Development and degeneration of retina in rds mutant mice: analysis of interphotoreceptor matrix staining in chimaeric retina

Chimaeric mice were produced by aggregating two morulae--one of homozygous rds mutant and another from a strain of mice with normal retina, which also differed in colour genes. The interphotoreceptor matrix in the retinal sections of these chimaeras was studied histochemically. In sections, stained with colloidal iron, regions with rds/rds photoreceptor layer, characteristically lacking the outer segments, showed more intense staining of the interphotoreceptor matrix, while regions with normal receptor outer segments showed less intense staining of the matrix. In sections, stained with toluidine blue, rds/rds regions showed more intense reaction along the pigment epithelial--photoreceptor interface and less intense reaction over the inner segments in comparison to the regions with normal photoreceptors. These differential staining reactions were independent of the overlying retinal pigment epithelial cell genotype and resembled the reaction patterns in the retina of pure strain controls of the same age. Small patches, showing rds/rds type staining were also observed within areas which appeared normal. We suggest that the altered properties of the interphotoreceptor matrix in the rds retina result from gene expression within the photoreceptor cells.     

Retinal damage by constant light in chimaeric mice: implications for the protective role of melanin

Adult chimaeric mice, containing varying proportions of albino and pigmented cells in their ocular tissues, were exposed to constant light for 5 weeks and the distribution of the surviving rod perikarya in the retina and of the pigmented cells in various eye tissues were compared. In chimaeras which were mostly albino, the retinal lesion was similar to that in pure strain albino mice; in chimaeras with relatively more pigmented cells in their ocular tissues, the retina was unaffected as in fully pigmented mice. In chimaeras with amounts of pigmented cells in their ocular tissues varying between these two ends, lesions of intermediate degrees could be observed. Surviving rod cells in such chimaeric retinas were always found in regions adjoining the periphery. The location of the rod perikarya in such regions did not show an exact correlation with that of the overlying pigmented cells but regions of the outer nuclear layer with surviving rod perikarya were generally located in the half or quarter of the retina in which the overlying pigment epithelium also contained more pigmented cells than in the other regions. The proportions of the surviving photoreceptor cells varied between such chimaeras. The lesion appeared to be less extensive in individuals with more pigmented cells in the epithelium but no exact correlation was recorded. The findings suggest that while pigmentation in the iris reduces the amount of light reaching the retina, melanin in the pigment epithelium, in addition to preventing light reflection, may also play an antitoxic role, possibly as an antioxidative agent.     

Development and degeneration of retina in rds mutant mice: photoreceptor abnormalities in the heterozygotes

Mice homozygous for the rds (retinal degeneration slow) gene fail to develop receptor outer segments and show a slow loss of visual cells that starts from 14-21 postnatal days and results in complete absence at 1 year. In the heterozygous rds/+ mice the development of receptor outer segments is initially retarded. Although a distinct layer of outer segments of moderate length is formed, the disc structures remain disarrayed and form irregular whorls. Autoradiograms of rds/+ retinas show reduced incorporation of [3H]-leucine. Scleral movement of label, resulting from the addition of newly formed discs, is also retarded and appears irregular in comparison with the normal. Phagosomes, containing newly shed disc structures, within the retinal pigment epithelium of rds/+ mice are much larger than normal. Counts taken at different times of the dark- and light periods have shown an abnormally high turnover of phagosomes in the pigment epithelium of the rds/+ mice, with higher than normal peak frequency near the end of the light period, in contrast with the peak frequency in the normal pigment epithelium recorded around the beginning of the light period. Starting at 2 months, a very slow loss of visual cells, much slower than in the homozygous mutants, progresses throughout life. As a result, the outer nuclear layer at the age of 18 months or more is reduced to less than half. Prior to the reduction of the outer nuclear layer, the relative frequencies of the rod and cone perikarya in the rds/+ retina are similar to the normal values. With loss of visual cells, a small increase in the relative frequency of the cone perikarya is recorded in older rds/+ mice. This increase is more noticeable in the central than in the peripheral retina. The significance of the partial expression of the rds gene in the retina of the heterozygous mice in comparison with the changes observed in the homozygous retina is discussed. It is concluded that dose-dependent variation in phenotypic expression is an essential feature in the working of the rds gene.     

Development and degeneration of retina in rds mutant mice: effects of light on the rate of degeneration in albino and pigmented homozygous and heterozygous mutant and normal mice

The effect of light on the rate of visual cell loss in mice afflicted by the rds (retinal degeneration slow) gene was analyzed by comparing the changes in the thickness of the outer nuclear layer. Visual cell loss in the pigmented, homozygous mutant mice, maintained in cyclic light, is slightly slower than in the albino mutant mice. In the pigmented mutant mice, exposed to constant light, and in the albino mutant mice, reared in darkness, rate of cell loss is not significantly altered. In the albino animals exposed to constant light, the rate of cell loss is faster in the homozygous mutant than in the normal, and intermediate in the heterozygous mutant retina. The accelerated cell loss in the mutant retina progresses from the centre to the periphery, and affects the rods earlier than the cones. This resembles the photic lesion in the normal retina but is unlike the genetic lesion in the mutant retina which appears to progress from the periphery to the centre and affects both rods and cones. It is concluded that the visual cells in the retina of rds mutant mice are more vulnerable to photic damage than those in the retina of normal mice.     

Localization of an endogenous substrate for cyclic AMP-stimulated protein phosphorylation in retina

Incubation of mouse or rabbit whole retina homogenates in the presence of [gamma 32P]-ATP and Mg2+ leads to the phosphorylation of various proteins, as demonstrated using SDS-polyacrylamide gel electrophoresis and autoradiography. The phosphorylation of one protein (ca. approximately equal to 31000 mol. wt) was increased by cyclic AMP in both species, with half-maximal stimulation at 5 x 10(-7)M. Cyclic GMP was also active, but much less potent. Protein phosphorylation patterns were compared in retina homogenates from normal mice (C57BL/6J), from adult C57BL/6J mice homozygous for the retinal degeneration gene (rd/rd) in which rod photoreceptor cells are absent, and from 21-day-old 020/Cpb mice homozygous for the retinal degeneration slow gene (rds/rds) in which only the outer segments of the rod photoreceptors are missing. The 31 K protein was only present in normal and in 21-day-old rds/rds mice. When rabbit retina was microdissected into outer segment, inner segment plus outer nuclear, and inner retina layers, cyclic AMP-stimulated phosphorylation of the 31 K protein was evident only in the inner segment plus outer nuclear layer. These data indicated the presence of a specific, endogenous substrate for a cAMP-dependent protein kinase which is found in the inner portions of rod photoreceptor cells.     

Effects of partial receptor cell loss on the electroretinogram of chimaeric mice

A series of chimaeric mice were produced by aggregating morulae from rd/rd and normal (+/+) mice. In the retina of chimaeric mice, produced by aggregating morulae of these two genotypes, loss of rd/rd photoreceptor cells results in a patchy distribution of the surviving normal receptor cells. The number of remaining receptor cells vary between individual chimaeras. The inner retinal layers in the chimaeras, as well as in the two parental genotypes remain intact. Electroretinograms were recorded from 16 chimaeric mice, and various parameters were compared with the amount of visual cells present as estimated by the average thickness of the outer nuclear layer. The amplitudes of the a- and b-wave, showed a linear reduction with decreasing thickness of the outer nuclear layer thickness. However, threshold of the b-wave increased only when the thickness of the outer nuclear layer fell to about 25% of the normal thickness while the time-to-peak of the waves did not change appreciably among the chimaeric individuals. These results suggest that the changes in the electroretinogram of the chimaeric individuals are related to the amount of visual cells present in the retina.     

A developmental study of interphotoreceptor retinoid-binding protein (IRBP) in single and double homozygous rd and rds mutant mouse retinae

Interphotoreceptor retinoid-binding protein (IRBP) was studied using immunochemical and immunocytochemical techniques in retinae of mice with allelic combinations at the rd and rds loci at different stages of development and degeneration. Until postnatal day 7 (P7), IRBP is located intracellularly in developing retinae of the different genotypes. Thereafter, IRBP is present mainly in the interphotoreceptor matrix. As previously noted, cell death is slowest in the heterozygous +/+,rds/+ mutant with loss increasing in order in +/+,rds/rds, rd/rd, rds/rds and rd/rd,+/+ animals. The IRBP content of the total retina also approximates this pattern, with lowest amounts by far in rd/rd, rds/rds and rd/rd,+/+ mutants (after P14). Interestingly though, IRBP loss significantly precedes visual cell loss in the rd/rd,rds/rds retina. In all the mutants, the remaining rod cells in the outer nuclear layer exhibit synthesis of intracellularly located IRBP at late stages of degeneration. In the single homozygous rd/rd,+/+ and the double homozygous rd/rd,rds/rds mutants, IRBP is present intracellularly during the entire degenerative process with somewhat less intracellular IRBP in the rd/rd,rds/rds mutant. Retinae of homozygous +/+,rds/rds and heterozygous +/+,rds/+ animals exhibit a normal distribution pattern of IRBP immunoreactivity until loss of photoreceptor cells becomes pronounced at later stages of the disease. Many of the remaining cells at this time are probably cone elements although they are structurally changed. Double labeling with IRBP and S-antigen demonstrates, in many but not all, the presence of both proteins in the same cell body. Immunocytochemistry clearly demonstrated the presence of IRBP in remaining photoreceptor cells at late stages of the disease. Thus, the biochemically measured loss of IRBP appears to be a complex process neither directly dependent on the loss of photoreceptor outer segments and reduced interphotoreceptor matrix space (e.g. there is a sustained IRBP level in rodless rds mutants) nor simply due to cell death (e.g. in the rd/rd,rds/rds mutant, IRBP loss significantly precedes cell loss). That this IRBP is mainly intracellular, however, may indicate an abnormality in secretion which, combined with other factors, induces a degenerated and less differentiated phenotype.     

Development and degeneration of retina in rds mutant mice: altered disc shedding pattern in the heterozygotes and its relation to ocular pigmentation

In the heterozygous mutant (rds/+) mice, receptor outer segments (ROS) are irregular in form and are shed as abnormally large phagosomes. In the albino rds/+ mice, peak frequency of pigment epithelial (RPE) phagosomes is higher than normal and is recorded near the end of the light period, instead of at the time of light onset as in the normal (+/+) albino mice. In pigmented mice of both genotypes, the maximum numbers of phagosomes in the RPE remain lower than in the albinos. In pigmented +/+ mice the number of phagosomes is already high at the time of light onset. The number rises to peak after one hour and then declines slowly. The lowest frequency is reached after the end of the light period. In pigmented rds/+ mice, the number of phagosomes in the RPE is lowest at the time of light onset. The number rises rapidly to peak level within two hours, then declines and remains low until light onset. If the dark period is prolonged, phagosome frequency in the rds/+ RPE remains lower than in +/+ RPE. If the light period is prolonged, phagosome frequency in the rds/+ RPE remains at a higher level than in the +/+ RPE. This differential response to altered light regimen in the rds/+ and +/+ mice is less pronounced in the pigmented than in the albino individuals. The phagosomes in the rds/+ RPE are larger than in the +/+ RPE in all light regimens. These results show that ocular pigmentation may modify the circadian pattern of ROS disc shedding in the rds/+ retina.     

Development and degeneration of retina in rds mutant mice: ultraimmunohistochemical localization of S-antigen

In the developing photoreceptor cells of the homozygous rds mutant mice S-antigen is localized over the ciliary protrusion as in the control mice, and to a lesser extent over the inner segments, perikaryal cytoplasma and the cell terminals. As the outer segments develop in the normal retina, the discs become increasingly immunoreactive. In the rds/rds retina the outer segments fail to develop but small membrane bound vesicles, immunoreactive for S-antigen are extruded and phagocytized by the retinal pigment epithelium. In the retina of older mutant mice, as the photoreceptor cells degenerate slowly, the surviving cells continue to show persistent immunoreactivity for S-antigen in the different regions of the photoreceptor cells. In the heterozygotes the outer segments are reduced and appear abnormal, but the localization of S-antigen is similar to normal. In the receptor region of the normal retina and in the deviant membranous structures in the mutant retina the localization of S-antigen is similar to that of opsin. However, some differences in the subcellular localization of these two photoreceptor specific proteins have been observed. It is concluded that the rds gene acts subsequent to the synthesis of these proteins and possibly at the site of disc assembly.     

Development of photoreceptor cells in vitro: influence and phagocytic activity of homo- and heterogenic pigment epithelium

Differentiation of photoreceptor cells and phagocytosis by the pigment epithelium were examined in vitro. Neural retina of postnatal 5- and 10-day-old mice was combined with the pigment epithelium and cultured for up to 15 days. Differentiation of the neural retina including lamellar membrane formation in the photoreceptor outer segments was normal for both 5- and 10-day-old albino mice (DD strain) in vitro. The retinas of dystrophic C3H mice when isolated to culture at postnatal day 5 underwent differentiation similar to that observed in the albino of the same age, but when isolated to culture at postnatal day 10, outer segments degenerated rapidly and were absent by 5 days in culture.When the albino 10-day pigment epithelium was cultured with dystrophic neural retina of the same age, or vice versa, photoreceptor outer segments were not formed in either case. However, the pigment epithelium in both cases could phagocytize the heterogenic outer segments. These results suggest, first, that the phagocytic function of the pigment epithelium of the dystrophic C3H mouse to outer segments was normal and second, that the outer segments of the dystrophic mouse were normal in so far as they were recognized and phagocytized by the pigment epithelium of the albino mouse.     

Development and degeneration of retina in rds mutant mice: ultraimmunohistochemical localization of opsin

In normal retina the developing photoreceptor cells first show presence of opsin over the distal ends of the ciliary protrusions. In a fully differentiated cell intense activity is seen over the rod outer-segment discs; some activity is also seen over the Golgi zone and near the distal ends of the inner segments but the other parts of the receptor cell appear negative. In the pigment epithelium opsin is seen only over phagosomes containing rod outer segment debris. In the homozygous rds mutant retina, developing receptor cells show opsin activity over the ciliary protrusions as in the normal. These ciliary protrusions grow in size and show increased opsin activity and presumably constitute the site of phototransduction in the mutant retina. Although typical disc structures remain lacking, variable amounts of immunopositive, irregular, membranous structures are occasionally observed. The inner segments in the mutant cells show very little immunoreactivity but the perikarya and the spherule terminals show increased immunoreactivity in comparison with the normal. At the onset of degeneration, some of the receptor cells in the mutant retina show extrusion of small, membrane-bound vesicles which are immunopositive for opsin. Some receptor cells undergoing lysis disintegrate and also add to the opsin-positive vesicular structures in the interphotoreceptor space. The vesicles are phagocytized by pigment epithelial cells. In older mutant mice at an advanced stage of degeneration, the receptor cells show reduced opsin activity. In heterozygous mutant mice the outer segments are reduced in length and the discs are abnormal in form. However, the intensity and the pattern of opsin localization in the outer segments and at other sites are similar to normal.     

Foveomacular Vitelliform Dystrophy,Adult Type: A Clinicopathologic Study Including Electron Microscopic Observations

Foveomacular vitelliform dystrophy, adult type (FVDAT), is a dystrophy of the retinal pigment epithelium (RPE) characterized in part by autosomal dominant inheritance, midlife onset, mild visual loss, and various patterns of foveal yellow deposits located at the level of the pigment epithelium. Detailed pathologic studies of two postmortem eyes from a 61-year-old woman with documented FVDAT disclosed striking abnormalities involving mainly the foveal RPE and overlying photoreceptors. By light microscopy, marked focal atrophy of the pigment epithelial cells in the foveolar area was bordered by hypertrophic RPE. Interposed between the atrophic RPE and Bruch's membrane were scattered eosinophilic, fusiform collagenous plaques. The sensory retina overlying the deranged RPE displayed marked atrophy of the outer nuclear layer with loss of photoreceptor inner and outer segments. Pigment-laden macrophages containing periodic acid -Schiff-positive material had migrated into the atrophic, outer sensory retina. Ultraviolet fluorescent microscopy demonstrated massive accumulation of lipofuscin pigment within the macular RPE as well as within macrophages in the atrophic outer retina. By scanning electron microscopy, a confluent area of flattened, atrophic retinal pigment epithelial cells was rimmed by taller, hypertrophic RPE cells..By transmission electron microscopy, the pigment epithelial cells contained myriad lipofuscin granules. Bruch's membrane and the intercapillary pillars were slightly thickened and the choriocapillaris was patent. The possible interrelationships between FVDAT and the other pattern dystrophies of the RPE are discussed.     

Transplantation of retinal pigment epithelial cells to immature and adult rat hosts: short- and long-term survival characteristics

Retinal pigment epithelial (RPE) cells isolated from 6-8-day-old pigmented Long-Evans rat eyes were successfully grafted onto Bruch's membrane in albino Sprague-Dawley hosts ranging in age from 10 days postnatal to young adulthood. Injections of RPE cells were made into the subretinal space using a lesion paradigm which penetrates through the dorsal surface of the eye cutting through the sclera and choroid. Suspensions of pigmented RPE cells were labeled with Nuclear Yellow prior to transplantation, and at 1 week after grafting, the transplanted RPE cells were found attached to previously denuded areas of Bruch's membrane. The grafted RPE cells were positively identified by double labeling-only those RPE cells with melanosomes in their cytoplasm showed fluorescent Nuclear Yellow labeling; host albino RPE cells showed no nuclear labeling. The grafted RPE cells developed a normal relationship with photoreceptor cell outer segments as seen by electron microscopy. When compared with intact retinas or host control areas there were no significant differences in the thickness of the outer nuclear layer or in the lengths of photoreceptor inner and outer segments underneath the grafted RPE cells for at least 3 months after transplantation, which was the longest survival time examined.     

Spatio—temporate Expression of P53,Bcl—2 and Fas in the Retina of RD,a

Purpose: To investigate the expression status of p53, Fas and bcl-2 in the developmentof retinal degeneration in C3H, rd and rds mice.Methods: Expression of p53, Fas and bcl-2 in the retina of rd,rds and C3H mice as wellas in normal C3B mice in different periods was examined by immunohistochemicaltechnique.Results: The expression of Fas and p53 was not detected in normal C3B mice. Fas andp53 were expressed in the ganglionic layer in the early stage and then in the innernuclear layer, while the scale and intense increased in the ganglionic layer. There is nodifference of bcl-2 expression between the normal mice and mice with retinaldegeneration.Conclusion: Fas and p53 may be involved in the retinal cell death in rd, rds, and C3B,but bcl-2 may not. It is unknown why p53 and Fas appeared first in ganglion layer butnot in the outer nuclear layer where retinal cells death was noticed in early stage. EyeScience 2000; 16: 158 ~ 162.     

Some cytological and initial biochemical observations on photoreceptors in retinas of rds mice

As a control for biochemical studies in progress, an ultrastructural study has been carried out on the deteriorating, 21-day photoreceptors of the 020/Cpb strain of mice, homozygous for retinal degeneration, slow (rds). At 21 postnatal days, outer segments were essentially lacking, but cilia erupting from the inner segments were common. A low percentage of cilia bore small cytoplasmic masses containing a few layered membranes, and rare inner segments possessed spherical aggregations of multilayered membranes. Pigment epithelial cells also possessed membranous aggregations in presumed phagosomes. While other parts of photoreceptors possessed the usual organelles of normal rods, inner segments were reduced in volume, and the layer of photoreceptor synaptic terminals was thinner. Mutant 21-day retinas possessed about two-thirds of the protein of normal 21-day retinas but 50% more protein than "rodless" (rd/rd) 21-day retinas. Surprisingly, while dark-adapted rds retinas possessed markedly lower levels of cyclic GMP as compared to controls, light-adaptation significantly reduced cyclic GMP and cyclic AMP levels, and biochemical data point to persistent light-modulated cyclic nucleotide levels in the photoreceptors.     

Retinal pigment epithelial cell transplantation after subfoveal membranectomy in age-related macular degeneration: clinicopathologic correlation

PURPOSE: To report the histopathology after retinal pigment epithelial cell transplantation and subfoveal membranectomy in age-related macular degeneration. METHODS: An 85-year-old white woman with bilateral choroidal neovascularization underwent subfoveal membranectomy combined with transplantation of a sheet of human adult retinal pigment epithelium (retinal pigment epithelium) under the foveal center in the right eye. The patient was immunosuppressed postoperatively with prednisone, cyclosporine, and azathioprine. The patient died from congestive heart failure 114 days after surgery. RESULTS: A patch of hyperpigmentation was visible at the transplant site under the foveola after surgery. Mound-like clusters of individual round, large densely pigmented cells were present in the subretinal space and outer retina in this area. There was loss of the photoreceptor outer segments and native retinal pigment epithelium in the center of the transplant bed, with disruption of the outer nuclear layer predominantly over regions of multilayered pigmented cells. Cystic spaces were present in the inner and outer retina. A residual intra-Bruchs membrane component of the original choroidal neovascular complex was present under the transplant site. CONCLUSIONS: The transplant site contained clusters of round, pigmented cells that did not form a uniform monolayer in most areas. The morphology at the transplant site is consistent with the lack of visual improvement seen after surgery in this patient.     

Transplantation of photoreceptors to light-damaged retina

We investigated the possibility of reconstructing light-damaged retinas by photoreceptor transplantation. The outer nuclear layer containing the photoreceptor cells was eliminated in adult albino rat retinas by exposure to constant high-level illumination. Photoreceptors for transplantation were harvested from neonatal rats using a novel isolation technique that maintained the cellular organization of the outer nuclear layer. Transplantation was accomplished using a transcorneal approach to the subretinal space, which minimized trauma to the eye. The retina reattached to the back of the eye with transplanted photoreceptors interposed between the retina and the overlying tissues. Prelabelling with fluorescent dye enabled positive identification of the transplanted cells. The transplanted photoreceptors appeared to survive transplantation for at least 6 weeks and were immunohistochemically reactive for opsin. The antibody staining for opsin identifies the transplanted cells as photoreceptors and indicates that they are still capable of producing visual pigment and therefore may have the capacity to transduce light. These findings indicate that photoreceptors can be transplanted to form a new outer nuclear layer in a damaged mature retina.     

Expression of the diabetes risk gene wolframin (WFS1) in the human retina

Wolfram syndrome 1 (WFS1, OMIM 222300), a rare genetic disorder characterized by optic nerve atrophy, deafness, diabetes insipidus and diabetes mellitus, is caused by mutations of WFS1, encoding WFS1/wolframin. Non-syndromic WFS1 variants are associated with the risk of diabetes mellitus due to altered function of wolframin in pancreatic islet cells, expanding the importance of wolframin. This study extends a previous report for the monkey retina, using immunohistochemistry to localize wolframin on cryostat and paraffin sections of human retina. In addition, the human retinal pigment epithelial (RPE) cell line termed ARPE-19 and retinas from both pigmented and albino mice were studied to assess wolframin localization. In the human retina, wolframin was expressed in retinal ganglion cells, optic axons and the proximal optic nerve. Wolframin expression in the human retinal pigment epithelium (RPE) was confirmed with intense cytoplasmic labeling in ARPE-19 cells. Strong labeling of the RPE was also found in the albino mouse retina. Cryostat sections of the mouse retina showed a more extended pattern of wolframin labeling, including the inner nuclear layer (INL) and photoreceptor inner segments, confirming the recent report of Kawano et al. [Kawano, J., Tanizawa, Y., Shinoda, K., 2008. Wolfram syndrome 1 (Wfs1) gene expression in the normal mouse visual system. J. Comp. Neurol. 510, 1-23]. Absence of these cells in the human specimens despite the use of human-specific antibodies to wolframin may be related to delayed fixation. Loss of wolframin function in RGCs and the unmyelinated portion of retinal axons could explain optic nerve atrophy in Wolfram Syndrome 1.     

Photoreceptor survival in transplantation of autologous iris pigment epithelial cells to the subretinal space

PURPOSE: To investigate photoreceptor survival in transplantation of non-cultured iris pigment epithelial (IPE) cells to the subretinal space in a prospective experimental study. METHODS: Upper iridectomies were carried out in the right eyes of 37 pigmented rabbits. Suspensions of freshly harvested autologous IPE cells (without culturing) were prepared and injected into the subretinal space of the same eye. Follow-up examinations were carried out using ophthalmoscopy and colour fundus photography. The rabbits were killed at 1, 2, 3 and 6 months, respectively, and the eyes examined with light and electron microscopy. RESULTS: On histological examination, the photoreceptor cells were found to be well-preserved in grafted areas at 1-3 months. At 6 months, the photoreceptors generally disclosed a normal nuclear layer and long outer segments when overlying areas with single cells or clusters of transplanted IPE cells. Multilayers of cells in abundance, including native RPE cells and macrophages (stained with RAM 11), particularly under microfolds of the neural retina, were occasionally associated with photoreceptor damage and nuclear drop out from the outer retinal layer. There was no inflammatory response in the choroid and the choriocapillaris remained patent. CONCLUSION: The experiments show that grafting freshly harvested autologous IPE cells to the subretinal space is feasible and that the photoreceptors generally survive for at least 6 months when overlying the transplanted areas. Multilayers of abundant cells in the subretinal space may induce adverse focal effects on adjacent photoreceptors.     

Morphological characterization of the retinal degeneration in three strains of mice carrying the rd-3 mutation

Retinal development in 3 strains of rd-3/rd-3 mutant mice, previously shown to have different rates of degeneration, was studied using light, electron, and immunofluorescence microscopy. The time course and phenotype of the degeneration as well as details on the mechanism of massive photoreceptor cell loss are compared with other known retinal degenerations in mice. Up until postnatal day (P) 10, the retinas of all three strains (RBF, 4Bnr, In-30) develop similarly to those of pigmented and nonpigmented controls. TUNEL-positive cells appear in the outer nuclear layer (ONL) by P14, and reach a maximum in all three mutant strains around P21. Scattered rods and cones form a loose, monolayered ONL by 8 weeks in the albino RBF strain, by 10 weeks in the albino 4Bnr strain, and by 16 weeks in the pigmented In-30 strain. Though the initial degeneration begins in the central retina, there is no preferred gradient of cell death between central and peripheral photoreceptors. Rods and cones are present at all ages examined. During development, stacks of outer segments (OS) form in all three strains though they never achieve full adult lengths, and often have disorganized, atypical OS. Rod opsin is expressed in the developing OS but is redistributed into plasma membrane as OS degeneration proceeds. Retinal pigment epithelial (RPE) cells of all mutant strains contain packets of phagocytosed OS, and their apical processes associate with the distal ends of the OS. At their synaptic sites, photoreceptor terminals contain ribbons apposed to apparently normal postsynaptic triads. As photoreceptors are lost, Müller cells fill in space in the ONL but they do not appear to undergo significant hypertrophy or migration, though during the degeneration, glial fibrillary acidic protein (GFAP) expression is gradually upregulated. Macrophage-like cells are found frequently in the subretinal space after the onset of photoreceptor apoptosis. As OS disappear, the RPE apical processes revert to simple microvilli. Late in the degeneration, some RPE cells die and neighboring cells appear to flatten as if to maintain confluence. In regions of RPE cell loss that happen to lie above retina where the ONL is gone, cells of the inner nuclear layer (INL), wrapped by Müller cell processes, may front directly on Bruch's membrane.