Distribution of 70 kD stress protein in the optic nerve. A comparative study between the normal and retinal dystrophic rat]

The immunolocalization of the 70 kD stress protein (SP70) was investigated and compared in the optic nerve of normal Sprague-Dawley (SD) rats and that of the Royal College of Surgeons (RCS) rat with inherited retinal dystrophy. At postnatal day 8, SP70 was present in the maturing glial cell bodies of both rat strains. At postnatal day 22, SP 70 was observed in the glial cell bodies and optic nerve fibers of both rat strains. At postnatal day 40 RCS rat, SP70 was diminished in the optic nerve fibers and glial cell bodies but was increased in the glial cell nuclei. This suggests that axonal transportation of SP70 from the retina may be reduced following retinal degeneration. In the SD optic nerve, the normal distribution of immunostaining for SP70 was preserved. Stress proteins are thought to play an important role in cellular development and survival mechanisms. It was suggested that optic nerve was damaged by the retinal degeneration in the RCS rat.     

Developmental study of chondroitin-6-sulphate in normal and dystrophic rat retina

The RCS rat is a widely studied model of human retinal dystrophies including retinitis pigmentosa. Chondroitin-6-sulphate (C6S) in the interphotoreceptor matrix was localised immunocytochemically in both the normal congenic and dystrophic strains of the RCS rat up to 65 days postnatally. From postnatal days 5 to 15 the distribution of C6S in both strains was similar, being localised in the interstices of developing inner and outer segments and adjacent to the RPE surface. In the normal rats, the distribution of C6S did not change with age. In the RCS rats, however, at postnatal days 20 to 35 staining was observed as a dense band at the junction of inner and outer segments and no staining was observed adjacent to the surface of the RPE. At postnatal day 45 onwards there was a decrease and a complete absence of C6S staining in these rats. This change in the pattern of staining correlated with the morphological observation of the progressive degeneration of photoreceptor cells suggesting that C6S may be important in photoreceptor degeneration in the RCS rat.Correspondence to: L.N. WalkerThis study was partially supported by the Australian Retinitis Pigmentosa Association and Appealathon Foundation of Western Australia, NH & MRC, ORIA and OPSM Research Foundations     

Pigment epithelium-photoreceptor interactions in the normal and dystrophic rat retina.

The pigment epithelium of normal and dystrophic rat retinas phagocytized carbon particles that were injected into the extra-cellular space between photoreceptors and pigment epithelium. The epithelium of both infant and adult rats displayed phagocytic activity. Radioactively-labeled outer segment fragments from normal rats were usually not phagocytized by the pigment epithelium of either normal or dystrophic rats, although in rare cases some phagocytosis was observed in both the normal and dystrophic retinas. It is suggested that, prior to phagocytosis in normal animals, the pigment epithelium recognizes specific sites on the plasma membrane of discarded outer segment discs. It is further suggested that these sites are frequently removed in artificially prepared outer segment fragments either through the washing away of soluble components or removal of the enveloping plasma membrane. Although the pigment epithelium of the dystrophic rat retina will not phagocytize its endogenous outer segment debris, we now know that it is capable of phagocytosis when presented with the appropriate material. Our results also imply that modification of normal outer segment fragments can lead to their rejection by the normal pigment epithelium.     

Prosaposin gene expression in normal and dystrophic RCS rat retina

PURPOSE: To identify proteins secreted by the retinal pigment epithelium (RPE) and to analyze their cellular distribution in normal and pathologic rat retinas at various stages of eye development. METHODS: A cDNA library was constructed with RNA isolated from porcine RPE sheets and screened by using the yeast signal sequence trap system. In situ hybridization, immunohistochemistry, and semiquantitative RT-PCR analysis were performed on rat retinas. RESULTS: The cDNA encoding prosaposin was isolated. This is the first time this gene has been shown to be expressed in the retina. Prosaposin mRNA was detected in the rat RPE cell monolayer and in ganglion cells 14, 21, and 45 days after birth. The amount of prosaposin mRNA increased between days 14 and 45 after birth in normal retinas (rdy+), but not in the pathologic retinas (rdy-) of RCS rats. CONCLUSIONS: Several techniques were used to determine the localization of prosaposin in rat retinas. The increase in the amount of prosaposin mRNA in normal retinas coincided with the maturation of photoreceptor cells and the beginning of the phagocytosis process. In addition, the RCS rdy- RPE cells, characterized by the abrogation of the ingestion phase of the photoreceptor outer segments, are deficient in prosaposin expression.     

Permeability of retinal pigment epithelial cell junctions in the dystrophic rat retina

We have studied permeability of retinal pigment epithelial (RPE) cell junctions in Royal College of Surgeons rats with inherited retinal degeneration, and their genetic controls, using the horseradish peroxidase and lanthanum nitrate electron microscope tracer techniques. We find that early in the dystrophic process, at two postnatal weeks in the pink-eyed retina and three postanatal weeks in the black-eyed retina, RPE cell tight junctions form a barrier to extracellular tracer. However, at three postnatal weeks in the pink-eyed retina, at about the same time that degenerating photoreceptor nuclei begin to appear, RPE cell tight junctions become permeable. The permeability increase occurs later in the black-eyed strain, but by six postnatal weeks junctions are permeable in both strains. By 72 postnatal days, when most photoreceptor nuclei have disappeared, many RPE cells are abnormal in shape, with an elongated and flattened appearance, and some appear to have lost their junctions entirely. In the horseradish peroxidase experiments, many pinocytotic vesicles filled with reaction product were observed in the dystrophic RPE after the junctional breakdown. This suggests that an increase in transcellular transport may also occur in the dystrophic RPE.     

Localization of IgG in the normal and dystrophic rat retina after laser lesions.

PURPOSE: To test the hypothesis that access to extravasated plasma protein IgG may influence photoreceptor survival following laser photocoagulation and to determine whether this correlates with the retinal glial reaction. METHODS: A total of 45 rats (18 Royal College of Surgeons (RCS) dystrophic and 18 RCS-rdy+ congenic control) were used for this experiment. Nine non-lasered littermates of same age were used as controls. The superior retinas of postnatal day 23 rats were irradiated with a grid pattern of 40 argon green laser lesions of 50 microm in diameter and two powers (150 and 300 mW) for 0.2 s. At various times after laser lesions (up to 14 days), animals were perfused, the retinas snap frozen and sectioned on a cryostat. A one-step immunohistochemical technique was used by incubating with rabbit anti-rat IgG conjugated directly to horseradish peroxidase. Adjacent sections were processed using an antibody to glial fibrillary acidic protein (GFAP) by the standard avidin-biotin complex method. RESULTS: The labelling pattern for extravasated IgG after laser lesion was very similar in both RCS and RCS-rdy+ rat retinas. At 6, 12 and 24 h after lesions, IgG immunoreactivity (IR) was very intense in the lesion core and flanks. The outer plexiform layer (OPL) and photoreceptor inner segments provided a ready pathway for lateral spread of IgG. However, in the outer nuclear layer (ONL), IgG localization was much more restricted. Despite very intense IgG IR in the ONL of the coagulated lesion core, there was always a very sharply delineated boundary where the label abruptly halted. The GFAP labelling in both RCS dystrophic and RCS-rdy+ congenic control rat retinas showed that this boundary was between normal and necrotic cells because there was a core where GFAP was not produced by Müller cells. By 2 days after lesions, the coagulated cells in the lesion core were being removed by phagocytic cells that were IgG IR. Labelled phagocytic cells were also found among the inner and outer segment region on the lesion flanks. There was still IgG IR in the lesion, but the label was faint. No IgG IR was found in the retina at 3, 4, 7 and 14 days after lesions. Absorption control with pure rat IgG showed the label to be specific. CONCLUSIONS: The extravasated IgG was derived from the choroidal circulation because at no stage was IgG localized around the retinal vasculature. The IgG labelling was surprisingly widespread and, therefore, did not correlate with photoreceptor sparing, although it preceded the widespread Müller cell expression of GFAP and may, therefore, trigger glial reaction.     

Localization of IgG in the normal and dystrophic rat retina after laser lesions*

Purpose: To test the hypothesis that access to extravasated plasma protein IgG may influence photoreceptor survival following laser photocoagulation and to determine whether this correlates with the retinal glial reaction. Methods: A total of 45 rats (18 Royal College of Surgeons (RCS) dystrophic and 18 RCS-rdy⁺ congenic control) were used for this experiment. Nine non-lasered littermates of same age were used as controls. The superior retinas of postnatal day 23 rats were irradiated with a grid pattern of 40 argon green laser lesions of 50 μm in diameter and two powers (150 and 300 mW) for 0.2 s. At various times after laser lesions (up to 14 days), animals were perfused, the retinas snap frozen and sectioned on a cryostat. A one-step immunohistochemical technique was used by incubating with rabbit anti-rat IgG conjugated directly to horseradish peroxidase. Adjacent sections were processed using an antibody to glial fibrillary acidic protein (GFAP) by the standard avidin–biotin complex method. Results: The labelling pattern for extravasated IgG after laser lesion was very similar in both RCS and RCS-rdy⁺ rat retinas. At 6, 12 and 24 h after lesions, IgG immunoreactivity (IR) was very intense in the lesion core and flanks. The outer plexiform layer (OPL) and photoreceptor inner segments provided a ready pathway for lateral spread of IgG. However, in the outer nuclear layer (ONL), IgG localization was much more restricted. Despite very intense IgG IR in the ONL of the coagulated lesion core, there was always a very sharply delineated boundary where the label abruptly halted. The GFAP labelling in both RCS dystrophic and RCS-rdy⁺ congenic control rat retinas showed that this boundary was between normal and necrotic cells because there was a core where GFAP was not produced by Müller cells. By 2 days after lesions, the coagulated cells in the lesion core were being removed by phagocytic cells that were IgG IR. Labelled phagocytic cells were also found among the inner and outer segment region on the lesion flanks. There was still IgG IR in the lesion, but the label was faint. No IgG IR was found in the retina at 3, 4, 7 and 14 days after lesions. Absorption control with pure rat IgG showed the label to be specific. Conclusions: The extravasated IgG was derived from the choroidal circulation because at no stage was IgG localized around the retinal vasculature. The IgG labelling was surprisingly widespread and, therefore, did not correlate with photoreceptor sparing, although it preceded the widespread Müller cell expression of GFAP and may, therefore, trigger glial reaction.     

Rod outer segment lipid-opsin ratios in the developing normal and retinal dystrophic rat

The outer segment membrane lipid and opsin contents were determined in photoreceptor cell rods isolated from the eyes of developing normal rats reared in cyclic light or dark environments and dark-reared dystrophic rats. In cyclic light-reared normals rhodopsin/eye increased 49% during the period 20–60 days. Total ROS lipid content, a measure of ROS length, increased 50% while the polyunsaturated fatty acid docosahexaenoate increased from 42–51 mol % during the same period. The phospholipid/opsin ratio of cyclic light reared rat ROS membranes was 67 mol/mol at 20 days and 68 mol/mol at 60 days. In young dark-reared normals the phospholipid/opsin ratio was the same as for cyclic light-reared rats. Although 60 day-old dark-reared normals had 30% more rhodopsin/eye than their cyclic light-reared counterparts, non-significant changes in ROS length (14% longer) and in the phospholipid/opsin ratio (8% lower) were measured in these rats. In addition, light deprivation had no significant effect on the concentrations of polyunsaturated fatty acids or the lipid composition of the isolated ROS. The eyes of dark-reared rats with retinal dystrophy accumulated two times more rhodopsin than dark-reared normals during the 20–60-day period. The phospholipid/opsin ratio of mutant rat ROS was only 7% lower than dark normal at 20 days and 13% lower at 34 days. However, by 60 days of age, the phospholipid/opsin ratio in dystrophic rat ROS was significantly lower than in ROS from either cyclic light-or dark-reared normals. Docosahexaenoic acid in mutant rat ROS lipids averaged 40 mol% during the developmental period. These levels were significantly lower than the levels of docosahexaenoate measured in dark normals at both the 34- and 64-day periods. The glycerophospholipid composition of dystrophic rat ROS was the same as normal at all ages but the cholesterol/phospholipid ratio was higher than in normals.The data show: (1) that the retina accomodates changes in rhodopsin content induced by environmental light, age and genetic differences by alterations in ROS opsin density and length: (2) that the content of ROS membrane polyunsaturated fatty acids (fluidity) increases during development in normals but not in dystrophic rats. The data also suggest that basal membrane synthesis and/or post sythetic membrane modification of ROS lipid are impaired as a function of age in dystrophic rats.     

The use of sugar-coated beads to study phagocytosis in normal and dystrophic retina

In this study, a technique is described for coupling the sugars to carboxylated latex beads for use in phagocytic studies of dystrophic and normal retinal pigment epithelium. The presence and distribution of the sugars, mannose and fucose, on the bead surfaces are visualized by transmission electron microscopy using the lectin-Ferritin conjugates. Concanavalin A-Ferritin (Con A-Fe) and Ulex-europeus-Ferritin (Ulex-Fe), respectively. Both lectin-Ferritin conjugates label the surfaces of the sugar coated beads. Control experiments to determine the extent of nonspecific lectin-Ferritin binding using Con A-Fe with fucose-coated beads, Ulex-Fe with mannose-coated beads, or beads without coupling reagent show little to no labeling. Lectin-Ferritin conjugates are also incubated with the appropriate sugar-coated beads and their competing sugars and little to no labeling is observed. Our data shows that when sugar-coated beads are presented to retinal pigment epithelial explants, differences in phagocytic uptake are observed. Uncoated beads are avidly taken up by normal and dystrophic tissue, but fucose-coated beads are not taken up by either. Twice as many mannose beads are phagocytized by the normal as compared to dystrophic tissue. It remains to be established whether these differences in bead uptake indicate a difference in ligand-receptor interactions between normal and dystrophic retinal pigment epithelium.     

Ascorbate and glutathione levels in the developing normal and dystrophic rat retina: effect of intense light exposure

Ascorbic acid and glutathione were measured in retinas excised from normal rats reared in a cyclic light or dark environment and in dystrophic rats from the dark environment. Similar measurements were made on retinas from age matched rats exposed to intense visible light for periods of up to 24 hours. In other rats, ascorbic acid was given for various periods before exposure to intense light and the degree of photoreceptor cell death determined subsequently by rhodopsin measurements. In non-intense light treated rats ascorbate and glutathione were 12.1 nmol/retina at 20 days of age and 13.3 - 15.9 nmol/retina in 60 day old animals. In dystrophic rat retinas glutathione was 4-8% higher and ascorbate 10-20% higher than in normal dark reared rats. Although the levels of ascorbate and glutathione per retina increased during development, the molar ratios of the antioxidant materials to rhodopsin decreased by 36% and 60% in normal and dystrophic rats respectively. The levels of glutathione in young cyclic light or dark reared normals were unaffected by intense light exposure of either short (2-4 hrs) or long (24 hrs) duration. However, in both 20 and 40 day old dystrophic rats, intense light exposure resulted in a significant increase in retinal glutathione. In contrast to glutathione, retinal ascorbate decreased in normal rats exposed to intense light for 24 hrs, in an age and prior light environment dependent fashion. At ages greater than 20 days, normal rats exposed to light had significantly lower retinal ascorbate levels than their non-light exposed counterparts. The levels of ascorbate in 21-40 and 41-60 day old dark reared rat retinas were also significantly lower than in comparable intense light treated-cyclic light reared rats. In the youngest dystrophic rats whole eye ascorbate (retina, RPE, choroid and sclera) was 20-30% lower than in non-light treated rats, but in older mutant rats (41-60 day) light had no effect on the level of ascorbate in the retina. As determined by the level of rhodopsin remaining in the eye two weeks after 24 hrs light exposure, cyclic light reared rats lost 50-55% of their visual cells. However, cyclic light rats supplemented with ascorbic acid before intense light exposure lost only 30-35% of their visual cells.     

Glycoproteins in the retinal pigment epithelium of normal and dystrophic rats

The apical membranes of retinal pigmented epithelium (RPE) were isolated from adult, normal (LE), and dystrophic (RCS) rats. The proteins of these RPE subfractions were separated through the use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The lectin-binding properties of glycoproteins were examined in western blots through the use of lectin-peroxidase conjugates. No differences were detected between RPE membrane proteins from normal and dystrophic rats in silver-stained gels. However, these two preparations showed significant differences with respect to their binding of the lectins, Lens culinaris (Lentil), Tetragonolobus purpurea (Lotus), and concanavalin A (Con A). In particular, a glycoprotein with a molecular weight of 86 kD in the RPE apical membrane from normal rats bound Lentil, Lotus, and Con A, but in the membrane from dystrophic rats these binding sites were absent or significantly reduced. Another glycoprotein with a molecular weight of 175 kD was recognized by Lotus in the normal membrane preparation but not in the dystrophic RPE membrane preparation. Developmental studies show that these lectin-binding anomalies appear after postnatal day 11 and are, therefore, most likely coincident with eye opening in RCS rats. These results demonstrate that the RPE glycoproteins (86 and 175 kD) are significantly modified in dystrophic rats. The data also confirm previous observations that differences in the oligosaccharide chains, but not the polypeptide chains, of RPE membrane glycoproteins can be detected between normal and dystrophic rats. To the authors' knowledge, this is the first study to correlate developmentally regulated alterations in specific membrane-associated molecules in the RPE of dystrophic rats with the breakdown in phagocytosis that occurs in these rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lipids of the retinal pigment epithelium in RCS dystrophic and normal rats

The lipid composition of retinal pigment epithelial cells was determined for normal cells which have full phagocytic ability and for a genetic variant with impaired phagocytic function. Retinal pigment epithelial cells from 9-14-day-old congenic strains of normal (RCS-rdy+) and dystrophic (RCS-rdy/rdy) rats were separated from intact retinas and homogenized in 0.08 M Tris base, pH 7.4. The lipids were extracted using 2:1 chloroform--methanol. Fatty-acid methyl esters identified by gas chromatography were: 16:0, 17:0, 18:0, 18:1, 18:2 omega 6, 20:0, 20:2, 22:0, 20:4 omega 6, 22:4, 22:5, 22:6 omega 3. Major fatty acids for both normal and dystrophic cells were: 16:0, 18:0, 20:4 omega 6, 22:6 omega 3. One- and two-dimensional thin-layer chromatography was used to determine phospholipid composition of pigment epithelial cells at two different age groups. The relative amount of phosphatidylethanolamine was significantly higher in dystrophic RPE cells compared with normal cells (20.7% for 9-11-day-old and 17.3% for 12-14-day-old dystrophic rats). Cells from normal animals contained a higher level of phosphatidylethanolamine in the older age group whereas RPE cells from dystrophic animals contained a lower level of phosphatidylcholine in the older group. Anomalous phospholipid composition of dystrophic pigment epithelial cells may be associated with a change in cellular membranes and a defect in the cellular processes involved in phagocytic function.     

Activity distribution of cytochrome oxidase in the rat retina. A quantitative histochemical study

Cytochrome oxidase (CYO) is a key enzyme in the respiratory chain. Therefore, CYO has an important role in the cell metabolism. In the present study CYO activity in the rat retina was identified by histochemical staining. The density of the staining, corresponding to the activity of the CYO, was evaluated quantitatively by densitometry. A high CYO activity was found in the retinal pigment epithelium, in the inner segment of the photoreceptors, in the outer plexiform layer and in the inner plexiform layer. In the outer segment of the photoreceptors, in the outer nuclear layer, and in the inner nuclear layer the CYO activity was relatively low. An analysis of variance demonstrated that the precision in an estimation of a mean depends on the number of animals and the number of retinal sections per animal.     

Mannose receptor is expressed in normal and dystrophic retinal pigment epithelium.

In normal retinas, the phagocytosis of shed photoreceptor outer segments is mediated in part through a mannose receptor protein located in the apical retinal pigment epithelium membrane. As dystrophic rats of the Royal College of Surgeons have a defect in which the retinal pigment epithelium (RPE) is unable to phagocytize the shed outer segments, it is hypothesized that mannose receptor expression will be lost with the progression of photoreceptor degeneration. Immunohistochemical and molecular techniques have been used to study the developmental expression of the mannose receptor in normal and dystrophic retinal pigment epithelium. By immunofluorescence, the mannose receptor is localized to the retinal pigment epithelium, apical membrane region, beginning around 5 days postnatally in both normal and dystrophic retinas. In immunoblots, bands at 175 kDa are labelled by an anti-mannose receptor antibody in apical membrane samples from both normal and dystrophic RPE at all developmental times sampled. RT-PCR analysis reveals that mannose receptor message is present in normal and dystrophic RPE samples at all developmental time points examined. The present study demonstrates that the expression of the mannose receptor begins prior to outer segment differentiation and the initiation of phagocytosis in both normal and dystrophic RPE. Expression of the mannose receptor continues to be unchanged during the progression of photoreceptor degeneration in the dystrophic retina.     

Lipid and protein density in the rat retina: a microradiographical study

Lipid and protein density in each retinal layer were determined microradiographically in retinal sections of 10 rats. Lipid in retinal sections was extracted with chloroform. The fraction of dry mass remaining after lipid extraction is mainly protein. The dry mass lost in chloroform processing was calculated to obtain the lipid density, which accounts for about 30% of the total dry mass density. The total dry mass density in each retinal layer is the sum of the lipid and protein density. Lipid should be regarded as an important X-ray absorber when determining the dry mass density in the rat retina with microradiography.