Ruth M. Darrow

Light-induced Oxidation of Photoreceptor Outer Segment Phospholipids Generates Ligands for CD36-mediated Phagocytosis by Retinal Pigment Epithelium A POTENTIAL MECHANISM FOR MODULATING OUTER SEGMENT PHAGOCYTOSIS UNDER OXIDANT STRESS CONDITIONS

Clearance by the retinal pigment epithelium (RPE) of shed photoreceptor outer segments (OSs), a tissue with one of the highest turnover rates in the body, is critical to the maintenance and normal function of the retina. We hypothesized that there is a potential role for photo-oxidation in OS uptake by RPE via scavenger receptor-mediated recognition of structurally defined lipid peroxidation products. We now demonstrate that specific structurally defined oxidized species derived from arachidonyl, linoleoyl, and docosahexanoyl phosphatidylcholine may serve as endogenous ligands on OSs for uptake by RPE via the scavenger receptor CD36. Mass spectrometry studies of retinal lipids recovered from dark-adapted rats following physiological light exposure demonstrate in vivo formation of specific oxidized phosphatidylcholine molecular species possessing a CD36 recognition motif, an oxidatively truncated sn-2 acyl group with a terminal γ-hydroxy(or oxo)-α,β-unsaturated carbonyl. Cellular studies using RPE isolated from wild-type versus CD36 null mice suggest that CD36 plays a role in engulfment, but not initial binding, of OSs via these oxidized phospholipids. Parallel increases in OS protein-bound nitrotyrosine, a post-translational modification by nitric oxide (NO)-derived oxidants, were also observed, suggesting a possible role for light-induced generation of NO-derived oxidants in the initiation of OS lipid peroxidation. Collectively, these studies suggest that intense light exposure promotes “oxidative tagging” of photoreceptor outer segments with structurally defined choline glycerophospholipids that may serve as a physiological signal for CD36-mediated phagocytosis under oxidant stress conditions.

Adaptive changes in visual cell transduction protein levels: Effect of light

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)

Light-induced Damage in the Retina: Differential Effects of Dimethylthiourea on Photoreceptor Survival, Apoptosis and DNA Oxidation

Abstract. In the rat, photoreceptor cell death from exposure to intense visible light can be prevented by prior treatment with antioxidants. In this study we subjected albino rats raised in dim cyclic light and rats made more susceptible to light damage by rearing in darkness to exposures of green light that led to similar losses of photoreceptor cells. Rhodopsin and photoreceptor DNA, indicators of the number of surviving photoreceptor cells, were determined at various times over a period of 14 days after light exposure. Fragmentation of DNA was determined over a similar time course by neutral and alkaline agarose gel electrophoresis. Apoptosis in retinal DNA was measured by quantitating the appearance of 180 base pair (bp) nucleosomal fragments. Oxidation of DNA was measured by electrochemical detection of the nucleoside 8‐hydroxydeoxyguanosine (8‐OHdG) after separation by high‐performance chromatography. For albino rats reared in dim cyclic light, 24 h of intense light exposure resulted in the loss of 50% rhodopsin and photoreceptor cell DNA. In dark‐reared rats, the losses were 40%, respectively, after only 3 h of intense light treatment. In both cases pretreatment with the antioxidant dimethylthiourea (DMTU) prevented rhodopsin and photoreceptor cell DNA loss. The kinetics of the light‐induced apoptosis depended markedly on the rearing environment of the rats. The DNA ladders appeared within 12 h of the onset of intense light in the rats reared in dim cyclic light. In these rats the 180 bp fragment was at two‐thirds of its maximum intensity immediately after 24 h of light exposure and reached the maximum 12 h later. Dimethylthiourea partially inhibited ladder formation in rats reared in dim cyclic light and delayed the time of appearance of the 180 bp maximum by 6 h. By contrast, in rats reared in darkness the 180 bp fragment was undetected immediately after 3 h of light exposure and

Endogenous ascorbate regenerates vitamin E in the retina directly and in combination with exogenous dihydrolipoic acid

Vitamin E (alpha-tocopherol) is the major lipid-soluble antioxidant of retinal membranes whose deficiency causes retinal degeneration. Its antioxidant function is realized via scavenging peroxyl radicals as a result of which phenoxyl radicals of alpha-tocopherol are formed. Our hypothesis is that alpha-tocopherol phenoxyl radicals can be reduced by endogenous reductants in the retina, providing for alpha-tocopherol recycling. The results of this study demonstrate for the first time that: (i) endogenous ascorbate (vitamin C) in retinal homogenates and in rod outer segments is able to protect endogenous alpha-tocopherol against oxidation induced by UV-irradiation by reducing the phenoxyl radical of alpha-tocopherol, (ii) in the absence of ascorbate, neither endogenous nor exogenously added glutathione (GSH) is efficient in protecting alpha-tocopherol against oxidation; (iii) GSH does not substantially enhance the protective effect of ascorbate against alpha-tocopherol oxidation; (iv) exogenous dihydrolipoic acid (DHLA), although inefficient in direct reduction of the alpha-tocopherol phenoxyl radical, is able to enhance the protective effect of ascorbate by regenerating it from dehydroascorbate. Thus, regeneration of alpha-tocopherol from its phenoxyl radical can enhance its antioxidant effectiveness in the retina. The recycling of alpha-tocopherol opens new avenues for pharmacological approaches to enhance antioxidants of the retina.

Intense light exposure changes the crystallin content in retina

Toward a better understanding of light-induced photoreceptor damage, the crystallin content of rat retina was examined following intense light exposure. Nine crystallin species were identified by mass spectrometric analysis of rat retina fractionated by 2D gel electrophoresis. The Coomassie blue staining intensity of all crystallin 2D gel components was 2- to 3-fold greater in light exposed than in control retinas. Following light exposure, anti-alphaB-crystallin immunoreactivity was increased in rod outer segments and retinal pigment epithelium. These findings support a possible role for crystallins in protecting photoreceptors from light damage.

Evidence for a Circadian Rhythm of Susceptibility to Retinal Light Damage

Abstract This study investigated a possible circadian rhythm of light damage susceptibility in photoreceptors of both cyclic light-reared and dark-reared rats. A single exposure to intense green light was administered, beginning either in the early light period, the late light period or the dark period. In some animals exposed in the dark period, the synthetic antioxidant dimethylthiourea was administered before or after the onset of intense light exposure. Retinas were examined either immediately after exposure or after 2 weeks of recovery in darkness. Rod outer segment length and outer nuclear layer thickness measurements were used to assess light damage, along with qualitative analysis of swelling and disruption of the outer retinal layers. In all animals, retinal light damage was the most severe when intense light exposure began during the dark period. However, this severe damage was significantly reduced by pretreatment with the antioxidant. In a separate set of unexposed animals, fluctuations in plasma adrenocorticotropic hormone (ACTH) and corticosterone concentrations followed the same time course, regardless of the light regime during rearing. Our data support the notion of a circadian rhythm of light damage susceptibility that peaks in the dark period and yet can be modulated by the exogenous administration of an antioxidant.

The effects of L- and D-ascorbic acid administration on retinal tissue levels and light damage in rats

To assess the protective effect of ascorbic acid in retinal light damage of rats, we have determined the uptake and retinal tissue distributions of its L- and D- stereoisomers following interperitoneal or intraocular injections. The effects of intense-intermittent light exposure and darkness on tissue ascorbate were compared by measuring its levels in retina and retinal pigment epithelial tissues at various times after administration. The protective effects of the two forms of ascorbate against retinal light damage were also compared by measuring rhodopsin levels 2 weeks after intense light exposure. After interperitoneal injection, both forms of ascorbic acid were higher in the retinal pigment epithelial-choroid-scleral complex (eye cup) than in the retina. Over a 2 hr post-injection period, L-ascorbate in the eye cup was 2 to 4 fold higher than normal (10-11 nmol); D-ascorbate levels were between 15 and 30 nmol. During the same period retinal L-ascorbate was just above normal (12-14 nmol), whereas less than 5 nmol of D-ascorbate was present. When ascorbate was given by the intraocular route the opposite effect was found. During the 2 hr post-injection period retinal L-ascorbate levels were 2 to 5 fold higher than normal; D-ascorbate was between 25 and 50 nmol/retina. Within 1 hr post-injection, L-ascorbate in the eye cup was near normal and D-ascorbate levels were 10 nmol or less. In uninjected rats perfused with normal saline, the endogenous L-ascorbate was distributed 55% in the retina with 9% and 36%, respectively, in the RPE-choroid and sclera. Ten-thirty min after interperitoneal peritoneal injection about 40% of the L-ascorbate was present in the retina with 17% and 44% in the RPE-choroid and sclera. Total ascorbate (L + D) levels in the same tissues of D- injected rats were similar to those found for rats given L-ascorbate. Following 7 hrs of darkness, tissue ascorbate levels in the injected rats decreased to approximately the same levels present in uninjected animals. For rats exposed to intense light average retinal ascorbate levels decreased further, while RPE-choroid and scleral levels were largely unchanged from the dark control levels. About 50% of the tissue ascorbate was present in the retina 10-30 min after intraocular injection. The RPE-choroid contained between 10 and 14% of the ascorbate, with 35-40% present in the sclera. Retinal ascorbate levels remained high in the injected eyes following 2.5 hrs of darkness, but decreased as a result of intense light treatment.(ABSTRACT TRUNCATED AT 400 WORDS)

Damage to Rat Retinal DNA Induced In Vivo by Visible Light

Abstract— Intense visible light can damage retinal photoreceptor cells by photochemical or thermal processes, leading to cell death. The precise mechanism of light‐induced damage is unknown; however, oxidative stress is thought to be involved, based on the protective effect of antioxidants on the light‐exposed retina. To explore the in vivo effects of light on retinal DNA, rats were exposed to intense visible light for up to 24 h and the time courses of single‐strand breaks in restriction fragments containing the opsin, insulin 1 and interleukin‐6 genes were measured. All three gene fragments displayed increasing single‐strand modifications with increasing light exposure. Treatment with the antioxidant dimethylthiourea prior to light exposure delayed the development of net damage. The time course of double‐strand DNA damage was also examined in specific genes and in repetitive DNA. The appearance of discrete 140–200 base‐pair DNA fragments after 20 h of light exposure implicated a nonrandom, possibly enzymatic damaging mechanism. The generation of nucleosome core‐sized DNA fragments, in conjunction with single‐strand breaks, suggests two phases of light‐induced retinal damage, with random attack on DNA by activated oxygen species preceding enzymatic degradation.

Genetic, age and light mediated effects on crystallin protein expression in the retina

Abstract To probe for possible relationships between retinal crystallins and retinal degenerations, protein expression was compared in normal Sprague-Dawley rats, treated or not with intense light, Royal College of Surgeons (RCS) rats and transgenic rats expressing rhodopsin mutations. Rats were reared in dim cyclic light for 21–75 days. Photoreceptor cell DNA levels were determined at various ages to assess the rates of visual cell loss. 1D- and 2D-gel electrophoresis was used to profile retinal protein expression. Crystallins were identified by western analysis and by tandem mass spectrometry. In normal rat retinas, α, β and γ crystallins were present, although αA- and γ-crystallins exhibited some increase with age. As measured by DNA levels, the rate of genetically induced photoreceptor cell loss was greater in rats with faster degenerating retinas (RCS, S334-ter Line 4, P23H Line 3) than in rats with slower degenerating retinas (S334-ter Line 9, P23H Line 2). In genetic models of retinal degeneration increased levels of immunoreactivity for all crystallins, especially αA-insert, correlated with the different rates of photoreceptor loss. In the light induced degeneration model αA-insert was unchanged, truncated αB- crystallin levels were increased and γ-crystallins were greatly reduced. In the RCS rat retina 16 different crystallins were identified. Our data suggests that an increase in crystallin expression occurs during various retinal degenerations and that the increases may be related to the severity, type and onset of retinal degeneration.

Proteomic Changes in the Photoreceptor Outer Segment upon Intense Light Exposure

Acute light-induced photoreceptor degeneration has been studied in experimental animals as a model for photoreceptor cell loss in human retinal degenerative diseases. Light absorption by rhodopsin in rod photoreceptor outer segments (OS) induces oxidative stress and initiates apoptotic cell death. However, the molecular events that induce oxidative stress and initiate the apoptotic cascade remain poorly understood. To better understand the molecular mechanisms of light-induced photoreceptor cell death, we studied the proteomic changes in OS upon intense light exposure by using a proteolytic (18)O labeling method. Of 171 proteins identified, the relative abundance of 98 proteins in light-exposed and unexposed OS was determined. The quantities of 11 proteins were found to differ by more than 2-fold between light-exposed OS and those remaining in darkness. Among the 11 proteins, 8 were phototransduction proteins and 7 of these were altered such that the efficiency of phototransduction would be reduced or quenched during light exposure. In contrast, the amount of OS rhodopsin kinase was reduced by 2-fold after light exposure, suggesting attenuation in the mechanism of quenching phototransduction. Liquid chromatography multiple reaction monitoring (LC-MRM) was performed to confirm this reduction in the quantity of rhodopsin kinase. As revealed by immunofluorescence microscopy, this reduction of rhodopsin kinase is not a result of protein translocation from the outer to the inner segment. Collectively, our findings suggest that the absolute quantity of rhodopsin kinase in rod photoreceptors is reduced upon light stimulation and that this reduction may be a contributing factor to light-induced photoreceptor cell death. This report provides new insights into the proteomic changes in the OS upon intense light exposure and creates a foundation for understanding the mechanisms of light-induced photoreceptor cell death.