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The retinotoxic drug diaminophenoxypentane (DAPP) was administered to rats to determine its long term effects on rhodopsin levels, retinal morphology and the retina's susceptibility to damage from visible light. In rats given 2 intraperitoneal injections of DAPP at doses of 65 mg/kg body wt, there was a dramatic and sustained loss of rhodopsin. One wk later visual pigment levels were 41% lower than in comparable dark maintained rats injected with saline. Rhodopsin levels in the DAPP treated rats remained lower than in control animals for the 13 wk period of the study. Morphologically, the ROS of rats 1-2 wks after DAPP treatment exhibited some disorganization and shortening; the RPE was unremarkable. Seven wks after DAPP treatment an occasional focal area of damage was seen in the RPE. Similarly, focal areas of degeneration were seen in the outer nuclear layer between the rows of photoreceptor cells. As determined by photoreceptor cell nuclear counts, the retinotoxic effect of DAPP persisted long after drug administration. In the treated rats the loss of visual cell nuclei was 11% at 7 wks; it was 22% 13 wks after DAPP treatment. Immediately after exposure to intense visible light, damage was seen in both the photoreceptor cells and RPE of DAPP treated rats. However, the effects of exposure in the treated rats were less dramatic than in retinas from rats without DAPP treatment. Following a 2 wk dark recovery period, the DAPP treated rats had a normal appearing retinal morphology and an intact RPE layer. The retinas of rats without DAPP treatment showed extensive visual cell and RPE loss.(ABSTRACT TRUNCATED AT 250 WORDS)

Transplantation of normal retinal pigment epithelium (RPE) into a diseased eye holds promise for treatment of several blinding disorders. Previous studies have involved immunosuppression and implantation of freshly isolated cells. We report here the successful transplantation of cultured human RPE cells into rabbits that were not immunosuppressed. A modified pars plana transvitreal technique was used for RPE transplantation. The cultured RPE cells, loaded with carbon as a marker, were transplanted into the denuded Bruch's membrane of albino rabbits. The animals were followed for from 1 week to 3 months. On histologic examination at 2 months, no infiltrating lymphocytes were found in the vitreous cavity or choroid, even though Bruch's membrane was damaged. At about 3 months there were some macrophages in the subretina of transplanted eyes, indicating that an immunoreaction does occur eventually. Electron microscopy of the transplanted RPE showed apical-basal polarity and gap junctions. Restored function was attested to by the presence of phagosomes and phagocytosed outer segments in the transplanted cells. Our findings suggest that there is a weak, delayed immunoreaction to human RPE cells transplanted beneath the retina of the rabbit; however, functional recovery of the transplanted cells occurs before this immune response develops.

Dysfunction of the retinal pigment epithelium (RPE) is a major component of many degenerative disorders of the retina. We have developed a modified pars plana technique for subretinal implantation of homologous, cultured, carbon-loaded RPE cells in the rabbit. The natural history and morphologic behavior of the implanted RPE cells for up to 6 weeks after transplantation were observed in successful procedures.

Long-term environmental light-mediated changes in visual cell transduction proteins were studied to assess the influence of rearing environment on their levels and their potential effects on intense light-induced retinal damage. The levels of rhodopsin, S-antigen and the alpha subunit of transducin were measured in whole eye detergent extracts, retinal homogenates or rod outer segments isolated from rats reared in weak cyclic light or darkness, and following a change in rearing environment. Rats changed from weak cyclic light to darkness had 22% more rhodopsin per eye than cyclic-light rats after 12-14 days in the new environment. Western trans-blot analysis of retinal proteins from these dark-maintained animals contained 65% higher levels of immunologically detectable alpha transducin; S-antigen levels were approximately 45% lower than in cyclic-light rats. In rats changed from the dark environment to weak cyclic light, rhodopsin levels decreased by 18% during a comparable period; retinal alpha transducin was 35% lower, S-antigen was 30% higher. At various times after the change in rearing environment, some rats were exposed to intense visible light to determine their susceptibility to retinal damage. Two weeks after an 8-hr exposure, cyclic-light reared rats had rhodopsin levels only 10% lower than control (2.1 nmol per eye). However, rhodopsin was 75% lower when cyclic-light rats were maintained in darkness for 2 weeks before intense light. For animals originally reared in darkness, rhodopsin was 78% lower following 8 hr of intense light, whereas only 30% rhodopsin loss occurred in dark-reared rats after previous maintenance for 2 weeks in weak cyclic-light.(ABSTRACT TRUNCATED AT 250 WORDS)

Visible light-induced photoreceptor cell damage resulting from exposure to multiple intermittent light-dark periods was compared with damage resulting from continuous light in albino rats maintained in a weak cyclic-light environment or in darkness before light treatment. The time course of retinal damage was determined by correlative measurements of rhodopsin and visual cell DNA at various times after light exposure, and by histopathological evaluation. The effect of intense light exposures on rhodopsin regeneration and on the level of rod outer segment docosahexaenoic acid was also determined. For rats previously maintained in weak cyclic light, 50% visual cell loss was measured 2 weeks after 12 1 hr light/2 hr dark periods, or following 24 hr of continuous light. A comparable 50% loss of visual cells was found in dark-reared rats after only 5 hr of continuous illumination or 2-3 hr of intermittent light. As judged by histology, cyclic-light-reared rats incurred less retinal pigment epithelial cell damage than dark-reared animals. In both experimental rat models intermittent light exposure resulted in greater visual cell damage than continuous exposure. Visual cell damage from intermittent light was found to depend on the duration of light exposure and on the number of light doses administered. Measurements of rhodopsin and DNA 2 hr and 2 weeks after light exposure of up to 8 hr duration revealed that visual cell loss occurs largely during the 2 week dark period following light treatment. The loss of docosahexaenoic acid from rod outer segments was also greater in rats exposed to intermittent light than in animals treated with continuous light. It is concluded that intermittent light exposure exacerbates Type I light damage in rats (involving the retina and retinal pigment epithelium) and the schedule of intense light exposure is a determinant of visual cell death.

An enzymatic procedure for the estimation of organic hydroperoxides has been adapted to biological tissues and applied to the measurement of hydroperoxides in the rat retina. Hydroperoxides are determined from the coupled activities of glutathione peroxidase and glutathione reductase as measured by the loss of NADPH absorbance. To minimize the effects of tissue catalyzed peroxide degradation, incubations were performed in the presence of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU); which inhibited the activity of retinal tissue glutathione reductase by 85%. For comparisons to the enzymatic technique, retinal tissue hydroperoxides were also estimated by the absorption of tissue extracts at 232 nm. Using the enzymatic procedure the hydroperoxide concentration in whole retina homogenates was significantly higher in 19-day-old rats than in either 35-day or adult animals. Hydroperoxides in the retina of young rats exposed to light for one hour were significantly lower than in non-exposed controls, while in adult rats, following light, hydroperoxides increased 13%. Fractionation of rat retinas into crude ROS and retina minus ROS components revealed that the ROS fractions contain at least twice the hydroperoxide concentration of the remaining retina. The concentration of hydroperoxides in the ROS fractions from dark-reared rats were significantly lower than in cyclic-light-reared animals. In both types of rats, one hour intense light exposure resulted in an increase in ROS hydroperoxides but the increases were not significant. ROS hydroperoxides were also found to be 85-90% water soluble. Estimates of the retinal hydroperoxide content obtained by absorption at 232 nm gave similar results to the enzymatic technique, but the levels were significantly lower. When retinas were maintained in vitro for one hour before analysis, hydroperoxides determined by either technique were significantly higher than in retinas assayed immediately, but A232 hydroperoxides were still significantly lower than hydroperoxides measured by the enzymatic procedure. It is concluded: (1) that the observed retinal hydroperoxide concentration depends upon animal age and the method of measurement; (2) that within the retina the photoreceptor cell contains at least a two-fold higher concentration of hydroperoxides than the remaining retina and that prior light history can affect those hydroperoxide levels (it appears that the photoreceptor cell is also a major site of hydroperoxide formation in the retina); (3) that during intense light exposure of short duration significant levels of hydroperoxides do not accumulate in the retinas of rats.

Vitamin E levels were measured in retina and retinal pigment epithelium from human eye bank donors of from 12-82 years of age. In comparison to an age group of 12-45 years, humans 59-82 years of age had a higher concentration of vitamin E in both retina and retinal pigment epithelium. Depending on age, the concentration of vitamin E in retinal pigment epithelium was from 4-7 times higher than in retina. Vitamin E accumulated in the human retinal pigment epithelium in an age dependent fashion, so that by 80 years it was from 3-4 times higher than in those 20 years old. The level of vitamin E in young human retinal epithelium, however, was higher than in comparable bovine tissue. The age-related increase in human tissue vitamin E levels does not appear to be affected by postmortem time.

It has been postulated that ascorbic acid may help to protect the retina from oxidative insult by light. To confirm this hypothesis, the authors compared light-damaged retinas of rats with or without ascorbate supplement by morphologic and morphometric studies at different time periods after light exposure. No dramatic morphologic differences were observed in the photoreceptor-retinal pigment epithelium complex between the two groups six hr after light exposure to 200 to 250-foot candles of visible light. Six to 13 days after 24 hr of exposure, the retina of rats that received ascorbate supplement showed significantly less severe damage than the retina of unsupplemented rats. The superior and temporal quadrants of the retina appeared to be most susceptible to the light damage when comparing rats with or without ascorbate supplement. These findings suggested that ascorbate ameliorates the photic injury in rat retina.

High pressure liquid chromatography was used to measure alpha-tocopherol in the retinas of rats reared in a cyclic light or dark environment. These measurements were performed on extracts of whole retinas during the developmental period, 18-60 days, and on isolated ROS from adult animals. Similar alpha-tocopherol determinations were performed on retinas and isolated ROS following exposure of rats to intense visible light for 24 hr periods. The results show that alpha-tocopherol is chromatographically separated from the vitamin A derivatives found in the retina and is pure, as judged by mass spectrometry. In the retinas of cyclic light and dark reared rats, alpha-tocopherol accumulates in an age dependent fashion, so that at 60 days the level is nearly double that of animals at 18-20 days of age (P less than 0.001). Because the age dependent accumulation of rhodopsin is greater in dark reared rats, the average molar ratio of rhodopsin to alpha- tocopherol in the retina of dark reared animals is 25% higher than in cyclic light rats. Following exposure of rats to intense visible light for 24 hr periods, alpha-tocopherol concentrations in the retina were unchanged from the levels in control animals. In adult animals the concentration of alpha-tocopherol in ROS is 2.5-3.5 times higher than in whole retina. ROS from adult cyclic light reared rats also contain an average of 43% more alpha-tocopherol per mg protein than ROS from dark maintained animals (P less than 0.005).(ABSTRACT TRUNCATED AT 250 WORDS)

The posterior segment of the eye was divided into four compartments: retinal cytosol (R), subretinal fluid on the retinal surface (S/R), retinal pigment epithelial (RPE) cytosol, and subretinal fluid on the RPE surface (S/RPE). The volume of each compartment was estimated from the dilution of creatinine (in the extraction buffer) by the endogenous tissue fluid. The ascorbate concentrations in R, S/R, S/RPE, and RPE were 20.6, 12.3, 3.7, and 5.8 mg/dl respectively. Dehydroascorbate was observed only in the RPE and S/RPE. The decreasing ascorbate concentration from the retina to RPE, and the distribution of dehydroascorbate suggest a movement of ascorbate from the vitreous cavity into the subretinal space. The permeability of retinal cell layers to ascorbate was confirmed by the high radioactivity observed in the subretinal space after an intravitreal injection of C14-ascorbate. The occurrence of dehydroascorbate in the RPE and the S/RPE indicates the presence of oxidative reaction of ascorbate in these compartments, where light induced free radicals are located.

Cyclic light and dark-reared rats were exposed to intense visible light for various periods and then rhodopsin-measured following recovery in darkness for up to 14 days. Animals were injected with ascorbic acid or ascorbate derivatives at various doses prior to light exposure in green Plexiglas chambers. The results show that ascorbic acid administration elevates retinal ascorbate and reduces the loss of rhodopsin and photoreceptor cell nuclei resulting from intense light. When given in comparable doses, L-ascorbic acid, sodium ascorbate, and dehydroascorbate were equally effective in preserving rhodopsin. The ascorbate protective effect in the retina is also dose dependent in both cyclic light and dark-reared rats and exhibits a requirement for the L-stereoisomer of the vitamin. Ascorbic acid is effective when administered before, but not after, light exposure, suggesting that protection from light damage in the retina occurs during the light period. In some experiments, rod outer segments were isolated from rats immediately after light exposure, lipids extracted, and fatty acid composition determined. As judged by the preservation of rod outer segment docosahexaenoic acid in rats given ascorbate, the vitamin may act in an antioxidative fashion by inhibiting oxidation of membrane lipids during intense light.

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.