Enhancing the efficacy of AREDS antioxidants in light-induced retinal degeneration.

PURPOSE: Light-induced photoreceptor cell degeneration and disease progression in age-related macular degeneration (AMD) involve oxidative stress and visual cell loss, which can be prevented, or slowed, by antioxidants. Our goal was to test the protective efficacy of a traditional Age-related Eye Disease Study antioxidant formulation (AREDS) and AREDS combined with non-traditional antioxidants in a preclinical animal model of photooxidative retinal damage. METHODS: Male Sprague-Dawley rats were reared in a low-intensity (20 lux) or high-intensity (200 lux) cyclic light environment for 6 weeks. Some animals received a daily dietary supplement consisting of a small cracker infused with an AREDS antioxidant mineral mixture, AREDS antioxidants minus zinc, or zinc oxide alone. Other rats received AREDS combined with a detergent extract of the common herb rosemary, AREDS plus carnosic acid, zinc oxide plus rosemary, or rosemary alone. Antioxidant efficacy was determined by measuring retinal DNA levels 2 weeks after 6 h of intense exposure to white light (9,000 lux). Western blotting was used to determine visual cell opsin and arrestin levels following intense light treatment. Rhodopsin regeneration was determined after 1 h of exposure to light. Gene array analysis was used to determine changes in the expression of retinal genes resulting from light rearing environment or from antioxidant supplementation. RESULTS: Chronic high-intensity cyclic light rearing resulted in lower levels of rod and cone opsins, retinal S-antigen (S-ag), and medium wavelength cone arrestin (mCAR) than found for rats maintained in low cyclic light. However, as determined by retinal DNA, and by residual opsin and arrestin levels, 2 weeks after acute photooxidative damage, visual cell loss was greater in rats reared in low cyclic light. Retinal damage decreased with AREDS plus rosemary, or with zinc oxide plus rosemary whereas AREDS alone and zinc oxide alone (at their daily recommended levels) were both ineffective. One week of supplemental AREDS plus carnosic acid resulted in higher levels of rod and cone cell proteins, and higher levels of retinal DNA than for AREDS alone. Rhodopsin regeneration was unaffected by the rosemary treatment. Retinal gene array analysis showed reduced expression of medium- wavelength opsin 1 and arrestin C in the high-light reared rats versus the low-light rats. The transition of rats from low cyclic light to a high cyclic light environment resulted in the differential expression of 280 gene markers, enriched for genes related to inflammation, apoptosis, cytokine, innate immune response, and receptors. Rosemary, zinc oxide plus rosemary, and AREDS plus rosemary suppressed 131, 241, and 266 of these genes (respectively) in high-light versus low-light animals and induced a small subset of changes in gene expression that were independent of light rearing conditions. CONCLUSIONS: Long-term environmental light intensity is a major determinant of retinal gene and protein expression, and of visual cell survival following acute photooxidative insult. Rats preconditioned by high-light rearing exhibit lower levels of cone opsin mRNA and protein, and lower mCAR protein, than low-light reared animals, but greater retention of retinal DNA and proteins following photooxidative damage. Rosemary enhanced the protective efficacy of AREDS and led to the greatest effect on the retinal genome in animals reared in high environmental light. Chronic administration of rosemary antioxidants may be a useful adjunct to the therapeutic benefit of AREDS in slowing disease progression in AMD.

Extreme retinal remodeling triggered by light damage: implications for age related macular degeneration.

PURPOSE: Our objective was to comprehensively assess the nature and chronology of neural remodeling in retinal degenerations triggered by light-induced retinal damage (LIRD) in adult albino rodents. Our primary hypothesis is that all complete photoreceptor degenerations devolve to extensive remodeling. An hypothesis emergent from data analysis is that the LIRD model closely mimics late-stage atrophic age relared macular degeneration (AMD). METHODS: Sprague-Dawley (SD) rats received intense light exposures of varied durations and survival times ranging from 0 to 240 days. Remodeling was visualized by computational molecular phenotyping (CMP) of a small molecule library: 4-aminobutyrate (gamma), arginine (R), aspartate (D), glutamate (E), glutamine (Q), glutathione (J), glycine (G), and taurine (tau). This library was augmented by probes for key proteins such as rod opsin, cone opsin and cellular retinal binding protein (CRALBP). Quantitative CMP was used to profile 160 eyes from 86 animals in over 6,000 sections. RESULTS: The onset of remodeling in LIRD retinas is rapid, with immediate signs of metabolic stress in photoreceptors, the retinal pigmented epithelium (RPE), the choriocapillaris, and Müller cells. In particular, anomalous elevated aspartate levels appear to be an early stress marker in photoreceptors. After the stress phase, LIRD progresses to focal photoreceptor degeneration within 14 days and extensive remodeling by 60 days. RPE and choriocapillaris losses parallel Müller cell distal seal formation, with progressive neuronal migration, microneuroma evolution, fluid channel formation, and slow neuronal death. The remaining retina in advanced light damage can be classified as survivor, light damage (LD), or decimated zones where massive Müller cell and neuronal emigration into the choroid leaves a retina depleted of neurons and Müller cells. These zones and their transitions closely resemble human geographic atrophy. Across these zones, Müller cells manifest extreme changes in the definitive Müller cell tauQE signature, as well as CRALBP and arginine signals. CONCLUSIONS: LIRD retinas manifest remodeling patterns of genetic retinal degeneration models, but involve no developmental complexities, and are ultimately more aggressive, devastating the remaining neural retina. The decimation of the neural retina via cell emigration through the perforated retina-choroid interface is a serious denouement. If focal remodeling in LIRD accurately profiles late stage atrophic age-related macular degenerations, it augurs poorly for simple molecular interventions. Indeed, the LIRD profile in the SD rat manifests more similarities to advanced human atrophic AMD than most genetically or immunologically induced murine models of AMD.

Isolation of plasma membranes from the bovine retinal pigment epithelium.

Retinal pigment epithelium plasma membranes have been isolated by differential and density gradient centrifugation of glass-bead-bound, collagenase-treated cells. Electron microscopic evidence indicates that the glass-bead-bound cells were devoid of red blood cells, rod outer segments and other ocular cell contaminants. The plasma membranes were recovered in 4-6 micrograms/eye yields and purified 10-fold by 5'-nucleotidase and alkaline phosphodiesterase I, and 6.5-fold by (Na+ + K+)-ATPase. Plasma membrane purity as measured by covalent labeling of the epithelial cell plasma membrane proteins with p-(diazonium) benzene[32S]sulfonic acid was 8-19-fold. In purified plasma membranes contamination by mitochondria was undetectable and lysosomal contamination reduced 100-fold, while endoplasmic reticulum was 2-fold enriched. SDS-polyacrylamide gel electrophoresis of the plasma membrane proteins revealed 23-26 major bands by Coomassie blue staining and 12-16 major bands by radioactive labeling. The plasma membranes exhibited a 3-fold lower concentration of docosahexaenoic acid, a 3-fold higher cholesterol/phosphate ratio, and were 10-fold enriched in cholesterol per micrograms protein when compared to the whole cell fraction. Retinal epithelial plasma membranes contain an average of 1 mol cholesterol per mol of lipid phosphorus, a high palmitic acid concentration (39 mol%) and a low concentration of docosahexaenoic acid (2 mol%). The lipid profile of the retinal pigment epithelial plasma membranes indicates that they are typical of plasma membranes from many other cell types and that they appear to be less fluid than total rod outer segment membranes.

Rhodopsin phosphorylation in rats exposed to intense light.

The damaging effects of intense light on the rat retina are known to vary depending on the time of day of exposure. The purpose of this study was to determine if rhodopsin phosphorylation patterns, a measure of the activity of the pigment, varied in a similar manner. After 10 min in strong light (1400 lux), all six threonine and serine sites in the rat rhodopsin C-terminus were phosphorylated, with mono- to tetraphosphorylation being substantially more prominent than penta- to hexaphosphorylation. The level and multiplicity of rhodopsin phosphorylations were reduced both with the duration of light exposure and the duration of subsequent darkness. Although showing vast differences in susceptibility to light damage, rats exposed at 5 P.M. or 1 A.M. showed similar rhodopsin phosphorylation levels and patterns. These data indicate that a process controlled by circadian rhythm other than rhodopsin phosphorylation is involved either in damaging or mediating the damage evoked by intense light exposure.

Ultrastructural, histochemical, and biochemical studies of the melanin metabolism in eye and skin of pallid mice.

The hair follicles and the eyes of pallid mice (C57/6J-Pa/Pa) and those of black mice (C57/6J-+/Pa) were examined ultrastructurally, histochemically, and biochemically to determine the cause of pigment dilution. The pigment cells in the hair follicles and the eyes of pallid mice have less mature melanosomes than those of black mice. In the hair follicles the pallid melanosomes were transferred into keratinocytes and became aggregated. In the eyes they were already aggregated within the pigment cells and were digested in acid phosphatase-positive lysosomes. The activity of acid phosphatase, a marker of lysosomal enzymes was significantly higher in pallid hair follicles and eyes than in black hair follicles and eyes. Dopa reactions at light and electron microscopical level indicated that the pigment cells in each tissue produced a large amount of Dopa oxidase when compared with those in each black counterpart. However, the rate of hydroxylation of L-tyrosine-3,5-3H was significantly lower in the pallid eyes than in black eyes, while this rate was significantly higher in pallid hair follicles than in black hair follicles. Immediate digestion of melanosomes within the pigment cells, i.e., autophagocytosis, seemed to explain the low activity in the pallid eyes. The diluted coat and eye colors of pallid mice are, therefore, not related to low Dopa oxidase activity but to immaturity of melanosomes and high activities of lysosomal enzymes; these enzymes seem to digest many of these immature melanosomes and contribute to the diluted coat and eye colors of pallid mice.

The rod outer segment phospholipid/opsin ratio of rats maintained in darkness or cyclic light.

Rhodopsin (opsin), lipid, and fatty acids were measured in rod outer segments (ROS's) of rats maintained for at least 2 weeks in continuous darkness or in 12 hours per day cyclic light. Average rhodopsin per eye was 1.8 nmol. for the 5 ft.-c. cyclic light groups compared to 2.4 nmol. for the dark groups of the same age. The phospholipid/opsin ratio was significantly higher after cyclic light maintenance, suggesting that slow adaptive processes control the opsin density of the ROS membranes. Estimates indicate that ROS length also depends on the long-term light environment. ROS lipid and fatty acid composition were not consistently different in dark and light groups.

Rhodopsin determinations in C57BL/6J-pallid strain mice.

The influence of light environment on rhodopsin concentration per eye was determined in littermate pigmented and nonpigmented C57BL/6J-pallid gene mice reared under cyclic light or continuous dark environments. Attempts to exacerbate a congenital manganese deficiency in pallid strain mice included dietary deprivation and supplementation with manganese and exposure to intense light followed by the determination of rhodopsin recovery rates in darkness. Homozygous pallid mice (pa/pa) reared in cyclic light had rhodopsin levels which were significantly lower than heterozygous (+/pa) or homozygous (+/+) black control mice. Dark-rearing resulted in a significant increase in rhodopsin per eye in pallid strain mice and equivalent levels in adult mice, but young pallid strain mice did not achieve the same rhodopsin concentration as young +/+ mice. Although dietary manganese deprivation or supplementation did not significantly alter rhodopsin levels among pallid mice, the deficient diet resulted in lower rhodopsin per eye in the young +/+ control animals. The recovery of rhodopsin in darkness following intense light exposure was equal and complete within 24 hr for most genotypes. However, recovery by pallid mice after 24 hr was significantly lower than by pigmented or albino genotypes.

Ascorbate treatment prevents accumulation of phagosomes in RPE in light damage.

In dark-reared albino rats, exposure to 2 or 3 hr of intense light interrupted by 2 hr dark periods resulted in extensive degeneration of photoreceptor cells and degeneration of the retinal pigment epithelium (RPE). Ascorbate (ie, vitamin C) administration prior to light exposure protected photoreceptors and the RPE from light damage. In the present study, ascorbate-treated and untreated rats were exposed to various cycles of intermittent light. Immediately after this light exposure, phagosome frequency in the RPE was morphologically evaluated in comparable 50 microns sections. In untreated rats, exposure to 2 or 3 hr of intermittent light resulted in a five- to sixfold increase in phagosome density compared to unexposed controls. In contrast, no increase in phagosome density was observed in ascorbate-treated rats. In these animals, under all lighting regimens, phagosome levels remained essentially identical to those in rats not exposed to light. After a single nondamaging light exposure, phagosome density remained at the level of dark controls in ascorbate-treated and untreated rats. These results indicate that phagosome frequency may serve as an index for light damage and that the protective effect of ascorbate may be linked to its capacity to prevent rod outer segment shedding and phagocytosis under intense light conditions.

Characterization of pigment epithelial cell plasma membranes from normal and dystrophic rats.

Retinal pigment epithelial cell plasma membranes were isolated from the eyes of normal and RCS-dystrophic rats by binding glass microbeads to the intact pigment epithelial cell layer, removal of the bead-bound cells from the eyes and subsequent sucrose density gradient centrifugation. Plasma membranes were recovered from the gradients in identical yields and characterized by membrane marker enzymes, lipid analysis and SDS-polyacrylamide gel electrophoresis. Membrane purification by alkaline phosphodiesterase I and 5'nucleotidase activities averaged 8-fold for normal rats and 5.5 for the dystrophic rats. The ratio of cholesterol per microgram protein indicated 6 to 7-fold purification for both types of plasma membranes. Na+K+-ATPase in the normal and mutant rat plasma membranes was purified 5- and 3.5-fold, respectively, but the specific activities of both Na+K+-ATPase and 5'nucleotidase were higher in the dystrophic rat membranes than in normal. Subcellular organelle contamination was low and relatively uniform in both types of membranes, while opsin contamination was less than 1%. By electrophoretic analysis the plasma membrane proteins were similar, with 30-40 identifiable bands present in each membrane type. The plasma membranes both contain high levels of cholesterol, sphingomyelin and phosphatidylcholine and low levels of polyunsaturated fatty acids. However, the dystrophic rat membranes had significantly higher levels of docosahexaenoic acid than normal, and significantly lower levels of arachidonic acid. The differences in these plasma membrane fatty acids and in the membrane-bound enzymes may affect the ionic balance of the interphotoreceptor matrix or otherwise contribute to degenerative changes in dystrophic rat photoreceptors.

The protective effect of ascorbic acid in retinal light damage of rats exposed to intermittent light.

Retinal light damage in dark-reared rats supplemented with ascorbic acid and exposed to multiple doses of intermittent light was studied and compared with damage in unsupplemented dark-reared and cyclic-light-reared rats. The extent of photoreceptor cell loss from intense light exposure was determined by whole-eye rhodopsin levels and retinal DNA measurements two weeks after light treatment. Two weeks after 3 or 8 hr of intermittent light, ascorbate-supplemented animals had rhodopsin and retinal DNA levels that were two to three times higher than in unsupplemented dark-reared rats. In both types of rats rhodopsin levels were influenced by the number of light doses, the duration of light exposure, and to a lesser extent, by the length of the dark period between exposures. Rhodopsin levels in the dark-reared ascorbate-supplemented rats were significantly higher than in unsupplemented dark-reared rats, and were similar to the levels in unsupplemented cyclic-light-reared animals. Ascorbate treatment had no effect on the rate of rhodopsin bleaching. However, regeneration was greater in supplemented rats after multiple 1-hr light exposures. Intermittent light also resulted in lower ascorbate levels in the retinas of supplemented and unsupplemented rats, with dramatic losses from the retinal pigment epithelium (RPE)-choroid in both types of animals. We conclude that ascorbic acid protects the eye by reducing the irreversible Type I form of light damage in dark-reared rats. Ascorbate appears to shift light damage to the Type II form typical of cyclic-light-reared animals.

The occurrence of glutathione-insulin transhydrogenase in the retina.

Insulin-degrading activity in the retina of cattle, rat, and rabbit has been studied. On the basis of activation by reduced glutathione, complete inhibition by N-ethylmaleimide, identification of one of the reaction products as the A chain of insulin, and reaction with antibody to purified beef pancreatic glutathione-insulin transhydrogenase, it is concluded that the insulin-degrading enzyme occurs in the retina. Preliminary studies showed that there is no difference in the levels of enzyme activity in retinal homogenates from cattle, rats, or rabbits and that the rat rod outer segments are probably devoid of the degrading activity.

Protection by dimethylthiourea against retinal light damage in rats.

The protective effect of dimethylthiourea (DMTU) against retinal light damage was determined in albino rats reared in darkness or in weak cyclic light. Rats maintained under these conditions were treated with DMTU at different concentrations and dosing schedules and then exposed for various times to intense visible light, either intermittently (1 hr light and 2 hr dark) or continuously. The extent of retinal light damage was determined 2 weeks after light exposure by comparing rhodopsin levels in experimental rats with those in unexposed control animals. To determine the effect of DMTU on rod outer segment (ROS) membrane fatty acids, ROS were isolated immediately after intermittent light exposure, and fatty acid compositions were measured. The time course for DMTU uptake and its distribution in serum, retina, and the retinal pigment epithelium (RPE)/choroid complex was determined in other rats not exposed to intense light. After intraperitoneal injection of the drug (500 mg/kg body weight), DMTU appeared rapidly in the serum, retina, and the RPE and choroid. In the ocular tissues, it was distributed 70-80% in the retina and 20-30% in the RPE and choroid. This antioxidant appears to have a long half-life because it was present in these same tissues 72 hr after a second intraperitoneal injection. For rats reared in the weak cyclic light environment, DMTU (two injections) provided complete protection against rhodopsin loss after intense light exposures of up to 16 hr. Only 15% rhodopsin loss was found in cyclic-light DMTU-treated rats after 24 hr of intermittent or continuous light. For rats reared in darkness, DMTU treatment resulted in a rhodopsin loss of less than 20% after 8-16 hr of continuous light and approximately 40% after similar exposure to intermittent light. Irrespective of the type of light exposure, rhodopsin loss in the dark-reared DMTU-treated rats was nearly identical to that found in uninjected cyclic light-reared animals. In rats from both light-rearing environments, DMTU treatment prevented the light-induced loss of docosahexaenoic acid from ROS membranes. As measured by rhodopsin levels 2 weeks later, DMTU was most effective when given as two doses administered 24 hr before and just before intense light exposure. As a single dose given during continuous light exposure, DMTU protected cyclic light-reared rats for at least 4 hr after the start of exposure but was ineffective in dark-reared animals if injected 1 hr after the start of light. It was also ineffective in both types of rats when given after light exposure.(ABSTRACT TRUNCATED AT 400 WORDS)

The protective effect of ascorbate in retinal light damage of rats.

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.

Amelioration of photic injury in rat retina by ascorbic acid: a histopathologic study.

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.

Retinal light damage in rats exposed to intermittent light. Comparison with continuous light exposure.

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.

Retinal light damage in rats with altered levels of rod outer segment docosahexaenoate.

PURPOSE: To compare retinal light damage in rats with either normal or reduced levels of rod outer segment (ROS) docosahexaenoic acid. METHODS: Weanling male albino rats were maintained in a weak cyclic light environment and fed either a nonpurified control diet or a purified diet deficient in the linolenic acid precursor of docosahexaenoic acid (DHA). Half the rats on the deficient diet were given linseed oil, containing more than 50 mol% linolenic acid, once a week to maintain ROS DHA at near normal levels. Diets and linseed oil supplementation were continued for 7 to 12 weeks. To replenish DHA in their ROS, some 10-week-old rats on the deficient diet were given linseed oil three times a week for up to 3 additional weeks. Groups of animals were killed at various times for ROS fatty acid determinations or were exposed to intense green light using intermittent or hyperthermic light treatments. The extent of retinal light damage was determined biochemically by rhodopsin or photoreceptor cell DNA measurements 2 weeks after exposure and morphologically by light and electron microscopy at various times after light treatment. RESULTS: Rats maintained for 7 to 12 weeks on the linolenic acid-deficient diet had significantly lower levels of DHA and significantly higher levels of n-6 docosapentaenoic acid (22:5n-6) in their ROS than deficient-diet animals supplemented once a week with linseed oil or those fed the nonpurified control diet. As determined by rhodopsin levels and photoreceptor cell DNA measurements, deficient diet rats exhibited protection against retinal damage from either intermittent or hyperthermic light exposure. However, the unsaturated fatty acid content of ROS from all three dietary groups was the same and greater than 60 mol%. In 10 week-old deficient-diet rats given linseed oil three times a week, ROS DHA was unchanged for the first 10 days, whereas 22:5n-6 levels declined by 50%. After 3 weeks of treatment with linseed oil, ROS DHA and 22:5n-6 were nearly the same as in rats supplemented with linseed oil from weaning. The time course of susceptibility to retinal light damage, however, was different. Hyperthermic light damage in rats given linseed oil for only 2 days was the same as for rats always fed the deficient diet. Six days after the start of linseed oil treatment, retinal light damage was the same as in rats given the linseed oil supplement from weaning. Morphologic alterations in ROS of linseed oil-supplemented rats immediately after intermittent light exposure were more extensive than in either the deficient-diet animals or those fed the control diet. The deficient-diet rats also exhibited better preservation of photoreceptor cell nuclei and structure 2 weeks after exposure. CONCLUSIONS: Rats fed a diet deficient in the linolenic acid precursor of DHA are protected against experimental retinal light damage. The relationship between retinal light damage and ROS lipids does not depend on the total unsaturated fatty acid content of ROS; the damage appears to be related to the relative levels of DHA and 22:5n-6.

Circadian-dependent retinal light damage in rats.

PURPOSE: To determine the relative susceptibility of rats to retinal light damage at different times of the day or night. METHODS: Rats maintained in a dim cyclic light or dark environment were exposed to a single dose of intense green light beginning at various times. Normally, light exposures were for 8 or 3 hours, respectively, although longer and shorter periods were also used. Some animals were treated with the synthetic antioxidant dimethylthiourea (DMTU) before or after the onset of light. The extent of visual cell loss was estimated from measurements of rhodopsin and retinal DNA levels 2 weeks after light treatment. The time course of retinal DNA fragmentation, and the expression profiles of heme oxygenase-1 (HO-1) and interphotoreceptor retinol binding protein (IRBP) were determined 1 to 2 days after exposure. RESULTS: When dark-adapted, cyclic light-reared or dark-reared rats were exposed to intense light during normal nighttime hours (2000-0800) the loss of rhodopsin or photoreceptor cell DNA was approximately twofold greater than that found in rats exposed to light during the day (0800-2000). The relative degree of light damage susceptibility persisted in cyclic light-reared rats after dark adaptation for up to 3 additional days. For rats reared in a reversed light cycle, the light-induced loss of rhodopsin was also reversed. Longer duration light treatments revealed that dim cyclic light-reared rats were three- to fourfold more susceptible to light damage at 0100 than at 1700 and that dark-reared animals were approximately twofold more susceptible. Intense light exposure at 0100 resulted in greater retinal DNA fragmentation and the earlier appearance of apoptotic DNA ladders than at 1700. The extent of retinal DNA damage also correlated with an induction of retinal HO-1 mRNA and with a reduction in IRBP transcription. Antioxidant treatment with DMTU was effective in preventing retinal light damage when given before but not after the onset of light. CONCLUSIONS: These results confirm earlier work showing greater retinal light damage in rats exposed at night rather than during the day and extend those findings by demonstrating that a single, relatively short, intense light exposure causes a circadian-dependent, oxidatively induced loss of photoreceptor cells. The light-induced loss of photoreceptor cells is preceded by DNA fragmentation and by alterations in the normal transcriptional events in the retina and within the photoreceptors. The expression profile of an intrinsic retinal factor(s) at the onset of light exposure appears to be important in determining light damage susceptibility.

Hyperthermia accelerates retinal light damage in rats.

PURPOSE: To study the time course of visual cell damage resulting from hyperthermic light exposure and the possible involvement of rod outer segment (ROS) lipids in the process. METHODS: Rats were acclimated in darkness for 2 hours in a hyperthermic chamber to elevate core body temperature and then exposed to intense green light for up to 4 hours during hyperthermia. After light exposure, the animals were either sacrificed immediately for biochemical or morphologic analysis of retinal light damage or returned to darkness for up to 2 weeks at ambient temperature before analysis. Rod outer segment lipid profiles were characterized, and visual cell loss was determined by rhodopsin and visual cell DNA measurements. Morphology was performed at the light and electron microscopic level. RESULTS: Retinal damage resulting from hyperthermic light exposure was found to be temperature, time, and light intensity dependent. At an elevated environmental temperature of 34.5 degrees, 50% visual cell loss was found after 1.5 hours of 1100 lux light exposure; the same degree of visual cell loss occurred after only 1 hour when rats were maintained at 37 degrees C. At ambient temperatures, 4 hours of light exposure had no effect on visual cell loss. Irrespective of environmental temperature, when rats were maintained in darkness no visual cell loss occurred. Whereas docosahexaenoic acid (22:6) was unchanged in the purest fraction of ROS isolated immediately after light treatment, a 5 mol% loss of the polyunsaturated fatty acid was found in ROS isolated 2 or 24 hours after light exposure. Rod outer segment lipid composition was largely unaffected by hyperthermic light exposure, but the density of some ROS increased. Morphologically, the ROS appeared to be nearly normal immediately after hyperthermic light exposure and structurally more abnormal 2 and 24 hours later. The retinal pigment epithelium exhibited damage immediately after exposure, which also increased 2 and 24 hours later. CONCLUSIONS: Hyperthermia in rats dramatically accelerates retinal light damage compared with light exposure under euthermic conditions. Over loss of ROS 22:6 does not occur during hyperthermic light exposure, but it is apparent during the 24-hour period after light treatment. This suggests that the disappearance of 22:6 from ROS occurs in tandem with the process of visual cell death resulting from retinal light damage.

Light history and age-related changes in retinal light damage.

PURPOSE: To determine the effects of age and long-term light- or dark-rearing environments on acute, intense-light-mediated retinal degeneration. METHODS: Male albino rats were maintained in a dim cyclic light environment or in darkness for as long as 1 year. When aged 2, 4, 8, and 12 months, some rats were given the synthetic antioxidant dimethylthiourea (DMTU) by intraperitoneal injection and were exposed to intense visible light for as long as 24 hours. Uninjected control rats were exposed to light at the same time. Other rats were treated with light of lower intensity for various periods. Two weeks after intense-light treatment, photoreceptor cell degeneration was estimated by determining the level of rhodopsin and by measuring the content of photoreceptor cell DNA. Light-induced changes in retinal DNA were analyzed immediately after exposure by neutral gel electrophoresis and by 8-hydroxy-deoxyguanosine measurements. Expression of the antioxidative stress protein heme oxygenase-1 (HO-1) was determined by northern blot analysis of mRNA in retinal extracts. RESULTS: At all ages, rats reared in cyclic dim-light conditions had lower rhodopsin levels than did rats reared in darkness; photoreceptor cell DNA levels were unaffected by the rearing environment. Senescent losses in rhodopsin and retinal DNA were significant after rats were 12 months old. Dim-light-reared rats exhibited an age-related increase in retinal light damage susceptibility, whereas dark-reared rats were equally susceptible to damage at all ages. In both types of rats, the mechanism of light-induced cell death involved an apoptotic process, visualized by the pattern of DNA fragments on electrophoretic gels. The process also induced the expression of HO-1 mRNA. Photoreceptor cell loss determined by biochemical measurement, DNA fragmentation, and HO-1 induction were dramatically reduced by the administration of DMTU. CONCLUSIONS: The age-related increase in susceptibility to retinal light damage in rats is influenced by their long-term daily light history. Decreasing retinal irradiance by dark-rearing eliminates the age-related increase in light damage, suggesting a correlation between light environment and retinal gene expression associated with damage. In all rats, retinal light damage resulted in a pattern of DNA fragmentation consistent with apoptotic cell death and in an increased expression of HO-1 mRNA. Antioxidant treatment greatly reduced apoptosis and HO-1 expression. This indicates that light damage involves an oxidative process that may also trigger apoptosis in the retina. The rat aging model may provide useful insights into the role of light environment associated with retinal degeneration in an aging human population.

Mass spectrometric analysis of rhodopsin from light damaged rats.

PURPOSE: It is well established that the retina is damaged by intense visible light. Rhodopsin has been proposed to be involved in this process. We therefore undertook to examine whether rhodopsin isolated from light damaged animals is structurally altered at the molecular level. METHODS: Dark reared and dim cyclic light reared 8 week old Sprague-Dawley rats were exposed to intense visible light and sacrificed immediately or 24 h after exposure together with unexposed control animals reared under the same conditions. Rod outer segments were isolated by sucrose gradient ultracentrifugation, their membranes treated with urea, then washed with Tris buffer. The rhodopsin preparations were then reduced, pyridylethylated, delipidated, and cleaved with CNBr. Reversed phase HPLC was used to separate the fragments, and the effluent was analyzed online with a Finnigan LCQ ion trap mass spectrometer. C-terminal phosphorylation was investigated following Asp-N cleavage. MALDI-TOF mass spectrometry was used for the identification of glycosylation. RESULTS: The rat rhodopsin protein was mapped with the exception of two single amino acid fragments. The reported sequence was confirmed with the exception of the controversial T/S320 residue, which was found to be a threonine. Mono-, di-, tri-, and tetraphosphorylated forms of rhodopsin were found in the light damaged animals. Three sites of phosphorylation were confirmed with MS/MS (tandem mass spectral) data. Single or double phosphorylations were found among these three sites, in various combinations. Dark adaptation completely reversed the phosphorylation in all light damaged animals. Other posttranslational modifications were as previously reported. CONCLUSIONS: Our results indicate that intense visible light exposure of rats does not lead to oxidative or other primary structural alterations in the rhodopsin protein of rod outer segments. We also report that the mutated rhodopsin (P23H) is present in rat rod outer segments from heterozygous animals and that residue 320 in both normal and mutated rhodopsins is threonine, not serine.