Experimental Eye Research 81 (2005) 561–569
www.elsevier.com/locate/yexer
The pig eye as a novel model of glaucoma
Javier Ruiz-Ederraa, Mónica Garcı́aa, Marı́a Hernándeza, Haritz Urcolaa,
Ernesto Hernández-Barbáchanoa, Javier Araizb, Elena Vecinoa,*
a
Department of Cellular Biology, Faculty of Medicine, University of the Basque Country, E-48940 Leioa, Vizcaya, Spain
Department of Ophthalmology, Faculty of Medicine, University of the Basque Country, E-48940 Leioa, Vizcaya, Spain
b
Received 17 November 2004; accepted in revised form 29 March 2005
Available online 9 June 2005
Abstract
We validated the pig eye as a model of glaucoma, based on chronic elevation of intraocular pressure (IOP). IOP was elevated by
cauterising three episcleral veins in each of the left eyes of five adult pigs. Right eyes were used as controls. Measurement of IOP was
performed during the experiment with an applanation tonometer (Tono-Pen). Five months after episcleral vein occlusion, retinal ganglion
cells (RGCs) from both cauterised and control eyes were retrogradely backfilled with Fluoro-Gold. Analysis of RGC loss and morphometric
as characterization of surviving RGCs was performed using whole-mounted retinas. Elevation of IOP was apparent after three weeks of
episcleral vein cauterisation and it remained elevated for at least 21 weeks (duration of the experiments). Analysis of RGC loss after chronic
elevation of IOP revealed that RGC death was significant in the mid-peripheral and peripheral retina, mainly in the temporal quadrants of
both retinal regions. Moreover the mean soma area of remaining RGCs was observed to increase and we found a greater loss of large RGCs in
the mid-peripheral and peripheral retina. We conclude that the pattern of RGC death induced in the pig retina by episcleral vein cauterisation
resembles that found in human glaucoma. On the basis of this study, the pig retina may be considered as a suitable model for glaucomarelated studies, based on its similarity with human and on its affordability.
q 2005 Elsevier Ltd. All rights reserved.
Keywords: fluoro-gold; intraocular pressure; porcine; tracer; RGC; glaucoma
1. Introduction
Glaucoma is the second most common cause of
blindness worldwide, after cataract (Weinreb and Khaw,
2004). This ocular disease is associated with a progressive
loss of the visual field, caused by retinal ganglion cell
(RGC) death. Increased intraocular pressure (IOP) constitutes one of the principal risk factors (Glovinsky et al.,
1991; Vickers et al., 1995; Wygnanski et al., 1995).
Consequently, most of the current therapies to treat
glaucoma are directed to lowering IOP, in order to
minimise cell death. Thus, useful models of glaucoma
inevitably involve a significant and sustained elevation of
IOP.
* Corresponding author. Address: Department of Cell Biology and
Histology, Faculty of Medicine, University of the Basque Country,
E-48940 Leioa, Vizcaya, Spain
E-mail address: gcpvecoe@lg.ehu.es (E. Vecino).
0014-4835/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.exer.2005.03.014
The only large mammal, which is currently being
employed for the induction of experimental glaucoma, is
the monkey (Kalvin et al., 1966; Glovinsky et al., 1991;
Morgan et al., 2000; Kashiwagi et al., 2003). Despite being
an excellent model, monkey availability is very low due to
ethical and economical reasons. Thus, it is of interest to
evaluate the suitability of the pig as a model of glaucoma,
since it is phylogenetically close to the human and is much
more available than the monkey. The pig eye/retina shares
many similarities with that of the human (Prince et al., 1960;
Beauchemin, 1974; Peichl et al., 1987; De Schaepdrijver
et al., 1990; McMenamin and Steptoe, 1991; Olsen et al.,
2002; Ruiz-Ederra et al., 2003, 2004; Garcia et al., 2005).
The porcine retina is even more similar to the human retina
than that of other large mammals such as the dog, goat, cow
or ox (Prince et al., 1960). Moreover the pig has recently
been used to genetically reproduce a retinitis pigmentosa
condition, similar to that found in human (Li et al., 1998).
Additionally, tools employed for diagnostics in ophthalmology, such as optical coherence tomography, corneal
topography imaging or multi-focal electroretinography can
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be applied to the pig eye, supporting the use of this animal as
a good model for ophthalmological studies (Kyhn et al.,
IOVS, 2004, 2, ‘ARVO E-abstract’, 4247; Maverick et al.,
IOVS, 2004, 2, ‘ARVO E-abstract’, 2876; Van Velthoven
et al., IOVS, 2004, 2, ‘ARVO E-abstract’, 2371). Finally,
studies of the pig aqueous outflow system showed that this
animal could be a suitable model for specific types of
glaucoma (McMenamin and Steptoe, 1991).
In a previous study, we reported the presence of three
classes of RGCs based on soma size (small, medium and
large) (Garcia et al., 2002) and performed a detailed study of
the pig RGC topography as a function of soma size. Our
study revealed that the distribution of the different sized
RGCs is very similar in the porcine and human retina
(Garcia et al., 2005). This information may be useful in
order to unravel the mechanisms implicated in the selective
death of some size groups of RGCs in glaucoma, since it is
generally accepted that large RGCs are more susceptible to
death during human or experimental glaucoma (Quigley
et al., 1987, 1988, 1989; Glovinsky et al., 1991; Vickers
et al., 1995).
In the present work, we have evaluated the pig eye as a
novel model of glaucoma. Our study indicates that the pig
eye is a suitable animal model for glaucoma experimentation, based on the similarity of the features observed in
human glaucoma and in the pig eye subjected to chronic
increased intraocular pressure, and on the more ready
availability of pig eyes in comparison to those of nonhuman primates.
2. Materials and methods
All experiments were conducted following the ARVO
Statement for the Use of Animals in Ophthalmic and Vision
Research. We induced a chronic elevation IOP within pig
eyes by means of episcleral vein occlusion. Measurements
of IOP as well as analysis of optic disc excavation were
performed through the experimental period. At the end of
this period, RGC death was measured by analysing RGCs,
which had been retrogradely back-filled with Fluoro-Gold.
The eyecups were fixed with 4% paraformaldehyde in 0.1 M
phosphate buffer saline (PBS, pH 7.4) for 4 hr at 48C and
then retinas were removed and flat-mounted with the retinal
ganglion cell layer being uppermost. They were then cover
slipped with PBS/glycerine (1:1).
2.1. Induction of experimental glaucoma
Five adult pigs (Sus scrofa) were used in the present
work. An increase in IOP was induced by cauterising three
episcleral veins of the left eyes of the animals following the
method described elsewhere (Shareef et al., 1995). Briefly,
pigs were deeply anaesthetised following the protocol
described above, with an intramuscular injection of
ketamine hydrochloride (Ketolar)Cxylazine (Diazepan)
(each 20 mg kgK1). An intravenous cannula was applied
to the ear in order to provide the animal with additional
anaesthetic (1 ml Propofol every 15 min), maintaining deep
anaesthesia throughout the operation. A life-support
machine was used to facilitate breathing and to monitor
vital functions during the operation. Three episcleral veins
(nasal, dorsal and temporal) were cauterised following the
protocol described by Shareef et al. (1995) in rats. Animals
were kept alive during 21 weeks after episcleral vein
occlusion.
2.2. Intraocular pressure measurement
IOP was measured with an applanation tonometer
(Tono-Pen XL; Medtronic, Jacksonville, FL, USA) under
light general anaesthesia (KetamineCXilazine), following
application of drops of tetracaine hydrochloride (1 mg mlK1)
Coxibuprocaine hydrochloride (4 mg mlK1) on the corneas
(Colircusı́, Alcon Cusı́, Spain). All measurements were
carried out at the same time and always before feeding the
animals. Forty-five days before the episcleral vein operation,
the IOP of both eyes was measured in order to obtain the
baseline values. One week post-operation, both right
(non-operated control) and left (cauterised) eyes were
measured at fortnight intervals, to evaluate the increase in
IOP. The tonometer was applied perpendicularly to the more
apical side of the cornea, until at least five or six independent
measurements were obtained (each of these IOP values was
the average of four IOP readings. The results of the IOP
reading were accepted if the confidence interval was
greater than or equal to 95%). The mean values of the IOP
measurements were eventually averaged, and results
were expressed as mean IOPGSEM. Five such measurements
were made.
2.3. Capture of eye fundus images
In order to follow-up the progression of the excavation of
the papilla, we captured images from the optic disc of
control and cauterised eyes, 1 week after the occlusion of
the episcleral veins and 1 week before the end of the IOP
increase period. Images were obtained under general
anaesthesia, using a hand held fundus camera. Optic disc
excavation was determined comparing cup/disc ratio values
at initial stages and at final stages of the glaucomatous
procedure. Measurements were performed using a digital
palette (Easypen, Genius) in combination with image
analysis software (Scion Image; Scion, Frederick, MD) for
digitised images.
2.4. RGC backfilling
RGCs from eight eyes were backfilled from the optic
nerve with 3% Fluoro-Gold (Fluorochrome, Englewood
CO, USA) diluted in a solution containing 0.9% NaCl and
0.1% dimethylsulfoxide. Forty microlitres of Fluoro-Gold
J. Ruiz-Ederra et al. / Experimental Eye Research 81 (2005) 561–569
563
was injected into the optic nerve around 4 mm from the
optic nerve head. Pigs were kept alive for two days postoperation to allow Fluoro-Gold to fill the entire population
of RGCs. Then animals were euthanised with an overdose of
anaesthesia, the eyes were enucleated and the lens and
vitreous were extracted by cutting the anterior chamber at
the level of the ora serrata. The eyecups were fixed with 4%
paraformaldehyde in 0.1 M phosphate buffer saline (PBS,
pH 7.4) for 4 hr at 48C and then retinas were removed and
flat-mounted with the retinal ganglion cell layer being
uppermost. They were then cover slipped with PBS/glycerine (1:1), so that shrinkage did not occur during the
processing of the tissue.
We divided the animals in two groups: the first one
consisted of two pigs, whose RGCs from their left
(cauterised) eyes were backfilled with Fluoro-Gold. RGC
topography pertaining to cauterise eyes was then compared
with that of a pool of control eyes from different pigs, which
we had analysed in previous studies. The second group
consisted of three pigs, whose RGCs from both left and right
eyes were backfilled from the optic nerve with the
fluorescent tracer; a paired analysis of RGC distribution
between fellow eyes was then performed. Since we only
found differences in the number and distribution of RGCs
when we compared fellow control vs. cauterised eyes, the
results in the present study regarding RGC topography will
refer to the paired study performed in this group.
both parameters were transferred to a data sheet for
subsequent statistical analysis.
RGC density, mean area and soma size distribution
(attending to major axis length in small, medium and large
RGCs: !15; 15–20 and O20 mm, respectively) were
analysed separately in the three major regions of the retina:
the visual streak, mid-periphery and periphery (Unpublished
results). We proceeded with further subdivision in quadrants
(nasal–dorsal (ND); nasal–ventral (NV); temporal–dorsal
(TD) and temporal–ventral (TV) only in those retinal
regions where we found significant differences with respect
to controls.
2.5. Capture of retinal ganglion cell images
The mean IOP in control eyes was 15.2G1.8mmHg.
Elevation of IOP after cauterisation of three episcleral veins
was apparent by the third week in all animals, when IOP
rose from 15.6G1.8mmHg in control eyes to 20.8G
2.4mmHg in cauterised eyes (1.3 fold-increase, pZ0.032).
Differences reached a maximum by the 16th week with a 1.4
fold-increase, pZ0.048 [21.0G4.1mmHg (cauterised) vs.
14.8G1.3mmHg (control)]. Elevation of IOP was maintained throughout the course of the experiment, with
significant differences still being apparent at week 20
(15.1G2.9mmHg in control vs. 19.8G5.9mmHg in cauterised eyes, pZ0.037) and week 21 (11.5G0.9mmHg in
control vs. 13.9G0.8mmHg in cauterised eyes, pZ0.021)
(Table 1).
Images from both the left and the right retinas of each
animal were obtained using an epifluorescence microscope
(Axioskop 2; Zeiss, Jena, Germany) coupled to a digital
camera (Coolsnap, RS Photometrics, Tucson, USA). The
images were captured in a systematic way using the optic disc
and the visual streak as reference points, as previously
described (Garcia et al., 2005). Briefly, we recorded one out
of four 40! microscope fields from the optic disc towards the
periphery, along both the X and the Y-axes of the retina. We
recorded 100–130 fields/retina, using the 40! objective. The
sampling area recorded represented 1.4% of the mean area of
the six retinas analysed. This sample size has been previously
reported to represent a significant percentage of the retina
(Peinado-Ramón et al., 1996).
2.7. Statistical analysis
RGC density, mean soma area and percentage of size
groups, as well as IOP measurements were compared
between glaucomatous and control eyes, by a paired Student
test, using SPSS software (SPSS Sciences, Chicago, IL).
Values are expressed as meanGSEM. The minimum level of
significant difference was defined as p!0.05.
3. Results
3.1. Intraocular pressure
Table 1
Intraocular pressure (IOP) in control and cauterised pig eyes
2.6. Morphometric analysis
Week
Mean IOP (mmHg)
Control
Cauterised
IOP fold-increase
Morphometric analysis was performed as described
elsewhere (Garcia et al., 2002). For each recorded retinal
field, we quantified the number of RGCs and the soma area
of each of the RGCs present. Analysis of these two
parameters was performed using a digital palette (Easypen,
Genius, Taipei, Taiwan) in combination with image analysis
software (Scion Image; Scion, Frederick, MD). Each RGC
soma was filled out directly on the computer screen, so that
area and major axis length could be measured. Values for
Baseline
3
9
12
16
20
21
15.2G2.1
15.6G1.7
15.0G1.7
15.5G3.2
14.8G1.5
15.1G2.3
11.5G0.7
15.1G2.2
20.8G2.9
16.5G2.2
17.4G4.1
21.0G2.9
19.8G4.7
13.9G0.7
–
1.33*
1.10
1.12
1.41*
1.31*
1.23*
Values are expressed as mean IOP (mmHg)GSEM. Results from statistical
analysis are represented by: *p!0.05, significant difference with respect to
control.
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Fig. 1. Photographs of the fundus showing the optic disc from the control eye (A) and from the corresponding cauterised eye (B) 5 months after cauterisation of
the episcleral veins. At this advanced stage of damage, morphological changes could be observed, such us a paler appearance and an increase in the cup–disc
ratio. Optic disc excavation was identified by the curving of the retinal blood vessels at the disc margin.
3.2. Eye fundus
Examination of the eye fundus showed a discrete
excavation of the optic disc of all glaucomatous eyes at
advanced stages of optic nerve damage (pZ0.055). Thus, the
cup/disc ratio varied from 0.58G0.04 one week after the onset
of IOP elevation to 0.63G0.04 at week 21 following vein
cauterisation. Optic disc excavation was apparent from the
curving of the blood vessels (especially the thinner ones) at the
disc margin in the glaucomatous eyes at advanced stages of
damage (Fig. 1). However, blood supply did not seem to be
affected since we could identify all the large and small blood
vessels after the period of IOP elevation, which were
indistinguishable from those of the control retinas (Fig. 1).
3.3. Pattern of retinal ganglion cell death
Chronic elevation of IOP led to a mean loss of 18G8%,
pZ0.0005 of RGCs in the glaucomatous retinas. RGC loss
did not take place homogeneously, but was more pronounced
in the peripheral and mid-peripheral retina, compared to the
more central retina where the visual streak (a high RGC
density region) is located. Moreover, a larger loss of RGCs
occurred in the temporal retina, which was associated with an
increase in the mean soma area of RGCs. The combination of
both phenomena resulted in an alteration in the proportion of
RGC size groups along the retina.
3.4. Retinal ganglion cell density loss
RGC loss in glaucomatous compared to control retinas
was significantly greater in peripheral regions. Thus, RGC
density in the control peripheral retina was 239G28 RGCs
per mm2, whereas in cauterised eyes, RGC density was
significantly reduced (188G23 RGCs per mm2) (21% loss,
pZ0.046). This situation was also observed in the midperiphery (727G27 vs. 838G34 RGCs per mm2 in control
eyes; a 13% loss, pZ0.0005). No significant decrease in
RGC density was observed within the visual streak (4216G
148 vs. 4285G139 RGCs per mm2 in control eyes)
(Fig. 2A).
Fig. 2. RGC density in pig retinas from control and episcleral vein
cauterised eyes. (A) RGC density in the different regions of the retina. (B,C)
RGC density in the different retinal quadrants of the mid- periphery and the
periphery, respectively. Values are expressed as mean density (RGCs per
mm2)GSEM. Results from statistical analysis are represented by: **p!
0.01, significant difference with respect to control. Abbreviations: ND,
nasal–dorsal; TD, temporal–dorsal; NV, nasal–ventral; TV, temporal–
ventral.
J. Ruiz-Ederra et al. / Experimental Eye Research 81 (2005) 561–569
A more detailed analysis of the different regions of the
mid-peripheral retina revealed that RGC loss was greater in
both temporal quadrants with significant cell loss in the
temporal–dorsal (TD) quadrant (1015G62 vs. 1310G
67 RGCs per mm2 in control eyes; 22.5% decrease, pZ
0.0005), and in the temporal–ventral (TV) quadrant (428G
40 vs. 645G78 RGCs per mm2 in control eyes; 33.6% loss,
pZ0.01) (Figs. 2B and 3A). No differences were observed
in the quadrants located in the nasal retina.
Although differences in RGC density in the peripheral
retina were found to be significant (Fig. 2A), no
565
significant differences were found when we analysed
each retinal quadrant separately, probably due to the small
number of RGCs located in the fields captured in the
periphery. However, the density of RGCs was found to be
reduced in the TD and TV quadrants of glaucomatous
retinas compared to controls. Thus, we observed an RGC
density of 270G70 vs. 377G87 RGCs per mm2 in control
eyes in TD quadrant (28.3% decrease, pZ0.30), and
154G73 vs. 282G77 RGCs per mm2 in controls eyes
(45% decrease, pZ0.45) in the TV quadrant (Figs. 2C
and 3B).
Fig. 3. Representative images from the mid-peripheral (A) and peripheral (B) retina corresponding to both temporal quadrants in control (left) and cauterised
(right) porcine eyes captured at an equivalent retinal location. Porcine retinal ganglion cells from both control and cauterised eyes were retrogradely backfilled
with Fluoro-Gold. Abbreviations: TD, temporal–dorsal; TV, temporal–ventral. Scale bar in B represents 20 mm for all images.
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3.5. Changes in retinal ganglion cell mean area
The mean area of RGC somata increased in the
experimental retinas with respect to controls. This increase
was observed in the mid-periphery where we detected a
mean RGC soma area of 235G3 mm2 in controls vs. 244G
4 mm2 in cauterised eyes (a 3.8% increase, pZ0.01) and in
the periphery (328G16 vs. 298G8 mm2 in controls; a 10%
increase, pZ0.048). No significant differences were
observed in mean RGC area in the visual streak (170G4
vs.179G4 mm2 in controls) (Fig. 4A).
Upon analyzing the mid-peripheral regions in detail, we
found a significant increase in the mean area of RGC somata
in quadrants located on the temporal side of the experimental retina. Thus, in the TD quadrant, we found a mean RGC
soma area of 249G5 vs. 229G4 mm2 in the same quadrant
in control eyes (8.7% increase, pZ0.004). In the TV
quadrant, we measured a mean RGC soma area of 289G16
vs. 228G10 mm2 in control eyes (27% increase, pZ0.003).
Fig. 4. RGC mean soma area in control and glaucomatous pig retinas. (A)
Among the different regions of the retina. (B) Among the different retinal
quadrants of the mid- periphery retina. (C) Among the different retinal
quadrants of the peripheral glaucomatous retina. Values are expressed as
RGC mean soma area (mm2)GSEM. Results from statistical analysis are
represented by: **p!0.01, significant difference with respect to control.
Abbreviations: ND, nasal–dorsal; TD, temporal–dorsal; NV, nasal–ventral;
TV, temporal–ventral.
Significant differences were not observed in the nasal retina
(Fig. 4B).
With respect to the peripheral retina, we observed an
increase of the mean area of RGC somata in quadrants
located in the temporal side of the glaucomatous retina, in
comparison with the corresponding controls. Thus, the mean
RGC soma area was 281G13 (cauterised) vs. 263G23 mm2
(control) in the TD quadrant (6.8% increase, pZ0.48) and
320G21 (cauterised) vs. 304G17 mm2 (control) in the TV
quadrant (5.3% increase, pZ0.31). However, as occurred
with the analysis of RGC density in the periphery, the small
number of RGCs present in fields located in the periphery
led to large variability, which did not allowed us to observe
significant differences. Differences were not observed in the
nasal retina (Fig. 4C).
3.6. Retinal ganglion cell loss as a function of soma size
We analysed RGC loss in terms of soma size, by
analyzing the density of large, medium and small RGCs as
well as the percentages of these groups of RGCs in the three
retinal regions and in the temporal quadrants. We observed a
significant loss of large RGCs in the visual streak, with
475G42 large RGCs per mm2 in controls vs. 333G42 large
RGCs per mm2 in cauterised eyes (29% loss, pZ0.004)
(Fig. 5). The percentage of large RGCs, which represents
11.1G1.5% of total RGCs in the control visual streak, was
significantly (pZ0.005) reduced in the visual streak of
cauterised eyes, representing 7.9G1.4% of total RGCs in
this retinal region (Table 2A).
Significant (pZ0.007) loss of large RGCs was also
observed in the mid-peripheral retina, with 266G9 large
Fig. 5. The density of small, medium and large RGCs in the different retinal
regions of control and cauterised porcine eyes. Values are expressed as
mean density (RGCs per mm2)GSEM. Statistical differences between
control and cauterised eyes are represented by: **p!0.01 for large RGCs,
and && for small RGCs.
J. Ruiz-Ederra et al. / Experimental Eye Research 81 (2005) 561–569
Table 2
Percentages of small, medium and large retinal ganglion cells within the
visual streak (A), and the different quadrants of the mid-periphery (B) and
periphery (C) of control and cauterised eyes
(A) Percentage of visual streak RGCs
VS
Large (O21 mm)
Medium size (15–20 mm)
Small (!14 mm)
(B) Percentage of mid-periphery RGCs
TD
Large (O21 mm)
Medium size (15–20 mm)
Small (!14 mm)
TV
Large (O21 mm)
Medium size (15–20 mm)
Small (!14 mm)
(C) Percentage of peripheral RGCs
TD
Large (O21 mm)
Medium size (15–20 mm)
Small (!14 mm)
TV
Large (O21 mm)
Medium size (15–20 mm)
Small (!14 mm)
Control
Cauterized
11.1G1.5
53.0G2.0
35.9G2.3
7.9G1.4**
53.6G2.0
38.5G2.2
32.8G1.2
40.3G1.6
26.4G1.8
33.5G2.1
40.1G3.3
26.5G4.5
37.6G1.9**
42.9G1.5
19.5G1.4**
49.1G4.5**
39.1G3.6
11.7G2.1**
53.1G9.9
39.4G8.8
7.5G4.2
54.1G9.9
30.3G7.7
15.6G6.8
65.9G11.1
17.3G7.9
16.9G6.9
73.8G11.1
16.8G8.4
9.4G5.5
Values are the mean value of the percentages of different sized RGCsGSEM.
Results from statistical analysis are represented by: **p!0.01 significant
difference with respect to control retinas.
RGCs per mm2 in controls vs. 242G9 large RGCs per mm2
in cauterised eyes. Moreover, we found a significant
(pZ0.0005) loss of small RGCs in this region after
cauterisation of episcleral veins, with 238G50 small
RGCs per mm2 in controls vs. 155G25 small RGCs per
mm2 in cauterised eyes (Fig. 5). A detailed analysis of
the mid-periphery showed that the greater changes in the
percentage of RGCs in terms of soma size took place in the
temporal quadrants (Table 2B).
Finally, although we did not observe significant differences in the density of small, medium or large RGCs in the
peripheral retina (Fig. 5), variations in the distribution of
percentages of RGC soma size (Table 2) followed a similar
trend to that observed in the mid-periphery after elevation of
IOP. However, differences were not significant probably
due to the small number of RGCs located in the fields
captured in this retinal region.
4. Discussion
In the present work, we have established a model of
glaucoma based on the occlusion of the episcleral vein
method, using the pig as a novel experimental animal.
Damage was considered to be glaucomatous due to the
presence of elevated IOP, altered eye fundus morphology
and RGC loss.
Elevation of IOP was observed after the third week with
values 1.3 times higher in cauterised than in control eyes.
By the 16th week, IOP was 1.4 times higher than in the
control eye. Differences in the IOP were maintained up to
567
the end of the experimental period, with significant
differences between glaucomatous and control eyes being
present at the 21st week. This IOP fold-increase is similar to
that reported by others for rat eyes with experimental
glaucoma induced via the same method to increase the IOP
(Laquis et al., 1998). These authors described a loss of
RGCs in association with elevated IOP. Previous studies of
monkey eyes with experimental glaucoma showed that the
IOP in hypertensive eyes was about 2.1 or 2.4 times higher
than the IOP in control eyes (Pease et al., 2000; Weber et al.,
1998). However, the method employed to increase the
monkey IOP was different to that used in the present study.
Additionally, it is quite possible that Schlemm’s canal in the
pig eye, which is made up of multiple collector vessels
(McMenamin and Steptoe, 1991) could at least in part,
correct the increase of IOP induced by the cauterisation of
the episcleral veins in the pig eye.
The glaucomatous eye fundus revealed optic disc
excavation, which is one of the most common events
associated with fibre loss in glaucoma. This was observed in
all glaucomatous animals analysed. The mean values of the
cup/disc ratio were 0.58G0.04 at the initial state of
glaucomatous damage and 0.63G0.04 at later, more
advanced stages. The increase in optic disc excavation
was not very prominent, if we compare with optic disc
excavation reported previously in monkey under experi
mental glaucoma (Weber and Zelenak, 2001). However, it
has been reported that topographic features of the optic disc
of patients of different races convey different information
with regard to assessing the risk of early glaucoma.
Therefore, it is possible that the changes in the optic disc
topography parameters associated with the elevation of IOP
are not indicative of the glaucomatous damage in the
porcine retina (Girkin et al., 2003).
4.1. Retinal ganglion cell loss in glaucomatous eyes
Elevation of IOP led to RGC loss, which was significant
in peripheral regions of the retina (the mid-periphery and the
peripheral retina). A greater loss of RGCs in the peripheral
retina of monkey and rat with experimental glaucoma has
previously been reported (Laquis et al., 1998; Vickers et al.,
1995). A more detailed analysis revealed that regions
located in the temporal quadrants showed a greater loss of
RGCs. This finding correlates with previous reports, in
which a greater loss of RGC fibres and loss of visual
function were reported for the temporal compared to the
nasal human retina (Fortune et al., 2002; Garway-Heath
et al., 2002; Takamoto and Schwartz, 2002).
4.2. Increase in the mean soma area of the remaining RGCs
We observed an increase in the mean area of RGC
somata in those regions of the retina, which presented
significant RGC loss. An increment in RGC mean soma
area, associated with a decrease in RGC density, has
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J. Ruiz-Ederra et al. / Experimental Eye Research 81 (2005) 561–569
previously been described to take place after experimental
insults affecting the retina (Rapaport and Stone, 1983;
Moore and Thanos, 1996). The same phenomenon has been
reported using the rat episcleral vein cauterisation glaucoma
model and the authors suggested that the increment of RGC
soma size intrinsically linked to soma density, was probably
a compensatory response to cell loss (Ahmed et al., 2001). It
is possible that the remaining RGCs are stimulated to
occupy the areas remaining after RGC death, increasing
their soma area and/or dendritic field, as described by other
authors (Perry and Linden, 1982; Kirby and Chalupa, 1986;
Ahmed et al., 2001).
Alternatively, the increase in RGC volume before death
could represent a loss of osmotic regulation, secondary to
RGC damage (Morgan et al., 2000). In contrast, a reduction
in the mean soma size of the remaining RGCs after
induction of experimental glaucoma in the monkey and in
the cat has been reported (Weber et al., 1998; Shou et al.,
2003). It is possible that the mean volume/area values of
glaucomatous RGCs are dependent on the stage at which the
disease is studied.
4.3. Selective RGC loss
Our analysis of RGC loss in terms of soma size points to a
selective death of large RGCs. This was especially evident in
the visual streak, in which a significant loss of large RGCs
was found. The fact that the mean area of RGCs located in the
visual streak did not increase after the experimental period
may possibly indicate that this region of the retina is
experiencing the initial stages of damage due to increased
IOP, while more advanced stages of tissue damage (and
increased mean RGC area) are evident in the peripheral
retina. RGC soma size increases with retinal eccentricity
(Dacey and Petersen, 1992; Yamada et al., 2001), indicating
that similar functional roles may be carried out by cells of
different sizes depending on their location within the retina.
The greater loss of large RGCs in the visual streak, which we
have observed, may indicate that alpha cells are more
sensitive to the increased IOP than other RGC types. Similar
to our findings in the porcine visual streak, a selective loss of
large RGCs in the human and monkey fovea has been
described during naturally occurring and experimentally
induced glaucoma (Asai et al., 1987; Glovinsky et al., 1993).
Selective death of large RGCs within the mid-peripheral
and peripheral retina was more difficult to evaluate, since
the size of surviving RGC soma within these retinal regions
increased after the period of elevation of IOP. Thus, in these
regions the death of large and/or medium-sized RGCs is
probably followed by a shift in cell size from small and
medium RGCs to medium and large RGCs in response to
the glaucomatous process. In the mid-periphery, we
observed a significant loss in number of large RGCs despite
the incorporation of previously medium-sized RGCs to this
size category. This observation supports the hypothesis of a
selective loss of large RGCs after glaucomatous damage.
The existence of a selective loss of large RGCs during
glaucoma is nonetheless controversial (Kalloniatis et al.,
1993; Graham et al., 1996; Morgan et al., 2000). Nevertheless, an increasing number of publications indicate that
large RGCs are the main type affected during naturally
occurring and experimentally induced glaucoma (Quigley
et al., 1988; Glovinsky et al., 1991, 1993; Vickers et al., 1995;
Anderson and O’Brien, 1997; Shou et al., 2003). The
extensive loss of large diameter axons (Quigley et al., 1987,
1988) and post-mortem studies of the lateral geniculate
nucleus of glaucoma patients or animals with experimentally
induced glaucoma (Dandona et al., 1991; Chaturvedi et al.,
1993; Weber et al., 2000) also point to a selective loss of large
RGCs.
5. Concluding remarks
The main goal of the present study was to evaluate the pig
eye as an experimental model of glaucoma. The pig eye was
chosen since it is similar in many respects to the human eye.
Moreover, the pig is an affordable animal and its use offers
few ethical problems. Additionally, in contrast to previous
studies involving the indirect analysis of monkey optic nerve
fibres or geniculate neurons using Nissl staining, in the
present study, we have directly analysed specifically labelled
RGCs with Fluoro-Gold. The advantage of using this novel
large mammal will be especially significant in experiments in
which a large number of animals would be required.
Acknowledgements
Grants from The Glaucoma Foundation, European
Community (QLK6-CT-2001-00385), Spanish Ministry of
Science and Technology (BFI 2003-07177) and the
University of the Basque Country (E-14887/2002;
15350/2003). MG holds an EC postdoctoral fellowship.
JRE holds a predoctoral fellowship from the UPV and from
the Jesus de Gangoiti Barrera Foundation MH holds a FPI
predoctoral fellowship. We want to sincerely acknowledge
Francisco Martı́n for his skillful help with the animals.
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