Camacho P, Lechleiter JD, Herman B. Measurement of intracellular
calcium. Physiol Rev 1999; 79(4):10891125.
Johnson I. Fluorescent probes for living cells. Histochem J 1998; 30(3):123140.
Vidal-Sanz M, Villegas-Perez MP, Bray GM, Aguayo AJ. Persistent retrograde labeling of adult rat retinal ganglion cells with the carbocyanine dye diI. Exp Neurol
1988; 102(1):92101.
Levin LA, Clark JA, Johns LK. Effect of lipid peroxidation inhibition on retinal
ganglion cell death. Invest Ophthalmol Vis Sci 1996; 37:27442749.
Kortuem K, Geiger LK, Levin LA. Differential susceptibility of retinal ganglion
cells to reactive oxygen species. Invest Ophthalmol Vis Sci 2000; 41:31763182.
Budd SL, Castilho RF, Nicholls DG. Mitochondrial membrane potential and hydroethidine-monitored superoxide generation in cultured cerebellar granule cells. FEBS
Lett 1997; 415(1):2124.
Scanlon JM, Reynolds IJ. Effects of oxidants and glutamate receptor activation on
mitochondrial membrane potential in rat forebrain neurons. J Neurochem 1998;
71(6):23922400.
15
Evaluation of Retinal Function:
Electroretinography
Marc Hebert
University of Laval
Quebec City, Quebec, Canada
Pierre Lachapelle
McGill University
I.
INTRODUCTION
The purpose of this chapter is to provide the reader with a working knowledge
of functional testing of the retina by describing the most common procedure used
for that purposenamely, the electroretinogram whether evoked to a diffuse
(ash ERG) or structured (pattern ERG and multifocal ERG) light stimulus. It
is not, however, the scope of this chapter to discuss in great details the origin of
the different components of the ERG, especially since for several of them this
is still an unresolved issue.
The electroretinogram (ERG) identies the biopotential that is generated
by the retina in response to a light stimulus. To date, the ERG still represents
the test of choice to investigate the functioning retina whether in its normal state
or altered either as a result of pathology, aging, or intoxication (medication or
otherwise), to name a few. Given the neural origin of the retina, its functioning
is often affected by neurotoxic or neuroactive components. On numerous occasions the ERG was shown to be a precious ally to the ophthalmologist and neuroscientist who wished (1) to detect the effect of neuroactive components on the
function of the retina; (2) investigate the long-term sequels that these components
had on the functional integrity of the retina; and (3) examine the possible revers249
250
Hebert and Lachapelle
ibility of the effect either spontaneously or as a result of therapy. Similarly, the

ERG was also shown to be a signicant adjunct to the clinicians armamentarium
as it not only permitted one to diagnose and characterize a pathology, even in
instances where there were no other obvious clinical signs, but also the information gathered was instrumental in identifying the possible site of malfunction. In
the following sections, we will describe the different techniques used to record
and analyze the electroretinogram. The accent will be on the human ERG because
the techniques used are now standardized. However, it should be noted that human techniques can be used with little modications on all sorts of animal species,
such as: dogs [1] rats [2], mice [3], pigs [4], birds [5], and rabbits [6], to name
a few. Although our aim is not to conduct a review of the literature to support
this claim, modied human ERG techniques were previously used to study the
toxicity of inhaled trichloroethylene on the rabbit retina [7,8], determine the
short- and long-term sequels of postnatal oxygen exposure in newborn rats (animal model of human retinopathy of prematurity) [9,10], evaluate the benecial
effect of an intravitreal injection of ciliary neurotrophic factor (CNTF) in rescuing
the degenerating photoreceptors in RDS mice [3], and, more recently, study the
potentially harming effect of Sildal (Viagra) on the retinal function of mice
affected with a retinal degeneration similar to a form of human retinitis pigmentosa (RP) [11]. Similarly, the electroretinogram is frequently used to quantify
the functional consequences of the new genotypes that are generated through
selective breeding or created through genetic manipulations [12].
II. GENERAL OVERVIEW OF

ELECTRORETINOGRAPHY
The electroretinogram (ERG) represents the electrical response that is generated
by the entire retina when stimulated by a ash of light of adequate energy. It is
often compared to the electrocardiogram (ECG) in that it is similarly composed
of a series of waves that are presumed to originate from different retinal cells.
However, unlike the ECG, which represents the ongoing activity of the myocardium, in order to generate an ERG response one must stimulate the retina with
light. The ERG is thus an evoked potential generated by the excited retina and
recorded at a distance (usually the cornea) from the latter. It represents a weighted
average of all the retinal cells excited by the ash of light.
It is fair to say that the ERG does not exist as such, unlike the ECG. It is
a creation that enables us to synchronize the retinal activity and consequently
greatly facilitate its study. This concept is of extreme importance if one wishes
not only to understand the origin of the ERG but also to appreciate the need to
normalize the ERG acquisition parameters in order to extract as much meaningful
functional information as possible. Continuing with the ECG analogy, in the latter
Evaluation of Retinal Function
251
case it is the electrode position that is critical for inter-laboratory comparisons,

whereas with the ERG it is the stimulating parameters that are of the utmost
importance.
Added to the above, one must also take into consideration the unique physiology of the retina, which permits it to work under a wide range of luminance
levels. This is for the great part due to the two types of photoreceptors: rods (for
night time or scotopic vision) and cones (for daytime or photopic vision), which
are normally found in the retinas of most higher vertebrates. One of the aims of
electroretinography, whether used as an investigative tool or as a diagnostic test,
is to separate the cone ERG from the rod ERG in order to distinctly assess the
function of each type of photoreceptor. As we will see later on, standardized
ERG protocols were written with that specic aim in mind.
III. COMPONENTS OF THE ERG

AND THEIR ORIGINS
The electroretinogram was the rst biopotential ever recorded. As early as 1865,
Holmgren published a study in which he reported the recording of the electrical
response of the eye of frogs upon excitation by a light stimulus [13]. Some 12
years later, Dewar was rst to report human ERG recordings [13]. It is, however,
Granit who contributed most to our understanding of the ERG wave genesis. In
his classic study, which made use of ether intoxication in cats, Granit showed
that the ERG was basically composed of three different processes, each of which
gives rise to a different wave [14]. The three processes, named PI, PII, and PIII
to reect their order of disappearance as the level of anesthesia deepened, were
shown to correspond to the c-wave, b-wave, and a-wave, respectively. With the
renement of the stimulating and recording techniques, new waves were added
to the three studied by Granit. However, it is customary in modern electroretinography to concentrate on the two major waves of the ERG: namely, the a- and
b-waves as well as the oscillatory potentials (OPs), which are components of
high frequency and low voltage normally seen on the rising limb of the b-wave
(see Fig. 1 for ERG waves identication) [15]. In the following paragraphs we
will briey review the postulated site of origin of these waves.
A. The a-Wave
As shown at Figure 1, the a-wave, which is negative in polarity, is the rst major
component of the ERG. To date, the consensus situates the generator of the awave at the level of the photoreceptors [13]. Depending on the state of retinal
adaptation, either the cones alone or the cones and the rods will contribute to the
genesis of the a-wave [13]. It should be noted that there is no pure rod ERG a-
252
Figure 1 Representative photopic broadband (recording bandwidth: 1500 Hz)

electroretinogram recorded from a sedated Guinea pig. The vertical arrow points
to the onset of the stimulus. Components of the ERG are identied as follows: awave (a), b-wave (b), and oscillatory potentials (OPs). The amplitude of the a-wave
is measured from the prestimulus baseline to the trough of the a-wave, whereas
that of the b-wave is measured from the trough of the a-wave to the peak of the
b-wave. Peak times are measured from ash onset to peak. Vertical calibration:
30 V. Horizontal calibration: 20 ms.
wave as such because in scotopic condition, an a-wave is recordable only in

response to ashes of light of intensities which are in the photopic range (see
Sec. V). These responses are usually referred to as mixed cone and rod ERG
because both photoreceptors are claimed to contribute to their genesis. Analysis
of the leading edge of the a-wave, using a computational approach, is often used
to further characterize the physiology of the photoreceptor response as measured
with the ERG [16]. It is of interest to note that in some animal species (such as
rats and mice, for example), the photopic (cone) ERG is devoid of a recordable
253
a-wave despite a large b-wave. The exact reason for this remains obscure to date.
By convention, the amplitude of the a-wave is measured from baseline to the
trough of the a-wave.
B. The b-Wave
This is the most prominent and consequently the most important component of
the ERG response. As shown in Figure 1, the b-wave is positive in polarity and
said to be generated as a second-order potential by the Muller cells, which are
glial cells running through the entire thickness of the retina, from the outer to
the inner limiting membrane [13]. Sieving et al. (1994) suggested that the photopic ERG would be generated as a result of the synchronized activation of the
ON-depolarizing bipolar cells (ON-DBC) and OFF hyperpolarizing bipolar cells
(OFF-HBC), each contributing in sequence to the shaping of the b-wave [17].
The ON-DBC provides the impetus to push the b-wave up (i.e., from the trough
of the a-wave to the peak of the b-wave) while the OFF-HBC limits the amplitude
of the b-wave by pulling the retinal potential from the b-wave peak to a baseline
value. This concept of ERG b-wave genesis is known as the PUSH-PULL hypothesis. By convention, the amplitude of the b-wave is measured from the trough
of the a-wave (when present) to the peak of the b-wave.
C. The Oscillatory Potentials
The oscillatory potentials (OPs) identify the high-frequency components of the
ERG, which are often seen as ripples on the ascending limb of the b-waves (Fig.
1) [18]. Fourier analysis reveals that while the a- and b-waves of the ERG are
of a frequency domain of about 60 Hz or less, the OPs are usually of a frequency
domain greater than 100 Hz. As we will see later, this difference in frequency
domain is put to a use when one wishes to selectively record the OPs with minimal contamination from the slower a- and b-waves of the ERG. Practically all
the retinal components, with the exception of the photoreceptors and the Muller
cells, have been suggested as possible candidate for the genesis of the OPs [18].
Although there is yet no consensus as to their exact origin, it is important to note
that there is more and more evidence to suggest that the terminology oscillatory
potentials is probably a misnomer, because there are studies published to date
that clearly show that the OPs do not (collectively) represent an oscillation such
as the vibration of a retinal element or membrane, as the name could suggest.
Past studies have shown that the number of OPs will vary as a function of stimulus
intensity [19] (see also Fig. 4), inter-stimulus interval [20], retinal adaptation
[21,22], pharmacological manipulation [23,24], and pathology [25,26], to name
a few. Results from the above-mentioned studies clearly suggest that each OP
would be generated by a different, and most probably functionally independent
254
retinal element. Compared to the other ERG components, the OPs were also
shown to be signicantly more sensitive to alteration in the retinal environment,
whether acquired or innate [18,25].
Finally, as it currently stands, ash-evoked ERG responses do not include
component(s) specically assigned to the activity of the ganglion cells or beyond.
There have been reports suggesting that some long-latency postb-wave components (photopic i-wave or long-latency OPs) might be attributable to the activation of the retinal ganglion cells and/or the optic nerve including the chiasm [27
29]. However, these claims were not veried elsewhere.
IV. RECORDING PROCEDURE

A.
Preparation of the Subject
As indicated at the onset, the electroretinogram is an evoked potential. Consequently, in order to record, one must synchronize the recording of the retinal
potential with the stimulus that will generate this response. To achieve this goal,
several criteria must be met, the most important of which being full cooperation
from the subject. Taking again the ECG analogy, the heart will continue to beat
and thus an ECG recording will be possible (maybe with some difculties)
whether the subject (human or animal) is compliant or not. This is somewhat
different for the ERG because the subject must cooperate to a certain degree
if one wishes to record good quality and reproducible responses. If the subject
voluntarily keeps its eyes shut, an adequate ERG recording is simply impossible.
It is for that reason that, in animal experimentation, the subjects are systematically
anesthetized for the procedure and not for the pain that might be involved, because it is, depending on the type of recording electrodes, an almost harmless
procedure. In this laboratory we have successfully used a mixture of ketamine
and xylazine to record ERGs from a variety of animal species including rats,
mice, Guinea pigs, rabbits, and birds. Human subjects, on the other hand, need not
be anesthetized or even sedatedeven newborns or young infants. At least this has
been our experience of more than 25 years of recording ERGs in a pediatric center.
B.
Electrode and Recording Parameters
1. Electrode Types and Position (active, reference,

ground)
According to the ERG standard of the International Society for Clinical Electrophysiology of Vision (ISCEV) [15], the active electrode should be corneal or as
close as possible to the cornea. It is for that reason that the electrode of choice
is the contact lens electrode (Henkes, Burian-Allen, etc.; see Fig. 2, top). The
255
reason for this is simple. As mentioned above, the ERG is an evoked potential
that is recorded on the surface of the eye but originates from the retina. Therefore,
the recording site is at some distance from the origin of the potential. Consequently, the closer we are from the source of the potential, the larger this potential
will be. This is true of all ERGs, human or animal, and for that reason contact
lenses have been developed for several animal species, including the mouse. Another advantage to the contact lens electrodes is the fact that their construction
also includes a blepharostat whose function is to keep the eyelids open during
the procedure, thus optimizing the delivery of light to the retina (see Fig. 2, top).
Contact lens electrodes must, however, be used with a topical anesthetic, as they
are painful to wear, and also with a viscous interface (usually methylcellulose)
in order to protect the cornea from a possible abrasion. These electrodes cannot
be worn for more than 2030 min because the risk of corneal abrasion increases
with time. Also, several sizes must be purchased in order to accommodate the
different sizes of eyes (especially when pediatric and adult patients are seen in
the same clinic) as well as the waiting time due to sterilization (in a clinical setup). The latter point is of importance as these electrodes are quite expensive. As
an alternative to the contact lens electrode, the ber electrodes are gradually
becoming more and more popular, the most popular one being the DTL ber
electrode, which is made of a nylon yarn coated with silver (Fig. 2, middle)
[30,31]. Fiber electrodes offer several advantages, such as painless even after
several hours of wear (in humans), no need for topical anesthesia and disposable.
They must, however, be used with great care and their positioning controlled as
even minor changes in position will have a signicant impact on the amplitude of
the resulting ERG. Regardless of the type of electrode used, with human subjects,
reference and ground electrodes (surface electrodes) are usually positioned at the
external canthi (or forehead) and earlobes (earclip electrodes) respectively; for
animal subjects, they are respectively placed in the mouth and inserted in the tail
(needle electrodes) or subcutaneously in the leg. Electrode impedance should be
minimized as much as possible (5K).
2. Delivering the Stimulus: The Ganzfeld
The electroretinogram being an evoked response, a good ERG examination,
whether for clinical or investigative purpose, will try to record the electrical response from as much retinal tissue as possible. It is for that reason that it is
recommended to use a light diffuser (or Ganzfeld: Fig. 2, bottom) whose purpose,
by design, is to diffuse the stimulus (and light adapting background light when
used) to retinal eccentricities as far as the ora serata [32]. This is even more
important in experimental situations where the rod function must be accurately
assessed because, as we know, rods are most numerous in the peripheral retina.
The same applies to animal experimentation where human Ganzfeld or stimulator
256
257
Table 1 Normative Data Obtained in 40 Normal Subjects (22 men; 18 women,

age: 582)
Amplitude (V)
average
(lower-upper limits)
Peak time (ms)

average
(lower-upper limits)
SF (Standard ash)
a-wave
b-wave
a-wave
b-wave
PHOTOPIC
28.8
(2038)
na
13.4
(11.514.8)
na
na
30
(2632)
26.7
(24.429.6)
67.4
(5782)
40.2
SF Single ash
2.5 log SF
(pure rod)
na
110
(85140)
90
(65120)
150
SF (mixed rodcone)
165
(110220)
250
14.2
(120270)
(160365)
(12.715.8)
SF Flicker 30hz
SCOTOPIC
(3246.3)
na: not applicable (e.g., wave not present in response).
specically designed for animal experimentation are used [33]. It is important to

stress here that an adequate stimulator with diffusing possibilities should always
be used if one seeks reproducible responses of high diagnostic potentials. Placing
the subject (human or animal) directly in front of the light source (ash or other)
is clearly insufcient.
3. Amplication and Recording Bandwidth
As stated above, the electroretinogram is a far eld potential in that it represents
the electrical activity evoked from the retina but recorded at the corneathat is,
in humans, about 25 mm from the source. The amplitude of the resulting biopoFigure 2 In order to obtain signals of the highest possible quality, it is imperative
that the recording electrode be in contact with the eye, whether the ERGs are
recorded from human or animal subjects. This is best achieved with the corneal
contact lens (top picture), which normally includes a built-in blepharostat in order
to keep the eyelids open. Use of these electrodes requires topical anesthesia and
thus limits the wearing time to some 30 min or so. Alternative ERG electrodes,
such as the DTL ber electrode (second picture: DTL ber is seen running on
margin of the lower lid: arrow head), placed deep in the conjunctival bag signicantly extends the recording time because they are basically painless as well as
harmless. Once the eye returns to its primary position (third picture), the electrode
does not interfere with the optical axis. Finally, ERGs should always be evoked in
full eld (Ganzfeld: bottom picture) or full eldlike condition in order to stimulate
as much of the retina as possible.
258
Figure 3 Typical ERG responses representative of the ISCEV standards. Vertical

line(s) indicate ash onset. Photopic responses (background set at 25 cd/m2: single ash (A), oscillatory potentials (B), icker 30 Hz (C). Scotopic responses: pure
rod (D), mixed rod-cone (E), and oscillatory potentials (F). Recordings were obtained using a UTAS-3000 system (LKC Technologies, Inc, Gaithersburg, MD)
along with DTL ber electrodes (DTL Plus ElectrodeTM, Retina Technologies,
Scranton, PA) and dilated pupils. Bandwidths were between 1 and 500 Hz for the
broadband signals (A,C,D,E) or 75 and 500 Hz for the OPs (B,F). SF ISCEV standard ash which was set at 3 cd/m2 /s. DA dark adaptation. Vertical calibration: 25
V (photopic tracings) and 50 V (scotopic tracings). Horizontal calibration: 25 ms.
tential is in the range of approximately 100 V. This value varies with the intensity of stimulation, the level of retinal adaptation (photopic usually yields smaller
responses compared to scotopic: see Table 1), the type of recording electrodes
(usually the largest responses are obtained with contact lens electrodes) [34] as
well as species. Given the relatively low voltage generated, it is necessary to
amplify this signal in order to obtain a measurable response. Similarly, it was
259
Figure 4 Photopic ERG luminance-response function. A gradual increase in the

intensity of the stimulus (bottom to top) augments the amplitude of the a- and bwaves of the broadband ERG (left column). However, while the amplitude of the
a-wave continues to grow regularly, that of the b-wave reaches a maximum at .64
log cdm2s, after which it decreases, a phenomenon known as the photopic hill.
Similarly, the timing of the a-wave shortens with brighter ashes, whereas that
the b-wave increases. The photopic waveforms also include another component
identied as the i-wave, the origin of which is still debated [27]. A similar increase
in ash energy will not only impact on the amplitude of the OPs but also on the
number. At threshold, the OP response includes only one major OPnamely, OP2
(OP1 identies the small notch seen prior to OP2 in some tracings). A gradual increase in intensity will add OP3 and OP4 to this initial response. Horizontal calibration: 25 ms. Vertical calibration: 50 (ERG) and 25 (OPs) V.
mentioned above that the ERG response includes several components, some of
which are easily differentiated with their temporal frequency domain. Consequently, in order to record as complete an ERG response as possible, it is recommended to amplify the retinal signal at least 1000 times within a bandwidth of
1500 Hz at least. However, if the selective recording of the OPs is sought, the
low frequency cutoff of the recording bandwidth should be increased to 75100
Hz while keeping the upper limit to 500 Hz. In the latter case, the amplication
should be augmented to 10,000 times to compensate for the attenuation in signal
that will result from restricting the recording bandwidth (see Figs. 3 and 4).
260
V.
PROTOCOLS FOR DIFFERENTIATION

OF THE CONE AND ROD SYSTEMS
(THE ISCEV STANDARDS)
One of the main objectives when using the ERG is the differentiation of the
cone and rod systems. According to the standards of the International Society
for Clinical Electrophysiology of Vision (ISCEV) [15], in order to reach this goal
at least ve basic responses should be acquired, two in the photopic state (lightadapted retina) and three in the scotopic state (dark-adapted retina):
1.
2.
3.
4.
5.
Photopic single ash response (standard ash)

Photopic 30 Hz icker response (standard ash)
Scotopic dim ash rod response (intensity 2.5 log unit below the standard ash)
Scotopic mixed rod-cone response (standard ash)
Scotopic oscillatory potentials (standard ash, bandpass 75500 Hz)
Examples of typical ERGs obtained according to the ISCEV standards are presented in Figure 3. A complete description of the ISCEV standards can be found
on the Societys website at www.iscev.org
A.
Photopic Single Flash Response
It should be noted that ISCEV does not require the use of a specic level of
background illumination nor a specic intensity of ash in order to generate the
standard ash. Rather the standard suggests a range of photopic background (17
34 cdm2) and ash intensity (1.53.0 cdm2s), an approach that was recently
questioned [35]. Responses illustrated in Figure 3 were evoked to a ash of 3
cdm2s delivered against a background set at 25 cdm2. It is recommended to
expose the retina to the rod desensitizing background at least 5 min before initiating the cone ERG evaluation. This period of adaptation should be increased to
10 min, at least, if the recording of the photopic responses immediately follows
the dark adaptation period in order to avoid the light adaptation effect that was
previously shown to affect the amplitude and the timing of most of the photopic
ERG components [21,22].
As shown in Figure 4, with an increase in the strength of the stimulus (from
bottom to top) there is a gradual increase in the amplitude of the ERG a- and bwaves. However, while the amplitude of the a-wave appears to grow steadily,
that of the b-wave reaches a maximum at approximately 0.64 log cdm2s, following which further increases in ash intensity will yield b-waves of gradually
smaller voltages. This unique feature of the photopic ERG luminance-response
function is known as the Photopic Hill [35,36]. Also of interest is to note that
261
while the peak time of the a wave shortens with brighter ashes, that of the bwave increases. Finally, as mentioned above (Sec. III.C), in response to progressively brighter ashes, the oscillatory potentials increase in number as well as
in amplitude.
B. Photopic Flicker Response

Although in most instances the single-ash photopic ERG response will be sufcient to identify most of the cone-related retinal disorders, the ISCEV standard
also recommends the use of a stimulus ickering at a rate of 30 ashes per second
as the best means to isolate a cone ERG free from any possible rod-mediated
contamination (Fig. 3, tracing C). Analysis of the resulting waveforms will include amplitude (from trough to peak) as well as peak time measurements (see
Table 1).
C. Scotopic ERG Responses

As shown in Figure 5, with time spent in dark-adaptation, there is a gradual
increase in the amplitude of the resulting ERG response as a result of the slow
regeneration of rhodopsin, which takes approximately 30 min (Fig. 5, tracing 1).
However, the ERG response becomes much more stable once the regeneration
of the rods photopigment is completed (Fig. 5, tracing 2). Consequently, in order
to assess the rod function specically, the intensity of the ash stimulus must be
signicantly attenuated in order to account for the gain in sensitivity brought by
the dark-adaptation process. The intensity recommended is one that is 2.5 logunit dimmer than that used in photopic condition. Typically, the ERG evoked to
this intensity of stimulation will be devoid of a recordable a wave (see Fig. 3,
tracing D, and Fig. 5, tracing 1), while use of an intensity of stimulation within
the photopic range will yield an ERG identied as the scotopic mixed rod-cone
response (Fig. 3, tracing E, and Fig. 5, tracing 2). It should be noted that if
averaging is considered, the interstimulus interval should be of at least 2 s for
the dimmer stimuli and at least 5 s when the brighter ashes are used. Also,
responses to the rst ash should be discarded as they often show a conditioning
ash effect [37,38]. The ISCEV standard also recommends that the OPs should
be obtained in scotopic condition (Fig. 3, tracing F) because they are usually of
larger amplitudes than those obtained in photopic condition. However, the latter
recommendation should not prevent one from recording the OPs against a
photopic environment as well, as there is a growing body of evidence supporting the view that photopic and scotopic OPs might originate from different retinal
structures or pathways [26,39].
262
Figure 5 Tracing 1 illustrates the progressive growth in amplitude of the b-wave

as a result of gradual dark-adaptation from onset of darkness (t 0) to 30 min (t
30). The waveform was obtained by the superposition of ERGs in response to
ashes of light (intensity: 3.5 log cdm2 s) delivered at the rate of 1 ash each
5 s. At tracing 2, the same procedure was adopted for the following period of 30
min [e.g., from 30 min (t 30) to 60 min (t 60) of dark adaptation]. The intensity
of the ash was also raised by 1 log-unit, thus explaining the different morphology.
As mentioned above, scotopic ERG waveforms evoked to dim ashes only

include a b-wave (Fig. 6, tracing 3.8 and dimmer). With increasing intensities,
the amplitude of the b-wave increases rapidly until a plateau is reached. This
plateau usually correlates with the appearance of an a wave suggestive of a cone
contribution to the response (tracing 3.0). When plotted on a graph (see Fig.
7), the relationship between the amplitude of the b-wave and the intensity of the
263
Figure 6 Representative scotopic luminance-response function obtained from a

normal human subject. As the intensity of the stimulus grows (from bottom to top),
the amplitude of the b-wave increases gradually but, unlike in photopic condition
(see Fig. 4), its peak time shortens. At threshold, the ERG includes only a b-wave;
the a-wave appearing only in responses evoked to brighter ashes (brighter than
3.0 log-unit). Again, as in photopic conditions, the a-wave peak time shortens
with gradually brighter ashes. Also, several oscillatory potentials are seen riding
on the ascending limb of the b-wave. Vertical arrow points to the onset of the ash.
Calibration: 50 ms (horizontal) and 100 V (vertical).
stimulus (luminance-response function curve) adopts the shape of a sigmoidal

function, which can be best described with the Naka-Rushton equation [40]
V/V max I n /(I n K n)
where V represents the amplitude of the b-wave evoked from a ash of intensity
I, n represents the slope of the function (which is usually close to 1 in scotopic
condition) and K (or retinal sensitivity) the intensity of stimulation necessary to
produce a b-wave half of maximal amplitude (Vmax). K and Vmax parameters have
been used on several occasions to further characterize the scotopic function.
These measurements were shown to be highly reproducible in test-retest conditions [41].
264
Figure 7 Scotopic luminance-response function curve (Naka-Rushton) obtained

by plotting the b-wave amplitude data (ordinate in microvolts) against the intensity
of the stimulus (abscissa in log intensity) with the use of sigmoidal curve t software. Data obtained from both eyes (OD: right eye, OS: left eye) were plotted to
illustrate high reproducibility. Vmax identies maximal rod b-wave amplitude (as per
equation) while K identies the retinal sensitivity which is the intensity of the stimulus needed to evoke a b wave half the amplitude of Vmax.
VI. FACTORS AFFECTING THE ERG

The ISCEV standard makes use of a very brief ashing stimuli (usually 5 ms).
Longer stimuli will add an OFF effect, which will contaminate the response and
thus complicate the analysis; that is of course unless the experimenter wishes to
separate the ON-ERG from the OFF-ERG [42,43]. Although the ISCEV standards are based on white light only, there are also specialized ERG protocols
specically aimed at isolating the activity of the short, medium, and long wavelength sensitive cones [44]. Use of these protocols might be helpful in both clinical and research settings. Normal aging also inuences the ERG parameters. In
humans, it is estimated the amplitude of the b-wave decreases by about 2.5 V
per year from age 10 to 70 [45]. A diurnal variation in the ERG b wave has also
been reported [46]. It is therefore suggested to record the ERGs at about the same
time of day, especially if a follow-up study is considered.
265
Retinal pathologies, whether acquired or inherited, which have an impact

on the normal processing of the visual stimulus can also alter the genesis of the
ERG response. In order to appreciate the possible consequences, it is suggested
to measure the amplitude and peak times of the major ERG components (a- and
b-waves and OPs) and compare these parameters with normative data. Previous
studies have shown that, depending on the retinal anomaly, the waves of the
ERG could be affected in amplitude and/or timing. Also, examining each ERG
component separately allows one to distinguish wave-specic anomalies. Previous studies have also shown that individual OPs can be specically affected by
a given disease process (or experimentally) while the remaining OPs are unaltered
[25,26].
VII. INTERPRETATION OF THE

ELECTRORETINOGRAM
The electroretinogram is used to assess the functional integrity of the retina. With
that in mind, one must remember that the retina contains two types of photoreceptorsnamely, the cones and the rodsand that the activity of each can be specically isolated with the use of specic stimulating conditions as advocated by
the ISCEV standards. This is of the utmost importance, given that most retinal
disorders (acquired or inherited) will often initially affect the normal functioning
of one of the two photoreceptor populations. In order to determine the normalcy
of the response, two parameters are considered: namely, the peak time and the
amplitude. The peak time is dened as the time separating the onset of the stimulus from the maximal amplitude (or peak) of the wave under consideration. For
example the peak time of the a wave refers to the time elapsed between the onset
of the ash and the rst major negative peak of the ERG response (or a-wave),
while the peak time of the b-wave identies the time separating the ash onset
and the highest positive peak of the ERG wave (or b-wave), and so on. The same
logic also applies to the individual oscillatory potentials (OPs). Similarly, the
amplitude of a given wave is always measured from the preceding trough to peak
except for the a-wave, which is measured from the prestimulus baseline. Thus,
the amplitude of the b-wave is measured from the trough of the a-wave to the
peak of the b-wave, while the amplitude of the OP3 for example is measured
from the trough between OP2 and OP3 and the peak of OP3. Once the amplitudes
and peak times of the different ERG waves and OPs are obtained, they are compared to normative data to ascertain normalcy.
If one considers only amplitude and peak time measurements, the following
diagnostic categories are possible: (1) normal amplitude and timing, (2) normal
amplitude and delayed timing, (3) low amplitude and normal timing, and (4) low
amplitude and delayed timing.
266
It should be noted that faster than normal responses are usually considered
as a variation of normal. The above four categories can be applied to any of the
ERG waves and OPs, although (as a rule) the analysis has been traditionally
limited to the b-wave (photopic and scotopic). It is only recently that the a wave
as well as the OPs (photopic and scotopic) have received increased attention and
that their use was shown to increase the diagnostic potential of the ERG. Use of
the above method of analysis of the ERG will not only allow the investigator to
identify cone- and/or rod-specic retinopathies but also localize the retinal site
most affected by comparing a- and b-wave measurements. Similarly, since individual OPs were previously shown to be differently affected either by the stimulus
parameters or pathologies, analysis of each OP individually is also strongly suggested in order to increase the diagnostic potential of the ERG.
VIII. OTHER ELECTRODIAGNOSTIC TECHNIQUES

WITH MORE SPECIFIC OR LIMITED USE
A.
The Pattern-ERG (PERG)
The pattern ERG is the retinal response evoked by viewing (mono or binocularly)
an alternating reversible checkerboard pattern. It is claimed to be generated at
the level of the retinal ganglion cell and/or optic nerve [47]. The PERG is made
of two principal components: P50 and N95 (see Fig. 8), which letters and numbers
correspond to the polarity (positive or negative) and usual timing of the peak or
trough of the response observed in normal individuals. P50 is sensitive to the
luminance and appears to be generated in part by the same generators of the fulleld ERG, whereas N95, which is sensitive to the contrast and spatial frequency
of the stimulus, is more specialized to the ganglion cells. The overall amplitude
of the PERG is relatively small and range from 0.5 to 8 V in normal individuals.
This low amplitude accounts for the difculty in achieving good recordings and
explains why the technique is used routinely only by a handful of laboratories.
PERG has received research attention because it can detect selectively macular
and inner retina dysfunctions that go undetected with the full-eld ERG. The
pattern ERG is considered a good test for macular function but requires very
good technical skills and experience with the technique. It should be noted that
the PERG needs an averaging of about 100400 responses in order to achieve
a good signal-to-noise ratio. Fixation and refraction is also very important, which
makes the technique difcult to use in infants and patients with low visual acuity
(see the ISCEV standards for proper PERG recording).
B.
The Multifocal ERG (mfERG)
The mfERG is a relatively new technique that allows for the assessment of localized retinal dysfunction within a 40 to 50 eld that covers approximately 23%
267
Figure 8 Representative example of a PERG obtained binocularly in a female

(26 years old) using a checkerboard pattern presented on a monitor with a eld
size of 16 16 and reversals of 4.5/s and average of 800 responses (UTAS
3000 LKC Technologies Inc., Gaithersburg, MD). The largest amplitude, observed
at N95, is close to 8 V.
of the total cone population [48]. The stimulus is a pattern (typically presented
on a computer screen that is composed of an array of hexagons that alternate
independently between black and white state in a pseudo-random sequence described mathematically as an m-sequence [49]. At any point in time, half of the
hexagons are white and half are black, which allows for a constant luminance.
The most common matrix is composed of either 61, 103, and 241 hexagons that
increase in size with distance from the center to compensate for the decrease of
cone density with eccentricity [50]. This allows the production of focal ERG
responses of similar amplitudes independently if they are recorded in the macula
or para-macula. At a fast rate of stimulation of 75 Hz, each hexagon is renewed
every 13.3 ms. For the viewer, the stimuli appear as little lights ickering in a
random manner. The electricity generated by the retina in response to the highspeed stimulation is recorded as a continuum (strand of ERGs) using the same
electrodes that are used for the full-eld ERG. Focal ERGS are extracted from
each location using a cross-correlation technique [51]. This is made possible as
each hexagon follows the same exact sequence of black and white presentation,
although each location begins at a different point in the cycle. From our experience, using a DTL electrode and a matrix of 61 hexagons, good results are
achieved with a 4-min protocol. However, because the patient cannot blink during
the stimulation and has to maintain a perfect xation at a cross in the center of
268
Figure 9 Representative example of a mfERG recording obtained with a matrix of

61 hexagons, which subtended a eld of about 40 (VERISTM, Science 4, ElectroDiagnostic Imaging, Inc, San Mateo, CA). In (A), a 3-D topographical map (scalar plot) of
local response density. Observe the central peak (fovea), which has the highest
density of photoreceptors as well as a noticeable depression caused by the blind
spot seen on the far left-hand side. In (B), a trace arrays of all 61 electroretinograms
are presented. Observe that the overall amplitude at each location is maintained
because the hexagons are scaled with eccentricity (larger in the periphery). Having
similar amplitudes allows the detection of area of abnormalities just by looking at the
trace arrays. Responses can also be averaged into rings, quadrants, or hemiretinal
areas, to name a few (not presented). The total recording was 3 min 38 s, achieved
using 16 slightly overlapping segments 15 s long (signal amplication: 100,000;
bandwidth: 10100 Hz). The mfERG recording was obtained from the left eye of a
male (35 years old) dilated with Tropicamide 0.8% using a DTL ber electrode.
the screen, the 4-min protocol is split into 16 segments of 15 s duration (30 s if
using a contact lens). Depending on how the patient handles that task, the 4-min
protocol can be completed in 5 to 15 min. In contrast to the full-eld ERG, which
necessitates minimal cooperation from the patients, cooperation is crucial to
achieve the mfERG. Therefore, it would be difcult to perform this type of recording in infants. As with the pattern ERG, refraction is also important.
Recently, ISCEV introduced the rst guidelines for the mfERG. In these
guidelines it is recommended that when the hexagons are in the white state they
should produce a luminance of 200 cd/m2, whereas when they are in the black
state they should be close to a luminance of 0 cd/m2. The background should be
269
set at 100 cd/m2, yielding to an overall mean luminance of 100 cd/m2. Amplication is usually 100,000 times and the bandwidth set at 1300 Hz or 10300 Hz.
Typical views of the recording are presented in Figure 9. Looking at Figure
9 (right-hand side), it is clear that mfERG response shares some resemblance
with the full-eld cone ERG as it is composed of a negative deection (N1)
followed by a positive deection (P1). However, in the mfERG, the peak of the
response occurs at an earlier time and it is devoid of the fast oscillation observed
in the full-eld ERG, namely, the oscillatory potentials. This difference in the
waveforms is likely due to the stimulation paradigm that is employed and how
the data analysis is performed. In contrast to the full-eld ERG in which the nal
response is composed of consecutive responses that are simply averaged together,
in the mfERG the nal response is composed of additions and subtractions of
preselected responses that depend on the analysis selected. For instance, with
the rst-order analysis (most commonly used), only white hexagons that were
followed by a black hexagon are averaged together at each location. With the
second-order analysis, we are measuring the impact of successive ashes on the
mfERG response [51]. Similar to the full-eld ERG, the generators of the rstorder analysis of the mfERG appear to be the photoreceptors and bipolar cells.
Higher order analyses that take into account the effect of consecutive ashes are
believed to be indicators of ganglions cell activity as well as optic nerve function
[51]. Studies are still needed to conrm the exact components and generators of
the mfERG. The research interest in mfERG is that it provides a spatial resolution
of the macula that cannot be achieved with the full-eld ERG. Of note, animal
research can be performed with mfERG, as shown by reports on monkeys, cats,
rats, and pigs.
IX. CONCLUDING REMARKS

The purpose of this chapter was to provide the reader with standardized means
to assess the retinal function in clinical or experimental conditions. Assessing
the functional status of the retina using noninvasive procedures is relatively simple. The methods described can yield a wealth of extemely valuable information
that can be used to make a diagnosis whether the retinal disorder was acquired
through experimentation or otherwise, or inherited. However, in order to achieve
the highest level of diagnostic accuracy possible, users are urged to create their
own sets of normative data for all the species and age ranges under analysis.
ACKNOWLEDGMENTS
This work was supported by grants-in-aid from McGill University-Montreal Childrens Hospital Research Institute, the Canadian Institutes of Health Research
270
(Grant MT-12153 and MT-13383), the FCAR-GRENE and the Vision Network
of the FRSQ. Thanks are due to Olga Dembinska, Julie Racine, and Marianne
Ruange for providing some of the illustrations.
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16
Evaluation of Visual Outcome
Pamela A. Sample
University of California, San Diego
La Jolla, California, U.S.A.
I.
INTRODUCTION
Until recently, clinical trials for glaucoma have focused on medical or surgical
techniques for lowering intraocular pressure (IOP). The outcome measure of
choice in many of these trials was a criterion reduction in IOP. The effectiveness
of a given intervention could be measured in a matter of a few months at most.
Very recently, potential new neuroprotection therapies designed to protect undamaged but at-risk axons and ganglion cells, or to rescue those that are marginally damaged, have been put forward [1]. Neuroprotection studies, however, may
take much longer to determine whether these treatments prevent slowly progressing glaucomatous damage. To determine whether ganglion cells have survived and maintained function due to neuroprotective therapy, outcome measures
of optic nerve appearance and visual function will be necessary. Lowering intraocular pressure cannot answer this question. Visual function testing is the only
thing that can assess effectiveness in the living human eye. Even if measures of
optic nerve structure show no progression, it does not guarantee that function is
spared or has recovered.
There are two approaches to determining the best tests to measure outcome
in neuroprotection trials. We need to know what are the best tests available to
us right now. This review will present several candidate tests currently available.
We also need to be aware that each of these tests has disadvantages that reduce
273
274
Table 1
Sample
Advantages and Disadvantages of Test Procedures
Availability
Standardized test
Optimized procedures
Normative database
Statistical analysis package
Monitors xation
Reliability indices
Self-calibrating equipment
Test time under 6 min
Patients prefer it
Validated by studies
Ganglion cell specic
Isolation known
Longitudinal studies
Early detection
Identies progression
Over/underclassies OHT
Correlates with optic nerve disease
Works for a variety of eye disease
Variability
Lens, pupil, refraction/affect
SAP
SWAP
FDT
HPRP
1
yes
yes
yes*
1*
yes
yes
yes
yes/SITA
3
yes
no
none
yes
no
3
under
3
3
3
2
2
yes
yes
yes
yes
yes
yes
yes
no
4
yes
yes*
yes*
yes
yes
1
no
1
1
4
4
3
yes
?
yes
yes
no
yes
yes
yes
2
yes
yes
no
no
yes
?
over
?
?
2
1
4
no
?
yes
4
no
yes
no
yes
1
yes
yes
no
yes
no
2
?
2
2
1
3
* obviously best test

14: Ranking best test to worst test
? not yet known
their ability to detect the small changes in visual outcome that are expected in
these trials. I will review the advantages and disadvantages of each test (summarized in Table 1) and highlight areas of research needed to correct the disadvantages.
II. CANDIDATE VISUAL FUNCTION TESTS

Measures of visual function include electrophysiology, visual acuity, and various
other psychophysical measures targeted at peripheral retina. Electrophysiological
measures, although promising [24], still need modication and continued study
to determine if they will be useful for detecting the subtle changes over time
required for assessing neuroprotection. In addition, the commercially available
275
testing equipment (VERIS, Tomey, Nagoya, Japan) is not currently present in

many of the centers and ofces participating in the clinical trials.
Visual acuity may be a useful outcome measure for studies of macular
degeneration or optic neuropathies that affect foveal function, but it is not the
measure of choice for primary open angle glaucoma because the central macula
is spared until the later stages of the disease. Currently, perimetry remains the
measure of choice for studies of glaucoma progression. This review will discuss
the rationale for different perimetric techniques and the potential for following
change in clinical trials of neuroprotection. The focus will be on commercially
available tests with standardized procedures. The advantages and disadvantages
of each are summarized in Table 1.
Ganglion cells and their axons, which form the optic nerve, are the primary
sites of damage due to glaucoma. There are roughly 1.25 million ganglion cells
in the human retina with a minimum of 22 subtypes [5]. The three major ganglion
cell typesmagnocellular, parvocellular, and small-bistratiedcompose up to
90% of all retinal ganglion cells in macaque and human eyes. Each of the three
ganglion cell types has distinctive features that have been used in devising tests
to detect and follow loss of vision due to glaucoma (Fig. 1). In addition, the
morphological differences among these three cell types may indicate that one
type is more amenable to neuroprotection than another, and appropriate measures
of each subtype could be crucial to determining if this is the case.
A. Psychophysical Tests of Peripheral Visual Field

1. Standard Achromatic Automated Perimetry
Standard achromatic automated perimetry (SAP) is the most commonly used version of perimetry for clinical diagnosis as well as in clinical trials. SAP utilizes
a small (0.47) 200 ms ash of white light as the target presented on a dim
background (10 cd/m2 or 31.5 asb). The target is randomly presented to several
locations (54 in program 24-2 and 76 in program 30-2) using a Humphrey Visual
Field Analyzer II (Humphrey Instruments, Dublin, CA). Similar test patterns are
available on Octopus perimeters (Interzeag AG, Zurich, Switzerland). Results in
this section will focus on those obtained with the Humphrey unit.
While a virtual congruence between visual eld damage and loss of optic
nerve bers has been assumed, signicant damage to the ganglion cells may occur
before the disease can be recognized by visual eld abnormalities [6,7]. By the
time visual eld loss is initially detected by Goldmann kinetic perimetry, 40% to
50% of retinal ganglion cells may have suffered irreversible damage [6]. Before
SAP rst demonstrates abnormalities, 20 to 40% of ganglion cells may be lost
[8,9]. These ndings spurred several studies designed to nd more sensitive measures of vision loss, measures that target specic subtypes of retinal ganglion cells.
276
Sample
Figure 1 An oversimplied schematic depicting the separation of visual pathways

serving different visual functions from cones through to the LGN. Cones are depicted by triangles at the top. Their inputs are combined at the retinal ganglion cell
level to form the blue-yellow opponent on ganglion cell (small bistratied), the redgreen opponent ganglion cell (midget), which also handles high resolution tasks,
and the achromatic black-white opponent cells (parasol). The axons from each
type of ganglion cell project to a different region in the lateral geniculate nucleus
(LGN) and that is how they get their other names, koniocellular ganglion cell, parvocellular ganglion cells, and magnocellular ganglion cells. The visual functions preferred by each ganglion cell are listed at the bottom of each pathway. Each pathway
can handle other visual functions, but not as efciently as it does those listed beneath it.
It can be argued that SAP is nonspecic for ganglion cell type, and detection of a white ash of light can be mediated through many types of retinal
ganglion cells. This allows a redundancy at a given retinal location. Meaning if
one type of ganglion cell is damaged, but the others are not, the signal can still
be detected. Each of the tests described below, on the other hand, attempts to
evaluate one type of visual function as a surrogate for isolating a specic ganglion
cell subtype (see Fig. 1). If successful, this means that when the subtype of ganglion cell under test is damaged, the other subtypes can not easily detect the
target. This has been referred to as reduced redundancy [10,11].
277
2. Short-Wavelength Automated Perimetry

Short-wavelength automated perimetry (SWAP) is a modication of SAP available on the Humphrey visual eld analyzer and the Octopus perimeter utilizing
the same test programs as SAP [12]. SWAP utilizes a 440 nm, narrow band, 1.8
target at 200 ms duration on a bright 100 cd/m2 yellow background to selectively
test the short-wavelength sensitive pathway. The test provides a dynamic range
of approximately 35 dB and 15 dB of isolation before the next most sensitive
mechanism can detect the target, most likely the middle-wavelength sensitive
pathway [12,13].
At the ganglion cell level, the patients response to this test is mediated by
the small bistratied blue-yellow ganglion cells. Their dendritic eld size, soma
size, and axon size are slightly smaller than that of the magnocellular cells in
humans [14]. Small bistratied ganglion cells are fewer in number and compose
610% of ganglion cells in peripheral retina [14]. The small bistratied cells
receive their input from the blue-cone bipolar cells [15]. These cells respond in
a sustained manner and have blue-yellow color opponency. They prefer shortwavelength stimuli, which are stressed in SWAP by adapting rods and the other
two cone inputs using the bright yellow background. The key word here is prefer. For example, ganglion cells that prefer high temporal frequencies can respond to lower temporal frequencies under certain stimulus conditions, but their
maximal response will be in the higher temporal frequency range and they will
be more sensitive than the other ganglion cell types to these stimuli.
SWAP has more than 13 years of longitudinal evaluation and has been
shown by several independent studies to be a more effective test than SAP for
early detection of glaucoma-related eld loss [1618]. SWAP also identies progression 13 years prior to detection by standard visual elds [1921], and works
well in advanced cases not complicated by the presence of cataracts [22]. Although SWAP has higher test-retest variability than SAP, and more than is desirable for long-term follow-up for progression of glaucomatous vision loss [23,
24], it has consistently been shown superior to SAP for identifying progression
[20,21].
3. High-Pass Resolution Perimetry
It was originally thought that SWAP was testing blue-yellow ganglion cells that
pass through the parvocellular pathways of vision. Since SWAP was developed,
it has been determined that the blue-yellow ganglion cells responsible for processing the stimuli of SWAP are most likely not parvocellular, but are the small
bistratied ganglion cells whose axons project to the interlaminar layers of the
lateral geniculate nucleus of the thalamus [14,2527]. Parvocellular ganglion
cells are the most numerous, making up about 70% of the total. These cells are
278
Sample
also referred to as midget ganglion cells. These cells are distributed throughout
the retina, and they have the smallest cell bodies, axons, and dendritic eld sizes
of the three types [14]. These cells handle acuity and resolution tasks. They prefer
stimuli with low temporal frequencies and high spatial frequencies, along with
color [28].
If we wish to include a test more likely to be detected by the parvocellular
pathway, we can look to High-Pass Resolution Perimetry. This test is a resolution
task. High-pass resolution visual elds were developed by Lars Frisen, M.D.,
and are tested using the Ophthimus High-Pass Resolution Perimeter (HighTech
Vision, Malmo, Sweden) [29]. The test presents spatially ltered rings across 50
test locations in a 30 visual eld. Fourteen different ring step sizes are used,
with the smallest subtending 0.8 visual angle and each successive larger ring
size spaced 0.1 log units apart. Thresholds are designated as the smallest ring
size that can be resolved by the patient. The ring targets consist of a dark border
(15 cd/m2) surrounding a light core (25 cd/m2) and are designed such that the
space-average luminance across the ring target is equiluminant with the background of the display (20 cd/m2). In this manner, resolution rather than detection
thresholds are measured and the target is both seen and resolved instantaneously
or not seen at all.
High-Pass Resolution Perimetry was found to be comparable to standard
elds for detecting vision loss [30] and superior for identifying change over time
[31]. It is a very patient-friendly test, taking about 5 min and giving feedback
for correct responses. The major drawback to its acceptance is the lack of standardization across test units.
4. Frequency-Doubling Technology Perimetry
Frequency-doubling technology perimetry (FDT) [32,33] is based on the frequency-doubling illusion [34,35], which occurs when viewing a counterphased
grating with a low spatial frequency and a high temporal rate. Above threshold,
the percept is double the spatial frequency of the actual physical grating [35].
This illusion has been attributed to a subset of the magnocellular ganglion cells,
which are nonlinear in their response properties [36].
Magnocellular ganglion cells are fewer in number and compose about 8
10% of the ganglion cell population. These cells are also referred to as parasol
ganglion cells. These cells are distributed across the retina, and compared to the
parvocellular cells they have larger cell bodies, axons, and dendritic eld sizes
with consequently larger receptive eld center sizes. The magnocellular cells
axons project to the magnocellular layers of the lateral geniculate nucleus [37].
They respond in a transient fashion to visual stimulation, and their axons have
a relatively faster conduction velocity compared to the other two cell types [38
41]. These cells prefer stimuli with high temporal frequencies, low spatial fre-
279
quencies, and motion. There is some debate about whether FDT at threshold is
measuring a small subset of nonlinear magnocellular cells (estimated at about
3% of the ganglion cells), or if the target is more likely detectable due to its
icker component [4244] by the full complement of magnocellular cells (still
only about 10% of the population). At threshold, the percept is not always of a
grating, either perceptually doubled or veridical, but sometimes is described as
a shimmering or ickering [45,46]. Either way, early evidence has shown
the test is sensitive to early glaucomatous defects and correlates well with SAP
for mean defect [32,4751].
Frequency-doubling perimetry is measured with a new instrument, the
Humphrey FDT Visual Field Instrument using Welch-Allyn Frequency doubling
technology (Skaneateles Falls, NY). The targets consist of a 0.25 cycle per degree
sinusoidal grating that undergoes 25 Hz counterphase icker. The test uses a
modied binary search staircase threshold procedure to measure the contrast
needed for detection of the stimulus. Each grating target is a square subtending
about 10 in diameter. Targets are presented in one of 17 test areas located within
the central 20 radius of visual eld (program C-20). With a shift in xation
point location, the range can be extended to 30 and two additional locations in
the nasal step area (program N-30).
FDT has some advantages over SAP and SWAP. The test time is about
one-half of the time required for a full threshold 24-2 eld, primarily due to the
smaller number of test locations used. The results are less affected by blur, pupil
size differences if always greater than 2 mm diameter, or bifocal correction [52],
and it has lower test-retest variability than SAP [53]. It is similar to SAP and
SWAP in that statistical analysis packages can be developed to give global indices, such as MD and PSD, and pattern deviation probability plots can be derived.
The major drawback to FDT for clinical trials that look for change over time is
that it is too new to have longitudinal data. We do not yet know if the small
number of test locations will provide sufcient resolution for following change
over time. Johnson and colleagues are currently working on a modication of
the test to increase the number of test targets [54]. While this may improve the
test ability to detect change over time, it will also increase the test time.
The advantages and disadvantages of each test are summarized in Table
1. If a test is obviously the best, an asterisk denotes it. For example, SAP has
the most extensive normative database and FDT is least affected by changes in
pupil size. If the tests could be ranked, they are ranked from best (1) to worst
(4). For example, SWAP has been found more useful than SAP for a variety of
eye diseases but it is most affected by lens opacities. If we do not yet know
something it is indicated by a ?. For example, we do not know if FDT will be
good for following change over time because it is too new for extensive longitudinal studies.
These advantages and disadvantages have had some inuence on whether
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Sample
these tests have been selected for use in current clinical trials. To my knowledge,
there is no study currently using HPRP. Although this test has numerous advantages, it has not received widespread acceptance due to the lack of standardized
test equipment and no self-calibrating feature. Results obtained on the same eye
may vary from one unit to another. The other three tests are all involved in clinical
trials at present. SWAP is not an outcome measure but is an ancillary test in the
NEI-sponsored Ocular Hypertension Treatment Study (OHTS) [55] and FDT is
a secondary outcome measure in an Allergan-sponsored clinical trial of the drug
Memantine. This study should help us determine the effectiveness of FDT in
longitudinal studies assessing change in visual function.
In nearly all clinical trials involving perimetry, SAP is the method of choice
to date. SAP is currently the standard of care for visual eld testing. The availability, standardization, and sophisticated statistical analyses are useful in clinical
trials. However, in clinical trials that must identify small changes in visual performance, it may be less than ideal. SWAP identies change 1 to 3 years sooner
than SAP [20,21] and the results indicating change are more likely to be repeatable [56,57]. However, the increased variability and full threshold only strategy
of SWAP have been detrimental to its acceptance. SITA (see below) for SWAP,
when available, should go a long way toward alleviating these problems as it has
reduced both test time and variability for SAP.
5. Swedish Interactive Thresholding Algorithm
Swedish interactive thresholding algorithm (SITA) is a new way of obtaining
threshold for SAP [58,59]. It has led to an increased interest in SAP despite evidence that the visual function specic subtests, SWAP and FDT, are much more
sensitive for detection [51] and SWAP and HPRP are better for following progression [20,21,31]. This new enthusiasm for standard elds is based on two attributes
of SITA. The test time for a visual eld is reduced in half relative to full threshold
SAP [5961] and the test-retest variability may also be reduced, which should
make the results more reliable for measuring change over time [6264]. Because
SITA is a change in threshold procedure, which utilizes information collected
during the test to better determine the intensity of the next ash at a given location
[65,66], it easily could be applied to other forms of perimetry, such as SWAP.
Studies comparing SITA with the original full threshold algorithm indicate
that SITA will show approximately 1 dB better thresholds than standard full
threshold testing, but with a statistically deeper defect on the pattern and total
deviation plots in glaucoma patients [60,62,66,67]. The smaller intersubject variance and greater reproducibility in patient eyes may mean shallower defects are
needed in SITA elds for statistical signicance to be reached than for the standard full threshold algorithm, but this has not been tested [62,63]. For trials that
have already begun testing with full threshold strategy, it is not advisable to
281
switch to SITA. Clinically, when switching from standard full threshold to the
SITA strategy to follow patients, it is necessary to obtain new baseline elds and
to make comparisons between the two types of algorithms based on the total and
pattern deviation probability plots rather than on absolute threshold values [64].
Clinical trials often initiate treatment after obtaining baselines, so there is no
opportunity to reassess baselines with SITA. However, trials that were initiated
after the development of SITA, such as the Neuroprotection in Ischemic Optic
Neuropathy Trial sponsored by Allergan, have selected SITA for use with SAP
as a secondary efcacy measure.
III. THE DEFINITION OF CHANGE IN CLINICAL

TRIALS
Because it is often necessary in clinical trials to determine if progression is occurring before a series of ve to seven elds can be obtained, linear regression
has not been the method of choice. Instead, the statistical methods for identifying
eld progression relative to two baseline visits provided by the GCP and global
indices of Statpac II have been incorporated into several large clinical trials: (1)
Normal Tension Glaucoma Study (NTG); (2) Early Manifest Glaucoma Trial
(EMGT); (3) Advanced Glaucoma Intervention Study (AGIS); (4) Collaborative
Initial Glaucoma Treatment Study (CIGTS); and (5) Ocular Hypertension Treatment Study (OHTS).
A. Normal Tension Glaucoma Study
The Normal Tension Glaucoma Study was developed to assess the effect of lowering intraocular pressure on the progression rate of normal tension glaucoma
[68]. To be eligible for this study, patient eyes had to have glaucomatous excavation of the optic disc and a eld defect, standard achromatic perimetry, consisting
of a cluster of three non-edge points depressed by 5 dB, with one of the points
also depressed by 10 dB. This defect had to be conrmed by two of three baseline
elds performed within a 4-week window. Patients with a history of IOP greater
than 24 mmHg were excluded.
Progression was suspected when (1) at least two contiguous points within
or adjacent to a baseline defect showed a reduction is sensitivity from baseline
of at least 10 dB, or three times the average baseline short-term uctuation for
that patient, whichever is greater; (2) the sensitivity of each suspected point is
outside the range of values observed during baseline testing; or (3) when a defect
occurs in a previously normal part of the eld. To reach a denitive decision of
progression, four conrmatory tests were required. This large number of conrmatory elds was a consequence of looking for smaller eld changes, which were
282
Sample
reliable indicators of glaucomatous visual eld loss and not just indicative of
physiologic long-term uctuation [68].
B.
The Early Manifest Glaucoma Trial
The Early Manifest Glaucoma Trial (EMGT) was developed to assess the effectiveness of reducing intraocular pressure in early, previously untreated open-angle
glaucoma. Because EMGT uses eld progression as a study endpoint, a progression algorithm was developed [69]. As mentioned previously, the GCP from Statpac was modied so scoring is based on the pattern deviation probability map.
For the EMGT, there is an initial screening, two pre-intervention eld tests,
and two baseline visits. The two baseline visits must have a GHT outside normal
limits due to the same sectors or a borderline GHT on two consecutive eld
exams with obvious localized change to the optic disc [69]. Progression requires
three or more points agged by the pattern deviation version of the GCP analysis.
Only if these same points are conrmed on two subsequent visual elds is progression veried.
C.
Advanced Glaucoma Intervention Study
The Advanced Glaucoma Intervention Study (AGIS) algorithm was developed

to determine eligibility for the AGIS study and to evaluate disease progression
in patients with more advanced glaucomatous visual elds [70]. The AGIS scoring system is based on the concepts that (1) multiple defects can occur in the
upper, lower, and nasal hemields; (2) a defect requires two or more adjacent
defective points; (3) the severity of depression must be greater than changes due
to variability; and (4) the defect must be caused by glaucoma. The AGIS score
is calculated by totaling the number of adjacent depressed test points in the upper,
lower, and nasal hemields compared with age-matched standardized normal
eyes in the total deviation printout of Statpac II. The score becomes larger with
increases in the number of depressed test sites and with increasing depth of defect.
The nal AGIS score ranges from 0 to 20. Two pre-intervention eld tests are
conducted less than 60 days apart to determine subject eligibility. An AGIS score
between 1 and 16 and a reliability score less than 3 are required on the rst eld
for subject inclusion. The second eld is used as the baseline for subsequent tests.
Progression is quantied as an increase in score from baseline reference by four
or more points on two consecutive reliable elds.
D.
Collaborative Initial Glaucoma Treatment Study
The Collaborative Initial Glaucoma Treatment Study (CIGTS) uses a scoring

system modied from AGIS [71]. In brief, scoring is based on (1) the total devia-
283
tion probability plot, which adjusts the total deviation values at each point relative
to the most normal region in the visual eld; (2) each abnormal test location must
be accompanied by at least two adjacent abnormal points; and (3) each abnormal
point is given a score from one to four based on the probability level (5% to
0.5%) of the three contiguous depressed points. The value for each of the 52
locations within the visual eld is combined to get a maximum possible score
of 208. The total score is then divided by a conversion factor (10.4) to get a nal
score ranging from 0 to 20. Two pre-intervention eld tests must be reliable
(reliability score 4); the GHT must read outside normal limits; there must
be at least three contiguous points on the total deviation plot with a p 0.02;
and if the points are in the nasal eld, they cannot cross the horizontal midline.
The two pre-intervention eld scores are averaged to create a baseline CIGTS
score. If the two baseline elds are more than 7 points apart, then a third eld
is conducted and the three are used to compute a baseline CIGTS score. Progression is quantied as an increase in score from baseline reference by three or more
points on two consecutive reliable elds.
E.
The Ocular Hypertension Treatment Study
The previously mentioned studies all involve assessment of change in an already

abnormal visual eld. The Ocular Hypertension Treatment Study (OHTS) [55]
looks for change from a designation of normal visual eld to abnormal visual
eld using SAP 30-2 full threshold visual elds. Fields are identied as abnormal
if they have a glaucoma hemield test results of outside normal limits or a
corrected pattern standard deviation at the 5% probability or worse with a cluster
of abnormal points consistent with glaucoma. Two conrming elds are required
to call the elds glaucomatous.
What these descriptions of progression or change make clear is that there
is no agreed-upon method for identifying progression and each clinical trial has
developed methods specic to the study population involved. A few studies have
compared some of the methods [72,73]. Regardless of the algorithm employed,
it is still difcult to identify and conrm change without sufcient follow-up.
IV. FOLLOWING CHANGE MORE EFFECTIVELY

In addition to the obvious need to reduce the variability inherent in psychophysical tests of visual function and to conrm changes seen, there are other strategies
that may assist us in identifying change more reliably. Each ganglion cell axon
crosses the retina and enters the optic nerve. These bers travel in bundles in a
specic pattern. Several studies have correlated the location of visual eld defect
to location of optic nerve damage with good results [7479]. We have found these
284
Sample
correlations hold for focal defects found on SAP, SWAP, and non-commercially
available motion automated perimetry (MAP) [46,80,81]. In addition, there is an
important and direct relationship between these measures of visual function and
location of damage. In a study comparing SAP, SWAP, FDT, and MAP, we
found that when a given individual had vision loss on more than one test, the
same area of the visual eld was affected [51]. Finally, in early glaucoma there
is little evidence for secondary effects of neural degeneration as demonstrated
by a lack of crossover of defect at the horizontal midline [82]. This does not rule
out the presence of secondary degeneration but may be indicative of the limitations of our current testing strategies.
Since we now have information about different visual functions, location
of visual eld loss and the relationship to optic nerve damage, how can we use this
information to improve tests for monitoring the effectiveness of neuroprotection
therapies in clinical trials? Here we are not only interested in detection of a defect
but also in change over time. Will those eyes receiving the neuroprotective agent
show less progression of glaucoma than those that do not receive neuroprotection?
For this, we also must understand something about how visual elds progress.
To determine typical patterns of visual eld progression and their relationship to the 24-2 eld grid, we prospectively followed, in a multicenter study,
115 glaucoma patients, who were experienced with elds, having two abnormal
baseline visual elds, abnormal optic nerves, and repeated testing for elds that
progressed [83]. Progression was categorized as (1) deepening of an existing
scotoma; (2) expansion of an existing scotoma; or (3) a new scotoma. Three
methods for classifying progression were used: (1) a clinically determined
method; (2) the glaucoma change probability (GCP) based on total deviation
(TD); and (3) the GCP based on pattern deviation (PD). Regardless of the classication method used, the glaucomatous visual elds progressed in the area of
the visual eld where baseline testing showed an existing scotoma. Because an
individuals location of defect overlaps on different visual function tests and progression occurs in this area, we can maximize nding repeatable change by concentrating testing on the defective areas determined at study entry.
V.
CONCLUSIONS
Tests of visual function and measures of optic nerve structure that take into account the location of damage at baseline for each individual and incorporate
this information in their designs for following change should be most successful
for determining the effectiveness of neuroprotection. Since there is no gold standard for progression, it is still necessary for each clinical trial to identify an
algorithm that best ts the needs of the study population under test. It would be
prudent to incorporate a variety of visual function tests in these studies, because
285
we do not know if one type of ganglion cell may be more susceptible to a given
neuroprotection therapy than another. It will be necessary to continue to improve
our methods for identifying change in visual elds. This includes improving visual eld techniques and improved analysis of results over time. As it stands
now, more time for follow-up will be necessary to determine the effectiveness
of a given neuroprotective agent than was needed in past studies to show the
effectiveness of IOP-lowering agents. This is because glaucoma progresses
slowly and possible change requires conrmation on more than one subsequent
visit.
ACKNOWLEDGMENTS
This work is supported by NEI EY08208 and a Lew R. Wasserman award from
Research to Prevent Blindness (PAS).
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17
Clinical Trials in Neuroprotection
Scott M. Whitcup
Allergan, Inc.
Irvine
and Jules Stein Eye Institute
David Geffen School of Medicine, UCLA
Los Angeles, California, U.S.A.
I.
INTRODUCTION
A clinical trial is a planned experiment in humans designed to assess the safety

and/or efcacy of a treatment. The well-designed clinical trial should control for
bias that can corrupt the interpretation of clinical data. Unfortunately, a great
deal of medical practice is based on anecdotal clinical reports or poorly designed
clinical studies. Much of the scientic dogma we read in textbooks is actually
based on a retrospective review of inconclusive data obtained from a handful
of patients. This is especially true of new therapeutic areas in medicine, where
experience and published data are lacking.
II. METHODS OF CLINICAL STUDIES

There are four basic types of clinical studies: case series, case-control studies,
cohort studies, and randomized clinical trials. Case reports or case series are
usually retrospective reviews that detail the clinical ndings and outcomes of
patients with a particular disease. Although these reports can help dene the
manifestations of a disease, they are prone to bias and can mislead the reader.
Since the data are collected retrospectively from patient charts, critical information is often missing. The disease may not be well dened in the report, and
291
292
Whitcup
some of the reported patients may actually have a different condition. There may
be bias in acquiring the patients, and the reported patients may be dissimilar from
the general patient population. Case series tend to be written by specialists who
treat patients with more severe or atypical diseases. Importantly, case series lack
a control group for comparison. For example, a physician could report two patients with non-arteritic ischemic optic neuropathy (NAION) who had substantial
improvement in their vision after starting multivitamins. It would be difcult to
conclude that vitamins improve vision in patients with this disease without knowing how many patients with NAION on vitamins had no improvement in vision.
Occasionally, investigators will try to compensate for the lack of an appropriate control group in a study by comparing their study results to a group of
historical controls. The investigators agree that a control group is needed, but
are still reluctant to randomly assign patients to the new treatment or to a standard
therapy or placebo. There are a number of reasons for this reluctance. First, it is
much more difcult to conduct a well-controlled, randomized clinical trial. A
protocol needs to be written, institutional review board (IRB) approval is required, and the methods for patient randomization, conduct of the trial, collection
of the data, and analysis of the results need to be detailed. Second, many investigators truly believe that the new treatment is better, and that it would be unethical
to keep patients from receiving the new treatment.
The main problem with the use of historical controls is that data from historical controls tend to be biased. Data from historical controls are often collected
differently from patients enrolled in a trial and followed prospectively (information bias). Patients in a trial can also differ clinically from the patients in a
historical control group (selection bias), not only in recognized important clinical
parameters like disease severity, but also in potentially unrecognized or undocumented parameters that could affect a clinical outcomefor example, diet or
other environmental factors.
There are numerous other sources of bias in clinical studies [1]. Observer
bias leads to a systematic alteration in the measuring of a response in patients.
Unvalidated or inappropriate instruments for measurement can also bias the results of a study. In a properly designed trial, controlling for confounding factors
can minimize bias. Randomization, for example, can help to balance these factors
in the treatment groups. Importantly, randomization helps to balance unrecognized sources of bias between groups.
In the case-control study the investigator compares a group of patients with
a given disorder to a control group without this condition. The clinical records
of both groups are then compared to see if certain factors occur more commonly
in one group. A classic example of a case control study would compare the smoking history of a group of patients with lung cancer and an age- and sex-matched
control group. One can then calculate an odds ratio that states the relative risk
for a condition like lung cancer given a specic risk factor like smoking. For
293
example, an odds ratio of 6.7 would mean that people who smoke are 6.7 times
more likely to develop lung cancer than people who do not smoke.
Case-control studies, although more powerful than case series, also rely on
a retrospective review of patient records. Again, bias may systematically alter the
data and lead to inappropriate conclusions. There may be a recording bias in the
information collected from patients and controls. For example, patients with cancer
may spend more time thinking about their medical history and reasons why they
might have developed their disease than would a person without the disease. Physicians may spend a great deal of time detailing clinical information from patients
that may not be collected from the controls. Despite this potential bias, well-conducted case-control studies can provide useful clinical information, especially when
standard procedures for data collection are followed. Furthermore, case-control
studies may be the only feasible method for studying certain rare conditions.
Cohort studies identify two groups of patients; for example, one cohort
receives a treatment and the other cohort does not receive the therapy. The two
groups of patients or cohorts are then followed prospectively for the development
of a specic outcome. However, because the treatment is not randomly assigned,
the two groups of patients may differ greatly in certain critical clinical parameters.
For example, maybe the treatment is given only to the most severely ill patients
who have nothing to lose. These patients may be unlikely to respond to any
treatment, no matter how effective.
Pharmaceutical drug development therefore includes a number of clinical
studies, but nal determination of safety and efcacy is based predominantly on
pivotal randomized clinical trials. Clinical studies during the development of a
new medicine are often divided into four phases. Phase 1 clinical trials are the
initial safety trials of a new medicine. These are usually conducted in normal
volunteers, often in males. In the eld of cancer, Phase 1 trials are often conducted
in more severely ill patients. The trials can be open label, where patients and
investigators are unmasked to the treatment allocation. Multiple doses may be
tested in a Phase 1 trial, often starting with the lowest dose and escalating to
higher dosages if they are tolerated.
Phase 2 trials are designed to study the safety and efcacy of a new medication. These trials are often double-masked, where both the patient and investigators do not know what treatment is being administered. Classically these studies
are called double-blind studies; however, in ophthalmology we prefer the term
double-masked, since it is difcult to get a patient with an eye disease to enroll
in a study with double-blind in the title. Phase 2 trials typically have more patients
than Phase 1 trials, are conducted in patients with the disease, but still may examine several dosages or treatment regimens.
The Phase 3 clinical trial is the pivotal clinical study for the approval of
the medication. These studies almost always are larger randomized clinical trials
comparing the new medication to the standard treatment or to placebo. The
294
Whitcup
United States Food and Drug Administration (FDA) usually requires two Phase
3 trials prior to the approval of a new drug.
Studies conducted after a medicine is approved and marketed are called
Phase 4 trials. These studies are conducted in patient populations for which the
medicine is intended and may compare the medicine to currently available therapies. These studies are also used to elucidate additional clinical data that supplements the results of the Phase 1, 2, and 3 trials.
The randomized clinical trial provides the most robust evidence about the
safety and efcacy of a new treatment. Because patients are randomly assigned
to the new treatment or to the control treatment, if the number of patients in the
study is large enough, the treatment groups are usually similar. This is a critical
point, since treatment outcome could be affected if the groups differed in clinically relevant parameters. Although one could try to control or compensate for
imbalances using certain statistical analyses, this only works for the parameters
that are thought to affect outcome and for which data are available. As stated
before, randomization is powerful because it controls for both known and unknown sources of bias.
III. ISSUES IN THE DESIGN AND CONDUCT

OF CLINICAL TRIALS
Table 1 lists the components of a well-designed clinical study. It is important
that the investigators be experienced in conducting clinical trials and have clinical
expertise in the disease being studied. The primary outcome variable of the study,
also known as the primary endpoint, should be clearly stated. This primary outcome variable is the main parameter on which the investigator plans to judge the
efcacy of the intervention. Therefore, it is important to prospectively choose
the primary outcome variable, even if multiple clinical outcomes are examined.
The procedure for enrolling patients into the study should be detailed, and the
inclusion and exclusion criteria listed, because the patient population will determine how generalizable the results are to a larger patient population outside of
the trial. For example, results of a potential neuroprotective medication studied
in patients with narrow angle glaucoma and intraocular pressures of 40 mmHg
or above may not be generalizable to patients with open angle glaucoma with
pressures in the range of 22 to 30 mmHg.
The treatment and dosing regimen should be clearly stated. Choice of the
control treatment is also critical to the value of the study. Patients in the control
group should be treated according to the current best standard of care. If no
proven treatment is available, a placebo could be considered. The dosage of the
control regimen should also be appropriately chosen. It would be inappropriate
to compare a new glaucoma medication to pilocarpine 1% dosed once daily.

Table 1
295
Components of a Randomized Clinical Trial
Study approved by Institutional Review Board.

Appropriate informed consent obtained from patients.
Disease well dened with specic diagnostic criteria.
Patient population well dened with specic inclusion and exclusion criteria.
Patients randomly assigned to new treatment and control treatment according to
standardized procedures.
Patients and investigators appropriately masked from treatment assignment.
Sample size accurately determined to control for type I and type II error.
Outcome measures specied and minimum differences to be considered as clinically
important detailed.
Procedures for the conduct of the trial well detailed.
Timing of study visits and collection of data strictly specied.
Statistical analysis plan specied prior to locking the database and unmasking of
treatment assignments. Results of an intent-to-treat analysis, where all randomized
patients are included in the analysis should be provided, even if additional analyses
performed.
It is extremely important to perform appropriate sample size calculations

for all clinical trials. Sample size is based not only on the event rate expected
in the two groups, but also on the desired level of protection against type I and
type II error. Type I error (alpha) occurs when the study falsely concludes that
the therapies tested are different when in fact they are the same. Especially when
a standard therapy for a disease currently exists, most clinical trials protect more
strongly against this type of error, since one would not want a new treatment to
be wrongly administered when an effective therapy is available. Most studies
limit the possibility of type I error to less than 0.05 (5%). Type II error (beta)
occurs when a study falsely concludes that there is no difference between the
treatments when in fact a difference exists. Typically, type II error for many
clinical trials is set at 0.2 (20%). This means that there is a 20% chance that the
treatments are different although the study shows no signicant difference. The
number of patients greatly affects type 2 error. Statistical power (1-beta) is the
chance of proving the difference between the two groups that is dened in the
sample size calculations. Many studies in the literature are underpowered. They
do not have sufcient patients to have a reasonable chance of detecting a meaningful difference between the two groups. A small study that concludes that there
is no signicant difference between two treatments can be extremely misleading.
The treatment effect could be 40% higher with the new treatment, but if the
numbers of patients are small and the variability of response high, this difference
may not be statistically signicant, yet clearly be clinically meaningful. In analyzing study results, look at the actual data, and do not be misled by an insignicant
296
Whitcup
P value, especially if the study is inadequately powered to detect a clinically

meaningful difference. Remember that clinical studies can never denitively
prove that two treatments are the same.
IV. CLINICAL TRIALS OF NEUROPROTECTION

IN OPHTHALMOLOGY
Proof of neuroprotection in ophthalmology will require a well-designed clinical
trial employing many of the strategies detailed above. The most denitive evidence will require a randomized clinical trial comparing the potential neuroprotective treatment to a control treatment or placebo. Although there are difculties
in conducting any clinical trial, studies of potential neuroprotective medications
will encompass several additional challenges.
There are very few randomized clinical trials of neuroprotective medications in medicine. One such study, the Glycine Antagonist in Neuroprotection
(GAIN) Americas trial, was a randomized, double-masked placebo-controlled
trial conducted to examine the efcacy of gavestinel, an antagonist of the glycine site of the N-methyl-d-aspartate receptor, as a neuroprotective therapy for
acute ischemic stroke [2]. The main outcome measure was functional capability
at 3 months, measured by the Barthel Index. This study concluded that gavestinel administered up to 6 h after an acute ischemic stroke did not improve functional outcome at 3 months. Memantine, an uncompetitive N-methyl-d-aspartate
(NMDA) antagonist has been studied in patients with severe dementia, both Alzheimer type and vascular type [3]. In this study, neuroprotection with memantine
led to functional improvement and reduced care dependence in severely demented
patients.
There are even fewer randomized controlled clinical trials of neuroprotection in ophthalmology. One example is the Ischemic Optic Neuropathy Decompression Trial (IONDT) [4]. This was a National Eye Institutesponsored
multicenter clinical trial designed to assess the safety and efcacy of optic nerve
decompression surgery compared with careful follow-up alone in patients with
nonarteritic ischemic optic neuropathy (NAION). Prior to this study, several nonrandomized trials showed benets of optic nerve decompression; however, none
of these were randomized studies [58]. Interestingly, results from the IONDT
showed that patients assigned to surgery did no better when compared with patients assigned to careful follow-up [4]. Improved visual acuity of three or more
lines of visual acuity was achieved by 23.6% of the surgery group compared
with 42.7% of the careful follow-up group. In fact, patients receiving surgery
had a signicantly greater risk of losing three or more lines of vision at 6 months:
23.9% in the surgery group worsened compared with 12.4% in the careful followup group.
297
The IONDT illustrates the need for a well-controlled clinical study to adequately assess neuroprotective therapy. Prior to this trial, improvement in visual
acuity in patients with NAION was thought to be rare: less than 10% [9,10]. In
the IONDT, 42.7% of patients in the careful follow-up group had a three line or
greater improvement in visual acuity [4]. Furthermore, optic nerve decompression
surgery was found to be ineffective and potentially harmful to patients with this
disease.
A. Endpoints
A critical factor in designing clinical trials for neuroprotection is endpoint selection. The primary outcome measures in the IONDT were gain or loss of three
or more lines of visual acuity on the New York Lighthouse chart at 6 months
after randomization [4]. This is a functional endpoint that has been used in a
number of ophthalmology trials and is felt to be clinically meaningful. Visual
acuity has also been used as a standard clinical endpoint for clinical trials in
macular degeneration, where central acuity can be affected early in the course
of the disease. Unfortunately, changes in central acuity may be an insensitive
endpoint for many ophthalmic diseases. For example, central visual acuity loss
occurs relatively late in the course of some diseases including glaucoma and
retinitis pigmentosa. Although visual eld loss can be used as a functional endpoint, visual eld loss progresses slowly and may require many years before
meaningful changes occur.
Identication and validation of surrogate endpoints will improve our ability
to assess neuroprotective therapies. In a sense, intraocular pressure has been used
as a surrogate endpoint for glaucoma treatments. Without well-controlled data,
lowering intraocular pressure was felt to reduce the risk of vision loss in patients
with glaucoma. Data from randomized clinical trials that support the benets of
lowering intraocular pressure are now becoming available. In a recent report from
the Advanced Glaucoma Intervention Study (AGIS), investigators showed that
eyes with intraocular pressure less than 18 mmHg for 100% of visits over 6 years
had mean changes from baseline in visual eld defect score close to zero during
follow-up, whereas eyes with intraocular pressure less than 18 mmHg for less
than 50% of visits had an estimated worsening over follow-up of 0.63 units of
visual eld defect score [11].
There are a number of new methods for assessing visual function that may
be useful as endpoints in clinical trials for neuroprotection. Depending on the
disease, endpoints may change at differing rates and have a large impact on length
of clinical trials and the required sample size. Other studies have documented
that rates of change differ among measures of visual function. In a trial of patients
with retinitis pigmentosa, investigators assessed changes in measures of visual
function in patients over time [12]. The smallest amount of change occurred for
298
Whitcup
visual acuity and hue discrimination, and the greatest amount of change occurred
for visual eld area.
Electrophysiologic testing, newer methods of visual eld testing, and contrast sensitivity are just a few examples of potential endpoints for clinical trials
in neuroprotection. The electroretinogram was used as the primary outcome measure in a randomized clinical trial of vitamin A and vitamin E supplementation
for retinitis pigmentosa [13]. This was a National Eye Institutesponsored randomized, double-masked trial to determine whether supplements of vitamin A
or vitamin E alone or in combination affect the course of retinitis pigmentosa. In
this study, the main outcome measure was the cone electroretinogram amplitude.
Patients receiving 15,000 IU/day of vitamin A were 32% less likely to have a
decline in amplitude of 50% or more from baseline than those not receiving
this dosage (P 0.03). Although not statistically signicant, similar trends were
observed for rates of decline of visual eld area. These data support the potential
benet of identifying endpoints that may be more sensitive or less variable in
clinical trials.
More accurate and sensitive measures of visual function will improve our
ability to test neuroprotective therapies. Use of the scanning laser ophthalmoscope to assess perimetry [14] or of the multifocal electroretinogram [15] may be
useful endpoints in trials of retinal disease. Similarly, new methods of assessing
glaucomatous damage such as the Heidelberg retinal tomograph (HRT), the GDx
nerve ber analyzer (GDx), and the optical coherence tomograph (OCT) may be
important measures of visual function in neuroprotection trials in patients with
glaucoma [1619]. New methods of measuring visual eld using short-wavelength automated perimetry or multifocal visual evoked potential could also improve our ability to accurate assess changes in visual function in these patients
[20,21]. However, it is important to remember that all of these endpoints need
to be extensively evaluated and validated before widespread use in clinical trials.
Increasingly, there is an interest in assessing the effect of new therapies
on quality of life. Questionnaires are often employed to determine quality of life;
however, it is important to make sure that the questionnaires are validated for
the disease of interest. The visual function questionnaire is one such quality of
life measure that has been validated in a number of diseases, including diabetes
mellitus, macular degeneration, glaucoma, and cytomegalovirus retinitis [22].
B.
Neuroprotection and Glaucoma
A number of scientists are trying to develop neuroprotective therapies for glaucoma. However, data suggest that lowering IOP can decrease visual loss, and
in a sense, provide neuroprotection. Therefore, in studying neuroprotection in
glaucoma, it will be important to assess preservation of vision while controlling
for intraocular pressure. For example, there are a number of laboratory studies
of glaucoma medications that demonstrate neuroprotection in animal models of
299
optic nerve disease. It is important to compare these drugs to control medications

that similarly lower IOP but reportedly have no neuroprotective effects. For example, in an animal model of ocular hypertension, systemic administration of
brimonidine or timolol had equivalent effect on IOP [23]. Nevertheless, brimonidine signicantly reduced the progressive loss of retinal ganglion cells by greater
than 50%, whereas timolol had no effect. These considerations should also apply
to human clinical trials. Two Phase 3 randomized, double-masked clinical trials
are in progress assessing the neuroprotective effects of memantine, a drug that
blocks the NMDA receptor. In this trial, the neuroprotective effects of the drug
will be assessed independently from IOP.
Although denitive proof of a neuroprotective medication in ophthalmology will probably require data from a randomized, clinical trial, there are some
instances where an open-label trial of a medication could lead to believable evidence. In diseases where the clinical outcome is well documented with little to
no variability, an open-label trial with historical controls could provide convincing data, using historical controls. Unfortunately, there are few such conditions.
One potential disorder is central retinal artery occlusion in patients lacking a
cilioretinal artery. If a treatment were studied that preserved 20/20 vision in even
a relatively small number of patients, the data could be fairly convincing, because
the visual outcome of this condition is usually catastrophic. However, there is
still the opportunity for bias, and one should be very careful about misinterpreting
data from trials without a concurrent control group and random assignment of
treatment.
V.
CONCLUSIONS
In conclusion, neuroprotection offers an exciting therapeutic approach to a number of diseases. Clearly, patients with disorders affecting the retina or optic nerve
may benet from neuroprotective medications. One must be careful in interpreting data from small, uncontrolled studies. As can be seen, there are a multitude
of issues that must be considered. Well-designed clinical trials with validated
endpoints will provide the best insight on the neuroprotective effects of medications and provide new treatment options that, one hopes, will save vision.
REFERENCES
1.
2.
Sackett DL. Bias in analytic research. J Chronic Dis 1979; 32:5163.

Sacco RL, DeRosa JT, Haley ED Jr, Levin B, Ordronneau P, Phillips SJ, Rundek
T, Snipes RG, Thompson JL. The Glycine Antagonist in Neuroprotection Americas
Investigators. Glycine antagonist in neuroprotection for patients with acute stroke:
GAIN Americas: a randomized controlled trial. JAMA 2001; 285:17191728.
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3.
Winblad B, Poritis HN. Memantine in severe demintia: results of the 9M-Best Study
(benet and efcacy in severely demented patients during treatment with memantine). Int J Geriatr Psychiatry 1999; 14:135146.
The Ischemic Optic Neuropathy Decompression Trial Research Group. Optic nerve
decompression surgery for nonarteritic anterior ischemic optic neuropathy (NAION)
is not effective and may be harmful. JAMA 1995; 273:625632.
Sergott RC, Cohen MS, Bosley TM, Savino PJ. Optic nerve decompression may
improve the progressive form of nonarteritic ischemic optic neuropathy. Arch Ophthalmol 1989; 107:17431754.
Kelman SE, Elman MJ. Optic nerve sheath decompression for non-arteritic ischemic
optic neuropathy improves multiple visual function parameters. Arch Ophthalmol
1991; 109:667671.
Spoor TC, Wilkinson MJ, Ramocki JM. Optic nerve sheath decompression for the
treatment of progressive nonarteritic ischemic optic neuropathy. Am J Ophthalmol
1991; 111:724728.
Spoor TC, McHenry JG, Lau-Sickon L. Progressive and static nonarteritic ischemic optic neuropathy treated by optic nerve sheath decompression. Ophthalmology 1993; 100:306311.
Boghen DR, Glaser JS. Ischemic optic neuropathy: a clinical prole and natural
history. Brain 1975; 98:689708.
Repka MX, Savino PJ, Schatz NJ, Sergott RC. Clinical prole and long-term implications of anterior ischemic optic neuropathy. Am J Ophthalmol 1983; 96:478
483.
The AGIS Investigators. The advanced glaucoma intervention study (AGIS): 7. The
relationship between control of intraocular pressure and visual eld deterioration.
Am J Ophthalmol 2000; 130:429440.
Holopigian K, Greenstein V, Seiple W, Carr RE. Rates of change differ among
measures of visual function in patients with retinitis pigmentosa. Ophthalmology
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Berson EL, Rosner B, Sandberg MA, et al. A randomized trial of vitamin A and
vitamin E supplementation for retinitis pigmentosa. Arch Ophthalmol 1993; 111:
761772.
Sunness JS, Schuchard RA, Shen N, Rubin GS, Dagnelie G, Haselwood DM. Landmark-driven fundus perimetry using the scanning laser ophthalmoscope. Invest
Hood DC, Seiple W, Hologigian K, Greenstein V. A comparison of the components
of the multifocal and full-eld ERGs. Vis Neurosci 1997; 14:533544.
Miglior S, Casula M, Guareschi M, Marchetti I, Iester M, Orzalesi N. Clinical ability
of Heidelberg retinal tomograph examination to detect glaucomatous visual eld
changes. Ophthalmology 2001; 108:16211627.
Zangwill LM, Bowd C, Berry CC, Williams J, Blumenthal EZ, Sanchez-Galena
CA, Vasile C, Weinreb RN. Discriminating between normal and glaucomatous eyes
using the Heidelberg retina tomograph, GDx Nerve Fiber Analyzer, and Optical
Coherence Tomograph. Arch Ophthalmol 2001; 119:985993.
Wollstein G, Garway-Heath DF, Fontana L, Hitchings RA. Identifying early glaucomatous changes. Comparison between expert clinical assessment of optic disc pho-
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Bosworth CF, Sample PA, Weinreb RN. Detecting early glaucoma by assessment
of retinal nerve ber layer thickness and visual function. Invest Ophthalmol Vis
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Hutchings N, Hosking SL, Wild JM, Flanagan JG. Long-term uctuation in shortwavelength automated perimetry in glaucoma suspects and glaucoma patients. Invest Ophthalmol Vis Sci 2001; 42:23322337.
Klistorner A, Graham SL. Objective perimetry in glaucoma. Ophthalmology 2000;
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Mangione CM, Lee PP, Gutierrez PR, Spritzer K, Berry S, Hays RD. Development
of the 25-item National Eye Institute Visual Function Questionnaire. Arch Ophthalmol 2001; 119:10501058.
WoldeMussie E, Ruiz G, Wijono M, Wheeler L. Brimonidine is neuroprotective to
ganglion cells in rats with laser-induced chronic ocular hypertension. Invest Ophthalmol Vis Sci 2001; 42:28492855.
18
Regulatory Issues in Clinical Trials
Anthony C. Arnold
Jules Stein Eye Institute
David Geffen School of Medicine, UCLA
Los Angeles, California, U.S.A.
I.
INTRODUCTION
Investigators who perform clinical trials on human subjects in the United States
must recognize certain established principles with regard to recruiting and maintaining subjects and must adhere to the policies of both local (Institutional Review
Board, IRB) and federal (Food & Drug Administration, FDA) regulatory agencies. Coordination with sponsoring pharmaceutical company policies is essential
in all aspects, particularly as compromises may be necessary to satisfy the requirements of all agencies involved.
II. RECRUITING AND MAINTAINING SUBJECTS

Recruitment of subjects is often one of the most difcult features of a clinical
trial. Regulatory issues connected with IRB review of recruitment will be discussed below. Key features for effectively maximizing entry of subjects into a
trial include:
1. Awareness of both referral sources and subjects. Informational pamphlets for patients and physicians, individual letters to referral sources, CME
lectures by investigators, publicity campaigns such as press releases and radio
and television interviews, may all increase awareness of the disease to be studied
and the specic clinical trial being developed. Professional publicists may be
303
304
Arnold
valuable assets in the development and implementation of an organized plan for

dissemination of information. Referral sources should be encouraged to utilize
their patient databases to identify subjects at risk or with the disease, but privacy
issues regarding contact with such patients (see below) must be addressed.
2. Motivation of referral sources and subjects. The disease being investigated may be currently untreatable, and referral to a trial may provide a possible
new hope for the subject as well as prestige for the referring physician by association with the center. However, both subjects and referring physicians must be
invested in answering the question of effectiveness rather than simply obtaining
a possible therapy, because a placebo or a standard therapy will be a possible
result of entry; receipt of the study medication is not guaranteed. Subjects may
be paid a reasonable amount for participation, but coercion by too high a sum
must be avoided. Similarly, referral sources may receive some perquisites for their
participation, including reimbursement for time in examination, but specic referral fees or kickbacks for referral of subjects are not acceptable. Referral sources
must be comfortable that patients will be returned to their care following the trial.
3. Availability of clinical center personnel. A critical factor in subject
recruitment is the ease with which subjects may be referred to the nearest Clinical
Center. A single telephone call to the center should allow the referring physician
to establish the subjects probable eligibility and obtain instructions for timely
evaluation of that subject without further demands on the referring physicians
time. This requires Center staff that is adequately trained, motivated to facilitate
entry of subjects, skilled at interpersonal relations, and available to devote the
necessary time to the trial. Additionally, ethical considerations in recruitment
become important for clinical trials.
1. Overzealous recruitment. There is, of course, ample motivation for an
investigator to recruit as many subjects for the trial as possible, for reasons related
to rapid completion of the trial, prestige of high recruitment, and nancial gain.
The risks of overzealous recruitment include
a. Coercion of subjects by counseling that slants information to encourage
entry into the trial;
b. Bias of data obtained (entry of subjects with incorrect diagnosis, poor
followup for subjects who lack true motivation due to inaccurate counseling, and entering subjects after a time window for treatment in acute
disease, thus biasing results of therapy negatively).
c. Exposure of subjects to undue risk in cases of incorrect diagnosis or
missed time window, when there may be no scientic basis for therapy.
Advertisements or yers must not be misleading. Examples of such improprieties
include claims of safety and efcacy, referring to new treatment or free medical
treatment, when in actuality the subject simply will not be charged for participating.
2. Treatment outside the trial. If a study drug is available outside the
study but is not FDA-approved for the studied disease or at the dosage being
Regulatory Issues
305
studied, its use for the disorder may not be supported by the FDA except under
special conditions. However, when the study drug is available and approved, for
example when the study is comparing it to another therapy, subjects must be
advised that it is available outside the trial. While such advice may impede recruitment, it is ethically required; the IRB will generally address this issue in the
informed consent process.
III. INSTITUTIONAL REVIEW BOARD ISSUES

Human medical research in the United States is guided by ethical principles developed and outlined in the World Medical Association Declaration of Helsinki
(1964) and in the Belmont Report, a 1979 document created by the National Commission for the Protection of Human Subjects of Biomedical and Behavioral Research. All research involving human subjects in the United States is subject to
Institutional Review Board (IRB) approval. The basis for IRB review of research
is contained in the Code of Federal Regulations, Title 45, Part 46 (45 CFR 46).
The IRB approval process is often complex and time-consuming, but is deemed
essential for the adequate protection of human subjects, particularly where scientic and nancial stakes are high. The investigator must become familiar with
IRB requirements of the institution sponsoring or supporting the research; these
may vary widely depending on the IRB constituency (private vs. public). Regarding the conduct of clinical trials, the following aspects are highlighted:
A. Informed Consent
Informed consent is considered to be a process, not an isolated, one-time counseling session between investigator and subject. This ongoing dialogue should provide continuing disclosure of risks and benets as new information becomes
available. It begins with the initial description of research activity in the recruitment phase and continues through the subjects nal participation in the research
activity, with updates as necessary regarding new ndings, risks, and benets
detected as the trial progresses. Such updates may include complications occurring during the trial, new treatment options that become available, or decisions
by sponsor to provide study drug to subjects without charge following the study.
The updates may require pre-approval by the IRB.
The informed consent form (ICF) is the documentation of this process. In
todays clinical research environment, many elements of the ICF are required,
and both IRBs and pharmaceutical sponsors develop templates for investigators
use in clinical research. A great deal of time and effort may be saved by reconciling differences in required wording that may exist between the two versions prior
to IRB submission.
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B.
Arnold
Recruitment
In addition to the recruitment issues discussed above, certain points are of signicant concern to the IRB, particularly with regard to privacy issues and the
possibility of coercion. Areas of focus in reviewing studies include:
Selection. Unless there is a specic medical justication that may be provided, subjects for a trial should not be preselected on the basis of age,
race, gender, or other potentially biasing feature.
Method of identication. The use of medical records or any information
that is not in the public domain to identify potential subjects on the basis
of existing medical conditions violates individual privacy rights. Similarly, the investigator should request that referring physicians approach
their own potential subject patients to inform them of possible research,
allowing them to make their own decision as to whether to contact the
investigator, rather than asking the referring physician to identify specic
potential subjects without their knowledge and prior approval. In some
cases, the use of a yer or information sheet, requiring the potential
subject to initiate contact with the investigator, may be required. Such
recruitment tools must not mislead potential subjects into believing that
a study treatment is superior to current therapy. These yers must have
IRB approval.
Avoiding coercion. This is especially critical in the common situation in
which potential subjects are under the care of the investigator and may
be concerned that their medical care may be jeopardized if they do not
participate in research. A statement is generally placed into the ICF to
explain the potential conict which may exist between an investigators
interest in a study versus an individual patients care.
Additionally, coercion in the form of excessively high payment to subjects
must be avoided. Amount and method of payment should be detailed in
the ICF.
C.
Denition of Minimal Risk
Minimal risk, as dened by federal regulations, is a specically described situation, where the probability and magnitude of harm or discomfort anticipated in
the proposed research are not greater, in and of themselves, than those ordinarily
encountered in daily life or during the performance of routine physical or psychological examinations or tests. It is important to note, in the preparation of IRB
and FDA documents, that this may not necessarily correspond to the investigators
intuitive concept of minimal risk. For example, even though a proposed research
procedure may entail little physical or psychological risk to the subject, if that
Regulatory Issues
307
risk is more than encountered in routine activities for that subject, it may involve
more than minimal risk and may require additional approval procedures.
D. Statement of Emergency Care
and Compensation
All research that presents more than the strict denition of minimal risk as
noted above requires a statement regarding Emergency Care and Compensation
for Injury. This statement generally outlines the responsibility of the institution,
such as a university, supporting the clinical center for a trial, in case of injury
to a subject during a trial. Specic wording is generally required by the institution,
and it may vary depending on whether the sponsor is a public (governmental
e.g., NIH) entity or a private (industrye.g., pharmaceutical company). The
sponsoring agency may also require specic wording, and the two may conict.
In clinical trials conducted by a pharmaceutical company in a university-based
clinical center, agreement must be reached between the two organizations regarding nancial responsibility for emergency care resulting from injury during the
conduct of a trial. It may either be outlined in the ICF or in the clinical trial
agreement (CTA) contract detailing the nancial reimbursement from the pharmaceutical company to the university for the conduct of the trial. It is not acceptable to require that such emergency care be provided by third-party payers.
E.
Reporting Adverse Events
An adverse event (AE) is dened as an undesirable and unintended, although

not necessarily unexpected, result of therapy or other intervention. It may or
may not be drug-related. Most IRBs require the reporting of such events, in writing, within a specied period of time, usually within 25 days, depending on the
severity of the event. Typical adverse events involve injury, problems in the consent process, and other violations of protocol. The investigator is responsible for
determining whether the event requires a change in the research protocol and/
or ICF, and recommending such changes when indicated. Subjects should be
instructed to report any unusual medical occurrence to the PI as soon as possible.
Determinations as to whether an incident is considered an adverse event,
whether an AE is considered serious, and whether it is causally related to study
drug are critical issues.
Incidents considered non-AE include those that occur before or later than
4 weeks after dosing and worsening of disease that is considered within
the normal variation of clinical course for the disease.
Serious AEs are specically dened by the FDA as those that result in or
prolong current hospitalization, produce persistent or signicant incapac-
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Arnold
ity or disability, result in congenital anomaly, are life-threatening, or

result in death. The severity of an AE is a separate issue. Other medical
events not specically meeting these criteria may be considered serious
if they jeopardize the subject. If life-threatening, these must be reported
to FDA by the pharmaceutical company within 7 days, and any SAE
must be reported within 15 days.
Causality for AEs is generally an issue for the FDA to decide, the guideline being a reasonable possibility that the event was caused by the
drug.
F.
Investigator Training and Certication in Human

Research
As of October 1, 2000, the NIH instituted a policy requiring all proposals for
contracts and grants for research involving human subjects to certify that all key
personnel have received education on the protection of human subjects. In many
university research environments, this same requirement is applied by the IRB
to all human research, including that sponsored by sources other than NIH. Both
live teaching sessions at research institutions and web-based training programs
are available to fulll this requirement. Certication is time-limited, with duration
for the current programs 2 years.
IV. FOOD AND DRUG ADMINISTRATION ISSUES

A.
Investigational New Drug Exemption
Federal law prohibits the distribution of new drugs, biologics, and medical devices until the Food and Drug Administration (FDA) has reviewed clinical data
and determined that a product is safe and effective for a specic use in human
patients. Testing of a new drug requires an exemption from that law, and the
sponsoring agent must apply to the FDA for an Investigational New Drug (IND)
exemption before tests with human subjects may begin. If the investigator, rather
than a pharmaceutical company, is the developer of the drug, that person or the
sponsoring institution may be responsible for submitting an IND application to
the FDA and providing the response to the IRB. In certain cases in which an
approved drug is being tested for a slightly modied use, the FDA may issue a
response indicating that an IND is not required. In general, however, approved
drugs tested for new indications or at new doses require an IND exemption. Any
study of such a drug must strictly adhere to the submitted protocol; changes must
be submitted in writing and approved by the FDA and IRB.
Regulatory Issues
309
B. Classication of Trials
The FDA classies clinical trials according to the following schema:
Phase 1. Initial introduction of an investigational new drug into humans.
Designed to determine pharmacologic and metabolic actions, side effects
with increasing doses (establish safe dosage), and gain early evidence
for effectiveness. Study is very closely monitored, often involves healthy
volunteers and a few patients with disease, usually 2080 subjects. The
goal is to gain sufcient data to design a valid Phase 2 study.
Phase 2. Controlled study to evaluate effectiveness for a specic indication
and to determine short-term risks and side effects. Closely monitored,
usually several hundred subjects.
Phase 3. Large-scale controlled study of the drug administered as it would
be when marketed, often involving several hundred to thousand subjects.
Performed after preliminary evidence of effectiveness has been obtained,
it is designed to evaluate effectiveness, safety, and appropriate dosage,
to provide a basis for labeling. Following this study, a sponsor typically
applies for FDA approval to market the drug. Most large-scale clinical
trials fall in this category.
Phase 4. Concurrent with approval, the FDA may seek agreement from the
sponsor to conduct post-marketing (Phase 4) studies to evaluate other
doses, indications, or duration for drug use.
C. Controlled Studies
The FDA describes ve categories of controls which may be used in drug trials:
1.
2.
3.
4.
5.
Placebo concurrent
Dose-comparison concurrent
No-treatment concurrent
Active-treatment concurrent
Historical
The FDA requires that the study design be adequate to obtain valid results, but
it does not indicate a preference for type of control. There is misunderstanding
regarding this issue. The use of placebo in a clinical trial may be unethical if
there is proven effective therapy for the disease and it is withheld in favor of
placebo. Many believe the FDA prefers placebo for comparison of effect, requiring it as a feature of randomized clinical trials. Placebo would be expected to
have an advantage over the use of an active-treatment control, as the difference
between effects of study drug and placebo may be greater than that between
study drug and active-treatment control, thus allowing smaller sample size to
310
Arnold
demonstrate signicance. The FDA has indicated that an active-treatment control

trial in which a nding of no difference between study drug and active-control
would be considered evidence of effectiveness of the new agent. It has also
observed that such a design may be incapable in certain settings of allowing a
conclusion to be drawn, due to the statistical difculty of proving no difference.
The FDA has not, however, as is commonly suggested, mandated the use of
placebo for the study of new drugs. The risks of the use of placebo or no-treatment
controls in specic diseases must be weighed against the risk that an activecontrol design may expose the subject to the risks of a trial without a reasonable
chance to obtain useful information.
D.
Financial Disclosure
Investigators who participate in clinical trials must disclose all nancial ties to the
sponsoring pharmaceutical company, whether relatively minor, such as limited
honoraria for lectures; more substantial, such as ongoing consulting agreements,
research grants or equipment purchases; or major, such as full-time employment
by the company or equity interest in the company. Proprietary interest in a test
product, such as patents, copyright, trademark, or licensing agreements must also
be disclosed. Such disclosure must be reported to the IRB, the FDA, and may
be required to be documented in the ICF. The FDA monitors data from investigators with such nancial links extremely carefully.
In certain universities, potential conict of interest or the appearance of
such conict is highly scrutinized. A positive nancial disclosure by an investigator may prompt detailed review for such conicts, and a determination as to
whether the investigator may take part in a sponsored trial or whether he or she
must sever all such nancial ties during the conduct of the trial may be made by
the university.
E.
Reporting Adverse Events
Specic procedures are outlined in the CFR for reporting the occurrence of an
adverse event during a clinical trial. Investigators must familiarize themselves
and comply with these regulations.
BIBLIOGRAPHY
1.
2.
18th World Medical Assembly. World Medical Association Declaration of Helsinki:

Recommendations guiding medical doctors in biomedical research involving human
subjects. Helsinki, Finland, June 1964.
National Commission for the Protection of Human Subjects of Biomedical and Be-
Regulatory Issues
3.
4.
5.
311
havioral Research. The Belmont Report: Ethical principles and guidelines for the
protection of human subjects of research. OPRR Reports, April 18, 1979.
Code of Federal Regulations. Title 45 (45 CFR 46): Protection of Human Subjects.
Revised June 18, 1991.
Code of Federal Regulations. Title 21 (21 CFR): Food and Drugs. Revised April 1,
1999.
FDA Information Sheets, October 1, 1995.
Index
Abducens nerve, 132, 133

Acepromazine, 24, 48, 52, 113
Acetazolamide, 51, 52, 121
Acinus, 227
Acridine orange, 210, 212
Ad libitum, 92, 130
Adeno-associated virus, 168, 170, 171,
174, 175, 176, 177, 180
Adenovirus, 62, 157, 158, 168, 170,
171, 172
Advanced Glaucoma Intervention Study,
281, 282, 297
Adverse event, 307, 308
Agarose, 98, 176
Age-related macular degeneration
(ARMD), 110, 113, 199
Alpha-crystallin, 67
Alphagan (brimonidine), 50, 51
Amacrine cell, 7, 55, 66, 132, 192, 206,
219
Aminoguanidine, 159
Antiglaucoma, 50, 62
Antisense, 230
Apoptosis, 1, 85, 97, 98, 120, 169, 225,
226, 227, 230, 231, 234, 235,
241, 242, 243
Apoptosis initiation factor (AIF), 226,
227
Apoptosis protease activating factor-1
(APAF-1), 226
Apoptosome, 226
Applanation, 27, 40, 50, 51

Aquamount, 229, 231, 232, 233, 234,
235, 237
Aqueous humor, 23, 32, 33, 37, 39, 44,
45, 47, 57, 60, 72, 154
Arcuate, 62
Area centralis, 195, 213, 214, 218, 219
Argon laser, 32, 49, 50
Arrestin, 99
Articial CSF, 209, 210, 211, 212
Ascorbate, 101, 102, 103
Ascorbic acid, 101, 102, 103
Aspartate, 2, 3
Astrocyte, 4, 67, 69, 111, 119, 175
Autouorescence, 231, 232
Axoclamp, 213
Axon, 6, 13, 14, 16, 17, 18, 19, 23, 32,
43, 63, 66, 67, 69, 130, 131,
132, 133, 140, 141, 145, 146,
192, 206, 207, 209, 212, 213,
214, 215, 217, 242, 273, 275,
277, 278, 283
Axotomy, 133, 146, 169, 172, 242
Azimuthal, 197
Bandpass, 260
Basic broblast growth factor, 169
Bax, 226, 233, 237
Bcl-2, 99, 169, 237
Benzolamide, 121
Betaxolol (Betoptic), 155
313
314
Biomicroscopy, 51, 68
Bipolar cell, 207, 247, 253, 269, 277
Bisbenziamide, 234
Bistratied cell, 207, 275, 277
Blepharostat, 255, 257
BODIPY, 235
Brain-derived neurotrophic factor
(BDNF), 3, 120, 156, 169, 217,
218, 219, 220
Bregma, 16, 19
Brimonidine (Alphagan), 50, 51, 62,
162, 299
Brn3a, 192
Brn3b, 192
Bullous, 113, 119, 122, 123
Buprenorphine, 68, 113
CA1, 147
Calbindin, 118, 192
Cannula, 38, 41, 42, 45, 114, 115, 144
Canthi, 255
Canthotomy, 36, 48
Capsid, 175
Carbocyanine, 133, 208
Carbonic anhydrase, 118, 121, 191
Carotenoids, 102
Caspase, 169, 226, 232
Cautery, 25, 68
CCD, 215
CD34, 179
Cecum, 161
Cell culture, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
173, 178, 234, 242, 244, 245, 246
Cell density, 4, 7, 190, 195, 196, 197,
200, 202, 219
Central retinal artery, 131
c-fos, 87, 99, 100
cGMP, 168
Chloromethyl-tetramethylrosamine
(CMTMR), 235, 236, 237, 238
Chondroitin, 67
Choriocapillaris, 110, 116
Choroid, 62, 66, 110, 111, 123, 131,
135, 136, 148, 154, 162, 210
Chromogen, 233
Chymotrypsin, 72
Index
Ciliary neurotrophic factor (CNTF), 3,
120, 169, 250
Cilioretinal, 299
c-jun, 100
Clinical trials, 99, 273, 274, 276, 279,
280, 281, 283, 284, 291, 292,
293, 294, 295, 296, 297, 298,
299, 303, 304, 305, 307, 309,
310
Cobalt chloride, 227, 229, 230
Co-culture, 4
Collaborative Initial Glaucoma Treatment Study (CIGTS), 281, 282,
283
Collagenase, 210
Cone photoreceptors, 62, 116, 117, 118,
121, 124, 190, 191, 192, 195,
199, 200, 251, 264, 265, 275
Confocal, 52, 69, 192, 193, 201, 214,
215, 228, 232, 238
Conjunctiva, 14, 26, 37, 257
Contamination, 8, 9, 154, 173, 177,
253, 261
Cornea, 5, 14, 27, 40, 42, 50, 51, 55,
60, 61, 114, 134, 136, 154, 155,
158, 250, 254, 255
Cosmid, 174
Cotransfection, 173, 176, 177, 179
Counterphase, 278, 279
Counterstain, 232
Coverslip, 134, 213, 229, 231, 232, 233,
234, 235, 237
Coxsackievirus, 172
Cribriform, 71
Cross-action forceps, 15
Cryosection, 191, 230
CsCl, 176
Culture media, 2, 3, 245
Cy5, 237
Cyanoacrylate, 117, 157
Cyclodialysis, 57, 60
Cyclopentolate, 113
Cynomologus monkey, 47, 48, 57, 61,
66, 69, 70, 116
Cystoid macular edema, 110, 122, 303
Cytochrome c, 226
Index
Cytochrome oxidase, 66
Cytomegalovirus, 172, 178
Cytotoxicity, 13, 171, 174
DAPI, 191, 232, 234, 238, 242, 243,
246, 247
Dehydroascorbic acid, 102
Dehydrogenase, 96, 230
Dendritic, 63, 207, 208, 209, 212, 214,
215, 216, 277, 278
Depo-Medrol, 68
Dexamethasone, 68, 69, 70, 130
Diaphorase, 66
DiAsp, 17, 19, 133, 139, 140, 142, 143, 144
Dichroic, 243, 244
Dihydroethidium (HEt), 243, 247
DiI, 208
Dimethyl sulfoxide (DMSO), 132, 202,
236, 244, 245
Dimethylformamide, 133
Dimethylthiourea (DMTU), 101, 102, 103
Disector, 199, 201
DNAse, 6
Docosahexaenoic acid, 3
Dorzolamide (Trusopt), 50, 51, 62
Dremel, 35
tool, 35
Dulbeccos modied Eagles media
(DMEM), 2, 3, 5
Dura, 17, 19, 68, 135, 137
dUTP, 229
Earclip, 255
Early Manifest Glaucoma Trial
(EMGT), 281, 282
Eclampsia, 111
Elastin, 67
Electrocardiogram, 250, 254
Electrode, 8, 19, 211, 212, 213, 251,
254, 255, 257, 267, 268
Electroretinogram (ERG), 54, 55, 95,
96, 97, 118, 119, 120, 145, 249,
250, 251, 252, 253, 254, 255,
257, 258, 259, 260, 261, 262,
263, 264, 265, 266, 267, 268,
269, 298
315
Embolization, 123
Encapsidation, 168, 171, 174, 176, 178
Endonuclease, 96, 227
Endophthalmitis, 156
Endothelin, 69
Enucleation, 154
Epiuorescence, 211, 212
Epiretinal, 122
Episclera, 24, 25, 26, 31, 32, 33, 34, 36,
39, 40, 45
episcleral vein, 24, 26, 31, 32, 33, 34,
36, 45
Episomal, 170, 172
Equiluminant, 278
Ethidium, 191, 245
Euthermic, 93, 94
Excitotoxicity, 3, 56, 61, 235, 279, 296,
299
Explant, 2, 5, 6, 9
cultures, 5, 6
Extracapsular, 114
Extracellular matrix, 1, 4, 5, 6, 67
Exudative, 110, 111, 199, 200
Fetal Bovine Serum (FBS), 5, 6
Fibroblast growth factor (FGF), 3, 120, 169
Fibronectin, 4
FK506, 161
Flavoprotein, 226
Fluorescein, 60, 117, 122, 139, 162
FluoroGold, 16, 17, 28, 132, 133, 139,
140, 141, 142, 144
Fluorophore, 16, 28, 132, 208, 237
Fluorophotometry, 57, 60
Flupirtine, 155, 156
Food and Drug Administration (FDA),
294, 303, 305, 306, 307, 308,
309, 310
Forceps, 4, 5, 14, 15, 25, 34, 36, 37, 38,
209, 210, 211
Forskolin, 62
Fovea, 52, 56, 63, 64, 86, 110, 121,
124, 190, 195, 196, 197, 202,
207, 211, 213, 214, 268, 274
Fundus, 51, 52, 53, 68, 113, 114, 134,
135, 136, 138, 144, 146, 155, 198
316
Funduscopy, 68
Fyrite, 9
Ganzfeld, 54, 95, 255, 257
GAPDH, 230, 231, 232, 237, 238
Gavage, 102, 159, 160, 161
Gavestinel, 296
GDx, 52, 298
GelMount, 229, 231, 232, 233, 234
Gene transfer, 28, 157, 167, 168, 169,
171, 173, 174, 175, 176, 178,
180
Glaucoma, 1, 23, 24, 28, 31, 32, 44, 45,
47, 48, 49, 50, 52, 53, 54, 55,
56, 57, 60, 61, 62, 63, 65, 66,
67, 69, 72, 147, 148, 153, 168,
169, 209, 214, 215, 273, 274,
275, 277, 278, 280, 281, 282,
283, 284, 294, 297, 298
hemield test, 282
Glia, 6, 32, 51, 66, 67, 111, 119, 169,
172, 175, 253
brillary acidic protein (GFAP), 67, 119
-derived neurotrophic factor (GDNF),
120
Gliosis, 43, 120
Gliotic, 123
Glucocorticoid, 69, 70
Glutamate, 2, 3, 61, 119, 120, 235
Glutamine synthetase, 97, 119
Glyceraldehyde, 230
Glycine, 229, 231, 235, 296
Glycoprotein, 3
Golgi, 208
Gonioftal, 134
Goniolens, 48
Gonioscopic, 61
Green uorescent protein (GFP), 178,
179
Growth factors, 1, 3, 6, 100, 155
Hanks balanced salt solution (HBSS), 5,
6, 8, 237, 245
Heidelberg retinal tomograph (HRT),
52, 298
Hemacytometer, 6
Index
Hematopoietic, 178, 179
Hemield, 282, 283
Hemiretinal, 268
Henle, 202
Heparan, 67, 176
HEPES, 245
Herpesvirus, 177
HEt, 242, 243, 244, 245, 247
Heterologous, 180
Heterozygote, 99
High-Pass Resolution Perimetry
(HPRP), 277, 278, 279, 280
Histochemistry, 118, 191
Hoechst dye, 8, 234
Homodimer, 191
Hyaluronic acid, 115, 117
Hyaluronidase, 112
Hydrogen peroxide, 228
Hydroxypropylmethylcellulose, 134
Hyperoxia, 120, 121
Hyperthermia, 91, 92, 93, 94
Hypertonic saline, 31, 32, 33, 34, 45
Hypertrophic, 111, 119
Hypotony, 33, 39, 40, 44, 49
Icosahedral, 171
Imaging software, 246, 247
MetaFluor, 244, 246
Metamorph, 193
Immune response, 23, 28, 171, 172,
173, 174, 176, 178
Immunoblotting, 232
Immunocytochemistry, 118, 132, 227,
230, 234, 236
Immunodecient, 172, 179
Immunohistochemistry, 7, 119
Immunoreactivity, 65, 66
Immunoreagents, 202
Immunostaining, 66
Immunosuppression, 170, 173
In situ end labeling, 227, 228, 230, 231,
234, 235
Incubator settings, 5, 6, 7, 9, 236, 243,
244, 245
Infarction, 147
Inner plexiform layer, 54, 132, 146
Index
Innervation, 68
Insertional, 175, 178
Institutional review board (IRB), 292,
295, 303, 305, 306, 307, 308,
310
Integrin, 172
Interlaminar, 277
International Society for Clinical
Electrophysiology of Vision
(ISCEV), 254, 258, 260, 261,
264, 265, 266, 268
Interneurons, 192
Interphotoreceptor, 118
Intracameral, 69, 71
Intraconal, 68
Intraocular pressure (IOP), 23, 24, 26,
27, 28, 31, 32, 33, 36, 39, 40,
41, 42, 43, 44, 45, 48, 49, 50,
51, 52, 53, 54, 57, 60, 62, 63,
65, 66, 67, 68, 69, 70, 71, 72,
114, 122, 131, 133, 134, 135,
144, 145, 146, 147, 153, 169,
273, 281, 294, 297, 298, 299
Intraorbital, 14, 17, 131, 133, 140, 143
Intraperitoneal, 24, 130, 158, 161, 162
Intraretinal, 209, 212, 214
Intravenous, 123, 158, 162, 163
Intravitreal, 56, 112, 120, 121, 153, 155,
156, 157, 173, 175, 178, 250
Inverted terminal repeat, 174, 176
Iodixanol, 176
Iodoantipyrine, 61
Ionophore, 246
IOP measurement, 28, 40, 41, 70
Iridocyclitis, 49, 72
Iridolenticular, 51
Ischemic Optic Neuropathy Decompression Trial (IONDT), 296, 297
ISEL, 227, 228, 230, 231, 234, 235
Isoeccentricity, 198
Isouorane, 68
Kainic acid, 7
Ketamine, 24, 48, 50, 51, 52, 113, 130,
136, 254
Koniocellular, 275
317
Lamina cribrosa, 50, 51, 64, 67
Laminin, 4, 5
Laser, 32, 48, 49, 50, 51, 52, 57, 60,
61, 62, 63, 69, 228, 232, 234,
237, 298
Latanoprost (Xalatan), 51, 62
Lateral geniculate nucleus (LGN), 4, 7,
14, 63, 64, 65, 66, 131, 275,
277, 278
Lectin, 4, 192
Lens, 5, 38, 40, 42, 48, 51, 72, 114,
116, 136, 146, 154, 156, 246,
254, 255, 257, 258, 268, 279
Lentivirus, 168, 170, 177
Light,
dark cycle, 39
damage, 9, 85, 86, 87, 88, 89, 91, 92,
93, 94, 95, 96, 97, 98, 99, 100,
101, 102, 103
exposure, 9, 85, 86, 87, 88, 89, 90,
91, 92, 93, 94, 95, 96, 97, 98,
99, 100, 101, 102, 123
Limbus, 19, 24, 33, 34, 35, 36, 37, 38,
40, 113, 114, 134, 135, 136,
139, 156
Linoleic, 3, 5
Lipophilic, 17, 19, 102, 133
Long posterior ciliary artery, 33, 36
Long-terminal repeats, 177, 179, 180
Macaca mulatta, 48
Macula, 58, 59, 64, 109, 110, 113, 116,
117, 118, 123, 168, 169, 190,
193, 196, 197, 202, 266, 267,
269, 274, 297, 298
edema, 110
Maculopathy, 191, 199, 200
Magnocellular (M-cell), 63, 65, 275,
277, 278
Mannitol, 51, 52
Media supplements, 3, 8
Memantine, 61, 279, 296, 299
Metabotropic, 121
receptors, 121
Metamorphopsia, 110
Methohexital sodium, 48, 52
318
Methylene blue, 208
Methylprednisolone, 68
Microelectrode, 19, 210
Microgel, 69, 71
Microglia, 140, 142, 145, 175
Microinstruments, 35
Microneedle, 34, 35, 36, 44
Microscissors, 24
Microspheres, 69, 71, 123
Microsyringe, 136
Microvessel, 26
Minipumps, 69, 173
Mitochondria, 95, 225, 226, 227, 234,
235, 236, 237, 241, 242, 243,
244, 245, 246, 247
permeability transition pore, 236,
241
Mitomycin, 62
MitoTracker, 235, 236, 237
Monolayer cultures, 5
Morphometry, 95, 96, 97, 192, 201
Motoneurons, 132, 133
Muller cell, 111, 119, 120, 169, 170,
178, 192, 253
Multifocal ERG, 56, 57, 58, 62, 69,
118, 249, 266, 268, 269
Multifocal VEP, 57
Multipolar, 7
Mycoplasma, 8
Mydriatic, 52, 87, 88, 94
Myelin, 43, 116
Myocilin, 69
NADPH, 66
ND4, 212
Neovascularization, 26, 51, 110
Nernstian, 236
Nerve ber analyzer, 298
Nerve growth factor, 3, 230, 235
Neurite, 2, 6, 7
Neurobasal, 3, 244
Neurobiotin, 212, 213, 215
Neurodegeneration, 47, 116, 168, 176
Neurobrillar, 208
Neurolament, 44
Neurogenetic, 192
Index
Neuroprotection, 1, 13, 16, 23, 28, 29,
31, 32, 44, 45, 61, 86, 100, 101,
103, 116, 120, 121, 123, 129,
130, 132, 136, 145, 146, 147,
148, 153, 155, 156, 157, 158,
159, 161, 162, 167, 168, 169,
170, 171, 172, 173, 175, 178,
179, 180, 189, 192, 193, 205,
206, 208, 215, 216, 217, 219,
220, 221, 273, 274, 276, 280,
284, 291, 294, 296, 297, 298,
299, 303
Neurotoxic, 249
Neurotracer, 16, 17
Neurotrophic factors, 100, 101, 129,
169, 173, 235
Neurotrophin, 3, 28, 100, 101, 168, 169,
172, 173
neurotrophin-3 (NT-3), 3
neurotrophin-4 (NT-4), 3
Nipradilol, 154, 155
Nitrergic, 66
Nitric oxide synthase (NOS), 159
NMDA, 56, 61, 296, 299
Nomarski, 193
Nonarteritic anterior ischemic optic neuropathy (NAION), 292, 296, 297
Nonresponder, 69, 70
Normal tension glaucoma (NTG), 31, 281
Normotensive, 58, 59
Normoxia, 120
Ocular Hypertension Treatment Study
(OHTS), 279, 281, 283
Oculoplastic, 67
Oligodendrocyte, 175
Oligonucleosome, 227
Oligonucleotides, 230
Ophthalmic vessels ligature, 133, 134,
135, 136, 137, 138, 144, 146,
148
Ophthalmoscopy, 52, 67, 68, 69
Opponency, 277
Opsin, 7, 62, 118
Optic nerve crush, 15, 16, 161, 218,
220
Index
Optic nerve head, 14, 25, 32, 51, 56,
61, 64, 67, 69, 131, 137, 155
Optic nerve transection, 28, 53, 55, 57,
67, 68, 69, 133, 140
Optical coherence tomograph (OCT),
52, 298
Ora serrata, 209
Orbitotomy, 67, 68
Oscillatory potentials (OPs), 54, 56,
251, 252, 253, 254, 258, 259,
260, 261, 263, 265, 266, 269
Outer nuclear layer (ONL), 95, 96, 119,
148, 191, 200, 201
Outer plexiform layer (OPL), 54, 122, 146
Outow, 23, 24, 32, 33, 39, 40, 44, 45,
49, 57, 60, 69, 112, 113
Overexpress, 99
Oversampling, 195
Palpebral, 48
Paracrystalline, 201
Paralm, 229, 231, 232, 235
Paraformaldehyde, 18, 19, 139, 158,
213, 234, 237
Parafovea, 63, 190, 198
Parvalbumin, 66
Parvocellular (P-cell), 63, 65, 66, 275,
277, 278
Parvoviridae, 174
Pattern electroretinogram (PERG), 266,
267
PC12, 230
pClamp6, 217
Penumbra, 147
Pericyte, 111
Perifoveal, 59
Perimacular, 58, 59, 66
Perimetry, 52, 56, 57, 197, 274, 276,
277, 278, 280, 281, 283, 298
frequency-doubling technology
(FDT), 278, 279, 280, 283
Perineural, 69
Periocular, 155
Peripapillary, 52, 53
Perivascular, 37
Permeability transition pore, 236, 241
319
Peroxidase, 233
PGF2, 62
PGF2-1-isopropylester, 50, 51
Phagocytosis, 63, 96, 99, 140, 225
Phenobarbital, 154
Phenylephrine, 52, 113
Phosphodiesterase, 168
Photoactivation, 123
Photochemically, 123
Photocoagulator, 123
Photoisomerized, 93
Photopic, 54, 56, 95, 119, 120, 251,
252, 253, 254, 258, 259, 260,
261, 263, 266
Photopigment, 261
Photoreceptor cultures, 7
Photostasis, 87
Photothrombosis, 123
Phototoxicity, 9, 85, 86, 87, 88, 89, 91,
92, 93, 94, 95, 96, 97, 98, 99,
100, 101, 102, 103
Pia mater, 132
Pigmentation, 87, 88
Pilocarpine, 62, 294
Planimetric, 190
Pneumotonography, 57
Pneumotonometry, 28, 51
Polarimeter, 52
Polyacrylamide, 69
poly-l-lysine, 3, 248
poly-l-ornithine, 3, 5
Polymerase chain reaction, 173
Potentiometric dye, 235, 236
Prelabel, 28, 208
Primary visual cortex, 19
Procaspase, 226
Proparacaine, 27
Propargylamines, 230
Proteinase, 228, 230, 235
Proteoglycan, 4, 67, 157, 176
Punctate, 140
Pyranine, 212
Pyruvate, 2
Radiolabeling, 154, 177
rds mouse, 168, 169, 250
320
Reactive oxygen species (ROS), 95, 96,
99, 236, 241
Reaggregation, 7
cultures, 7
Recti muscle, 25, 26, 68, 132, 133
Retina, 2, 4, 5, 6, 14, 16, 18, 19, 28,
54, 56, 58, 61, 62, 63, 64, 66,
68, 85, 86, 87, 95, 96, 97, 98,
99, 100, 101, 102, 110, 111,
112, 113, 114, 115, 116, 117,
118, 119, 120, 121, 122, 123,
129, 130, 131, 132, 133, 135,
136, 138, 139, 141, 142, 143,
144, 145, 146, 147, 148, 153,
154, 155, 156, 157, 162, 167,
168, 169,172, 173, 175, 176,
178, 180, 189, 190, 191, 192,
193, 194, 195, 196, 197, 200,
201, 202, 205, 206, 207, 208,
209, 210, 211, 212, 213, 214,
215, 216, 217, 218, 219, 220,
221, 228, 231, 232, 234, 242,
249, 250, 251, 253, 255, 257,
258, 260, 265, 266, 267, 269,
274, 275, 277, 278, 283, 299
blood ow, 61, 62, 69, 131, 133, 134,
135, 136, 138, 146, 148, 155
detachment, 109, 110, 111, 112, 113,
114, 115, 116, 117, 118, 119,
120, 121, 122, 123, 124, 156,
169
ganglion cell (RGC), 4, 7, 8, 13, 14,
15, 16, 17, 18, 19, 23, 28, 54,
57, 59, 61, 63, 129, 130, 131,
132, 133, 134, 136, 139, 140,
141, 142, 143, 144, 145, 146,
147, 148, 161, 162, 169, 170,
172, 175, 192, 205, 208, 209,
214, 217, 220, 231, 241, 242,
243, 244, 245, 246, 247, 254,
266, 275, 276, 299
cultures, 7
ischemia, 69, 110, 129, 133, 134,
135, 136, 138, 140, 144, 146,
147, 148, 235
Index
pigment epithelium (RPE), 5, 94, 95,
96, 99, 102, 111, 113, 116, 117,
118, 119, 121, 122, 123, 170,
172, 175, 178, 179, 199, 200
reattachment, 110, 114, 116, 117,
118, 121, 123
slice cultures, 6, 7
whole-mount, 18, 141, 142, 189, 191,
192, 193, 196, 201, 202, 205,
217, 220
Retinaldehyde, 119
Retinitis pigmentosa, 100, 168, 250, 297, 298
Retinocollicular, 130
Retinogeniculate, 64
Retinopexy, 110
Retinorecipient, 132, 192
Retinotomy, 117
Retinotopic, 56
Retrobulbar, 15, 61, 113
Retrograde labeling, 16, 130, 132, 133,
139, 140, 192, 208, 209, 219, 242
dyes,
DAPI, 191, 232, 234, 238, 242,
243, 246, 247
DiAsp, 17, 19, 133, 139, 140, 142,
143, 144
DiI, 208
FluoroGold, 16, 17, 28, 132, 141, 142
Retrolaminar, 66
Rhegmatogenous, 110, 111, 112, 117,
118, 121, 122, 123
Rhodamine, 236, 237
Rhodopsin, 62, 87, 88, 89, 92, 93, 94,
96, 97, 99, 100, 102, 118, 119,
168, 169, 175, 261
Ribozyme, 168
RNAse, 228, 231, 232, 235
Rod photoreceptors, 8, 117, 118, 121,
190, 191, 198, 199, 200, 251,
255, 277
Rose bengal, 122, 123
Rostral, 16
Royal College of Surgeons (RCS) rat,
87, 88, 96, 99, 169
RPMI, 3
Index
Sampling, 61, 193, 194, 195, 196, 199,
200, 213, 219
Schlemms canal, 23, 27, 33, 34, 35,
38, 63, 71
SCID, 179
Sclera, 5, 25, 87, 113, 131, 136, 155,
156, 157, 158, 210, 211, 213,
217
Sclerosing, 34
Scotoma, 284
Scotopic, 54, 55, 95, 119, 120, 251,
252, 258, 260, 261, 262, 263,
264, 266
Secondary degeneration, 13, 283
Selenite, 3
Sialic acid, 3
Sigmoidal, 263, 264
Sildal, 250
StatPac, 281, 282
Stereology, 193
Stereotactic, 16, 19
Subconjunctival, 26, 62, 157, 158
Subretinal, 110, 111, 112, 113, 114,
115, 116, 117, 121, 122, 172,
173, 175, 178, 179
Superior colliculi, 14, 16, 19, 131, 132,
141, 142, 172, 208, 242
Superoxide, 241, 242, 243, 244
Swedish Interactive Thresholding Algorithm (SITA), 280
Synaptogenesis, 1
Synechiae, 51, 63, 72
Syneresis, 117
Tectum, 14
Tenon, 37, 155, 157, 158
Terminal deoxynucleotidyl transferase
(TdT), 227, 228, 229, 230, 235
Tetrodotoxin, 56
Thermistor, 244
Thiols, 236
Timolol (Timoptic), 50, 51, 62, 155, 299
TMRM, 236
Tonography, 57
Tonometry, 40, 41, 42, 43, 50, 51
321
Tonopen, 27, 39, 40, 41, 42, 43, 51
TopSS, 52
Trabecular, 32, 33, 34, 38, 39, 48, 62, 63
Trabeculectomy, 62
Trans-corneal, 153, 155, 157
Transfection, 174, 177, 179
Transferase, 227, 228
Transferrin, 3
Transgenic, 86, 89, 99, 156, 157, 158,
167, 170, 171, 172, 173, 174,
175, 176, 177, 178, 179, 180
Trans-scleral, 33, 153, 157
TRITC, 237
Trituration, 6
Trophic factor, 129, 235
Trophic withdrawal, 235
Tropicamide, 52, 113, 134, 268
Trusopt (dorzolamide), 50, 51
Trypsin, 6, 8, 9
TUNEL, 118, 120, 227
Tween, 231, 232, 237
Ultrastructure, 43, 168
Ultrathin, 192, 201
Undersampling, 195
UniBlitz shutter, 244
Uveoscleral, 49, 57, 60
Valinomycin, 246
Vanadate, 62
Vannas scissors, 34
Vasoconstriction, 155
Vasodilation, 155
Vectastain, 213
Venous plexus, 24, 25, 27, 33
VERIS, 268, 274
Vervet, 48
vesicular stomatitis virus (VSV), 179
Vibratome, 7
Virion, 177, 179
Viscoelastic, 61, 69
Visual evoked potential (VEP), 18, 57, 298
Vitrectomy, 110, 111, 114, 117, 122
Vitreoretinal, 117, 122
Vitreoretinopathy, 110, 111, 119
322
Vitreous, 5,
112,
122,
156,
218
Vmax, 263,
Index
18, 19, 53, 61, 110, 111,
113, 114, 116, 117, 119,
131, 136, 139, 154, 155,
172, 173, 200, 209, 210,
Xalatan (latanoprost), 51, 62

XIAP, 169
Xylazine, 24, 113, 130, 136, 254
Xylene, 202, 228, 233
Xylocaine, 113
264
Wistar, 23, 24, 26, 28
YOYO-1, 228, 230, 234, 235, 236, 237,

238
About the Editors
LEONARD A. LEVIN is Associate Professor of Ophthalmology and Visual Sciences, Neurology, and Neurological Surgery at University of Wisconsin Medical
SchoolMadison, where he studies mechanisms of retinal ganglion cell death.
He primarily focuses on the role axonal damage plays in inducing loss of retinal
ganglion cells, an area common to both neuro-ophthalmology and glaucoma, and
is the principal investigator on a National Eye Institutesupported grant to study
this topic. Dr. Levin is a Research to Prevent Blindness Dolly Green Scholar and
an associate editor of the Archives of Ophthalmology. He received the A.B. degree (1980) in applied mathematics, the Ph.D. degree (1988) in neurobiology,
and the M.D. degree (1988) from Harvard University, Cambridge, Massachusetts.
He did a residency in ophthalmology and a fellowship in neuro-ophthalmology
at the Massachusetts Eye and Ear Inrmary.
ADRIANA DI POLO is Assistant Professor of Pathology and Cell Biology at
the Universite de Montreal, Quebec, Canada. A major focus of her laboratory is
to develop gene therapy strategies to promote cell survival and regeneration of
adult retinal ganglion cells, the neuronal population that dies in glaucoma. She
received the B.Sc. degree (1989) from the Universidad Central de Venezuela,
Caracas, and the Ph.D. degree (1995) from the University of California School
of Medicine, Los Angeles.

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We sincerely thank all the contributorsleading scientists in their elds

II. In Vivo Models of Optic Nerve Damage

3. Intraocular Pressure Elevation: Vein Cauterization

4. Intraocular Pressure Elevation: Injecting Hypertonic Saline

III. In Vivo Models of Retinal Damage

Light-Induced Retinal Degeneration

IV. Neuroprotective Strategies

Recombinant Viral Vectors

V. Analysis of Neuronal Survival and Function

Quantication of Retinal Cells

Ex Vivo and Whole-Mount Retinal Preparations

Detection of Single-Cell Apoptosis

Imaging of Retinal Ganglion Cells

Evaluation of Retinal Function: Electroretinography

VI. Clinical Trials

17. Clinical Trials in Neuroprotection

18. Regulatory Issues in Clinical Trials

Anthony C. Arnold, M.D. Neuro-Ophthalmology Division, Department of

Mount Sinai School of Medicine, New York,

Wright State University, Dayton, Ohio, U.S.A.

Adriana Di Polo, Ph.D. Department of Pathology and Cell Biology, University

Pierre Lachapelle, Ph.D. Department of Ophthalmology, McGill University,

Department of Ophthalmology, Universidad

Leonard A. Levin, M.D., Ph.D. Departments of Ophthalmology and Visual

Department of Ophthalmology, Universidad

Department of Ophthalmology, Universi-

Sansar C. Sharma, Ph.D. Department of Ophthalmology, New York Medical

Kenneth R. Sloan, Ph.D. Department of Computer and Information Sciences,

Mount Sinai School of Medicine, New York, New York,

Department of Ophthalmology, Universidad

Mara P. Villegas-Perez, M.D., Ph.D.

Department of Ophthalmology, Uni-

The Weizmann Institute of Science, Rehovot, Israel

II. DESIGN CONSIDERATIONS

Culture of Retinal Neurons

III. SELECTED METHODS

Isolation of Chick Retina

Culture of Retinal Neurons

rinses (changes) in CMF-HBSS, a solution containing 0.25% trypsin and 1.85

IV. ALTERNATIVE CULTURE FORMATS

Retinal Explant Cultures

Retinal Slice Cultures

Culture of Retinal Neurons

Puried Retinal Ganglion Cell Cultures

1 and the absence of expression of an antigen recognized by anti-macrophage

Abnormal performance of solutions used in tissue culture procedures can reect

Inactivation of Tissue Culture Reagents

Culture of Retinal Neurons

Examination of cultures using an inverted microscope is often the best way to

Culture of Retinal Neurons

pigment epithelial cells during mechanical and pharmacologic stimulation. Invest

Optic neuropathies are chronic neurodegenerative diseases of the optic nerve

Schwartz and Yoles

Figure 1 Anatomy of the rat visual system.

II. ANATOMY OF THE RAT VISUAL SYSTEM

III. PARTIAL CRUSH INJURY OF THE RAT OPTIC

Surgical Exposure of the Optic Nerve

Crush Injury of the Optic Nerve

Figure 2 Cross-action forceps.

IV. SEVERE CRUSH INJURY OF THE MOUSE

Schwartz and Yoles

RETROGRADE LABELING OF RETINAL

Labeling of All Retinal Ganglion Cells Prior