Ribozyme rescue of photoreceptor cells in a transgenic rat model of autosomal dominant retinitis pigmentosa.

Ribozymes, catalytic RNA molecules that cleave a complementary mRNA sequence, have potential as therapeutics for dominantly inherited disease. Twelve percent of American patients with the blinding disease autosomal dominant retinitis pigmentosa (ADRP) carry a substitution of histidine for proline at codon 23 (P23H) in their rhodopsin gene, resulting in photoreceptor cell death from the synthesis of the abnormal gene product. Ribozymes can discriminate and catalyze the in vitro destruction of P23H mutant mRNAs from a transgenic rat model of ADRP. Here, we demonstrate that in vivo expression of either a hammerhead or hairpin ribozyme in this rat model considerably slows the rate of photoreceptor degeneration for at least three months. Catalytically inactive control ribozymes had less effect on the retinal degeneration. Intracellular production of ribozymes in photoreceptors was achieved by transduction with a recombinant adeno-associated virus (rAAV) incorporating a rod opsin promoter. Ribozyme-directed cleavage of mutant mRNAs, therefore, may be an effective therapy for ADRP and also may be applicable to other inherited diseases.

Kinetics of rod outer segment renewal in the developing mouse retina.

The kinetics of rod outer segment renewal in the developing retina have been investigated in C57BL/6J mice. Litters of mice were injected with [(3)H]amino acids at various ages and killed at progressively later time intervals. Plastic 1.5 microm sections of retina were studied by light microscope autoradiography. The rate of outer segment disk synthesis, as judged by labeled disk displacement away from the site of synthesis, is slightly greater than the adult level at 11-13 days of age; it rises to more than 1.6 times the adult rate between days 13 and 17, after which it falls to the adult level at 21-25 days. The rate of disk disposal, as measured by labeled disk movement toward the site of disposal, is less than 15% of the adult level at 11-13 days of age; it rises sharply to almost 70% of the adult level by days 13-15 and then more gradually approaches the adult rate. The net difference in rates of synthesis and disposal accounts for the rapid elongation of rod outer segments in the mouse between days 11 and 17 and the subsequent, more gradual elongation to the adult equilibrium length reached between days 19 and 25. The changing rate of outer segment disk synthesis characterizes the late stages of cytodifferentiation of the rod photoreceptor cells.

Photoreceptor-pigment epithelial cell relationships in rats with inherited retinal degeneration. Radioautographic and electron microscope evidence for a dual source of extra lamellar material.

Protein synthesis and displacement in photoreceptor and pigment epithelial cells of inbred normal (Fisher) and mutant (RCS) rats with inherited retinal degeneration has been studied by light and electron microscope radioautography. Groups of animals 14, 15, 17, 19, 27, 35, and 50 days of age were injected with amino acids-H(3) and killed at subsequent time intervals. In normal rats, radioactive protein synthesized in the rod inner segments was incorporated into outer segment saccules and displaced outward; the total renewal time of outer segments at all ages was approximately 9 days. In RCS photoreceptors, outer segment displacement was slowed from the normal rate before day 17 and at all subsequent stages. Most of the newly synthesized protein appeared to migrate only into the basal third of the outer segments. Labeling of pigment epithelial cells in RCS rats was always heavier than in controls. Labeled protein was displaced as early as 1 hr postinjection from pigment epithelial cell somas into the apical processes, and by 2 hr postinjection was located in the adjacent lamellar whorls characteristic of the mutant rat retina. After 1 day, radioactivity was present in the 14, 15, 17, and 19 day series of RCS rats in the apical third of the outer segment layer (occupied mainly by extra lamellar material) while there were few silver grains in the middle third of the layer (occupied mainly by distal parts of outer segments). The RCS pigment epithelial cells thus have an unusual synthetic role and appear to be a source of the extra lamellar material. Electron microscope examination revealed that many intact pigment epithelial cell processes were incorporated into the large whorls of extra lamellae. In addition, many disorganized outer segment saccules were observed in continuity with longer membranous lamellae and large lamellar whorls. The extra lamellar material therefore appears to be derived from both rod outer segments and pigment epithelial cells.

Rod outer segment disk shedding in rat retina: relationship to cyclic lighting.

When albino rats are reared in cyclic light, a burst of rod outer segment disk shedding occurs in the retina soon after the onset of light. The number of large packets of outer segment disks (phagosomes) in the pigment epithelium at this time is 2.5 to 5 times greater than at any other time of day or night. The subsequent degradation of large phagosomes to smaller structures within pigment epithelial cells proceeds rapidly. The burst of disk shedding follows a circadian rhythm for at least 3 days, since it occurs in continuous darkness at the same time without the onset of light.

Inherited retinal dystrophy: primary defect in pigment epithelium determined with experimental rat chimeras.

Chimeric rats were produced by the aggregation of embryos of the pinkeyed, retinal dystrophic RCS strain with those of pigmented, normal rats. In the mosaic eyes, patches of neural retina with abnormal and degenerated photoreceptors were present only opposite patches of nonpigmented, mutant pigment epithelium. This indicates that the retinal dystrophy gene acts in the pigment epithelial cell rather than in the photoreceptor cell.

Retrograde axonal transport in the central nervous system.

When horseradish peroxidase is injected into the optic tectum of a chick, axons of ganglion cells transport it centripetally to their cell bodies in the retina at a rate of about 72 millimeters per day. After intraocular injections in the young chick, the peroxidase is transported centripetally along efferent axons, and is concentrated in cell bodies within the isthmo-optic nucleus. This retrograde movement of protein from axon terminal to cell body suggests a possible mechanism by which neurons respond to their target areas.

Light-evoked changes in the interphotoreceptor matrix.

The normal function of vertebrate photoreceptor cells depends on multiple interactions and transfer of substances between the photoreceptors and the retinal pigment epithelium (RPE), but the mechanisms of these interactions are poorly understood. Many are thought to be mediated by the interphotoreceptor matrix (IPM), a complex extracellular matrix that surrounds the photoreceptors and lies between them and the RPE. Histochemical, immunocytochemical, and lectin probes for several IPM constituents revealed that components of the IPM in the rat undergo a major shift in distribution or molecular conformation after the transition between light and dark. In the light, various IPM constituents concentrated in bands at the apical and basal regions of the outer segment zone; in the dark, they distributed much more uniformly throughout the zone. The change in IPM distribution was triggered by the light-dark transition; it was not a circadian event, and it was not driven by a systemic factor. The light-evoked change in IPM distribution may facilitate the transfer of substances between the photoreceptors and the RPE.

Quantitative genetics of age-related retinal degeneration: a second F1 intercross between the A/J and C57BL/6 strains.

PURPOSE: Previously, several quantitative trait loci (QTL) that influence age-related retinal degeneration (ageRD) were demonstrated in a cross between the C57BL/6J-c(2J) and BALB/cByJ strains (B x C). In this study, as a complementary approach to ongoing recombinant progeny testing for the purpose of identifying candidate quantitative trait genes (QTG), a second test cross using the A/J and the pigmented C57BL/6J strains (A x B) was carried out. The albino A/J strain was selected because it had the most amount of ageRD among several inbred strains tested, and the pigmented C57BL/6J strain was selected because along with its coisogenic counterpart C57BL/6J-c(2J) it had the least amount of ageRD. Thus, the effect of pigment on ageRD could be tested at the same time that the C57BL/6 genetic background was kept in common between the crosses from the two studies for the purpose of comparison. METHODS: A non-reciprocal F1 intercross between the A/J and C57BL/6J strains produced 170 F2 progeny. At 8 months of age after being maintained in relatively dim light, F2 mice, control mice and mice of other strains were evaluated for retinal degeneration by measurement of the thickness of the outer nuclear layer of the retina. The F2 mice were genotyped with dinucleotide repeat markers spanning the genome. Correlation of genotype with phenotype was made with Map Manager QTX software. RESULTS: Comparison of several strains of mice including the pigmented strains 129S1/SvImJ and C57BL/6J and the albino strains A/J, NZW/LacJ, BALB/cByJ and C57BL/6J-c(2J), showed significant differences in ageRD. The greatest difference was between the albino A/J strain and the pigmented C57BL/6J strain. However, there was no significant difference between the pigmented C57BL/6J and its albino coisogenic counterpart C57BL/6J-c(2J). Neither was there significant difference between the pigmented and albino F2 mice from the A x B cross. On the other hand, F2 males had a small but significantly lower amount of ageRD than females. Several QTL were identified in the A x B cross but surprisingly none of the 3 major QTL present in the original B x C cross (Chrs 6, 10, and 16) was present. There were minor QTL on proximal Chr 12 and proximal Chr 14 in common between the two crosses, and the proximal Chr 12 QTL was present in a previous light damage study involving the B and C strains. At least one sex-limited QTL was present on the X chromosome with a peak in a different location from that of a sex-limited QTL in the previous B x C study. In addition, the protective X allele was from the BALB/cByJ strain in the B x C cross and from C57BL/6J in the A x B cross. In both crosses, the C57BL/6J X-chromosome allele was recessive. CONCLUSIONS: Significant differences were observed in ageRD among several inbred strains of mice maintained in relatively dim light. AgeRD was not influenced by pigment but was influenced by gender, albeit to a small degree. The presence of the same QTL in one light-induced and two ageRD studies suggests at least partial commonality in retinal degeneration pathways of different primary cause. However, the three main QTL present in the B x C cross were absent from the A x B cross. This suggests that the genetic determinants responsible for the greater sensitivity to ageRD of BALB/cByJ and A/J relative to C57BL/6J are not the same. This is supported by the presence of sex-limited X-chromosome QTL in the two crosses in which the C57BL/6J allele is protective relative to the A allele and sensitive relative to the C allele. The findings in the two studies of differing allelic relationships of QTG, and differing QTL aid the identification of candidate genes mapping to critical QTL. Identifying natural modifying genes that influence retinal degeneration resulting from any causal pathway, especially those QTG that are protective, will open avenues of study that may lead to broad based therapies for people suffering retinal degenerative diseases.

Light and inherited retinal degeneration.

Light deprivation has long been considered a potential treatment for patients with inherited retinal degenerative diseases, but no therapeutic benefit has been demonstrated to date. In the few clinical studies that have addressed this issue, the underlying mutations were unknown. Our rapidly expanding knowledge of the genes and mechanisms involved in retinal degeneration have made it possible to reconsider the potential value of light restriction in specific genetic contexts. This review summarises the clinical evidence for a modifying role of light exposure in retinal degeneration and experimental evidence from animal models, focusing on retinitis pigmentosa with regional degeneration, Oguchi disease, and Stargardt macular dystrophy. These cases illustrate distinct pathophysiological roles for light, and suggest that light restriction may benefit carefully defined subsets of patients.

Role of neurotrophin receptor TrkB in the maturation of rod photoreceptors and establishment of synaptic transmission to the inner retina.

Brain-derived neurotrophic factor (BDNF) acts through TrkB, a receptor with kinase activity, and mitigates light-induced apoptosis in adult mouse rod photoreceptors. To determine whether TrkB signaling is necessary for rod development and function, we examined the retinas of mice lacking all isoforms of the TrkB receptor. Rod migration and differentiation occur in the mutant retina, but proceed at slower rates than in wild-type mice. In postnatal day 16 (P16) mutants, rod outer segment dimensions and rhodopsin content are comparable with those of photoreceptors in P12 wild type (WT). Quantitative analyses of the photoreceptor component in the electroretinogram (ERG) indicate that the gain and kinetics of the rod phototransduction signal in dark-adapted P16 mutant and P12 WT retinas are similar. In contrast to P12 WT, however, the ERG in mutant mice entirely lacks a b-wave, indicating a failure of signal transmission in the retinal rod pathway. In the inner retina of mutant mice, although cells appear anatomically and immunohistochemically normal, they fail to respond to prolonged stroboscopic illumination with the normal expression of c-fos. Absence of the b-wave and failure of c-fos expression, in view of anatomically normal inner retinal cells, suggest that lack of TrkB signaling causes a defect in synaptic signaling between rods and inner retinal cells. Retinal pigment epithelial cells and cells in the inner retina, including Müller, amacrine, and retinal ganglion cells, express the TrkB receptor, but rod photoreceptors do not. Moreover, inner retinal cells respond to exogenous BDNF with c-fos expression and extracellular signal-regulated kinase phosphorylation. Thus, interactions of rods with TrkB-expressing cells must be required for normal rod development.

Rods and cones in the mouse retina. I. Structural analysis using light and electron microscopy.

Rods and cones of the C57BL/6J mouse retina have been examined by light and electron microscopy to distinguish the structural features of the two photoreceptor types. By light microscopy, cone nuclei are conspicuously different from rod nuclei in 1-2 micrometer plastic sections. Cone nuclei have an irregularly shaped clump of heterochromatin that appears in single sections to be one to three clumps, whereas rod nuclei are more densely stained and have one large, central clump of heterochromatin. Cone nuclei make up approximately 3% of the photoreceptor nuclei in both the central and peripheral retina at all ages examined up to 267 days. Cone nuclei are confined to the outer half of the outer nuclear layer, and more than 50% of the cone nuclei lie adjacent to the outer limiting membrane. By electron microscopy, cones in the mouse retina meet virtually every morphological criterion of mammalian cones. The outer segments are conically shaped. Many, if not all of the outer segment discs are continuous with the outer plasma membrane, whereas almost all of the rod discs are not. Cone outer segments are only about half the length of the rod outer segments, and they are contacted by long, villous pigment epithelial cell processes. The cone inner segment diameter is greater than the outer segment diameter, and the accumulation of mitochondria present at the apical end of the inner segment forms a more conspicuous ellipsoid than in rods. The internal fiber or axon of the cone is larger in diameter than that of the rod, and it terminates in a large synaptic pedicle with multiple ribbon synapses, whereas the rod terminal is a smaller spherule with only a single ribbon synaptic complex.

Rods and cones in the mouse retina. II. Autoradiographic analysis of cell generation using tritiated thymidine.

The period of cell genesis of rod and cone photoreceptor cells has been determined in the retinas of C57BL/6J mice. Embryonic mice were exposed to a single dose of 3H-thymidine at embryonic day (E) 10--18 by injecting pregnant mice intraperitoneally. Animals at postnatal ages were injected subcutaneously once between postnatal day (P) 0--10. The eyes were removed at one to three months of age. After fixation, they were embedded in glycol methacrylate, sectioned at 1.5 micrometers and prepared for autoradiographic analysis. All of the cone cells are generated over a relatively short time interval during the fetal period. In the posterior retina, the peak of cone cell genesis occurs at E13-E14, and no cones are generated after E16. The rods, by contrast, are generated later and over a longer time period. They first begin to be generated in the posterior retina on E13, but the peak of cell genesis is not reached until the day of birth, and some rods are generated as late as P5. For both rods and cones the peaks of cell genesis in the peripheral retina occur two to three days later than in the posterior retina. The findings demonstrate that rods and cones are developmentally distinct cell types in the mouse retina.

Genesis of neurons in the retinal ganglion cell layer of the monkey.

We have analyzed the genesis of various neuronal classes and subclasses in the ganglion cell layer of the primate retina. Neurons were classified according to their size and the time of their origin was determined by pulse labeling with 3H-thymidine administered to female monkeys 38 to 70 days pregnant. All offspring were sacrificed postnatally, and their retinas processed for autoradiography. The somata of cells in the retinal ganglion cell layer generated on embryonic day (E) 38 ranged from 9 to 14 microns in diameter. Between E40 and E56, the minimum soma diameter remained around 8-9 microns, while the maximum gradually increased to 22 microns. As a consequence, the means of the distributions of labeled cells also increased with age, from 11.8 microns diameter for cells generated on E38 to 14.6 microns diameter at E56. Over this period the percentage of labeled cells in the 10.5-16.5 microns and greater than 16.5 microns diameter range gradually increased. The proportion of the labeled cells in the less than 10.5 microns diameter range decreased from E38 to E45, but subsequently increased rapidly. At the end of neurogenesis in the retinal ganglion cell layer, around E70, most labeled cells were considerably smaller (7-9 microns) than those generated earlier. Our results indicate that within the ganglion cell layer of the macaque, neurons of small caliber are generated first, followed successively by medium sized cells. Large, putative P alpha cells are generated late. The production between E56 and E70 of cells with the smallest somata suggests that the last-generated neurons in the ganglion cell layer are predominantly displaced amacrine cells. Within the same sector of retina, different classes of neurons in the ganglion cell layer of the rhesus monkey appear to have a sequential schedule of production.

Genesis of the retinal pigment epithelium in the macaque monkey.

The development of the retinal pigment epithelium (RPE) was studied in rhesus monkey (Macaca mulatta) fetuses, neonates, and juveniles exposed to a pulse of 3H-thymidine (3H-TdR) between embryonic day (E) 25 and postnatal day (P) 204 and examined at short and long intervals after the injection of the isotope. The RPE develops from the outer layer of the optic cup which by E45 consists of a multistratified epithelium. The outer layer appears immature near the retina's edge and gradually becomes monostratified and more mature centrally. Even at this early stage, all cells contain pigmented melanosomes, although peripherally the pigment is limited to the apical portion of the cells. Examination of autoradiograms from animals allowed to survive for several postnatal months shows that monkey RPE cell genesis begins just after E27, increasing to a peak frequency of 0.38 cells/mm at E43. Between E30 and E85 the density of radiolabelled cells varies within a restricted range of from 0.2 to 0.4 cells/mm (mean = 0.25 +/- 0.09). From the density of radiolabelled cells, and data on the overall density of RPE cells in the juvenile retina, we determined the labelling index. During the first half of gestation, between 0.38% and 0.99% (mean = 0.65 +/- 0.22) of RPE cells are generated during the short interval of isotope availability after pulse injection. Approximately 5% of RPE cells were generated by E33, and 50% by E71. After E85, RPE cytogenesis begins gradually to decrease, and 95% of the cells have been generated by the time of birth. Continued, very low density (0.01 cells/mm) cytogenesis in the RPE is seen at P17, and persists at least until seven months postnatally. RPE cell genesis begins near the fovea, and proceeds towards the periphery. Cell division largely ceases in both foveal and perifoveal regions by E56, at which time labelled cells first begin to appear peripheral to the equator. Besides the timing differences, RPE genesis in the central retina differs from that in the peripheral retina in that it proceeds at a higher rate, and lasts for a shorter time period. A prolonged postnatal period of low density RPE cell genesis persists in both central and peripheral retina. Comparison of the pattern of expansion of the area containing radiolabelled cells in the RPE and neuroretina demonstrates a remarkable spatial and temporal correspondence. Close analysis suggests that at any point on the retina, the last cells are generated in the neuroretina slightly before the last cells in the RPE.

Retinal degeneration in the pcd cerebellar mutant mouse. II. Electron microscopic analysis.

The cerebellar ataxia in Purkinje cell degeneration (pcd) mutant mice results from the rapid loss of Purkinje cells between 3 and 5 weeks after birth. The loss of photoreceptors in these mutants begins about the same time but proceeds slowly, with most photoreceptors being lost by 1 year of age. In this study the retinas of pcd/pcd mice and their littermate controls from the age of 10 postnatal days to 15 months were analyzed by electron microscopy. The first signs of photoreceptor cell degeneration are apparent in the region of photoreceptor inner segments as early as postnatal day 13, and more prominently at day 18. During this time, the degeneration is characterized by a large number of vesicles, ranging in diameter from 150 to 350 nm, which are located in the extracellular space adjacent to the photoreceptor inner segments. Analysis of serial sections shows that most of these membrane-bound degeneration profiles are tubular in shape and some are continuous with the cell membrane of the inner segment. Therefore, these "profiles" are thought to arise from tubular outpocketings of the inner segments which cleave off to form isolated membrane-bound profiles. This represents a new and unusual form of photoreceptor degeneration. While the most obvious abnormality in the retina is degeneration of photoreceptor cells, Müller cells also appear to be affected, with swollen apical processes often seen coursing through the outer nuclear layer.

Retinal degeneration in the pcd cerebellar mutant mouse. I. Light microscopic and autoradiographic analysis.

The Purkinje cell degeneration (pcd) mutant mouse rapidly loses cerebellar Purkinje cells between 3 and 5 weeks after birth and slowly loses retinal photoreceptor cells during the first year of life. In the present study, the retinal degeneration in the pcd mouse was analyzed by light microscopy and autoradiography throughout the first 15 months of age. By day 25 there is an abundance of pyknotic photoreceptor nuclei and many outer segments are clearly disorganized. Thereafter, as the photoreceptor cells are lost, their outer segments slowly become shorter and more variable in length; this slow change in length is explained by an almost proportional reduction in both rod outer segment renewal and disc shedding rates. At about 2 months of age, the rate of rod outer segment renewal is slightly less than half that in littermate controls, and the number of large phagosomes in pigment epithelial cells during the burst of disc shedding soon after light onset is one-half or less than that seen in littermate controls. Between 2 and 10.5 months of age, the retina in the inferior hemisphere of the eye shows substantially more-advanced photoreceptor degeneration than does the superior hemisphere, particularly in the far peripheral retina. A central-to-peripheral gradient of degeneration is conspicuous in the superior hemisphere; a similar but less obvious gradient of degeneration is also seen in the inferior hemisphere of the eye. Loss of photoreceptor cells and their synaptic terminals results in a predictable thinning of the outer synaptic layer. However, the inner nuclear layer shows no measurable thinning and the inner synaptic layer is reduced in thickness by only about 5-15%. Beginning at about 10 months of age, foci of thinned pigment epithelial cells are evident, and by 12 months there is some vascularization of the pigment epithelium by retinal capillaries.

Assessment of possible transneuronal changes in the retina of rats with inherited retinal dystrophy: cell size, number, synapses, and axonal transport by retinal ganglion cells.

In pigmented RCS rats with inherited retinal dystrophy, most photoreceptor cells disappear between postnatal days 20 and 100. We have examined the time course of the degeneration of photoreceptor nuclei and synapses and determined whether transneuronal changes occur in the inner nuclear layer (INL), inner plexiform layer (IPL), and retinal ganglion cells following loss of photoreceptor cells in these animals. Electron microscopic photomontages of the entire thickness of the IPL of dystrophic (RCS-p+) and control (RCS-rdy+ p+) rats 334 to 515 days old were prepared, and synapses were counted and identified as either conventional (amacrine) or ribbon (bipolar) types. Neither the incidence of synapses in the IPL nor the ratio of conventional to ribbon synapses differed in the dystrophic and control retinas. Ganglion cell diameter, perimeter, area, and density were measured from drawings of wholemount preparations of dystrophic and control rats 105 days and older. Diameter, perimeter, area and number of ganglion cells were not significantly different in the two genotypes. Anterograde axonal transport was measured by studying the displacement of labeled material as it traveled along ganglion cell axons and accumulated in the superior colliculus. The normal and dystrophic rats showed no significant difference in (1) the rates of rapidly moving components (approximately 110-180 mm/day) and slowly moving components (1.7-2.5 mm/day) or (2) the amount of radioactive material transported to the superior colliculus. The absence of transneuronal changes in retinal ganglion cells of RCS rats contrasts with results obtained earlier in rd mice (Graftstein et al., '72). Unlike the RCS rat, retinal degeneration in rd mice occurs before the maturation of the retina. We hypothesize that the ganglion cells may be more affected by loss of input early in development, and, therefore, ganglion cells of retinal dystrophic rats are less affected despite little or no synaptic input for several months. Furthermore, any reduction in the electrical activity of retinal ganglion cells that might follow loss of photoreceptor cells does not result in a significantly decreased rate of axonal transport.

Retinal degeneration in the nervous mutant mouse. II. Electron microscopic analysis.

Nervous mutant mice (nr/nr) show a rapid loss of most of cerebellar Purkinje cells between the ages of 3 and 7 weeks, as well as a progressive photoreceptor cell degeneration that occurs most rapidly between postnatal days (P) 13 and 19, but with a much slower attrition during the subsequent months. We have carried out an electron microscopic analysis of nr/nr and littermate control mice at representative ages to characterize the subcellular cytopathological changes in this form of retinal degeneration, to gain insight into photoreceptor cell degeneration mechanisms by comparing these changes to those in other rodent forms of retinal degeneration, and to compare the photoreceptor changes with those of cerebellar Purkinje cells. Ultrastructural observations were limited to rod photoreceptors, since the number of cones was limited in our micrographs. The retinas of nr/nr mutant mice can be distinguished from those of normal littermates as early as postnatal day (P) 6. At this time, some of the mitochondria in rod inner segments are larger and more rounded than normal. This represents the earliest cytopathological change thus far observed in this mutant. As early as P9 and thereafter, the volume and integrity of rod outer segment membranes are reduced from normal. In the inner segments of some rod photoreceptor cells, there is a reduction in the volume or number of polyribosomes as early as P11, a reduction in rough endoplasmic reticulum as early as P13, and reduced incidence and less organized Golgi membranes as early as P16. Qualitative evaluation and quantitative stereological analysis show that the enlarged mitochondria in rod inner segments never become normal in shape or size. No changes are seen in the inner retinal layers at any age. Despite similarities with inherited retinal dystrophy in the Royal College of Surgeons rat, as noted in the original description of retinal degeneration in nr/nr mice, ultrastructural features clearly distinguish these mutants. Moreover, nr/nr mice can be distinguished from all other murine forms of retinal degeneration by electron microscopy.

Ganglion cell loss in RCS rat retina: a result of compression of axons by contracting intraretinal vessels linked to the pigment epithelium.

In the dystrophic Royal College of Surgeons (RCS) rat retina, there is a progressive loss of photoreceptors. As a result, the retinal circulation becomes apposed to the retinal pigment epithelium (RPE) and neovascular formations develop. RPE and inner nuclear layer cells migrate along these vessels towards the retinal ganglion cell (RGC) layer. The retinal layers gradually become disrupted, and some of the RGC axon bundles involute into the retina. These bundles are always associated with blood vessels, and there is evidence of axon damage where they juxtapose. In wholemount preparations of dystrophic retinae (> or =6 months of age), abrupt changes are observed in the trajectory of RGC axon bundles, where they are crossed by circumferential vessels. Degenerative profiles can be seen at these locations. Visualisation of RGCs with Fluoro-gold shows wedge-shaped sectors in the dystrophic retina devoid of labelling, initially in the ventral retina but later spreading dorsally. It is hypothesised that the vessels supplying the neovascular formations contract and pull surface vessels into the retina, thus displacing any axon bundles that lie beneath them into the inner plexiform layer. The contractility may be an intrinsic property of the vessels or it may be conferred by the cells migrating along them. Axonal transport becomes blocked at the points of tension, thereby causing retrograde degeneration of the parent RGCs. Because RGC loss is also a feature of human retinitis pigmentosa, the RCS rat may provide a model to test interventions devised to prevent such loss following photoreceptor degeneration. This model also may be useful for testing methods designed to control blood vessel and matrix formation.

Retinal degeneration in the nervous mutant mouse. I. Light microscopic cytopathology and changes in the interphotoreceptor matrix.

Nervous is an autosomal recessive mutation in mice (gene symbol, nr) that produces a progressive cerebellar and retinal degeneration. We have examined various cytopathological features of the photoreceptor degeneration by light microscopy. An increase in the number of pyknotic photoreceptor nuclei in the outer nuclear layer (ONL) is first seen at postnatal day (P) 11. Between P13 and P19 there is a rapid loss of photoreceptors, with the ONL about 60% the thickness of littermate controls at P19. Between P19 and 2.5 months of age, photoreceptor cell loss is minimal, and there is a relatively slow loss of these cells between 3 and 7.5 months of age. At 7.5 months, the ONL consists of single row of nuclei, most of which are lost over the ensuing months, although a few photoreceptor nuclei persist at 17 months of age and older. Both rods and cones are lost at comparable rates for the first 2 months of life, but rods are somewhat preferentially lost at later ages. A very slight central-to-peripheral gradient of photoreceptor degeneration exists in the nr/nr retina, but no superior-inferior hemispheric differences are evident. The rate, spatiotemporal gradient, and hemispheric similarity in photoreceptor degeneration are the same in albino nr/nr mice reared either in cyclic light or in the dark, and in pigmented nr/nr mice. Autoradiographic analysis of rod outer segment renewal shows that outer segment membranes are synthesized in nervous homozygotes. Rhythmic outer segment disc shedding and phagocytosis by the retinal pigment epithelium occur at approximately normal rates in nr/nr mice. Histochemical and immunocytochemical study of the interphotoreceptor matrix (IPM) reveals the exclusion of stainable IPM from the outer segment zone by lamellar whorls of outer segment membrane, accumulation of stainable IPM in the basal region of the outer segment zone, and the absence of an intense band of stainable IPM at the apical surface of the retinal pigment epithelium. These changes in the IPM are similar to those seen in the Royal College of Surgeons rat. However, comparison of cytopathological changes in these two mutants reveal that the IPM defect probably is not the primary cause of photoreceptor cell death in nr/nr mice, and that similar phenotypic appearance does not necessarily signify similar pathological processes.