Oxygen-induced retinopathy in the rat. Vitamins C and E as potential therapies.

Oxygen-induced retinopathy (OIR) was produced by subjecting newborn albino rats to a 60% oxygen atmosphere for 14 days before they were killed and retinal analysis was done. The extent of OIR was measured by estimating the severity of retinal vasoobliteration in ink-perfused flat-mounted retinas. This was done with the aid of a digitizing camera and an image-analysis system designed to create binary images of the retinal blood vessels. Retinal levels of several antioxidant molecules also were measured. Alpha-tocopherol and ascorbic acid were reduced in oxygen-exposed rats by 34% and 20%, respectively, compared with room air-raised control animals. Retinal glutathione reductase, S-transferase, and peroxidase showed no differences between oxygen-treated and -untreated rats. Attempts to increase the newborn rats' retinal ascorbic acid by administering daily subcutaneous injections (5 g/kg body weight) to the mother rats were unsuccessful. However, the level of retinal alpha-tocopherol of newborn rats could be altered by dietary manipulation of the mothers. The mothers were fed diets containing either 1 g alpha-tocopherol acetate/kg food or none, starting 21-25 days before the birth of their litters and lasting throughout the exposure period. This treatment resulted in three- to fourfold differences in the retinal alpha-tocopherol levels of the pups. The combination of dietary and oxygen treatments also resulted in significant differences in retinal glutathione peroxidase activity, with the vitamin E-deprived, oxygen-exposed group having highest levels. Newborn rats both supplemented with and deprived of alpha-tocopherol had less vasoobliteration than did those nursed by mothers fed rat chow.

Effect of light history on the rat retina: timecourse of morphological adaptation and readaptation.

Sprague Dawley rats were born and raised under either 5 or 800 lux cyclic light (12L:12D) and were sacrificed at 1, 2, 3, 6, 12, 16, and 28 weeks of age. At each time point outer nuclear layer (ONL) area and rod outer segment (ROS) length were measured. The former is an estimation of photoreceptor number, and the latter is an estimation of the photon-catching integrity of the retina, both of which are known to be dependent on the light environment. Regression analysis revealed an ONL area reduction with time of 0.003 mm2/wk for 5-lux-reared rats and 0.009 mm2/wk for 800-lux-reared rats. ROS length was relatively constant in the dim light group, but showed a decline in 800 lux rats of 0.5 microns/wk. Rats moved from 800 to 5 lux at 9 and 21 wks of age showed no significant change in ONL area after 3 wks. ROS length in these rats increased at a prodigious rate, and in the 12-wk-olds (9 wks at 800 lux, followed by 3 wks at 5 lux), ROS length exceeded that of age-matched rats raised in 5 lux for life.

Predisposing factors to light-induced photoreceptor cell damage: retinal changes in maturing rats.

Retinal changes occurring during the period of growth and maturation of Long Evans pigmented rats were examined to obtain a better understanding of the basis for the age-dependency of light-induced photoreceptor cell damage. Susceptibility to light damage increased markedly between 30 and 60 days of age and to a lesser extent between 60 and 90 days. Although the retinal antioxidant vitamins E and C, and taurine showed a significant increase during the age-period studied, retinal lipid phosphorus and total protein increased by similar amounts indicating that the concentration of these nutrients was not changing. In contrast, rhodopsin content of the retina increased progressively by 44% between 30 and 90 days of age. While ROS length showed no appreciable change with age, rhodopsin per ROS length increased by 31% between 30 and 60 days of age and by 48% between 30 and 90 days. Determinations of ROS phospholipid to rhodopsin ratio and disks per ROS length indicated that rhodopsin did not become more concentrated in photoreceptor cells between 30 and 90 days. However, the 12% increase in ROS diameter between 30 and 90 days of age may partially account for the rhodopsin difference. These findings demonstrate an age-dependent association between greater rhodopsin per ROS length and increased susceptibility to retinal light damage. An increased metabolic demand on photoreceptor cells with greater rhodopsin may be an important factor influencing their destruction by light.

Photoreceptor physiology in the rat is governed by the light environment.

Albino rats were born and raised in one of three cyclic (12L: 12D) lighting conditions: (1) 5 lux for 9 weeks, (2) 800 lux for 9 weeks, or (3) 800 lux for 9 weeks, followed by 5 lux for 3 weeks (800:5). After the treatment, the following were determined: (i) retinal function as measured by the electroretinogram (ERG); (ii) retinal morphology, including rod outer segment (ROS) length and outer nuclear layer area; (iii) rhodopsin levels in whole retina and ROS preparations; (iv) fatty acid profile of ROS membranes and (v) retinal antioxidant levels. After 9 weeks, rats raised in 800 lux sustained an irreversible loss of photoreceptors which could not be reversed by then placing them in 5 lux. However, these rats displayed significant alterations in all other parameters measured after the 3 weeks in dim cyclic light. ERGs showed a 60% increase of b-wave maximum amplitude in 800:5 rats at 12 weeks over the value at the time of their change to a dim environment, while a-wave amplitude in 800:5 rats increased more than 2.5 times. This increase can be explained by a combination of increased ROS length and increased ROS membrane concentration of rhodopsin during the three weeks in 5 lux. Polyunsaturated fatty acids predominated in the ROS of 5 lux rats and 800:5 rats, but not in 800 lux animals. Measurements of retinal glutathione enzyme activity and vitamin E and C levels were relatively low in 800:5 rats. Some rats from the 800:5 group were exposed to 2000 lux for 24 hr. Retinas of these rats sustained 50% loss of photoreceptors from this exposure. Comparisons are made to previous studies concerning the effect of cyclic light environments on the retinas of albino rats.

Effects of oxygen rearing on the electroretinogram and GFA-protein in the rat.

Albino rats were maintained in 60% oxygen from birth through 14 days of age. Control rats were simultaneously maintained in room air. At the end of the exposure period some animals from both groups were prepared for electroretinography and, following this analysis, were sacrificed for histological scrutiny of retinal vasculature. The rest of the rats were removed to room air and allowed to remain for various periods of time before electroretinography and sacrifice. Some of the retinas from the latter group were analyzed for the presence of glial fibrillary acidic protein (GFAP) by immunocytochemistry. Oxygen exposed rats had a permanent reduction of the b-wave of the electroretinogram (ERG). The a-waves of the two groups were indistinguishable. Control rats showed GFAP reactivity in the retinal astrocytes at all times. The oxygen-treated rats showed positive GFAP-staining in astrocytes at all times and in Müller cells from 2 weeks post-treatment through 8 weeks post-treatment, the last time point. Immunoblot analysis confirmed that the anti-GFAP reacted with a protein having a molecular weight and solubility characteristics like those of GFAP. These results indicate that Müller cells produce GFAP in response to oxygen-rearing in newborn rats and that this production occurs in the absence of any detectable neuronal cell death.

Synergism between environmental lighting and taurine depletion in causing photoreceptor cell degeneration.

Experiments were conducted to examine the possible interaction between retinal taurine depletion and environmental lighting in causing photoreceptor cell degeneration. Albino rats were raised from birth in either dim (2 lx) or relatively bright (300 lx) cyclic light. Beginning at weaning, half the animals from both light environments were taurine-depleted by treating them for 10 weeks with guanidinoethyl sulfonate as a 1% solution in their drinking water. The remaining animals were given ordinary tap-water and served as controls. In the 2 lx light environment, taurine depletion caused a decrease in electroretinogram (ERG) a- and b-wave amplitude of 36 and 46%, respectively; however, no photoreceptor cells were lost in this group. Tap-water controls kept in the 300 lx light environment had a 59- and 43% decrease in ERG a- and b-wave amplitude, respectively, and a 21% reduction in the number of photoreceptor cells. In contrast to the other groups, animals that were taurine-depleted in the 300 lx environment showed a marked retinal degeneration. ERG a- and b-wave amplitude was decreased by 94- and 89% respectively and there was a 62% loss of photoreceptor cells. The greatest cell loss occurred in the central superior region of the retina, in which the outer nuclear layer was typically reduced to one to two rows of nuclei. The results of a two-way analysis of variance applied to the data indicated that the effects of taurine depletion on the retina were greater in the 300 lx as compared with the 2 lx environment in terms of loss of photoreceptor cells and reduction in log ERG a- and b-wave amplitude. These findings demonstrate a synergism between environmental lighting and taurine depletion in causing photoreceptor cell degeneration.