Rabbit retinal ganglion cell survival after optic nerve section and its effect on the inner plexiform layer

Structural modifications of the inner retina were studied after optic nerve section (ONS) in the rabbit. Retinal ganglion cells (RGC) were labelled by injection of Fast Blue into the optic nerve, and counted under fluorescent light in control retina and retina 7, 14, 21 and 26 days post-axotomy. Studies on retinal cross-sections were also performed. For this purpose, retinal sections were stained with haematoxylin-eosin and immunohystochemistry for alpha1 and beta2/beta3 sub-units of the GABA(A) receptors.One week after axotomy, there was no significant loss in the number of ganglion cells with respect to control counts (1086+/-173cellsmm(-2) in the visual streak and 119+/-46cellsmm(-2) in the periphery, mean+/-SD, n=5). At 14 days post-axotomy, 271+/-46cellsmm(-2) remained in the visual streak and 33+/-6cellsmm(-2) in the periphery, corresponding to a mean survival of 27%. The number of ganglion cells decreased further on the following days, reaching 7.54% 1 month after ONS. A significant reduction in the thickness of the inner plexiform and ganglion cell layers was also observed in retinal cross-sections. Immunocytochemical studies show a remarkable disorganization of the layer stratification in the inner plexiform layer (IPL). We conclude that after ONS, RGC death occurs mainly between 7 and 14 days post-axotomy and a progressive death up to 26 days, causing a decrease in the thickness of the IPL and subsequent disorganization of its layers.     

Nitric oxide synthase expression in ischemic rat retinas

PURPOSE: To investigate the expression of nitric oxide synthase (NOS) in the ischemic retina. METHODS: Retinal ischemia was induced in rats by bilateral common carotid artery occlusion (BCCAO) for various lengths of time. Using the retina after BCCAO, expression of neuronal NOS (nNOS) and inducible NOS (iNOS) and identification of their positive cells were studied by histological and immunohistochemical examinations. RESULTS: Histological examinations revealed significant reduction in the thickness of the inner plexiform layer and the outer plexiform layer of the retina. Expression of nNOS was detected in retinal ganglion cells, amacrine cells, and Müller cells after BCCAO. The expression of nNOS and iNOS detected in Müller cells became stronger and persisted long after BCCAO. CONCLUSIONS: In the ischemic retina, Müller cells and retinal ganglion cells expressed nNOS and iNOS. These phenomena may be involved in the ischemic damage to the retina.     

Quantitative confocal imaging of the retinal microvasculature in the human retina

Purpose. We investigated quantitatively the distribution of blood vessels in different neural layers of the human retina. Methods. A total of 16 human donor eyes was perfusion-fixed and labeled for endothelial f-actin. Retinal eccentricity located 3 mm superior to the optic disk was studied using confocal scanning laser microscopy. Immunohistochemical methods applied to whole-mount and transverse sections were used to colocalize capillary networks with neuronal elements. Capillary morphometry, diameter, and density measurements were compared among networks. Results. Four different capillary networks were identified and quantified in the following regions: Nerve fiber layer (NFL), retinal ganglion cell (RGC) layer, border of the inner plexiform layer (IPL) and superficial boundary of the inner nuclear layer (INL), and boundary of the deep INL and outer plexiform layer. The innermost and outermost capillary networks demonstrated a laminar configuration, while IPL and deep INL networks displayed a complex three-dimensional configuration. Capillary diameter in RGC and IPL networks were significantly less than in other networks. Capillary density was greatest in the RGC network (26.74%), and was significantly greater than in the NFL (13.69%), IPL (11.28%), and deep INL (16.12%) networks. Conclusions. The unique metabolic demands of neuronal sub-compartments may influence the morphometric features of regional capillary networks. Differences in capillary diameter and density between networks may have important correlations with neuronal function in the human retina. These findings may be important for understanding pathogenic mechanisms in retinal vascular disease.     

A histological analysis of the protective effect of ischemic preconditioning in the rat retina

PURPOSE: Ischemic preconditioning (IP) protects the retina from the damaging effect of subsequent ischemia in vivo. We aimed to investigate the histological alterations induced by the protective effect of IP to the retina. METHODS: The eyes of the rats were rendered ischemic by intra-ocular pressure (IOP) elevation. IP procedure consisted of producing ischemia for 5 minutes. Sham operation was similar to IP procedure except the pressure elevation. The operational eyes of sham and IP group underwent 60 minutes of ischemia 24 hours after the first procedure. The eyes contralateral to the experimental eyes made up the control group. The eyes were histologically analysed one week after the ischemia. RESULTS: The total retinal thickness of the sham group was significantly less than total retinal thickness of the control group (p < 0.001). There was not a significant difference between control and IP group regarding the total retinal thickness (p > 0.05). The thickness of the inner retinal layers of the sham group were significantly less than corresponding retinal layers of the control group (p < 0.001). The inner plexiform layer (IPL) and inner nuclear layer (INL) thickness values of the sham group were significantly less than same layers of the IP group (p < 0.001). Ganglion cell layer (GCL) thickness of the IP group was significantly less than GCL thickness of the control group (p < 0.001). IPL thickness of the IP group was significantly less than that of control group's (p < 0.05). The GCL and total retinal thickness of the IP group were significantly more than thickness of the corresponding layers of the sham group (p < 0.05). CONCLUSION: IP considerably protects inner retinal layers from subsequent ischemic damage in a high IOP ischemic model. This endogenous process could further be utilized to tailor specific neuroprotective strategies for retinal cells.     

Protective effects of catalase on retinal ischemia/reperfusion injury in rats

Retinal ischemia/reperfusion (I/R) injury causes profound tissue damage, especially retinal ganglion cell (RGC) death. The aims of the study were to investigate whether catalase (CAT) has a neuroprotective effect on RGC after I/R injury in rats, and to determine the possible antioxidant mechanism. Wistar female rats were randonmized into four groups: normal control group (Control group), retinal I/R with vehicle group (I/R with vehicle group), retinal I/R with AAV-CAT group (I/R with AAV-CAT group), and normal retina with AAV-CAT group (normal with AAV-CAT group). One eye of each rat was pretreated with recombinant adeno-associated virus containing catalase gene (I/R with AAV-CAT group or normal with AAV-CAT group) and recombinant adeno-associated virus containing GFP gene (I/R with vehicle group) by intravitreal injection 21 days before initiation of I/R injury. Retinal I/R injury was induced by elevating intraocular pressure to 100 mmHg for 1 h. The number of RGC and inner plexiform layer (IPL) thickness were measured by fluorogold retrograde labeling and hematoxylin and eosin staining at 6 h, 24 h, 72 h and 5d after injury. Hydrogen peroxide (H₂O₂), the number of RGC, IPL thickness, malondialdehyde(MDA), 8-hydroxy-2-deoxyguanosine (8-OHdG), CAT activity and nitrotyrosine were measured by fluorescence staining, immunohistochemistry and enzyme-linked immunosorbent assay analysis at 5 days after injury. Electroretinographic (ERG) evaluation was also used. Pretreatment of AAV-CAT significantly decreased the levels of H₂O₂, MDA, 8-OHdG and nitrotyrosine, increased the catalase activity, and prevented the reduction of a- and b- waves in the I/R with AAV-CAT group compare with the I/R with vehicle group (p < 0.01). Catalase attenuated the I/R-induced damage of RGC and IPL and retinal function. Therefore, catalase can protect the rat retina from I/R-induced injury by enhancing the antioxidative ability and reducing oxidative stress, which suggests that catalase may be relevant for the neuroprotection of inner retina from I/R-related diseases.     

视细胞移植术后RCS鼠视网膜谷氨酸的分布

目的 通过纯视细胞移植,观察重建视网膜神经传导通路中兴奋性神经递质谷氨酸的分布和变化。方法取同龄Wistar鼠和皇家外科学院鼠(RCS)为供／受体,准分子激光切削法制备视细胞层,外路途径移入RCS鼠的视网膜下腔,术后两周取RCS鼠手术眼,手术对照眼,RCS对照眼,Wistar鼠眼。分别做冰冻切片,免疫组织化学染色法染色,普通光学显微镜下观察。结果在视细胞移植术后两周,移植视细胞存活,外丛状层重建,与手术对照眼、RCS对照眼相比,受体RCS鼠重建视网膜中在内丛状层和节细胞层Wistar鼠谷氨酸免疫反应阳性的神经纤维相对密度增多。结论视细胞移植不仅可以使RCS鼠视网膜重建正常的解剖结构,而且在移植后的视网膜中观察到兴奋性神经递质谷氨酸的分布接近正常。     

腺病毒载体介导的色素上皮衍生因子对大鼠视网膜缺血再灌注损伤的保护作用

目的:观察重组腺病毒介导的色素上皮衍生因子(Ad-PEDF)对大鼠视网膜缺血再灌注损伤的保护作用及机制。方法:选用健康大鼠96只,随机分为正常组、缺血再灌注组、缺血再灌注+Ad-CMV组,缺血再灌注+Ad-PEDF组,以前房加压的方法制备大鼠视网膜缺血再灌注模型,缺血再灌注+Ad-CMV组,缺血再灌注+Ad-PEDF组分别玻璃体腔注射Ad-CMV或Ad-PEDF1μL(滴度3.8×109/PFU),每组按照时间点12,24,72,168h,为4亚组,光学显微镜观察视网膜组织切片情况,并测量视网膜内层厚度及神经节细胞层神经节细胞数量。以TUNEL方法观察大鼠视网膜神经节细胞凋亡情况。结果:Ad-PEDF组视网膜内层厚度均超过缺血组及缺血+Ad-CMV组,Ad-PEDF组神经节细胞数目多于缺血组及Ad-CMV组,Ad-PEDF组视网膜神经节细胞凋亡细胞少于缺血组及Ad-CMV组,凋亡程度减轻,上述差异均具有显著性(P<0.05)。结论:腺病毒介导的色素上皮衍生因子玻璃体腔注射能够恢复大鼠视网膜缺血再灌注损伤所致的视网膜内层厚度降低,神经节细胞密度减少,具有保护作用。     

金雀异黄酮对大鼠视网膜缺血-再灌注损伤的保护作用

目的　探讨金雀异黄酮（GEN）对大鼠视网膜缺血-再灌注（I/R）损伤的保护作用。方法　选取30只健康SPF级大鼠，按照随机数字表法将其分为假手术组、I/R组和I/R+GEN组，每组各10只。I/R组和I/R+GEN组大鼠采用前房灌注生理盐水升高眼压法制备视网膜I/R损伤大鼠模型，I/R组大鼠术后每天给予注射生理盐水（1 mL·kg^-1·d^-1）；I/R+GEN组大鼠术后每天给予注射GEN （40 mL·kg^-1·d^-1）；假手术组大鼠行相应假手术后每天予以注射生理盐水（1 mL·kg^-1·d^-1），连续7 d后，颈椎脱臼法处死各组大鼠，取眼部组织。HE染色观察各组大鼠视网膜的形态以及内丛状层（IPL）、内核层（INL）和神经节细胞层（GCL）厚度；免疫荧光分析观察各组大鼠视网膜神经节细胞（RGC）的存活率；TUNEL染色检测各组大鼠视网膜细胞凋亡水平；检测各组大鼠视网膜组织中过氧化氢酶（CAT）、丙二醛(MDA)及超氧化物歧化酶(SOD)水平以反映视网膜氧化应激状态；Western blot分析三组大鼠视网膜中NLRP3、ASC、Caspase-1的蛋白表达。结果　I/R组、I/R+GEN组和假手术组大鼠视网膜总厚度分别为(114.37±7.32）μm、（155.31±6.83）μm和（178.98±13.65）μm（t=14.284，P<0.01），I/R组低于假手术组和I/R+GEN组，差异均有统计学意义（均为P<0.01）。I/R组大鼠视网膜IPL、INL和GCL厚度分别为（18.95±5.06）μm、（17.62±4.69）μm和（19.03±4.74）μm，I/R+GEN组大鼠视网膜IPL、INL和GCL厚度分别为（20.69±8.13）μm、（25.74±6.78）μm和（26.71±7.85）μm，假手术组大鼠视网膜IPL、INL和GCL厚度分别为（11.73±3.15）μm、（11.97±3.56）μm和（12.59±3.24）μm（均为P<0.05），I/R组与假手术组和I/R+GEN组三个指标比较差异均有统计学意义（均为P<0.01）。I/R组、I/R+GEN组和假手术组大鼠视网膜中RGC相对存活率分别为（26.87±3.12）%、（73.46±7.80）% 和（100.00±5.64）%（t=16.825，P<0.01），I/R组RGC相对存活率低于假手术组和I/R+GEN组，差异均有统计学意义（均为P<0.01）。I/R组、I/R+GEN组和假手术组大鼠视网膜中CAT、MDA、SOD含量总体比较差异均有统计学意义（均为P<0.01），I/R组与I/R+GEN组和假手术组三个指标比较差异均有统计学意义（均为P<0.05）。Western blot检测结果显示，三组大鼠视网膜中NLRP3、ASC、Caspase-1的蛋白表达总体比较差异均有统计学意义（均为P<0.01），组间两两比较差异均有统计学意义（均为P<0.05）。结论　GEN可通过提高视网膜细胞抗炎和抗氧化能力抑制视网膜细胞凋亡，从而起到对大鼠视网膜I/R损伤的保护作用。     

Interleukin-6 in retinal ischemia reperfusion injury in rats

PURPOSE: To study the role of interleukin (IL)-6 after retinal ischemia-reperfusion (I/R) injury in rats. METHODS: Intraocular pressure of adult male Lewis albino rats was raised to create retinal ischemia for 1 hour. Retinal reperfusion was reestablished, and the animals were killed at various time points after the injury. Their eyes were enucleated and processed for immunohistochemistry to detect IL-6 and ED-1 (a marker of microglial/phagocytic cells), enzyme-linked immunosorbent assay (ELISA) of IL-6 protein, and semiquantitative real-time RT-PCR for IL-6 mRNA. The neuroprotective effect of IL-6 was evaluated by giving intravitreal injections of 150 or 300 ng rat recombinant IL-6 to eyes immediately after I/R injury and counting cresyl violet-stained retinal ganglion cell layer cells (RGCLCs) and fluorochrome-labeled retinal ganglion cells (RGCs) on flat preparations of retinas at 7 days. RESULTS: IL-6-positive cells appeared after I/R injury in the inner plexiform layer (IPL) and the inner nuclear layer (INL). Their numbers were significantly higher 18 hours after the injury, and most of these cells were also ED-1 positive. ELISA showed noticeable increases in endogenous retinal IL-6 protein levels 8 hours after I/R injury. Semiquantitative real-time RT-PCR showed significant increases in endogenous retinal IL-6 mRNA levels between 2 and 18 hours. Exogenously added IL-6 prevented between 50% and 70% of RGC loss after I/R injury. CONCLUSIONS: IL-6 is upregulated after retinal I/R injury, and its expression by microglia/phagocytic cells may protect RGC layer neurons from I/R injury. Exogenously added IL-6 protects the inner retina after I/R injury.     

The structural effect of intravitreal Brilliant blue G and Indocyanine green in rats eyes

Purpose

To compare the potential retinal toxicity of two commercially Brilliant blue G dyes (Brilliant Peel and Ocublue Plus) and Indocyanine green (ICG) at usual clinical concentration.

Methods

Brilliant Peel 0.025% (n=9), Ocublue Plus 0.025% (n=9), and ICG 0.05% (n=9) were injected intravitreally into Sprague–Dawley rat left eyes with balanced salt solution injected in the contralateral eyes as control. Evaluation of the effect of the dyes on retinal architecture was done by histological analysis of neurosensory retinal thickness and retinal ganglion cell (RGC) counts 7 days after intravitreal injection. Paired t-test was done to detect the presence of biologically significant thinning in neurosensory retina and five retinal layers for each dye (paired t-tests). One-way ANOVA and Tukey''s Honestly Significant Difference test were used to assess whether different dyes caused significant thinning in mean neurosensory retinal thickness and reduction of mean RGC density.

Results

Eyes treated with ICG had significantly thinner mean total neurosensory retinal thickness compared with the control eyes (P-value=0.01), followed by those treated with Ocublue Plus (P-value=0.03). Brilliant Peel did not cause significant thinning in any of the five retinal layers (all P-values>0.05). No significant difference in mean thinning of the total retinal thickness was detected between dyes (P-value=0.11). The mean thickness of the photoreceptor outer segment and outer plexiform layers were significantly reduced in ICG-injected eyes when compared with the control eyes (P-value=0.02). No significant difference in mean thinning between the three dyes was detected at all five retinal layers using one-way ANOVA (all P-values>0.35). RGC density was significantly reduced for ICG (P-value=0.01) but only marginally for Ocublue Plus (P-value=0.05). No significant reduction in RGC density was observed for Brilliant Peel (P-value=0.2).

Conclusion

Intravitreal Brilliant Peel is safe to rats retina. The retinal thinning and reduction in RGC density induced by Ocublue Plus requires further studies to determine the safety profile of this product. Potential retinal toxicity is seen with ICG 0.05%.     

Complex neurodegeneration in retina following moderate ischemia induced by bilateral common carotid artery occlusion in Wistar rats

Bilateral common carotid artery occlusion (BCCAO) produces moderate levels of ischemia in the retina of rats, which may simulate the inflow disturbances in severe carotid artery disease. ERG changes following acute BCCAO have been well described, but the effects of chronic BCCAO on the histopathology of the retina remain to be characterized in a reproducible model. Chronic BCCAO was induced in halothane-anaesthetized male Wistar rats and the retina fixed after 3, 6, or 24 hr, 1 week, and 2, 4, or 6 months. Cell counts and measurements of retinal layers were performed in H&E stained paraffin sections. Immunohistochemistry with a panel of fourteen antibodies served to examine the survival of different retinal cell class, astrocytic reactions and the expression of acute stress response proteins. A lectin method was used to label activated microglial cells. Microglial activation, heme oxygenase-1 upregulation and caspase-3 cleavage occurred during the first 24hr in the absence of overt cell death of retinal ganglion cells (RGC). Three waves of neurodegeneration followed. RGCs were affected after 1 week, followed by neurons in the inner nuclear layer at 2 months, and finally photoreceptors at 4 months. Immunomarkers indicated acute damage to horizontal cells and prolonged survival of amacrine cells. In conclusion, chronic BCCAO produced delayed neuronal death in the retina of adult male Wistar rats. The window of moderate changes of at least 1 day may facilitate molecular studies on retinal ganglion cell loss.     

Long-term effect of retinal ganglion cell axotomy on the histomorphometry of other cells in the porcine retina

PURPOSE: To determine the effect of retinal ganglion cell axotomy on the thickness of inner plexiform, inner nuclear, and outer plexiform layers, as well as the densities of short- and middle-to-long-wavelength cones, in the porcine retina. METHODS: Unilateral retinal ganglion cell axotomy was performed in seven domestic pigs by either surgical optic nerve section or peripapillary argon laser photocoagulation. Damage to the retinal vasculature was ruled out with fluorescein angiography. Histologic examination of the retinal tissue was performed nine months later. Cone densities were determined immunohistochemically with the anti-visual pigment antibodies COS-1 and OS-2. Image analysis of semithin retinal cross sections was used to measure the thickness of the retinal layers. The effect of axotomy was quantified by optic nerve axon counts and estimations of retinal ganglion cell counts. The data were compared between the eyes with axotomy and the contralateral normal eye using the nonparametric Wilcoxon rank sum test. RESULTS: Treatment of the peripapillary retina with the argon laser resulted in a median decrease in axon counts and retinal ganglion cell density estimates of 31%. No optic nerve axons and cells resembling retinal ganglion cells were found in the eyes with transected optic nerves. There was no significant difference in either the thickness of any retinal layers or cone densities between axotomized and normal control eyes. CONCLUSION: No signs of retrograde transsynaptic degeneration were observed in porcine retinas nine months after retinal ganglion cell axotomy.     

Effect of gestational nicotine treatment on newborn rat retina: a histopathological and morphometric analysis

BACKGROUND: Smoking is a significant risk factor in several debilitating and fatal diseases. It has been implicated in bilateral tobacco-toxic and Leber's hereditary optic neuropathies. Although it has been demonstrated that smoking has a cumulative effect on retinal and optic nerve functions and causes diffuse and localised retinal sensitivity decrease in healthy chronic heavy smokers, the affected retinal layer has not been identified and there is no experimental study investigating the effect of nicotine exposure during gestation on the newborn rat retina. PURPOSE: This experimental investigation evaluated histologically the influence in vivo of maternal nicotine treatment during pregnancy on the newborn rat retina. Different dosages of the test compound simulated the range of low, moderate, and heavy smokers in humans. METHODS: Experimentally naive, adult female Wistar-albino rats weighing 200-250 g were mated with adult male rats over 2 days for copulation in the proportion of two females for every male animal. After confirming pregnancy with vaginal smear method, 40 gravid rats (dams) were then randomly assigned into four equal groups (three experimental and one control; n = 10 in each). On day 9 of gestation, groups 1, 2, and 3 experimental dams were treated with intraperitoneal (i.p.) (-)-nicotine tartrate at doses of 0.5, 1, and 2 mg kg body weight-1 day-1, respectively during pregnancy from gestational day 9-21. Group 4 control dams were given i.p. saline solution daily for the same period. After normal delivery, the newborn litters were sacrificed at postnatal day 1 or day 30. The eyes were enucleated for histopathologic and morphometric analysis of the retinas. Nicotine-induced neuronal changes were measured by morphometric analyses on cell counts of ganglion cell layer (linear cell density in number per unit length of retina) and thickness of the various retinal layers. RESULTS: The litters in control group 4, and experimental groups 1 and 2 had normal retinal findings. On the other hand, morphometric analysis of retinal sections in experimental group 3 eyes demonstrated a 20.7% decrease in the number of surviving ganglion cells (40.7 +/- 2.0) compared with controls (51.3 +/- 1.1; p < 0.001). The thickness of whole retina (126.6 +/- 5.4 microm) was also reduced by 13.5% compared with controls (146.3 +/- 4.5 microm; p = 0.007). The main site of retinal atrophy was the inner plexiform layer (30.1 +/- 1.6 microm vs 43.5 +/- 1.3 microm; p < 0.001) with almost no change in the other retinal layers. CONCLUSIONS: Gestational nicotine treatment induces marked changes in the organisation of the developing retina in newborn rats histopathologically. Quantitative morphometric analysis clearly demonstrated that the two most affected structures were the retinal ganglion cells and the inner plexiform layer, both of which are supplied by central retinal artery.     

A model to study differences between primary and secondary degeneration of retinal ganglion cells in rats by partial optic nerve transection

PURPOSE: To use a rat model of optic nerve injury to differentiate primary and secondary retinal ganglion cell (RGC) injury. METHODS: Under general anesthesia, a modified diamond knife was used to transect the superior one third of the orbital optic nerve in albino Wistar rats. The number of surviving RGC was quantified by counting both the number of cells retrogradely filled with fluorescent gold dye injected into the superior colliculus 1 week before nerve injury and the number of axons in optic nerve cross sections. RGCs were counted in 56 rats, with 24 regions examined in each retinal wholemount. Rats were studied at 4 days, 8 days, 4 weeks, and 9 weeks after transection. The interocular difference in RGCs was also compared in five control rats that underwent no surgery and in five rats who underwent a unilateral sham operation. It was confirmed histologically that only the upper optic nerve had been directly injured. RESULTS: At 4 and 8 days after injury, superior RGCs showed a mean difference from their fellow eyes of -30.3% and -62.8%, respectively (P = 0.02 and 0.001, t-test, n = 8 rats/group), whereas sham-operation eyes had no significant loss (mean difference between eyes = 1.7%, P = 0.74, t-test). At 8 days, inferior RGCs were unchanged from control, fellow eyes (mean interocular difference = -4.8%, P = 0.16, t-test). Nine weeks after transection, inferior RGC had 34.5% fewer RGCs than their fellow eyes, compared with 41.2% fewer RGCs in the superior zones of the injured eyes compared with fellow eyes. Detailed, serial section studies of the topography of RGC axons in the optic nerve showed an orderly arrangement of fibers that were segregated in relation to the position of their cell bodies in the retina. CONCLUSIONS: A model of partial optic nerve transection in rats showed rapid loss of directly injured RGCs in the superior retina and delayed, but significant secondary loss of RGCs in the inferior retina, whose axons were not severed. The findings confirm similar results in monkey eyes and provide a rodent model in which pharmacologic interventions against secondary degeneration can be tested.     

Effects of acute delivery of endothelin-1 on retinal ganglion cell loss in the rat

The vasoconstrictive peptide, Endothelin-1 (ET-1) has been found at elevated levels in glaucomatous eyes. In this study, a single 5mul intraocular injection of ET-1 was injected into the rat eye in order to characterize an in vivo retinal ganglion cell (RGC)-specific cell death model. The most effective concentration of ET-1 at inducing RGC loss at 2 weeks post-injection was determined using 5, 50 and 500mum concentrations of ET-1. The density of surviving RGCs was determined by counting Fluorogold labelled RGCs. A significant loss (25%) of RGCs was observed using only the 500mum concentration when compared to PBS-injected controls. GFAP immunohistochemistry revealed an increase in GFAP expression in Müller cell end-feet, as well as a total increase in GFAP expression (80%), following ET-1 treatment. These changes in GFAP expression are indicative of glial hyperactivity in response to stress. The specificity of ET-1 mediated cell death for RGCs was determined by measuring the changes in retinal thickness and TUNEL labeling. Retinal thickness was quantified using confocal and light microscopy. In confocal measurements, Yo Pro-1 was used to stain nuclear layers and the thickness of retinal layers determined from reconstructions. No significant loss in thickness was observed in any retinal layers. The same observations were seen in semi-thin sections when viewed by conventional transmitted light microscopy. The lack of significant thickness changes in the outer nuclear, outer plexiform or inner nuclear layer suggests that there was no significant cell loss in the retina other than in the RGC layer. Exclusive co-localization of TUNEL-labelled nuclei with Fluorogold-labelled cytoplasm provided additional evidence for RGC-specific death that most likely occurs via an apoptotic mechanism. A cell death time course was performed to determine RGC loss over time. RGC losses of 25, 25, 36 and 44% were observed at 1, 2, 3 and 4 weeks post-ET-1 injection, compared to PBS-injected controls. The total number of remaining RGC axons was determined by multiplying the number of optic nerve (ON) axons per unit area, by the cross-sectional area. There was a 31% loss in total ON axons in ET-1 treated eyes at 3 weeks post injection. Functional integrity of the visual system was determined by observing changes in the pupillary light reflex. ET-1 treatment resulted in a slowing of the pupil velocity by 31% and an average increase in the duration of contraction of 1.85sec (32% increase). These experiments provide evidence that acute ET-1 injections can produce RGC-specific cell death and many cellular changes that are similar to glaucoma. This potential glaucoma model leaves the optic nerve intact and may be used in subsequent experiments, which are involved in increasing RGC survival and functional recovery.     

急性高眼压大鼠视网膜RhoA的分布及表达

目的:探讨急性高眼压大鼠视网膜RhoA的分布及表达变化。方法:将56只SD大鼠随机分成正常对照组、急性高眼压后1,3,7d共4组,用免疫组化和半定量RT-PCR方法检测大鼠视网膜组织中的RhoA的分布及表达情况。结果:正常及急性高眼压后1d,RhoA主要分布于视网膜神经纤维层及神经节细胞层;3d分布于神经节细胞层及内丛状层;7d分布于神经节细胞层、内丛状层、内核层及外丛状层。半定量RT-PCR提示:在正常大鼠视网膜组织中,RhoA mRNA仅有微量表达,急性高眼压后1,3,7d,RhoA表达均显著高于正常组(P<0.05),7d组表达量最高。结论:大鼠急性高眼压损伤后,视网膜RhoA蛋白的分布及表达显著增加,其在介导抑制性信号阻断轴突再生的抑制过程中发挥着重要作用。     

视细胞移植术后RCS鼠视网膜 γ-氨基丁酸的分布观察

目的通过纯视细胞移植, 观察重建视网膜神经传导通 路中抑制性神经递质γ氨基丁酸(γ-Aminobutyric Acid,GABA)的分布和变化。方法取同龄Wistar/皇家外科学院鼠(royal college of surgeon rat,RCS)为供/受体，准分子激光切削法制备视细胞层，外路途径移入RCS鼠的视网膜下腔，术后2周取RCS鼠手术眼，手术对照眼，RCS鼠对照眼，Wistar鼠眼。分别做冰冻切片，免疫组织化学染色法染色，普通光学显微镜下观察。测量内丛状层(inner plexiform layer,IPL)和节细胞层(ganglion cell layer,GCL)的光密度，计数无长突细胞的数量， 方差分析组间差异,配对t检验RCS鼠手术眼组内差异。结果在视细胞移植术后2周，移植视细胞存活，外丛状层重建 ，与手术对照眼，RCS对照眼相比，受体RCS鼠重建视网膜中内丛状层GABA免疫反应阳性 的神经纤维光密度测量值差异有显著性的意义 P < 0．001，而与Wi star鼠眼比较无明显区别。和手术对照眼，RCS对照眼，Wistar鼠组比，GABA能无长突细胞 (amacrine cell)明显增多(P<0．05)。节细胞层GABA阳性免 疫反应光密度测量值和上述几组比变化不明显(P>0．05)。接受移植的RCS鼠各部位的无长突细胞在移植部二侧和对侧赤道，后极部之间的差异无显著性 的意义( P = 0．3436)。结论视细胞移植不仅可以使RCS鼠视网膜重建正常的解剖结构，而且在移植后的视网膜中观察到抑制性神经递质GABA的分布接近正常，无长突细胞反应性增生。(中华眼底病杂志,2001,17:128-131)     

Expression of kynurenine aminotransferases in the rat retina during development

The study investigates the cellular expression of kynurenine aminotransferases (KAT I and II) in the rat retina during development. At P1 (the day of birth) and P7 (the 7th day after birth), KAT I expression was observed in the inner plexiform layer (IPL), the fiber layer (FL), and in vertically running processes in the ganglion cell layer (GCL) (but not in the cell bodies). At P14 (the 14th day after birth) a strong KAT I immunoreactivity was observed in Müller cell endfeet. KAT II was expressed in the IPL, the FL, and in cells in the GCL at P1 and P7. From P14 on, KAT II expression in the IPL decreased. Double labeling revealed that KAT I was expressed in Müller cell endfeet, whilst KAT II both on retinal ganglion cells (RGC) and Müller cell endfeet. In conclusion, KAT I and II are present in the rat retina during development. The heterogeneity of the KAT developmental profiles possibly reflects a neuromodulatory role in the retinal differentiation.     

Neuroprotective effect of citicoline on retinal cell damage induced by kainic acid in rats

PURPOSE: To examine whether citicoline has a neuroprotective effect on kainic acid (KA)-induced retinal damage. METHODS: KA (6 nmol) was injected into the vitreous of rat eyes. Citicoline (500mg/kg, i.p.) was administered to the rats once before and twice a day after KA-injection for 3- and 7-day intervals. The neuroprotective effects of citicoline were estimated by measuring the thickness of the various retinal layers using hematoxylin-eosin (H&E) staining. In addition, immunohistochemistry was conducted to elucidate the expression of endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS). RESULTS: Morphometric analysis of retinal damage in KA-injected eyes showed significant cell loss in the inner nuclear layer (INL) and inner plexiform layer (IPL) of the retinas at 3 and 7 days after KA injection, but not in the outer nuclear layer (ONL). At 3 days after citicoline treatment, no significant changes were detected in the retinal thickness and immunoreactivities of eNOS and nNOS. The immunoreactivities of eNOS and nNOS increased in the retina at 7 days after the KA injection. However, prolonged treatment for 7 days significantly attenuated the immunoreactivities and the reduction of thickness. CONCLUSIONS: The results indicate that citicoline has a neuroprotective effect on KA-induced neurotoxicity in the retina.     

Neuroprotective Effects of Granulocyte Colony-Stimulating Factor on Ischemia-Reperfusion Injury of the Retina

Purpose: It has been reported that granulocyte colony-stimulating factor (G-CSF) provides neuroprotection in models in which neuronal cell death is induced. This research was designed to investigate the effects of G-CSF on neurodegeneration of the inner retinal layer in a rat model of ischemic reperfusion (I/R) injury. Materials and Methods: Retinal ischemia was induced by increasing the intraocular pressure to 110 mm Hg for 45 min in the left eyes of the rats. A sham operation was carried out on the right eyes. G-CSF (100 μg/kg/day in 0.3 ml saline) or the same volume of saline was intraperitoneally injected just before the operation and continued for 4 consecutive days (a total of 5 consecutive days). Morphological examinations, including the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, were performed 7 days after I/R induction. The expression of phosphorylated AKT in the retina was examined by Western blot analysis and immunohistochemistry. Results: Cell loss in the ganglion cell layer was more significantly reduced in the I/R-induced eyes of the G-CSF-injected rats than in the I/R-induced eyes of the saline-injected rats (20.3 vs. 6.6%). The inner retinal thickness ratios, such as the inner plexiform layer to the inner limiting membrane/outer nuclear layer and the inner nuclear layer/outer nuclear layer, were significantly better preserved in the I/R-induced eyes of the G-CSF-injected rats than in the I/R-induced eyes of the saline-injected rats. TUNEL assays showed fewer apoptotic cells in the retinal sections of the I/R-induced eyes of the G-CSF-injected rats. The phosphorylation of AKT (p-AKT/AKT) was upregulated in the retinas of the I/R-induced eyes of the G-CSF-injected rats. Conclusion: Our results demonstrated that systemic injection of G-CSF can protect retinal ganglion cells and inner retinal layers from I/R injury. The effects could be associated with the activation of AKT.