Retinal degeneration in the rd mouse in the absence of c-fos

PURPOSE: Apoptosis is the final common death pathway of photoreceptors in light-induced retinal degeneration and in several animal models for retinal dystrophy. To date, little is known about gene regulation of apoptosis in the retina. The expression of the immediate early gene c-fos is upregulated concomitant with apoptosis in light-induced photoreceptor degeneration and in the rd mouse, an animal model for inherited retinal degeneration. In a recent study it was shown that c-Fos is essential for light-induced apoptosis of photoreceptors in vivo. To determine whether c-Fos is also involved in the apoptotic pathway of inherited retinal degeneration, rd/rd, c-fos -/- double-mutant mice have been generated. METHODS: Double-mutant mice (rd/rd, c-fos -/-) were crossbred from c-fos+/- mice and rd/rd mice. Their genotype was determined by polymerase chain reaction analysis of genomic DNA. Wild-type control mice and homozygous rd mice were killed at 2-day intervals from postnatal day (P)9 through P21. Double-mutant mice were killed at postnatal days P9, P11, P13, P15, and P21. To determine levels of apoptosis in the retina, eyes were enucleated and processed for light microscopy and in situ nick-end labeling. Total retinal DNA was extracted from isolated retinas for DNA fragmentation analysis. RESULTS: Morphologic, histochemical, and biochemical analyses showed that the time course of apoptosis and the outcome of photoreceptor degeneration in rd/rd, c-fos-/- double-mutant mice was indistinguishable from that in rd mice carrying functional c-fos. CONCLUSIONS: These data suggest that in contrast to its role in light-induced photoreceptor degeneration, c-Fos is not essential for apoptosis in the rd mouse.     

Photoreceptor allografts in a feline model of retinal degeneration

BACKGROUND: Photoreceptor transplants provide a potential means to restore function in a degenerate retina and/or rescue degenerating host photoreceptors by trophic influences. We have examined photoreceptor allografts in the Abyssinian cat model of hereditary photoreceptor degeneration to determine the viability and influence of such transplants on the host retina. METHODS: Small pieces of 3- to 5-day-old normal kitten retina containing undifferentiated photoreceptors were injected into the subretinal space of adult Abyssinian cats at an early stage of retinal degeneration using standard vitreo-retinal surgical techniques. The retinas were examined by ophthalmoscopy and fundus photography, then by light and electron microscopy at different times after surgery. RESULTS: Such allografts survive for at least 6 months after surgery. The photoreceptors develop outer segments, invariably in rosettes. The transplants gradually integrate with the host retina but detach the host photoreceptor layer from the retinal pigment epithelium (RPE), which tends to reduce the number of host photoreceptors over the transplant. There is no slowing of the photoreceptor degeneration in neighboring non-detached retina. Inflammation or rejection was not detected. CONCLUSION: Undifferentiated, neonatal photoreceptor allografts survive and develop outer segments in the subretinal space of the Abyssinian mutant feline retina. The allografts gradually integrate with the host neural retina without inducing rejection. In the vicinity of the transplant there is increased loss of host photoreceptors, considered to be due to their detachment from the RPE layer. There is no evidence of any rescue of host photoreceptors elsewhere in this mutant retina.     

Review article: glycyrrhizin as a potential treatment for chronic hepatitis C

The morphologic response of neonatal mouse retina to the alkylating agent N-methyl-N-nitrosourea (MNU) was examined at different periods of retinal development. A dose of 60 mg/kg N-methyl-N-nitrosourea was injected intraperitoneally to neonatal C57BL mice at 0, 3, 5, 8, 11, 14, 17, and 20 days of age and to C3H mice at 0 days of age, and the retinas were examined sequentially. In the C57BL mice, MNU evoked a time-dependent occurrence of retinal dysplasia and retinal degeneration. With MNU treatment at day 0 and day 3 (the stage of retinal cell proliferation), retinal dysplasia characterized by the progressive disorganization of neuroblasts, which led to the formation of rosettes, was found in the outer neuroblastic/nuclear layer above the normal pigment epithelial cells during days 8-20, but decreased at day 50. The rosettes were surrounded by photoreceptor segments and Müller cell processes, and by photoreceptor nuclei. The MNU response was related to retinal differentiation; following MNU treatment at day 5 or 8 (the stage of retinal cell differentiation) the cells were much less sensitive (i.e. no retinal response was found). However, with MNU treatment at days 11, 14, 17, and 20 (after cellular differentiation), retinal degeneration characterized by selective photoreceptor apoptosis was seen. These results suggest that there is a critical period for the time of MNU administration in the development of mouse retinal lesions. In C3H (rd/rd) mice, MNU treatment at day 0 resulted in retinal degeneration with only slight rosette formation at the peripheral retina.     

Descemet''s membrane as membranous support in RPE/IPE transplantation

PURPOSE: The correct orientation of retinal pigment epithelium (RPE) cells is necessary for the integrity and proper function of the retina. For transplantation of RPE/iris pigment epithelium (IPE) grafts to the subretinal space in age-related macular degeneration, this cellular orientation is most effectively provided by a membranous support. The goal of this study was to establish an autologous or homologous membrane as a substratum for the growth of RPE/IPE. METHODS: Porcine and bovine RPE and IPE were placed in primary culture on a dissected sheet (5 x 5 mm) of autologous porcine and bovine Descemet's membrane in slide chambers and grown to confluence. RESULTS: RPE and IPE cells cultured on Descemet's membrane form an intact monolayer. Light and electron microscopy showed the formation of both an intact monolayer and microvilli in both cell types. CONCLUSION: Since the slow host-graft rejection appears to play an important role in the failure of RPE transplantation in the subretinal space, it is critical to be able to transplant autologous materials. The techniques presented here establish a novel means to culture RPE or IPE cells on autologous Descemet's membrane where they form a "cell monolayer patch," consisting of a fragment of Descemet's membrane with cultured RPE or IPE, which can be easily manipulated and transplanted, using an established glass pipette method.     

The Royal College of Surgeons rat: an animal model for inherited retinal degeneration with a still unknown genetic defect

The Royal College of Surgeons (RCS) rat is the first known animal with inherited retinal degeneration. Despite the fact that the genetic defect is not known, the RCS rat is widely used for research in hereditary retinal dystrophies. This review tries to summarize observations which have been made in the RCS rat and to make an attempt to formulate candidate genes which may the cause for the retinal degeneration in this rat strain. The genetic defect in RCS rats causes the inability of the retinal pigment epithelium (RPE) to phagocytose shed photoreceptor outer segments. In normal rats or humans, this circadian process is regulated by both the cyclic adenosine monophosphate (cAMP) and the calcium/ inositol phosphate systems. The calcium/inositol phosphate system seems to be linked to the phagocytosis receptors which recognize photoreceptor outer membranes to initialize phagocytosis. The cAMP system appeared as modulator of the regulation of phagocytosis. An increase in the intracellular cAMP concentration is an 'off' signal for phagocytosis. In RPE cells from RCS rats many observations have been made which indicate a changed second messenger metabolism concerning both the cAMP and the calcium/inositol phosphate systems. The genetic defect seems to concern a protein which is involved in the initialization of a second messenger pathway. We conclude that the genes coding for the phagocytosis receptor or for proteins which are linked to receptors (for example G proteins) are good candidates for defective genes in RCS rats.     

Retina and retinal pigment epithelial cell autoantibodies are produced during murine coronavirus retinopathy

The murine coronavirus, mouse hepatitis virus (MHV), JHM strain, induces a biphasic retinal disease in adult BALB/c mice. In the early phase of the disease, day 1 to 7, a retinal vasculitis is noted and is associated with the presence of virus particles. In the late phase of the disease, day 10 to 140, a retinal degeneration is observed and is associated with the absence of both virus particles and inflammatory cells. We show that the retinal degenerative process is also associated with the presence of antiretinal autoantibodies. In total, 22 of 23 sera collected from 10 to 70 days after JHM virus inoculation contained antiretinal autoantibodies. These autoantibodies are not found in sera from normal or mock-injected mice. Antibodies to retinal tissue were identified as two distinct patterns of immunoperoxidase staining on frozen sections of normal rat eyes, retinal autoantibodies and retinal pigment epithelium (RPE) autoantibodies. The antiretinal autoantibodies first appeared as IgM class antibodies that shifted to IgG class autoantibodies. The anti-RPE cell autoantibodies were predominantly of the IgG class. Sera that were positive for these autoantibodies did not stain with liver or kidney sections but 2 of 3 did react with rat brain sections. A second mouse strain, CD-1, was also evaluated because these animals respond to JHM virus inoculation by developing only the early phase of this disease, i.e. vasculitis. On day 10 postinoculation, the retina architecture has a normal appearance. In these mice, which are free of a retinal degeneration, antiretinal autoantibodies are not produced. However, just as is noted in the BALB/c mice, antivirus neutralizing antibodies are produced in the infected CD-1 mice. These findings suggest a role for autoimmunity in the pathogenesis of murine coronavirus induced retinal degeneration. This study establishes an animal model for the study of humoral autoimmune responses in human retinal degenerations.     

Correlation between scanning laser ophthalmoscope microperimetry and anatomic abnormalities in patients with subfoveal neovascularization

PURPOSE: The purpose of the study is to identify the anatomic abnormalities associated with an absolute scotoma and the location and stability of fixation in patients with subfoveal neovascularization in age-related macular degeneration, presumed ocular histoplasmosis syndrome, and other disorders. METHODS: Scanning laser ophthalmoscope microperimetry was superimposed on color fundus photographs and fluorescein angiograms of 21 eyes with subfoveal neovascular membranes secondary to age-related macular degeneration (14 eyes) and presumed ocular histoplasmosis syndrome (7 eyes). The authors determined the location and the area occupied by the absolute scotoma and each of the following subretinal lesions: subretinal hemorrhage, neurosensory retinal detachment, retinal pigment epithelial (RPE) atrophy, RPE hyperplasia, atrophy of the choriocapillaris, hard exudates, and the subfoveal neovascular membrane. The area of absolute scotoma determined by scanning laser ophthalmoscope microperimetry was superimposed on the anatomic lesions. The authors calculated the relative risk ratio (RR) of an absolute scotoma occurring in regions corresponding to each anatomic abnormality, and determined the preferred location and stability of fixation in each eye. RESULTS: An absolute scotoma was present in areas of chorioretinal scar (RR = 107.61), RPE atrophy (RR = 9.97), subretinal hemorrhage (RR = 2.88), and the neovascular membrane (RR = 1.86). Fixation was stable in all patients with presumed ocular histoplasmosis syndrome but only 29% of patients with age-related macular degeneration. Fifty-five percent of patients with stable fixation fixated over an area of RPE hyperplasia. CONCLUSION: The relative risk of an absolute scotoma is highest over areas of chorioretinal scars, RPE atrophy, subretinal hemorrhage, and the neovascular membrane. Fixation is more stable in patients with subfoveal neovascularization from presumed ocular histoplasmosis syndrome than with age-related macular degeneration and frequently is present over an area of RPE hyperplasia.     

Pathophysiology and management of subretinal hemorrhage

Subretinal hemorrhage can arise from the retinal and/or choroidal circulation. Significant subretinal hemorrhage occurs in several conditions, but most commonly is associated with age-related macular degeneration, presumed ocular histoplasmosis, high myopia, retinal arterial macroaneurysm, and trauma. Released toxins, outer retinal shear forces, and a diffusion barrier created by subretinal hemorrhage all contribute to photoreceptor damage and visual loss. The use of tissue plasminogen activator and improvements in surgical instrumentation have facilitated surgical drainage and have made it a useful option in the management of selected cases. Mechanisms of subretinal hemorrhage formation, underlying etiologies, diagnostic evaluation, and the histopathology of damage are summarized. Published surgical series are reviewed and surgical advances are summarized. The value of surgically removing subretinal hemorrhages to improve visual outcome remains unestablished, because definitive studies have not been performed. Guidelines for selecting candidates for surgical intervention are proposed.     

A microtiter plate assay for cytochrome c oxidase in permeabilized whole cells

A new mouse retinal degeneration that appears to be an excellent candidate for modeling human retinitis pigmentosa is reported. In this degeneration, called rd-3, differentiation proceeds postnatally through 2 weeks, and photoreceptor degeneration starts by 3 weeks. The rod photoreceptor loss is essentially complete by 5 weeks, whereas remnant cone cells are seen through 7 weeks. This is the only mouse homozygous retinal degeneration reported to date in which photoreceptors are initially normal. Crosses with known mouse retinal degenerations rd, Rds, nr, and pcd are negative for retinal degeneration in offspring, and linkage analysis places rd-3 on mouse chromosome 1 at 10 +/- 2.5 cM distal to Akp-1. Homology mapping suggests that the homologous human locus should be on chromosome 1q.     

Albumin movement out of the subretinal space after experimental retinal detachment

PURPOSE: The subretinal fluid of serous retinal detachments contains protein, but little is known about its origin and fate. The authors designed experiments to study the rate and route of albumin movement out of the subretinal space. METHODS: Experimental retinal detachments were made in Dutch rabbits by injecting Hanks' balanced salt solution containing serum levels (approximately 30 mg/ml) of fluorescein isothiocyanate (FITC) albumin into the subretinal space through a micropipette. Subretinal, vitreous, and serum fluid samples were withdrawn 0 to 4 hours later through a similar micropipette and were analyzed for osmolality, FITC albumin content (by fluorophotometry) and FITC+native albumin content (by gel electrophoresis). Sodium iodate was injected intravenously in some rabbits to damage the retinal pigment epithelium (RPE). RESULTS: Albumin injected into the subretinal fluid diffused steadily into the vitreous, and its concentration decreased by approximately 5% per hour. This rate was unaffected by RPE damage. Albumin did not move into the bloodstream unless the RPE was damaged with sodium iodate, and then it crossed the RPE at approximately 25% of the rate at which it moved into the vitreous. Subretinal fluid osmolality remained within the range of 293 to 294 mOsm/kg despite protein movement and the continual absorption of fluid from the detachments. CONCLUSIONS: These results show that albumin in the subretinal space diffuses readily into the vitreous, and subretinal osmolality changes are rapidly equilibrated with the vitreous. Albumin does not cross normal RPE, and it crosses iodate-damaged RPE more slowly than it crosses retina. Thus, there must be a constant supply of albumin if high subretinal concentrations are to be sustained in clinical serous detachments.     

Cellular response in subretinal neovascularization induced by bFGF-impregnated microspheres

PURPOSE: To determine the sequence of cellular changes associated with a new rabbit model of subretinal neovascularization (SRN) induced by subretinal injection of basic fibroblast growth factor (bFGF)-impregnated microspheres. METHODS: bFGF-impregnated gelatin microspheres, prepared by forming a polyion complex between gelatin and bFGF, were subretinally implanted into rabbit eyes. The eyes were studied by immunochemistry at 3 days to 8 weeks after implantation. Antibodies to CD4, CD8, cytokeratin, CD31, glial fibrillary acidic protein (GFAP), and RAM11 were used. RESULTS: Cytokeratin-positive retinal pigment epithelial (RPE) cells appeared on day 3 and continued to increase in number in the subretinal space throughout the growth of the SRN membrane, becoming the predominant cell type. Macrophages (RAM11-positive) appeared early, but most disappeared within 7 days. GFAP-positive Müller cells were evident early in the retina but migrated into the subretinal space after 7 days; the gliotic adhesion they formed between the retina and the SRN membrane was prominent at 8 weeks. CD31-positive endothelial cells were first evident at 14 days and formed neovascular channels that were still present for up to 8 weeks. CD4- and CD8-positive lymphocytes appeared in the early stages but were few in number. CONCLUSIONS: SRN membranes are primarily composed of RPE cells and vascular endothelial cells. The membrane adheres to the retina by a gliotic band. The cellular components involved in the membrane of this model resemble those found in SRN membranes removed from patients with age-related macular degeneration.     

Immunoreactivity for calcium-binding proteins in the claustrum of the monkey

The sequence of degenerative changes in the retinal pigment epithelium (RPE) and the choroid of retinal degeneration (rd)-mice was studied in correlation with photoreceptor changes. Three weeks to 26-month-old animals were investigated using light and transmission electron microscopy, enzyme histochemistry and quantitative morphology. Changes in the choriocapillaris (CC) were additionally studied by scanning electron microscopy of corrosion cast preparations. In 3-week-old mice, in which most of the outer segments of photoreceptors in the central portion of the retina had disappeared but remnants of the cells were still present, the RPE was enlarged and showed elongated microvilli. In 8-week-old animals, the photoreceptors were completely absent in large areas of the posterior pole region. In these areas the RPE was also completely lost. Quantitative evaluation performed in histological serial sections showed that loss of RPE measured as length of RPE-free Bruch's membrane, continuously increased up to the age of 20 months. In 8-week-old animals, CC adjacent to degenerating RPE showed loss of fenestration. In 10-week-old animals, the CC disappeared in those areas where the RPE was already lacking. The loss of CC increased with increasing age and in 20-month-old animals 5-10% of the entire CC was lacking. Loss of the related arterioles and nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d)-positive nerve fibers occurred only in approximately 2-year-old rd-mice. Compared to other animal models, RPE and CC defects in rd-mice are relatively large. The rd-mice might therefore provide a good tool to study factors involved in CC degeneration.     

Adenovirus-mediated delivery of rhodopsin-promoted bcl-2 results in a delay in photoreceptor cell death in the rd/rd mouse

Gene transfer to retinal cells may provide a means to retard photoreceptor cell death and thus prevent blindness in diseases such as retinitis pigmentosa. We tested the possibility of interfering with apoptotic photoreceptor cell death in the rd mouse through subretinal delivery of a recombinant replication-defective adenovirus containing the human cDNA for bcl-2, Ad.2.5HRPbcl-2. Photoreceptor-specific transgene expression was accomplished through incorporation of the 2.5 kb human rhodopsin upstream fragment (HRP). Ad.2.5HRPbcl-2 was injected alone or in combination with Ad.CMVPDE beta. Ad.CMVPDE beta contains a cDNA encoding the beta subunit of cGMP phosphodiesterase (PDE beta). Recombinant viruses containing lacZ (driven either by the cytomegalovirus (CMV) promoter/enhancer or HRP) and of Ad.CMVPDE beta and vehicle alone were injected in contralateral eyes as control. Injection of Ad.2.5HRPbcl-2 in the rd mouse resulted in histologically detectable rescue lasting 6 weeks after birth. Extent of rescue was not as large as after delivery of wildtype PDE beta, the gene defective in the rd mouse. However, delivery of genes which prevent apoptotic cell death may have broad application to gene therapy of retinal degenerative diseases.     

Intraocular gene transfer of ciliary neurotrophic factor prevents death and increases responsiveness of rod photoreceptors in the retinal degeneration slow mouse

Several mutations causing both photoreceptor degeneration and malfunction have been identified in humans and animals. Although intraocular injection of trophic factors has been shown to reduce photoreceptor death in a few conditions of rapid photoreceptor loss, it is unclear whether long-term beneficial changes in functional properties of affected photoreceptors can be obtained by treatment with these factors. The rds/rds mouse is a spontaneous mutant bearing a null mutation in the rds/peripherin gene, which is linked to many forms of dominant retinal degenerations in humans. Here, we report that intraocular adenovirus-mediated gene transfer of ciliary neurotrophic factor (CNTF) in this mutant reduces photoreceptor loss, causes a significant increase in the length of photoreceptor segments, and results in a redistribution and an increase in the retinal content of the photopigment rhodopsin. These effects are accompanied by a significant increase in the amplitude of the a- and b-waves of the scotopic electroretinogram. These results suggest that continuous administration of CNTF could potentially be useful for the treatment of some forms of retinal degeneration.     

Non-cell-autonomous photoreceptor degeneration in rds mutant mice mosaic for expression of a rescue transgene

The inherited retinal dystrophies represent a large and heterogenous group of hereditary neurodegenerations, for many of which, the molecular defect has been defined. However, the mechanism of cell death has not been determined for any form of retinal degeneration. The retinal degeneration slow (rds-/-) mutation of mice is associated with nondevelopment of photoreceptor outer segments and gradual death of photoreceptor cell bodies, attributed to the absence of the outer segment protein rds/peripherin. Here, we examined the effects of a transgene encoding normal rds/peripherin that had integrated into the X-chromosome in male and female rds-/- mutant retinas. In 2-month-old transgenic males and homozygous-transgenic females on rds-/-, we observed virtually complete rescue of both the outer segment nondevelopment and photoreceptor degeneration. In contrast, hemizygous-transgenic rds-/- female littermates showed patchy distributions of the transgene mRNA, by in situ hybridization analysis, and of photoreceptor cells that contain outer segments. This pattern is consistent with random inactivation of the X-chromosome and mosaic expression of the transgene. Surprisingly, we observed significant photoreceptor cell loss in both transgene-expressing and nonexpressing patches in hemizygous female retinas. These observations were supported by nuclease protection analysis, which showed notably lower than predicted levels of transgene mRNA in retinas from hemizygous females compared with male and homozygous female littermates. This phenotype suggests an important component of non-cell-autonomous photoreceptor death in rds-/- mutant mice. These results have significance to both the etiology and potential treatment of human inherited retinal degenerations.     

Host response to mycobacterial infection in the alcoholic rat: male and female dimorphism

BACKGROUND: The intravitreal injection of ornithine produces selective damage to the retinal pigment epithelium (RPE) and results in a loss of RPE, choriocapillaris and photoreceptor cells. To elucidate the mechanism of secondary retinal atrophy, we investigated the presence of apoptotic cells in a rat model of ornithine-induced retinopathy. METHODS: At 6 and 12 h and 1, 2, 4, 7, 14 and 28 days after an intravitreal injection of L-ornithine hydrochloride in rat eyes, we removed the eyes and subjected them to histopathological examination. We detected apoptotic cells by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate digoxigenin nick end labeling (TUNEL) assay, which stains the 3'-OH ends of fragmented DNA. We used electron microscopy to detect the apoptotic cells morphologically. RESULTS: RPE cells were selectively damaged immediately after ornithine administration. TUNEL-positive photoreceptor cells appeared exclusively in the photoreceptor cell layer 12 h after ornithine administration. The number of TUNEL-positive cells increased throughout the 2 days following the injection, then decreased markedly. TUNEL-positive cells remained until 28 days, when the photoreceptor cells had disappeared. The ganglion cell layer, inner nuclear layer and damaged RPE cells were negative for TUNEL staining during all stages. The electron microscopic study also revealed the pyknotic nuclei of apoptotic photoreceptor cells. CONCLUSION: An intravitreal injection of ornithine caused primary damage to the RPE, and subsequently some of the photoreceptor cells revealed apoptosis by TUNEL assay. These findings suggest the dysfunction of the RPE causes photoreceptor cell death according to the intrinsic program of an apoptotic mechanism.     

Clinicopathologic correlation of localized retinal pigment epithelium debridement

PURPOSE: To characterize changes in the retina, retinal pigment epithelium (RPE), and choriocapillaris with fluorescein angiography (FA) and histology after hydraulic or abrasive RPE debridement in 26 domestic short-haired cats. METHODS: Hydraulic debridement was produced by injecting balanced salt solution forcefully into the subretinal space. For abrasive debridement, RPE were removed with a silicone-tipped cannula after creating a localized retinal detachment. The FAs were performed after surgery, and tissue was prepared for light microscopy (LM) and scanning electron microscopy (SEM). RESULTS: Sixty-seven blebs were examined by FA 1 hour after surgery, and RPE debridement was confirmed by SEM or LM in 15 blebs from 10 animals. Hyperfluorescence and variable central fluorescein leakage were seen 1 week after surgery in 52 of 53 blebs (which includes all 27 blebs from the 1-week timepoint and 26 of 29 blebs from the 4-week timepoint that were studied by FA 1 week after surgery). Choriocapillary filling delays were seen in no hydraulic debridements, but in 11 of 14 abrasive blebs, especially in areas showing leakage late in the angiogram. In 1 of 13 hydraulic and 12 of 14 abrasive debridements, areas of late dye leakage had no RPE with outer retinal degeneration. At the 4-week timepoint, 1 of 17 hydraulic and 10 of 12 abrasive debridements had foci of delayed or absent choriocapillary perfusion by FA, with degenerated outer retina, no RPE, and choriocapillary atrophy by histologic analysis. CONCLUSIONS: Abrasive debridement is more commonly associated with abnormal FAs and with incomplete RPE repopulation, choriocapillaris atrophy, and outer retinal degeneration than is hydraulic debridement. This clinicopathologic study may give insight into FA interpretation after choroidal neovascular membrane removal in human patients.     

Retinal degeneration induced by N-methyl-N-nitrosourea in Syrian golden hamsters

BACKGROUND: The sequential retinal changes in Syrian golden hamsters induced by N-methyl-N-nitrosourea (MNU) have not been studied. METHODS: Female hamsters received a single intraperitoneal injection of 90 mg/kg MNU at 50 days of age, and the retina was examined light and electron microscopically, immunohistochemically and by the TdT-mediated dUTP-digoxigenin nick end labeling (TUNEL) method until 20 weeks after the treatment. RESULTS: The retinal changes were as follows: (1) Photoreceptor apoptosis occurred 1 day after the treatment and resulted in photoreceptor loss at day 7. During the degeneration, Müller cell proliferation was conspicuous at day 5. (2) After the photoreceptor cell loss, migration of the pigment epithelial cells in all layers of the retina which were in contact with blood vessels occurred. Due to the Müller cell proliferation, gliosis was prominent at the later stage. CONCLUSIONS: The MNU injection caused photoreceptor apoptosis followed by pigment epithelial cell migration around the blood vessels, accompanied by gliosis. The primary event and the course of this disease closely resemble those of retinitis pigmentosa in humans.     

Cloning and localization of RPE65 mRNA in salamander cone photoreceptor cells1

RPE65 is a potential retinoid-processing protein expressed in the retinal pigment epithelium. Mutations in the RPE65 gene have been shown to cause certain inherited retinal dystrophies. Previous studies have shown that salamander cone photoreceptor cells have a unique retinoid processing mechanism which is distinct from that of rods. To determine whether RPE65 is expressed in photoreceptors, the RPE65 cDNA was cloned from a salamander retinal cDNA library. The deduced protein consists of 533 amino acids and is 85% identical to human and bovine RPE65. The RPE65 mRNA was detected in all of the single cone cells isolated from the salamander retina, as well as in the retinal pigment epithelium by RT-PCR, but not in the isolated rods. The RT-PCR products have been confirmed to be RPE65 by DNA sequencing. The results indicate that this potential retinoid processing protein is expressed in the cone photoreceptor cells but not in rods. Therefore, this protein may contribute to the unique retinoid processing capabilities in salamander cones.