Experimental Eye Research 83 (2006) 758e770

www.elsevier.com/locate/yexer

A rat model of glaucoma induced by episcleral vein ligation

Saiyuu Yu, Teruyo Tanabe*, Nagahisa Yoshimura
Department of Ophthalmology and Visual Sciences, Graduate School of Medicine, Kyoto University, 54 Shogoin Kawahara-cho,
Sakyo-Ku, Kyoto 606-8507, Japan
Received 10 October 2005; accepted in revised form 21 March 2006
Available online 16 May 2006

Abstract

To establish a reliable animal model of glaucoma, we examined if episcleral vein ligation in rat eyes can induce intraocular pressure (IOP) el-
evation and concomitant characteristic morphological features of glaucoma. IOP elevation was detected on the next day (30.1
4.4 mmHg:
operated eyes; 21.0
1.8 mmHg: control eyes) and persisted at least 7 months after the procedure (24.5
2.3 mmHg: operated eyes;
19.7
1.9 mmHg: control eyes). These results suggest that episcleral vein ligation can induce very mild IOP elevation immediately after the
operation, which can last over several months. Furthermore, it appears there was little variability in the patterns of IOP elevation among the
individual eyes treated with episcleral vein ligation. Morphological changes were detected selectively in the retinal ganglion cell (RGC) layer
and optic disc excavation was evident in the late stage of chronic IOP elevation. RGCs were selectively lost by apoptotic death. The number of
RGCs was reduced by 18% at 12 weeks and eventually by 35% at 8 months postoperatively. Müller cells downregulated the expression of
p27Kip1 and appeared to be partially in a reactive state even at the advanced stages of glaucoma. The expression of basic fibroblast growth
factor and ciliary neurotrophic factor, which are neurotrophic factors implicated in the control of cell survivals and neuroprotection, significantly
declined at the advanced stages. Taken altogether, these observations indicate that the episcleral vein ligation model based on the simple ligation
procedure reproducibly provides a reliable glaucoma model and contributes to give insights into the underlying molecular and cellular bases of
human glaucoma and to devise the new medication upon the disease.
Ó 2006 Elsevier Ltd. All rights reserved.

Keywords: glaucoma; rat model; episcleral vein ligation; retinal ganglion cell; apoptosis; trophic factor; Müller cell

1. Introduction
Glaucoma is an optic neuropathy characterized by a slow pro-
gressive loss of retinal ganglion cells (RGCs) and excavation of
the optic disc (Quigley and Green, 1979; Quigley et al., 1988;
Glovinsky et al., 1991). Elevation of intraocular pressure
(IOP) has been implicated as the most critical risk factor in
the generation of glaucomatous optic neuropathy. Several ani-
mal models have been devised to elucidate the molecular and
cellular bases of how IOP elevation triggers RGC death and
how RGC death progresses to glaucoma, and also to explore
treatments to inhibit the death of RGCs. A model of glaucoma
using monkey is beneficial in that the gross anatomical bases
* Corresponding author. Fax: þ81 75 752 0933.
E-mail address: tanabet@kuhp.kyoto-u.ac.jp (T. Tanabe).
of retina and optic disc are the most similar to human; however,
the costs are higher in preparing the samples compared to other
species (Pederson and Gaasterland, 1984; Glovinsky et al.,
1991; Quigley et al., 1995). In contrast, models using rodents
are advantageous in that large numbers of samples can be pre-
pared and handled at a time. In addition, spontaneous or genet-
ically manipulated mutant mice, which exhibit elevation of IOP,
are available (John et al., 1998; Grozdanic et al., 2003b;
Mabuchi et al., 2004). However, these mice only manifest the
glaucoma phenotype after a long period of time and current
IOP measurement techniques in mice are invasive. On the other
hand, in rats, operational procedures are applicable and IOP
measurement are non-invasive; thus, a wide variety of rat glau-
coma models, using hypertonic saline injection, cauterizing
episcleral veins or laser photocoagulation, have been devised
and analyzed (Shareef et al., 1995; Morrison et al., 1997;
Ueda et al., 1998; Levkovitch-Verbin et al., 2002). Every model

0014-4835/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.exer.2006.03.014
 S. Yu et al. / Experimental Eye Research 83 (2006) 758e770 759

shows a significant IOP elevation, glaucomatous changes in the
retina and is valuable as a glaucoma model; however, some
models require special devices or techniques to induce repro-
ducible IOP elevation.
To establish a reliable rat model that exhibits reproducible
and consistent IOP elevation over a long period of time with
simple procedure, we performed episcleral vein ligation and ob-
served whether IOP was elevated consistently over a long pe-
riod, and if any morphological changes, including RGC loss,
resembling glaucomatous phenotypes in human, were induced.
2. Materials and methods
2.1. Animals
All the animal experiments were performed in compliance
with the ARVO Statement for the Use of Animals in Ophthalmic
and Vision Research. Adult female Wister rats, 9 weeks of age,
were used in this study. Rats were maintained in an environmen-
tally controlled room with 14-h light/10-h dark daily cycles.
2.2. Rat model of chronic ocular
hypertension (glaucoma)
Rats were lightly anesthetized by intraperitoneal injection of
a mixture of ketamine (25 mg/kg) and xylazine (10 mg/kg); the
eyes were further anaesthetized locally with a topical applica-
tion of 0.5% proparacaine (Santen) eyedrop. Small incisions
into the conjunctiva and Tenon’s capsule covering the episcleral
veins in each quadrant of the right eye were made and the vein
was exposed. Ligation was applied to three trunks of the epis-
cleral veins and a branch of the other episcleral veins in the right
eye (Fig. 1) using 10-0 nylon (Alcon surgical). On the left eye,
sham surgery was performed in the same manner except for li-
gating episcleral veins. Antibiotic ointment was applied topi-
cally after each procedure. When the mean value of IOP
measured in the operated eyes was found to be less than
25 mmHg at 2 weeks or at 4 weeks after the initial ligation,
we further ligated newly formed collateral veins through which
blood flow escapes and thus subdivided all the treated samples
into three groups [Group 1, single treatment (n ¼ 29); Group 2,
retreatment at 2 weeks (n ¼ 18); Group 3, retreatment at
4 weeks (n ¼ 24)] to analyze the level of IOP.
2.3. Intraocular pressure monitoring
IOP was measured using a digital tonometer (Tonopen XL,
Mentor; Norwell, MA, USA) under general and local anesthe-
sia as described above. General anesthesia is known to affect
the level of IOP (Krupin et al., 1980; Jia et al., 2000). There-
fore, to circumvent the potential effect of general anesthesia,
we conducted IOP measurements as soon as rats were lightly
sedated by anesthesia. The automatic average values of accept-
able several consecutive measurements, with a coefficient of
variation less than 5%, was recorded three times and averaged.
Furthermore, we also evaluated the Tonopen XL readings in
relation to the transducer pressure according to the method
previously described (Mermoud et al., 1994). A regression
line calculated was y ¼ 2.8533 þ 0.8232x (r2 ¼ 0.959). The
values used in this study are actual readings by Tonopen and
no correction with a pressure transducer was performed. We
measured IOP of both eyes on the next day, day 3, weekly
for the first 3 months and monthly afterwards. All measure-
ments were started at 10:00 h during the follow-up period
and were basically conducted by one examiner. Another exam-
iner selected samples randomly and measured IOP in a blind
fashion. The difference in IOP values between two exam-
iners/the average IOP of two examiners were 3.5
2.1%.
2.4. RGC labeling and counting
Retrograde labeling and counting of RGCs was performed
according to the procedure described previously (Vidal-Sanz

Fig. 1. Anatomical bases of the operation of episcleral vein ligation in rat eyes. Topographical arrangement of episcleral/orbital vein (thick red line), limbal vein
(thin red line surrounding cornea), cornea, and rectus muscles (double thin black lines) is indicated in scheme on the left. The site of ligation is indicated by double
thick black lines on the episcleral vein. Picture on the right shows an example of operated eyes in which an episcleral/orbital vein was ligated. The central side of
the ligated vein became congested. This congestion is a criterion showing if the vein is ligated sufficiently to obstruct the blood flow. SR, superior rectus muscle;
SO, superior oblique muscle; MR, medial rectus muscle; LR, lateral rectus muscle; IR, inferior rectus muscle; IO, inferior oblique muscle.
760 S. Yu et al. / Experimental Eye Research 83 (2006) 758e770
et al., 1988). At various time points after episcleral vein liga-
tion (6, 12, 18, 24 and 32 weeks; n ¼ 5 per each time point),
the rats under deep anesthesia (mixture of ketamine
(37.5 mg/kg) and xylazine (15 mg/kg)), were immobilized ste-
reotaxically and were microinjected with 2.0 ml of a 5% solu-
tion of fluorescent tracer 1,10 -dioctadecyl-3,3,30 ,30 -tetramethyl
indocarbocyanine perchlorate ((DiI; Molecular Probes, Eu-
gene, OR, USA) in distilled water bilaterally into the superior
colliculus (SC). Seven days after DiI injections, eyes were har-
vested after perfusion with 4% PFA, and the isolated retinas
were flat-mounted, with vitreous side up, on a glass slide,
air-dried and mounted in Fluorosave (Calbiochem, La Jolla,
CA, USA). Peripapillary (PP: 1 mm from the optic disc), mid-
dle (M: 2 mm from the optic disc) and peripheral (PR: 3 mm
from the optic disc) retinal areas per each retinal quadrant, 12
areas in total, were subjected to RGC counting. Each rectangu-
lar area measured was 0.3257  0.3257 mm. The DiI-labeled
RGCs were analyzed by a confocal microscope (CLSM;
Carl Zeiss) and counted with a computer-assisted image-
processing unit and the ‘‘count-measure’’ operating software
(Image-Pro Plus software; Media Cybernetics, Silver Spring,
MD, USA). To exclude the microglia, which are fluores-
cence-labeled by phagocytosing dying labeled RGCs, DiI-
labeled cells smaller than 10 mm2 or with a spindle-shape
were not counted (Naskar et al., 2002). Data represents the
number of RGCs per square millimeter.
2.5. TUNEL (TdT-dUTP terminal nick-end
labeling) assay
After 6, 12 and 18 weeks of the initial episcleral vein liga-
tion, a TUNEL assay was performed on the treated and control
retinas that were retrogradely labeled by DiI 1 week before the
assay (n ¼ 5 per each time point). Eyes were fixed by perfu-
sion and DNA nick-end labeling was performed on flat-
mounted retina using a Mebstain apoptosis kit (MBL, Nagoya,
Japan), according to the manufacturer’s protocol. TUNEL-
positive DiI-labeled cells were analyzed by a confocal micro-
scope (CLSM; Carl Zeiss) and the number of apoptotic cells in
the entire retina was analyzed quantitatively with a computer-
assisted image-processing unit and ‘‘count-measure’’ operat-
ing software (Image-Pro Plus software; Media Cybernetics).
2.6. Histological and immunohistochemical analyses
Ten, 20 and 30 weeks postoperatively, the treated and control
eyes (n ¼ 3 per each time point, respectively) were paraffin-
embedded and histological changes were analyzed by light
microscopy, as described previously (Miyawaki et al., 2004).
Immunohistochemical analyses for retinal cell-specific markers
were performed on control and glaucoma retinas 32 weeks after
the treatment (n ¼ 3, respectively), while analyses for neurotro-
phic factors were performed on 10-, 20- and 30-week glaucoma
and control retinas (n ¼ 4 per each time point, respectively). Im-
munohistochemistry was performed on cryosections according
to the method described previously (Miyawaki et al., 2004).
The antibodies used were: mouse monoclonal antibody against
CD11B (OX42) (1:1000; Chemicon international, Temecula,
CA, USA), human polyclonal antibody against p75NGF
(1:1000; Promega, Madison, WI, USA; Cappel, Durham, NC,
USA), mouse monoclonal antibody against protein kinase C
(PKC) (1:100; Sigma, St. Louis, MO, USA), mouse monoclonal
antibody against opsin (1:8000; Sigma), rabbit polyclonal anti-
body against calbindin D-28K (1:200; Chemicon International),
mouse monoclonal antibody against glial fibrillary acidic pro-
tein (GFAP) (1:1000; Sigma), mouse antibody against
p27Kip1 (1:200; BD Transduction Laboratories, San Jose,
CA, USA), goat polyclonal antibody against Brn-3b (C-13)
(1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA),
sheep polyclonal antibody against fibroblast growth factor, basic
(bFGF) (1:1000; Chemicon), sheep polyclonal antibody against
brain derived neurotrophic factor (BDNF) (1:1000; Chemicon),
sheep polyclonal antibody against glial derived neurotrophic
factor (GDNF) (1:1000; Chemicon), mouse monoclonal anti-
body against ciliary neurotrophic factor (CNTF) (1:100; Chem-
icon). For secondary antibodies, Alexa Fluor 488-conjugated
anti-mouse, anti-rabbit, anti-sheep and anti-goat IgG (Molecu-
lar Probes) were used. TO-PRO-3 (Molecular Probes) was
used for the nucleic acid stains. Immunofluorescence was ana-
lyzed by confocal microscopy (Carl Zeiss).
2.7. Real-time RTePCR
Ten, 20 and 30 weeks postoperatively, rats were anesthetized
with ketamine/xylazine and perfused intracardially with medi-
cal grade physiological saline (n ¼ 5 per each time point). The
eyes were enucleated, anterior segments were removed, and
whole retinas were isolated. Total RNA was extracted from ret-
inas with the Qiagen RNeasy mini columns (Qiagen Inc.,
Valencia, CA) according to the manufacture’s protocol. The
concentration and purity of the extracted RNA were evaluated
by optical density measurements at 260 nm and 280 nm. Ran-
dom-primed cDNA was synthesized from 1 mg total RNA using
the First-Strand cDNA Synthesis kit (Amersham Pharmacia
Biotech, Uppsala, Sweden). Quantitative PCR was carried out
in 96-well optical reaction plates using real-time TaqManÒ
technology and the results were analyzed with a Model 7000
Sequence Detector System (Applied Biosystems, Foster City,
CA). The mixed real-time PCR solution, 25 ml in total, con-
tained 2 TaqMan Universal PCR Mastermix (Applied Biosys-
tems), 1 mM of each primer, 250 nM of TaqMan probes, and
25 ng of each cDNA. The thermal cycling conditions comprised
50  C for 2 min, 95  C for 10 min, and 45 cycles of 95  C for
15 s plus 60  C for 1 min, using the standard protocol of the
manufacturer. Quantitative normalization in each sample was
performed using the expression of rodent glyceraldehyde-
3-phosphate dehydrogenase (GAPDH) gene as an internal con-
trol. The primers and probes for bFGF were designed with
primer-express software (Applied Biosystems) as follows:
bFGF. Forward, 50 -GAACGCCTGGAGTCCAATAACTA-30 ,
reverse, 50 -CCCGTTTTGGGATCCGAGTTT-30 , detection probe,
50 -ACACTTACCGGTCACGGAAATACTCCAGTT-30 . The primers
and probes used for GFAP, p27kip1, GDNF, CNTF, BDNF
and GAPDH were from Applied Biosystems.
 S. Yu et al. / Experimental Eye Research 83 (2006) 758e770
2.8. Statistical analysis
Data are expressed as mean
S.D. (IOP recordings) and
mean
S.E.M. (number of RGC and real-time RTePCR).
Data between groups were compared with Student’s t-test or
repeated measures ANOVA with Dunnett’s multiple compari-
son tests. Statistical significance was declared for P < 0.05.
3. Results
3.1. Intraocular pressure was consistently elevated by
episcleral vein ligation
We first examined the temporal changes of IOP over several
months in the operated and sham-operated groups, and analyzed
the level, persistency and reproducibility of IOP elevation
(Fig. 2). All the values presented in this study are the actual read-
ings measured by Tonopen XL. The baseline IOP before treat-
ment was 19.9
1.9 (mean
S.D.) mmHg. On the next day
after the surgery, all treated eyes showed elevated IOP
(30.1
4.4 mmHg) compared to controls (21.0
2.0 mmHg).
On the third day after the surgery, all treated eyes showed
elevated IOP (27.7
3.6 mmHg) compared to controls
(20.9
1.9 mmHg). At 1 week after1 the surgery, all treated
eyes showed elevated IOP (27.8
1.3 mmHg) compared to con-
trols (20.2
1.5 mmHg). In 40.8% of glaucoma model speci-
mens we examined, the initially elevated IOP persisted for as
long as 7 months (Table 1). On the other hand, in 59.2% of spec-
imens, initially elevated IOP showed a significant decline
(25 mmHg) within 2e4 weeks, probably because of the newly
formed collateral vessels. Further ligation was performed in
those specimens at the second or fourth week after the first liga-
tion. These subsequent ligation procedures induced persistent
IOP elevation afterwards in all samples (Table 1). The level, per-
sistency and temporal changes of IOP elevation observed in the
‘‘double-treated’’ group were essentially similar to those ob-
served in the ‘‘single-treated’’ group during the chronic phase
of IOP elevation. At 7 months, the IOP measured was
24.7
2.2 mmHg in the operated eyes (single or double treated)
and 19.7
1.9 mmHg in the sham-operated eyes (P < 0.001).
Fig. 2. Temporal patterns of IOP elevation after the initial episcleral vein liga-
tion. Averages of IOP levels in the operated eyes (single and double treated) 1,
3, 6, 12, 24 and 32 weeks after treatment were 27.8, 26.6, 27.1, 27.1, 26.5 and
22.4 mmHg, respectively. IOP of the glaucoma eyes (black line) are signifi-
cantly higher than those of the sham-operated control eyes (dotted line) until
7 months after operation. By the end of 32 weeks, IOP of operated eyes de-
clines to control levels. Data expressed are mean
S.D. (mmHg).
761
3.2. Morphological changes of the retinas with
elevated IOP
We next examined if there were any morphological changes
in retinas in relation to the elevated IOP induced by episcleral
vein ligation (Fig. 3). The posterior eye segments (treated
eyes: n ¼ 6; control eyes: n ¼ 6), including retinas and optic
discs, were histologically analyzed by light microscopy at early
(10 weeks), middle (20 weeks) and late (30 weeks) stages of
chronic IOP elevation. In samples analyzed 10 weeks after
treatment, both the retina and optic disc of treated eyes showed
no apparent morphological difference from the control eyes
(Fig. 3AeD). At 20 weeks postoperatively, the reduction in
the number of RGCs was detected histologically, while the mor-
phology of optic disc was similar to the control (Fig. 3EeH). At
30 weeks after treatment, a marked decrease in RGC numbers
was observed (Fig. 3I,J). In addition, a significant excavation
was observed in the optic disc of the treated eye (Fig. 3K,L).
Apart from the morphological changes observed in the ganglion
cell layer (GCL), the outer and inner nuclear layers (ONL and
INL) did not exhibit any difference between the control and
treated eyes throughout the follow-up period (Fig. 3).
3.3. Spatial and temporal patterns of reduction
in the number of RGCs
To monitor the extent and the time course of the loss of RGC
in a more quantitative manner, RGCs were selectively labeled
by retrograde dye tracing from the SC using DiI and the number
of RGCs was analyzed by DiI fluorescence. We performed
quantitative analyses of the number of DiI-labeled RGCs that
are located in each quadrant of retinas of treated and control
eyes derived at 6, 12, 18, 24 and 32 weeks after the treatment
(n ¼ 5 per each case) (Fig. 4, Table 2). TUNEL assay was
also performed on whole-mount retinas to see if the loss of
RGC is mediated by apoptosis (Fig. 5). When the number of
RGCs at 6 weeks was analyzed, no significant change in the
number was observed between the treated and control retinas
(P ¼ 0.68). At 12 weeks, the number of RGCs in the treated ret-
inas gradually decreased, by 18% in total. It appeared there was
no regional difference for the RGC loss between quadrants of
the retina (Fig. 5A). In contrast, the extent of RGC loss is pref-
erentially high in the peripapillary retina (20.52
1.70% re-
duction) compared to the peripheral retina (13.12
2.83%
reduction) (Fig. 4, Table 2). At 24 weeks, nearly 35% reduction
in the RGC number was detected in the treated retinas com-
pared to the controls, and it appeared that there was no further
reduction in the number of RGC from 24 weeks onward. Our
results show that the number of TUNEL-positive DiI-labeled
RGCs detected in the treated retinas was significantly high
(32.8
9.5, 26.4
4.7 and 30.8
3.8 cells per entire retina
at 6, 12 and 18 weeks, respectively: P ¼ 0.001) compared to
the controls (8.4
1.0, 6.8
1.4 and 2.4
0.5 cells per entire
retina at 6, 12 and 18 weeks, respectively) (Fig. 5B).
We further analyzed quantitatively if there is any size-
dependent preferential loss of RGC in the glaucomatous ret-
inas induced by episcleral vein ligation at 12 and 24 weeks
762 S. Yu et al. / Experimental Eye Research 83 (2006) 758e770

Table 1
Postoperative IOP of episcleral vein ligation model (mmHg
S.D.)
Treatment type Postoperative IOP (mmHg
SD)
1 week 2 weeks 4 weeks 12 weeks 28 weeks
Group 1 G 27.8
1.2 29.1
1.8 27.5
1.4 27.4
1.2 24.3
2.3
(single treatment: n ¼ 29) C 20.1
1.3 20.0
1.5 19.9
1.1 19.9
1.3 19.8
1.5
Group 2 G 28.1
1.3 24.3
0.6 28.1
0.8 26.9
1.7 26.2
1.5
(retreatment at 2 weeks: n ¼ 18) C 20.5
1.7 20.4
1.5 20.1
1.5 20.3
0.8 20.1
1.5
Group 3 G 27.6
1.4 28.3
1.4 24.6
0.6 26.9
1.4 24.6
2.1
(retreatment at 4 weeks: n ¼ 24) C 19.9
1.5 20.2
1.7 20.2
1.7 20.1
1.0 19.8
1.3
G, glaucoma model; C, control.

postoperatively (Fig. 6). The cell loss was preferentially ob-
served in RGC population with the diameter range between
5 and 15 mm in the treated retinas compared to the controls
at 12 and 24 weeks. In contrast, RGCs with the diameter larger
than 15 mm did not show any decrease in their numbers by the
treatment. A similar tendency of size-dependent RGC loss was
observed at early (12 weeks) and late (24 weeks) stages of IOP
elevation (Fig. 6). At 24 weeks, RGCs with diameter less than
5 mm showed statistically significant decrease in the treated
retinas compared to the controls.
3.4. Selective impairment of RGCs and Müller cells
revealed by immunohistochemical analyses and
real-time RTePCR
To further analyze if there was any other cellular defects in
the retina at later stages of IOP elevation, immunohistochem-
ical analysis was performed using antibodies that are specific
for each retinal cell type after 32 weeks of surgery (Fig. 7).
The intensity of expressions of p75 and Brn3b, specific molec-
ular markers for RGCs, was decreased at 32 weeks, consistent
with the reduction in the number of retrogradely labeled
RGCs. In contrast, PKC (a bipolar cell marker), opsin (a rod
photoreceptor marker) and calbindin (an amacrine and a hori-
zontal cell marker) were detected in a similar pattern in their
expression in the treated retina compared to the control.
Among the glial cell markers, the expression of GFAP, an as-
trocyte marker that is expressed in reactive Müller glia under
pathological circumstances, appeared to be increased com-
pared with control retina. In addition, the expression of
p27Kip1, a cyclin-dependent kinase inhibitor that is expressed
by normal Müller glia but is downregulated under the reactive
condition, appeared to be decreased in the treated retina. To
further analyze the level of GFAP and p27Kip1 in a quantita-
tive manner, we performed a series of real-time PCR experi-
ment to analyze the level of each mRNA expressed in
treated and control retinas after 32 weeks of surgery. Our re-
sults showed that there was no difference in the expression

Fig. 3. Light microscopic analysis of control and glaucoma model eyes at 10 (AeD), 20 (EeH) and 30 (IeL) weeks after treatment. See details in the text. During
the 10e30-week period, a gradual loss of RGCs (B, F, J) and an augmentation of excavation of optic disc (D, H, L) in the operated eyes are detected. Sham-
operated control eyes showed no apparent morphological changes (A, C, E, G, I and K). Optic disc excavation (*) is evident in 30w glaucoma eye (L). GCL,
retinal ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; Gla, glaucoma model. Scale bar: 50 mm.
 S. Yu et al. / Experimental Eye Research 83 (2006) 758e770 763

Fig. 4. Temporal patterns of RGC loss in each region of the retina in operated and control eyes after treatment. RGCs (per square millimeter) were counted by ret-
rogradely transferred fluorescent dye. (A) Changes in the number of RGCs in the peripapillary (PP) region surrounding the optic disc at 1 mm radius. (B) Changes in
the number of RGCs in the midperipheral (M) region surrounding the optic disc at 1 mm width located 1e2 mm apart from the center of the optic disc. (C) Changes in
the number of RGCs in the peripheral (PR) region surrounding the optic disc at 1 mm width located 2e3 mm apart from the center of the optic disc. (D) Changes of the
total number of RGCs from the peripapillary through the peripheral region of retinas. From 12 weeks onward, a significant reduction in the number of RGCs is de-
tected in every region. Nearly 35% of RGCs were lost in total by 32 weeks. Data expressed are mean
S.E.M. (cell number/mm2).

level of GFAP in the treated and non-treated retinas, whereas
the level of p27Kip1 was significantly reduced in the treated
retinas (Fig. 8).
3.5. Changes in the expression pattern of
neurotrophic factor
To monitor if there were any changes in the expression of
neurotrophic factors, we next analyzed the expression of neu-
rotrophic factors, BDNF, CNTF, GDNF and basic FGF, which
are implicated in the control of RGC survival in previous stud-
ies (Johnson et al., 1986; Mey and Thanos, 1993; Sapieha
et al., 2003; Ji et al., 2004; Wu et al., 2004) (Fig. 9). We fo-
cused our analyses on retinas that were derived from early
(10 weeks), middle (20 weeks) and late (30 weeks) stages of
chronic IOP elevation. When control retinas were immunohis-
tochemically analyzed, there was no change in the expression
patterns of trophic factors throughout the follow-up period
(Fig. 9A,E,I,M). In contrast, in glaucoma retina, some of the
trophic factors exhibited changes in their expressions during
the period of IOP elevation. CNTF expression, preferentially
detected in the GCL in the control retinas, is detected also
in the INL at 10 weeks but declined at 30 weeks in the glau-
coma retina (Fig. 9AeD). Consistent with the decline in the
CNTF expression at 30 weeks, the quantitative analyses of
CNTF mRNA using real-time PCR showed a significant de-
crease at 30 weeks after IOP elevation (Fig. 10). Basic FGF
expression, detected in the NFL, GCL and INL in the control
retina, showed no change at 10 weeks (Fig. 9F), declined in
the GCL but maintained in the INL and NFL at 20 weeks
(Fig. 9G), and was markedly reduced in all the layers at
30 weeks (Fig. 9H). Consistent with this observation, total
amount of basic FGF mRNAs decreased significantly at
30 weeks postoperatively (Fig. 10). BDNF and GDNF, both
of which are mainly expressed in the GCL, did not change
their expressions throughout the entire period of IOP elevation
(Fig. 9IeP), which is consistent with the quantitative analyses
of BDNF and GDNF mRNA expressions (Fig. 10).

Table 2
% Reduction of RGC in glaucoma model compared to control retinas
Area % Reduction of RGC
6 weeks 12 weeks 18 weeks 24 weeks 32 weeks
PP 3.62
3.02 20.52
1.70 26.17
2.55 33.89
2.74 33.26
2.39
M 0.98
2.72 19.00
1.65 26.10
2.02* 32.84
2.37 36.01
2.11
PR 4.73
4.47 13.12
2.83** 16.64
4.70 29.76
3.40 30.07
3.29
Total 3.12
1.95 17.55
1.26 22.97
1.93 32.16
1.61 33.11
1.51
Data are the mean
S.E.M. *P < 0.05, between M and PR; **P < 0.05, between PP and PR.
764 S. Yu et al. / Experimental Eye Research 83 (2006) 758e770

Fig. 5. Apoptosis in glaucoma model retina detected by terminal dUTP nick-end labeling (TUNEL). (A) Confocal microscopic analyses of RGC death detected by
a combination of TUNEL staining (green) and retrograde fluorescent dye tracing (red). Each picture represents a region of whole-mount retina of 18 weeks after the
treatment. N, nasal; S, superior; T, temporal; I, inferior; PR, peripheral; PP, peripapillary. TUNEL-positive cells are confined to the retrogradely labeled RGCs in
the glaucoma retina (bar ¼ 50 mm). (B) Table showing the number of apoptotic cells detected in the control and glaucoma model retina at 6, 12, and 18 weeks after
treatment. The number of TUNEL-positive cells in glaucoma rat retinas is more than 10 times than that in sham-operated control retinas (n ¼ 5).

4. Discussion the cause of IOP elevation. Instead of cautery burn, we utilized

4.1. Features of the episcleral vein ligation
glaucoma rat model
Previous in vivo studies of glaucoma utilized many ap-
proaches to induce IOP elevation in rat eyes. These studies
showed that most procedures were able to induce a significantly
high IOP, a more than 1.5 times increase compared to the con-
trol eyes (Shareef et al., 1995; Morrison et al., 1997; Ueda
et al., 1998; Levkovitch-Verbin et al., 2002), which mostly
lasted for 1e3 months (Morrison et al., 1997; Levkovitch-
Verbin et al., 2002) and are correlated with morphological
changes similar to those observed in human glaucoma subjects.
In addition, these glaucoma models provided further insights
into the cellular defects induced by chronic IOP elevation
and beneficial for our understanding of the process of glau-
coma. Among the glaucoma models, Shareef’s model is one
of the most established glaucoma rat models, which obstructs
two or three trunks of episcleral veins by cauterization to
induce high IOP. Several laboratories have conducted studies
on glaucoma using this model; however, the IOP profiles
in those studies varied between laboratories (Sawada and
Neufeld, 1999; Mittag et al., 2000; Grozdanic et al., 2003a;
Kanamori et al., 2004) and it was technically difficult to pro-
duce this model in our experience. These observations promp-
ted us to establish another glaucoma model.
Our glaucoma model is essentially similar to the episcleral
vein cautery model in that the obstruction of episcleral vein is
a 10-0 nylon suture to obstruct the episcleral vein. One of the
most distinguished features of our model is the very mild ele-
vation of IOP that can be induced immediately after the oper-
ation and can be persisted over several months after the single
or double treatments. Furthermore, there was little variability
in the patterns of IOP elevation among the eyes treated with
episcleral vein ligation. Our glaucoma model was capable of
inducing mild IOP elevation on the next day immediately after
the treatment and maintaining its elevation for as long as
7 months: w25 mmHg in the treated eyes and w20 mmHg
in the control eyes. The levels of IOP at chronic phase were
maintained at a similar extent among the operated samples.
Nearly 40% of the operated eyes showed elevated IOP consis-
tently only by a single ligation procedure. The rest (w59.2%)
of the operated eyes, in contrast, did not exhibit IOP elevation
continuously after initial treatment, however, a subsequent re-
ligation procedure was sufficient for the achievement and
maintenance of the IOP elevation in all the cases. Other
models also reported the requirement of multiple retreatment
to maintain IOP elevation (Morrison et al., 1997; Ueda
et al., 1998; Mittag et al., 2000): 94.7% of our model showed
consistent IOP elevation for at least 7 months with one addi-
tional treatment. Indeed, the 10-0 nylon suture, which was
visible through the conjunctiva, enabled us to distinguish col-
lateral veins that needed to be ligated at the time of re-
treatment from the ones that have been ligated previously.
Compared to laser photocoagulation or hypertonic saline
injection models (Morrison et al., 1997; Levkovitch-Verbin
 S. Yu et al. / Experimental Eye Research 83 (2006) 758e770
Fig. 6. Size distribution of DiI labeled RGCs in control and treated (Gla
model: 12w, 24w) retinas as a function of the cell diameter. RGCs with the
diameter ranging between 5 and 15 mm appear to be preferentially decreased
in the treated retinas compared to the controls. In contrast, RGCs larger than
15 mm in diameter did not show significant decrease in their numbers by the
treatment. This tendency of size-dependency of RGC loss was observed
both at 12 and 24 weeks postoperatively. At 24 weeks, RGCs with diameter
less than 5 mm showed statistically significant decrease in the treated retinas
compared to the controls.
et al., 2002), the suturing approach utilized in our model did
not induce any visible inflammation in the eyes even with re-
treatment (data not shown).
4.2. Episcleral vein ligation model recapitulates
characteristic morphological features of glaucoma
Our model based on the episcleral vein ligation model ex-
hibits phenotypes that are essentially identical to that observed
by other models, and appears to recapitulate characteristic mor-
phological features of glaucoma. Correlated with chronic IOP
elevation, our results showed a RGC loss and optic disc excava-
tion. Previous studies, using other glaucoma models, showed
that there was a 1.4e6% reduction of RGCs per week and ex-
hibited significant cell loss immediately after IOP elevation
(Garcia-Valenzuela et al., 1995; Laquis et al., 1998; Sawada
and Neufeld, 1999; Naskar et al., 2002; Ji et al., 2004). In con-
trast, the loss of RGC in our ligation model is 1.1e1.5% per
week and appears to progress more slowly compared to other
procedures. The late onset and less severe extent of RGC loss
most likely reflect the mild IOP elevation induced in our model.
765
A previous study showed that there was a preferential cell
loss in the peripheral regions of retinas (Garcia-Valenzuela
et al., 1995; Laquis et al., 1998; Sawada and Neufeld, 1999).
Other studies showed a preferential RGC loss in the temporal
upper and lower regions of retinas (Morrison et al., 1997; Mit-
tag et al., 2000). In contrast, our ligation model exhibited the
susceptibility for the cell loss in the parapapillary retina at
early stage after IOP elevation. These apparent distinct fea-
tures of preferential regional sensitivity for cell loss in our
model might be due to the mild extent and consistency of
IOP elevation observed in our model. In addition to the re-
gional preference regarding RGC loss in glaucoma, the sus-
ceptibility of RGCs to IOP elevation depending on the size
is also the important issue. Several groups have shown that
larger ganglion cells are more vulnerable to IOP elevation in
a variety kind of species (Glovinsky et al., 1991; Danias
et al., 2006; Levkovitch-Verbin et al., 2002). However, the
preferential loss of RGCs in large size was nor detectable in
our episcleral vein ligation model. Further studies are needed
to see how regional distinct susceptibility is induced by the
different approaches to elevate IOP and the different pattern
of IOP elevation.
Previous studies using episcleral vein cautery model sug-
gested that the occlusion of episcleral vein might lead to the
impairment of ocular blood flow and the choroidal insuffi-
ciency, which may cause secondary effect on retina, especially
on photoreceptors. Grozdanic et al. reported the change in
ERG parameters; however, these changes were observed
only during the period of IOP elevation and there was no
change in the cytoarchitectures of treated retinas (Grozdanic
et al., 2003a). Bayer et al. also reported the changes in the sco-
topic ERG in their modified episcleral vein ligation model, but
suggest that changes in the patterns of ERG appear to be
mostly mediated by IOP elevation but not by the impairment
of ocular blood flow (Bayer et al., 2001). Other studies on
the episcleral vein cautery model also report that only RGCs
are affected. In our present study, by ligating episcleral vein,
we cannot rule out the possibility of impairment of ocular
blood flow and choroidal insufficiency. However, we observed
that there was a pronounced blood flow through the unligated
veins after the treatment, which might reflect the existence of
compensational shunt of blood flow via the unligated veins to
overcome the occlusion of the blood flow in the treated retinas.
This compensational increase of blood flow through the intact
vein in response to the occlusion might enable retinal tissue to
escape from the ischemic damage. Moreover, we detected no
change in the photoreceptors and the thickness of the outer nu-
clear layer between the treated and control retinas even at ad-
vanced stages of IOP elevation, which is consistent with the
idea that there is no ischemic change in our model.
Our analyses using TUNEL assays showed that RGC loss
was induced by apoptosis, consistent with previously reported
glaucoma models (Morrison et al., 1997; Ishii et al., 2003;
Fortune et al., 2004; Kanamori et al., 2004). TUNEL-positive
cells were shown to be confined to retrogradely labeled RGCs
in whole-mount preparations, suggesting that RGC is selec-
tively impaired during the chronic phases of IOP elevation
766 S. Yu et al. / Experimental Eye Research 83 (2006) 758e770

Fig. 7. Immunohistochemical analyses of retinal cells in sham-operated control retinas (A, C, E, G, I, K, M) and in glaucoma model retinas (B, D, F, H, J, L, N) at
32 weeks after operation, showing a decreased expression of p75 (A, B) and Brn3b (C, D) in the GCL in glaucoma retina. Calbindin (E, F), PKC (G, H) and Opsin
(I, J) showed no obvious difference between the glaucomatous retinas and controls. The expression of GFAP (K, L) was detected in a pattern that spans the retina
from INL to GCL in both the control and the glaucomatous retina. The expression of p27Kip1 (M, N) in the INL was decreased in glaucoma retinas. GCL, retinal
ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. Scale bar: 50 mm.

in our model. Although there are several reports showing that
photoreceptors are also affected in glaucoma (Bayer et al.,
2001; Grozdanic et al., 2003a), our immunohistochemical
analyses of the expression patterns of specific molecular
Fig. 8. Quantitative analyses of GFAP and p27Kip1 mRNAs at 30 weeks. Data
are expressed as mean
S.E.M. GFAP expression did not exhibit statistically
significant difference, but the expression of p27kip1 showed statistically sig-
nificant decrease in the glaucoma retinas (Gla model) compared to the controls
(Control). *P < 0.05.
markers of each retinal neuron as well as histological analyses
of retinal morphology showed that the retinal cells that were
lost in this model were restricted to RGC, and there was no
sign of involvement of photoreceptors. Only Müller glial cells
showed a change in the marker expression. With many types
of insult to the eye, Müller cells were demonstrated to upregu-
late GFAP expression and also to dedifferentiate and prolifer-
ate at an early stage, and gradually reduced (Kim et al., 1998;
Wang et al., 2000; Bringmann and Reichenbach, 2001). Previ-
ous studies on glaucoma models also showed that GFAP ex-
pression is increased in Müller cells at least at early stages
of IOP elevation, but whether GFAP upregulation persists
into advanced stages remained obscure (Wang et al., 2000;
Thanos and Naskar, 2004; Woldemussie et al., 2004). In this
ligation glaucoma model, we focused on the advanced stage
of glaucoma retina. GFAP expression in Müller cells appeared
to be increased in the treated retina, but we could not detect
a significant difference in GFAP mRNA expressions compared
to control retinas. In contrast, p27Kip1, a molecule that plays
a critical role in exiting from the mitotic cell cycle (Dyer and
Cepko, 2001) in Müller cells, was significantly downregulated
 S. Yu et al. / Experimental Eye Research 83 (2006) 758e770 767

Fig. 9. Immunohistochemical analyses of spatial and temporal patterns of neurotrophic factors, CNTF, bFGF, BDNF, and GDNF in the control (A, E, I, M) at
10 weeks and glaucomatous retina (Gla) at 10 weeks (B, F, J, N), 20 weeks (C, G, K, O) and 30 weeks (D, H, L, P). CNTF: AeD; bFGF: EeH; BDNF: IeL;
GDNF: MeP. See the details in the text. Note that the expression of CNTF in 10w glaucoma retina is upregulated compared to control retina. At advanced stage
of glaucoma (30 weeks after the treatment) all the factors including CNTF showed the decline in those expression. Sections of the retina were counterstained with
TO-PRO-3 (blue). GCL, retinal ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. Scale bar: 50 mm.

in the treated retinas. Altogether, these results are partly
consistent with the idea that, even at advanced stage of glau-
comatous retina, Müller cells might be under control of dedif-
ferentiation and proliferation mechanism to work in the
regenerating process in response to IOP elevation. Further
studies are needed to see if reactive Müller cells might contrib-
ute to compensational repair of RGC loss in the glaucomatous
condition.
4.3. Neurotrophic factors expression in elevated IOP
Neurotrophic factors play a central role in maintaining and
controlling the function of retinal cells in the developing and
mature retina (Von Bartheld, 1998; Vecino et al., 2002). Previ-
ous studies have suggested that one potential reason of RGC
loss observed in glaucoma might be the deprivation of neuro-
trophic factors that control the survival of RGCs (Johnson
et al., 1986; Ko et al., 2000). Indeed, applications of neurotro-
phic factors, including BDNF, CNTF, and GDNF, via various
approaches has been shown to be beneficial for rescuing RGCs
from death in glaucoma eyes (Naskar et al., 2000; Martin
et al., 2003; Ji et al., 2004). However, the spatial and
temporal changes in expression patterns of neurotrophic fac-
tors by chronically elevated IOP are not fully understood
(Pease et al., 2000; Ji et al., 2004; Rudzinski et al., 2004)
and need to be examined to devise more efficient rescue
approaches.
Our ligation model allowed us to analyze the expression
patterns of neurotrophic factors that might contribute to pre-
venting RGC loss. Immunohistochemical analyses for the
768 S. Yu et al. / Experimental Eye Research 83 (2006) 758e770
Fig. 10. Quantitative analyses of CNTF, bFGF, BDNF and GDNF mRNAs at 10, 20 and 30 weeks after the treatment. Datas are glaucoma retina/control retina ratio
at each point, and expressed as mean
S.E.M.(%). The expression level of CNTF and bFGF significantly decreased at 30 weeks, whereas that of BDNF and
GDNF did not change during the period of IOP elevation. *P < 0.05.
expression of growth factors at various time points revealed
a unique pattern for each of the factors examined. In the con-
trol retina, there was no obvious change in the expression of
growth factors throughout the follow-up time course (data
not shown). Previous studies showed that the expression of
BDNF in the retina was increased in the early glaucomatous
stage (Rudzinski et al., 2004). In our glaucoma model, how-
ever, we could not detect any increased expression of BDNF
in the GCL, possibly because of the difference in the timing
of analyses. GDNF (Naskar et al., 2000) also did not show
any increased expression during the chronic phase of IOP el-
evation. Another trophic factor, bFGF, that was normally pres-
ent in the GCL, INL, ONL and RPE cells (Connolly et al.,
1992), was shown to be upregulated in Müller cells by light
exposure and ischemia (Miyashiro et al., 1998; Walsh et al.,
2001). In contrast, in this ligation model, there was no appar-
ent upregulation of bFGF and the expression of bFGF declined
in the late glaucoma stage. Among the growth factors exam-
ined by immunohistochemical analyses, CNTF expression
alone appeared to be increased especially in the INL at an
early glaucoma stage. A similar expression pattern of changes
in CNTF expression was also detected in the optic nerve tran-
section model and NMDA- and kainic acid-induced neuronal
death (Honjo et al., 2000; Casson et al., 2004). Although we
could not detect increase in the expression of CNTF by real-
time PCR approach, these observations may, therefore, suggest
CNTF as a protective factor that is especially dedicated to the
loss of RGCs under several pathological circumstances (Chun
et al., 2000; Sarup et al., 2004). Taken together, the results of
our study on the expression patterns of neurotrophic factors es-
pecially highlight the potential role of CNTF in the control of
RGC survival during the early stage of IOP elevation. Further
analyses and identification of other trophic factors, which are
spatially and temporally regulated during the course of IOP el-
evation, may provide potential insights into understanding way
of reversing RGC defects in glaucoma.
In conclusion, we reported here an animal model of glau-
coma with episcleral vein ligation in rat eyes. Our analyses
of this animal model have revealed that IOP was elevated to
a mild extent from the early postoperative stage and persisted
reproducibly for several months after the procedure. A gradual
increase of RGC-specific cell death preceding optic disc exca-
vation was observed after several weeks of IOP elevation. Our
episcleral vein ligation model reiterated many critical charac-
teristics of human glaucoma and may, potentially, provide in-
sights into the underlying molecular and cellular bases of
glaucomatous optic neuropathy.
Acknowledgements
The authors thank Yoshinaga Tochikawa of Fujimoto
Pharmacentical Corporation for instructions in RGC labeling
and counting methods, and Mari Dezawa and Hiroto Ishikawa
of Kyoto University for their advice on histological and immu-
nohistochemical analyses.
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