Characteristics of Optic Nerve Damage Induced by Chronic Intraocular Hypertension in Rat

Purpose:To set up the Sharma‘s chronic intraocular hypertension model and investigate the intraocular pressure (IOP) as well as the optic nerve damage of this model in rat.Methods :The operations of the chronic intraocular hypertension model were performed as described by Sharma in 60 male Lewis albino rats. IOP was measured using the TonoPen XL immediately after surgery and then at 5 day, 2 week or 4 week intervals. Cresyl violet staining of whole-mounted retinas was used to label retinal ganglion cells (RGCs),then RGCs were counted. Paraphenylenediamine (PPD) staining was performed in the semi-thin cross sections of optic nerve of rat, in order to know whether the axons of optic nerve were degenerated or not.Results:There were 47 rats with higher IOP after the episcleral veins cauterized in 60 rats. The ratio of elevated IOP was 78.3%. The IOPs were stable in 4 weeks. After cresyl violet staining, the RGCs loss was 11.0% and 11.3% was found in the central and peripheral retina respectively after 2 weeks of increased IOP. After 4 weeks of increased IOP, the loss of RGCs was 17% for the central retina and 24.6% for the peripheral retina. In the retinas without higher IOP, there was no loss of RGCs. PPD staining showed that optic nerve of rat with about 5.3% damage of axons located at the superior temporal region. Region of affected optic nerve 1 mm posterior to the globe by light microscope showed evidence of damaged axons with axonal swelling and myelin debris.Conclusion:Sharma‘s chronic intraocular hypertension model is a reproducible and effective glaucoma model, which mimics human glaucoma with chronically elevation IOP and induced RGCs loss and damage of optic nerve.     

Survival of axotomized retinal ganglion cells in peripheral nerve-grafted ferrets.

PURPOSE: Peripheral nerve (PN) grafting to the optic nerve stump stimulates not only axonal regeneration of the axotomized retinal ganglion cells (RGCs) into the grafted PN but also their survival. The purpose of the present study was to determine the number, distribution, and soma diameter of only surviving RGCs without regenerated axons and surviving RGCs with regenerated axons in PN-grafted mammals. METHODS: A segment of PN was grafted to the optic nerve stump of adult ferrets. Two months after the PN grafting, surviving RGCs with regenerated axons were retrogradely labeled with granular blue (GB) and stained with RGC-specific antibody C38. Surviving RGCs without regenerated axons were identified as C38-positive cells without GB labeling. RESULTS: Twenty-one percent of RGCs survived axotomy after PN grafting in the area centralis (AC), whereas 47% survived in the peripheral retina. Twenty-six percent of surviving RGCs in the AC exhibited axonal regeneration, which was higher than that in the peripheral retina. Soma diameter histograms revealed that RGCs with regenerated axons showing both GB and C38 positivity were in the large soma diameter ranges. In contrast, the soma diameter distribution of surviving RGCs that did not have regenerated axons showed a peak in the smaller soma diameter ranges. CONCLUSIONS: The present data suggest that PN grafting promotes survival of axotomized RGCs more effectively in the peripheral retina than in the AC. Among surviving RGCs, the larger cells exhibited axonal regeneration into the grafted PN, whereas the axons of smaller cells did not to regenerate in either the AC or the peripheral retina.     

Ganglion cells in the juvenile chick retina and their ability to regenerate axons in vitro.

Ganglion cells in the chicken retina fail to regenerate their axons upon mechanical injury. In order to determine whether this failure to regenerate axons is intrinsic to the neurons or is mediated by the environment, we asked whether ganglion cells possess an ability to regrow their injured axons in the absence of their natural environment, namely in vitro. Since the retina contains morphologically different types of ganglion cells, it became desirable to investigate whether all types of ganglion cells contribute to regeneration of axons. Ganglion cells were labelled post-mortem with the fluorescent dye DiI and described morphologically. Morphometric parameters like the sizes of their perikarya, their dendrites, and the patterns of dendritic ramification and stratification were considered for grouping cells. Although a strong classification of the cells could not be achieved because of the high diversity among this population of neurons, the chick retinal ganglion cells could be separated into seven regular groups which have their somata located within the ganglion cell layer and into one group with the somata located within the inner nuclear layer (displaced ganglion cells). The experimental procedure for regeneration combines crush injury of the chick optic nerve in situ with explanation of retinal pieces 1 week later for organ cultures in a serum-free medium. Under these conditions, the ganglion cells extended axons 1 day after explanation on polylysine/laminin. The densities of ganglion cells contributing to the axonal regrowth reached up to 1447 cells mm-2 (mean 1028 cells mm-2; S.D. 237). This density corresponds to 13% of the ganglion cell density in the normal retina, averaged across the total retina area. Although the dendrites of some cells whose axons had regenerated were altered in comparison with the normal ganglion cells, all morphological types of ganglion cells including those with displaced cell bodies contributed proportionally to the regrowth of axons from the explants.     

Optic nerve head axonal transport in rabbits with hereditary glaucoma

Rabbits with hereditary glaucoma develop ocular changes that resemble human congenital glaucoma and buphthalmia. The inheritance is autosomal recessive (bu). Previous research was performed primarily on albino bu/bu rabbits that were unhealthy and bred poorly. We have bred pigmented bu/bu rabbits to determine if this would improve hardiness and provide a better model for the disease in humans. First-generation offspring from matings of bu/bu albino with bu/bu pigmented rabbits were all affected, indicating that the bu gene is found at the same locus in both strains. The pigmented bu/bu offspring had a high degree of mortality, as reported previously for albino bu/bu rabbits. Newborn bu/bu rabbits initially had normal intraocular pressure (IOP; 15-23 mmHg); after 1- to 3 months, the IOP increased to 26-48 mmHg. The eyes became buphthalmic and the IOP returned to normal or sub-normal levels after 6-10 months. Since the lamina cribrosa is absent or poorly formed in the rabbit optic nerve head (ONH), this model was used to test the role of mechanical factors in the etiology of ONH pathology caused by increased IOP. Orthograde axonal transport was evaluated in both eyes from eight normal and 24 bu/bu rabbits of different ages, using intravitreal injections of [3H]leucine to mark orthograde axonal transport, followed by light- and electron-microscopic radioautography of the ONHs and superior colliculi. Normal rabbits of all ages showed no blockage of axonal transport in the ONH. All optic axons from young bu/bu rabbits with normal IOP and most axons from older buphthalmic rabbits that previously had elevated IOP were normal morphologically. Small zones of transport blockage occurred in bu/bu eyes while IOP was elevated; most affected axons lay immediately adjacent to ONH connective tissue beams that radiate outward from the central retinal vessels to the optic-nerve sheath. Thus, the rabbit, which lacks a true lamina cribrosa, does not show marked blockage of axonal transport as occurs in the LS of the monkey and cat ONH when IOP is elevated acutely. This anatomic difference appears to be protective against axonal damage, since bu/bu rabbits with chronic IOP elevation did not show significant loss of optic axons. These results are consistent with the proposed 'mechanical' theory of ONH damage resulting from increased IOP. Electron-microscopic radioautography revealed that chronically elevated IOP in bu/bu rabbits, which caused small foci of blocked ONH axonal transport against ONH beams, also caused degeneration of a few optic nerve terminals in the superior colliculi as the disease progressed.(ABSTRACT TRUNCATED AT 400 WORDS)     

Retinal ganglion cell numbers and delayed retinal ganglion cell death in the P23H rat retina

The P23H-1 rat strain carries a rhodopsin mutation frequently found in retinitis pigmentosa patients. We investigated the progressive degeneration of the inner retina in this strain, focussing on retinal ganglion cells (RGCs) fate. Our data show that photoreceptor death commences in the ventral retina, spreading to the whole retina as the rat ages. Quantification of the total number of RGCs identified by Fluorogold tracing and Brn3a expression, disclosed that the population of RGCs in young P23H rats is significantly smaller than in its homologous SD strain. In the mutant strain, there is also RGC loss with age: RGCs show their first symptoms of degeneration at P180, as revealed by an abnormal expression of cytoskeletal proteins which, at P365, translates into a significant loss of RGCs, that may ultimately be caused by displaced inner retinal vessels that drag and strangulate their axons. RGC axonal compression begins also in the ventral retina and spreads from there causing RGC loss through the whole retinal surface. These decaying processes are common to several models of photoreceptor loss, but show some differences between inherited and light-induced photoreceptor degeneration and should therefore be studied to a better understanding of photoreceptor degeneration and when developing therapies for these diseases.     

Neuroprotection of the Inner Retina Also Prevents Secondary Outer Retinal Pathology in a Mouse Model of Glaucoma

PurposeWe examined structural and functional changes in the outer retina of a mouse model of glaucoma. We examined whether these changes are a secondary consequence of damage in the inner retina and whether neuroprotection of the inner retina also prevents outer retinal changes.MethodsWe used an established microbead occlusion model of glaucoma whereby intraocular pressure (IOP) was elevated. Specific antibodies were used to label rod and cone bipolar cells (BCs), horizontal cells (HCs), and retinal ganglion cells (RGCs), as well as synaptic components in control and glaucomatous eyes, to assess structural damage and cell loss. ERG recordings were made to assess outer retina function.ResultsWe found structural and functional damage of BCs, including significant cell loss and dendritic/axonal remodeling of HCs, following IOP elevation. The first significant loss of both BCs occurred at 4 to 5 weeks after microbead injection. However, early changes in the dendritic structure of RGCs were observed at 3 weeks, but significant changes in the rod BC axon terminal structure were not seen until 4 weeks. We found that protection of inner retinal neurons in glaucomatous eyes by pharmacological blockade of gap junctions or genetic ablation of connexin 36 largely prevented outer retinal damage.ConclusionsTogether, our results indicate that outer retinal impairments in glaucoma are a secondary sequalae of primary damage in the inner retina. The finding that neuroprotection of the inner retina can also prevent outer retinal damage has important implications with regard to the targets for effective neuroprotective therapy.     

Axonal transport deficit in the optic nerve of rats with inherited retinitis pigmentosa and experimentally induced glaucoma

Background  Aim of this study was to investigate the retrograde axonal transport from optic nerve (ON) to retinal ganglion cell (RGC) in two animal models: in Royal College of Surgeons (RCS) rats, a rat model for retinal degeneration, and in a rat model for glaucoma induced by elevated intraocular pressure (IOP). Methods  To carry out this study, dextran tetramethylrhodamine (DTMR- an hydrophilic neurotracer dye) was injected into the ON; 24 hrs later, the retina was removed and the number of labeled RGCs of the experimental rats was counted and compared. Results  The results of these studies showed that the number of fluorescent-labeled RGCs in RCS rats and in rats with elevated IOP was reduced compared to the number of labeled RGCs of their respective controls. Conclusion  Our findings suggest that RCS rats are characterized not only by loss of photoreceptor cells but also by functional deficits of RGCs. Valentina Sposato and Alfonso Iovieno contributed equally to this work.     

Retinal ganglion cell numbers and delayed retinal ganglion cell death in the P23H rat retina

The P23H-1 rat strain carries a rhodopsin mutation frequently found in retinitis pigmentosa patients. We investigated the progressive degeneration of the inner retina in this strain, focussing on retinal ganglion cells (RGCs) fate. Our data show that photoreceptor death commences in the ventral retina, spreading to the whole retina as the rat ages. Quantification of the total number of RGCs identified by Fluorogold tracing and Brn3a expression, disclosed that the population of RGCs in young P23H rats is significantly smaller than in its homologous SD strain. In the mutant strain, there is also RGC loss with age: RGCs show their first symptoms of degeneration at P180, as revealed by an abnormal expression of cytoskeletal proteins which, at P365, translates into a significant loss of RGCs, that may ultimately be caused by displaced inner retinal vessels that drag and strangulate their axons. RGC axonal compression begins also in the ventral retina and spreads from there causing RGC loss through the whole retinal surface. These decaying processes are common to several models of photoreceptor loss, but show some differences between inherited and light-induced photoreceptor degeneration and should therefore be studied to a better understanding of photoreceptor degeneration and when developing therapies for these diseases.     

Photoreceptor degeneration in the RCS rat attenuates dendritic transport and axonal regeneration of ganglion cells

PURPOSE: Photoreceptor loss in the Royal College of Surgeons (RCS) rat deprives the retinal ganglion cells (RGCs) of sensory input, which could interfere with RGC physiology. Whether axonal and dendritic transport is altered, and whether RGCs retain their capacity to regenerate their axons, both in vivo and in culture, was ascertained. METHODS: The study was conducted at postnatal days (P) 30 (while most photoreceptors are still intact), P90 (photoreceptors being almost completely absent), and P180 (approximately 3 months after photoreceptor disappearance). RGCs were studied with retrograde transport of the fluorescent dye 4Di-10ASP. Dendritic transport was also studied with 4Di-10ASP that is transported from the cell bodies into the RGC dendrites. Regeneration of RGC axons in vivo was monitored in the grafting paradigm of replacing the cut optic nerve (ON) with a sciatic nerve (SN) piece. Cell counts were performed in retinal wholemounts. Axonal regrowth in vitro was assessed in organotypic cultures of retinal stripes. RESULTS: Photoreceptor dystrophy did not adversely affect retrograde axonal transport but attenuated dendritic transport compared with the wild-type control rats. Axons of RGCs were able to regenerate if provided with a SN graft, and regeneration was observed to be similar between RCS and wild-type rats at P30 but differed significantly at P90 and P180. In addition to an age-dependent decline in the regenerative ability, seen also in control animals, the number of RCS RGCs able to regenerate declined drastically beginning at 3 months. It is plausible that the intraretinal reorganization, as a consequence of photoreceptor disappearance, interferes with the regenerative ability of the RGCs. CONCLUSIONS: The findings suggest for the first time that diminution of photoreceptor sensory input does not induce detectable death of RGCs until P180, but that it attenuates certain ganglion cell functions like intraretinal dendritic transport and propensity for axonal regeneration.     

内源性睫状神经营养因子对青光眼鼠视网膜的保护作用

目的 探讨睫状神经营养因子(CNTF)蛋白在慢性高眼压性青光眼大鼠视网膜中的定位及表达情况.方法采用水下电凝巩膜静脉建立大鼠慢性高眼压性青光眼模型,在1、3、7、14、28 d分别摘取眼球,运用免疫组织化学和Western blot法检测大鼠视网膜CNTF蛋白的定位及表达变化.结果对照组大鼠视网膜神经节细胞(RGCs)层有少量CNTF蛋白表达,青光眼大鼠视网膜CNTF蛋白的表达显著增加,建立模型后第7～14 d表达量达到高峰,此时除了神经节细胞层外,内外核层亦发现有CNTF蛋白的表达,之后表达减少.结论青光眼大鼠视网膜内源性CNTF表达增加,可能与促进损伤的RGCs的存活和轴突再生密切相关.     

Evidence for functional regeneration of the visual pathway in adult rats

Mature nerve cell axons do not spontaneously regrow within their natural environment unless they are stimulated by external application of growth-promoting measures. In the present study, axonal regrowth and restoration of visual function was studied in adult rats. The optic nerve was completely cut, and its proximal and distal stumps were realigned and sutured back together. During the same surgical procedure, the lens was lesioned in order to induce secondary intraocular inflammation, which is known to strongly support the survival of retinal ganglion cells (RGCs) and to promote axonal regeneration within the distal segment of the optic nerve.The neuroanatomical data showed that cut axons can regenerate over long distances within the white matter of a central nerve like the adult optic nerve, spanning over 11mm to the chiasm and between 12 and 15mm to the thalamus and midbrain. Responsiveness of the pupil to light was restored five weeks after injury, thus indicating reinnervation of the pretectal nuclei. Restoration of the ascending pathway between the retina and visual cortex was assured by recording flash visual evoked potentials (FVEPs). As expected, no FVEPs could be recorded during the postsurgical period of axonal growth throughout the optic nerve and tract. FVEPs could be recorded after two months, indicating that synaptic transmission in higher visual areas was established.The recording findings and the restoration of pupillary responses suggest, for the first time, that lentogenic stimulation of RGCs is sufficient to induce the formation of growth cones that can override putative inhibitors at the site of injury.     

Survival and axonal regeneration of retinal ganglion cells in adult cats

Axotomized retinal ganglion cells (RGCs) in adult cats offer a good experimental model to understand mechanisms of RGC deteriorations in ophthalmic diseases such as glaucoma and optic neuritis. Alpha ganglion cells in the cat retina have higher ability to survive axotomy and regenerate their axons than beta and non-alpha or beta (NAB) ganglion cells. By contrast, beta cells suffer from rapid cell death by apoptosis between 3 and 7 days after axotomy. We introduced several methods to rescue the axotomized cat RGCs from apoptosis and regenerate their axons; transplantation of the peripheral nerve (PN), intraocular injections of neurotrophic factors, or an antiapoptotic drug. Apoptosis of beta cells can be prevented with intravitreal injections of BDNF+CNTF+forskolin or a caspase inhibitor. The injection of BDNF+CNTF+forskolin also increases the numbers of regenerated beta and NAB cells, but only slightly enhances axonal regeneration of alpha cells. Electrical stimulation to the cut end of optic nerve is effective for the survival of axotomized RGCs in cats as well as in rats. To recover function of impaired vision in cats, further studies should be directed to achieve the following goals: (1) substantial number of regenerating RGCs, (2) reconstruction of the retino-geniculo-cortical pathway, and (3) reconstruction of retinotopy in the target visual centers.     

The pig eye as a novel model of glaucoma

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.     

Ocular hypertension impairs optic nerve axonal transport leading to progressive retinal ganglion cell degeneration

Ocular hypertension (OHT) is the main risk factor of glaucoma, a neuropathy leading to blindness. Here we have investigated the effects of laser photocoagulation (LP)-induced OHT, on the survival and retrograde axonal transport (RAT) of adult rat retinal ganglion cells (RGC) from 1 to 12 wks. Active RAT was examined with fluorogold (FG) applied to both superior colliculi (SCi) 1 wk before processing and passive axonal diffusion with dextran tetramethylrhodamine (DTMR) applied to the optic nerve (ON) 2 d prior to sacrifice. Surviving RGCs were identified with FG applied 1 wk pre-LP or by Brn3a immunodetection. The ON and retinal nerve fiber layer were examined by RT97-neurofibrillar staining. RGCs were counted automatically and color-coded density maps were generated. OHT retinas showed absence of FG⁺ or DTMR⁺RGCs in focal, pie-shaped and diffuse regions of the retina which, by two weeks, amounted to, approximately, an 80% of RGC loss without further increase. At this time, there was a discrepancy between the total number of surviving FG-prelabelled RGCs and of DMTR⁺RGCs, suggesting that a large proportion of RGCs had their RAT impaired. This was further confirmed identifying surviving RGCs by their Brn3a expression. From 3 weeks onwards, there was a close correspondence of DTMR⁺RGCs and FG⁺RGCs in the same retinal regions, suggesting axonal constriction at the ON head. Neurofibrillar staining revealed, in ONs, focal degeneration of axonal bundles and, in the retinal areas lacking backlabeled RGCs, aberrant staining of RT97 characteristic of axotomy. LP-induced OHT results in a crush-like injury to ON axons leading to the anterograde and protracted retrograde degeneration of the intraocular axons and RGCs.     

Downregulation of Thy1 in retinal ganglion cells in experimental glaucoma

PURPOSE: Thy1 is a surface glycoprotein uniquely expressed in retinal ganglion cells (RGCs) in retina. The aim of this study was to investigate the expression change of Thy1 in a model of experimental glaucoma. METHODS: The change of protein and mRNA levels of Thy1 in the retina were studied using stereological counts of back-labeled RGCs, Western blot analysis, immunohistochemistry, and laser capture microdissection (LCM) of RGCs with quantitative PCR analysis of mRNA in a model of experimental glaucoma. LCM after optic nerve crush was also performed to evaluate Thy1 mRNA levels after a different injury. RESULTS: After 10 days of elevated IOP, there was a 34% loss of RGC number, Thy1 protein decreased 60% in eyes with elevated intraocular pressure (IOP), and Thy1 mRNA levels decreased 51% in RGCs. Both protein and mRNA level change of Thy1 is to a much greater extent than RGC number loss. CONCLUSIONS: The current results confirm that Thy 1 mRNA levels do not reflect the number of RGCs present and extend this to include a parallel decrease in Thy1 protein levels. These results suggest that Thy1 serves as an early marker of RGC stress, but not a marker of RGC loss, in models of retinal damage.     

Increase in dephosphorylation of the heavy neurofilament subunit in the monkey chronic glaucoma model

PURPOSE: To investigate the phosphorylation of the heavy neurofilament subunit (NF-H), which could be deeply involved in axonal transport of retinal ganglion cells (RGCs), in an experimental glaucoma model of chronic elevation of intraocular pressure (IOP) in monkeys. METHODS: One eye in adult monkeys was randomly selected for laser treatment, and IOP was maintained between 30 and 40 mm Hg throughout the experiment. The eyeballs with the optic nerve and optic chiasm were enucleated as one tissue and were subject to immunocytochemical observation, using two NF-H-specific antibodies, NF-200 and SMI31. NF-200 reacts with both phosphorylated and dephosphorylated NF-H, whereas SMI reacts only with phosphorylated NF-H. Ratios of SMI31-positive to NF-200-positive areas were calculated for quantitative evaluation of phosphorylation status. Specimens from the retina, lamina cribrosa (LC), post-LC, and optic chiasm were evaluated separately. Phosphorylation of NF-H at the retina and optic nerve head was compared between specimens from temporal retina and nasal retina, or between temporal and nasal regions of the optic disc. The status of phosphorylation was confirmed by Western blot analysis. RESULTS: An enlargement of the disc cup was observed on the temporal side, and the superior and inferior poles were preferentially involved in the neuronal damage in laser-treated eyes. Most NF-Hs in the control eyes were phosphorylated in all investigated regions, whereas those in the glaucomatous eyes were significantly dephosphorylated, and NF-Hs in the temporal region were significantly dephosphorylated compared with those in the nasal region. At the optic chiasm, NF-Hs in axons traveling from laser-treated eyes were highly dephosphorylated, and the extent of NF-H dephosphorylation corresponded to the degree of glaucoma-induced axonal damage. Western blot analysis showed the change in the phosphorylation of NF-Hs. CONCLUSIONS: NF-Hs in RGC axons are dephosphorylated by elevated IOP, which may be deeply involved in glaucoma-induced damage to axonal transport.     

青光眼发病机制:从临床复杂表型剖析到基本科学问题探索

青光眼是一类复杂疾病综合征,其共同临床表现为"特征性视神经病变",实际病因则千差万别。青光眼的发病机制亟待剖析,重点在于梳理其错综复杂的临床表型,围绕眼压改变、视网膜神经节细胞(RGCs)/轴突损伤、非眼压因素等进行合理分型,还原为基础研究手段可以系统探索的基本科学问题。本文将青光眼分为两大类型:Ⅰ型:真实眼压显著升高,直接导致大量RGCs/轴突原发性损害,并在继发性神经免疫炎症参与下造成青光眼视神经病变(GON)。Ⅱ型:检测到的眼压不能或者不足以直接导致大量RGCs/轴突损害和GON,此时神经免疫炎症可能在青光眼的起始发病机制中起到更重要的作用。根据病因机制不同,本文又将前者进一步细分为5型,后者进一步细分为4型。     

Progressive optic axon dystrophy and vacuslar changes in rd mice

PURPOSE: To examine how the vascular plexuses in the rd mouse retina are affected by the loss of photoreceptors and how this compares with the Royal College of Surgeons (RCS) rat. To examine whether the profound effects of vascular pathology on retinal ganglion cells (RGCs) and their axons seen in RCS rats are also found in rd mice. METHODS: Vascular patterns were studied in flatmounted and sectioned retinas using either nicotinamide adenine dinucleotide phosphate(NADPH)-diaphorase histochemistry or vessel filling with horseradish peroxidase. Optic axons were visualized using RT97 (an antibody against the 200-kDa neurofilament subunit), and RGCs were labeled by retrograde transport of fluorescence label, the Fluorogold, applied to the superior colliculus. RESULTS: The present study showed that in the rd mouse, similar to the RCS rat, vascular complexes developed in association with retinal pigment epithelial cells at the outer border of the retina. The number and distribution of complexes were very different from the rat, but as in the rat, progressive axonal dystrophy was seen in the optic fiber layer. RGC loss, rather than being local was more broadly distributed, but some, at least, appeared to be secondary to axonal dystrophy caused by vessels supplying vascular formation. CONCLUSIONS: Photoreceptor loss in the rd mouse leads to RGC axonal dystrophy and loss. The lesser degree and different distribution of RGC loss caused by abnormal vasculature associated with vascular formations in the outer retina in the rd mouse may be due to the early atrophy of the deep vascular plexus in this animal.     

High intraocular pressure-induced ischemia and reperfusion injury in the optic nerve and retina in rats

• Purpose: The purpose of this paper is to describe the damage caused to the retina and the axons of the optic nerve by acute ischemia-reperfusion injury and the extent to which optic nerve damage correlates with the duration if ischemia due to high intraocular pressure (IOP). • Methods: Acute ischemia in the retina and optic disc was induced in albino rats by increasing the IOP to 110 mmHg for a period of 45–120 min. Thereafter, the eyes were reperfused at normal IOP after 7 days. The retina and optic nerve were examined by light and electron microscopy, and morphometrical counts of the optic nerve axons were performed. • Results: After 45 min of ischemia, electron microscopic examination revealed swelling of mitochondria and degeneration of neurotubules on axons in cross sections of the optic nerve. The axonal counts in eyes subjected to 45 min of ischemia were 29% lower than in control eyes. After 60 min of ischemia, there were distinct disruptions of mitochondria and degeneration of the axons. After 90 min of ischemia, numerous axons showed degeneration with disordered myelin sheaths. Neuronal cell death was seen in the retina, mainly in the ganglion cell layer. • Conclusion: Damage to the retinal ganglion cell layer and the optic nerve was evident after only 45 min of ischemia in normal eyes. This experiment suggests that seriously injured eyes must be protected from high IOP; if IOP elevation is required during vitrectomy, it is essential to reduce the duration of interruption of blood flow to a minimum.