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