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.

Purpose: Zinc oxide effectively reduces visual cell loss in rats exposed to intense visible light and is known to slow the rate of disease progression in advanced stages of age-related macular degeneration. Our goal was to determine the efficacy of zinc oxide in combination with novel and well-established antioxidants in an animal model of light-induced oxidative retinal damage.Methods: One group of male Sprague-Dawley rats was pretreated with zinc oxide with or without a detergent extract of rosemary powder and then exposed to intense visible light for 4-24 h. Another group of animals received zinc oxide combined with rosemary oil diluted with a mixture of polyunsaturated fatty acids (ROPUFA) and a third group was given an antioxidant mineral mix containing zinc oxide, as recommended by the Age Related Eye Disease Study group's first clinical trial (AREDS1). Visual cell survival was determined 2 weeks after intense light treatment by measuring rhodopsin and photoreceptor cell DNA levels and confirmed by retinal histology and agarose gel electrophoresis of DNA. Western analysis was used to determine the effects of zinc and antioxidants on the oxidative stress markers, glial fibrillary acidic protein (GFAP), heme-oxygenase-1 (HO-1), and carboxyethylpyrrole (CEP). Rod and cone opsin and arrestin levels were used as markers of photoreceptor cell function.Results: Dark-reared rats treated with 1.3 mg/kg zinc oxide and 17 mg/kg rosemary extract, or with one-half those doses, and exposed to moderate intensity green light retained 75%-85% of the rhodopsin and retinal DNA measured in unexposed rats. These levels were significantly higher than found for zinc oxide or rosemary treatment alone. Rosemary oil was also effective when combined with zinc oxide, but ROPUFA alone was no more effective than the detergent vehicle. Prolonged intense green light led to increases in retinal GFAP and HO-1 levels and to decreases in cone cell opsin and rod and cone arrestins. Rosemary plus zinc treatment reduced the expression of oxidative stress protein markers and enhanced visual cell survival, as shown by improved photoreceptor cell morphology and by decreased retinal DNA degradation. Using higher intensity white light for exposures in cyclic light-reared rats, treatment with an AREDS antioxidant/mineral mixture was found to be ineffective, whereas rosemary extract plus an equivalent dose of zinc oxide was significantly more effective in preserving visual cells. CEP protein adduct formation was reduced by all antioxidant treatments, but rosemary plus zinc oxide also prevented the loss of cone cell opsin and arrestin more effectively than AREDS.Conclusions: In the rat model of acute retinal light damage, zinc oxide combined with a detergent extract of rosemary powder or rosemary oil is more effective than treatment with either component alone and significantly more effective than an AREDS mixture containing a comparable dose of zinc oxide. Light-induced oxidative stress in animal models of retinal degeneration can be a useful preclinical paradigm for screening novel antioxidants and for testing potential therapeutics designed to slow the progression of age-related ocular disease.

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 damaging effects of visible light on the mammalian retina can be detected as functional, morphological or biochemical changes in the photoreceptor cells. Although previous studies have implicated short-lived reactive oxygen species in these processes, the termination of light exposure does not prevent continuing damage. To investigate the degenerative processes persisting during darkness following light treatment, rats were exposed to 24 h of intense visible light and the accumulation of DNA damage to restriction fragments containing opsin, insulin 1 or interleukin-6 genes was measured as single-strand breaks (ssb) on alkaline agarose gels. With longer dark treatments all three DNA fragments showed increasing DNA damage. Treatment of rats with the synthetic antioxidant dimethylthiourea prior to light exposure reduced the initial development of alkali-sensitive strand breaks and allowed significant repair of all three DNA fragments. The time course of double-strand DNA breaks was also examined in specific genes and repetitive DNA. Nucleosomal DNA laddering was evident immediately following the 24 h light treatment and increased during the subsequent dark period. The increase in the intensity of the DNA ladder pattern suggests a continuation of enzymatically mediated apoptotic processes triggered during light exposure. The protective effects of antioxidant suggests that the light-induced DNA degradative process includes both early oxidative reactions and enzymatic processes that continue after cessation of light exposure.

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.

Purpose: Light has been shown to modulate protein nitration in rat retinas. To better understand the role of protein nitration in photoreceptor cell death induced by intense light, we examined retinal protein nitration and identified target proteins in rod outer segments (ROS).Methods: Cyclic light-reared rats, treated or not with the antioxidant, dimethylthiourea (DMTU), were exposed to intense green light for 8 h. A subset of these rats was kept in the dark for 24 h after 8 h of light exposure. Western analysis of ROS proteins with an anti-nitrotyrosine antibody was performed to examine changes in protein nitration. 2D-immunoblots with anti-nitrotyrosine antibody followed by liquid chromatography tandem mass spectrometry was used to identify nitrated proteins in ROS. The expression levels of three nitric oxide synthase ( NOS) isoforms, inducible, neuronal-, and endothelial-NOS were semi-quantified by immunoblot analysis.Results: Western analysis revealed that the level of ROS protein nitration increased during the dark recovery period after 8 h of light treatment in both DMTU treated and untreated rats. However, DMTU effectively reduced protein nitration in ROS during light exposure and during the subsequent dark recovery period. Using 2D-immunoblotting followed by liquid chromatography tandem mass spectrometry analysis, we identified ten ROS proteins as nitration targets. Most of these proteins were glycolytic enzymes. The level of inducible-NOS in the retina was increased by light exposure.Conclusions: The effect of DMTU in reducing ROS protein nitration during and after light suggests the involvement of protein nitration during light-induced photoreceptor cell death. Nitration of glycolytic enzymes specifically may alter their activities. Increased levels of iNOS during and after intense light exposure suggest that this isoform is responsible for intense light induced protein nitration in ROS during the dark recovery period. The limited nitration seen in ROS during light exposure may reflect a quenching effect by endogenous antioxidants on the generation of reactive oxygen and nitrogen species.

PURPOSEDysregulation of neurturin (NTN) expression has been linked to photoreceptor apoptosis in a mouse model of inherited retinal degeneration. To investigate the extent to which any such dysregulation depends on the nature of the apoptotic trigger, the expression of NTN, glial cell line-derived neurotrophic factor (GDNF), and their corresponding receptor components were compared in a rat model of light-induced retinal degeneration.METHODSRetinal expression of NTN, GDNF, their corresponding receptors GFRalpha-2 and -1, the transmembrane receptor tyrosine kinase (Ret), and cSrc-p60, a member of the cytoplasmic protein-tyrosine kinases family, were analyzed by Western blot analysis and immunocytochemistry in cyclic light- and dark-reared rats in the presence and absence of intense light exposure.RESULTSAll components for NTN-mediated signaling activation are present in rat photoreceptors and retinal pigment epithelium, the cells primarily affected by light-induced damage. The expression levels of GDNF, its receptor components, and NTN, were not affected by light-induced stress. However, GFRalpha-2 expression strikingly increased with the extent of retinal damage, especially at the photoreceptors, in contrast to decreased levels that were observed previously in an inherited degeneration model.CONCLUSIONSThe present study indicates that the expression of receptors of the GDNF family is independently regulated in normal and light-damaged rat retina, and in conjunction with previous work, suggests that the pattern of modulation of these genes during photoreceptor degeneration is determined by the nature of the apoptotic trigger. Such differential responses to different modes of retinal degeneration may reflect influences of the neurotrophic system on photoreceptor survival or in the regulation of neuronal plasticity.