RESUMO
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.
Assuntos
Extratos Vegetais/farmacologia , Retina/efeitos dos fármacos , Retina/patologia , Rosmarinus/química , Óxido de Zinco/farmacologia , Animais , Western Blotting , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Eletroforese em Gel de Poliacrilamida , Ácidos Graxos Insaturados/farmacologia , Masculino , Óleos Voláteis/farmacologia , Substâncias Protetoras/farmacologia , Ratos , Ratos Sprague-Dawley , Retina/efeitos da radiaçãoRESUMO
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.
Assuntos
Luz , Células Fotorreceptoras de Vertebrados/química , Proteômica , Animais , Cromatografia Líquida , Masculino , Estresse Oxidativo , Ratos , Ratos Sprague-DawleyRESUMO
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.
Assuntos
Proteínas do Olho/metabolismo , Luz , Nitratos/metabolismo , Segmento Externo da Célula Bastonete/metabolismo , Segmento Externo da Célula Bastonete/efeitos da radiação , Animais , Antioxidantes/farmacologia , Western Blotting , Cromatografia Líquida , Escuridão , Masculino , Nitratos/antagonistas & inibidores , Óxido Nítrico Sintase/metabolismo , Ratos , Ratos Sprague-Dawley , Segmento Externo da Célula Bastonete/efeitos dos fármacos , Espectrometria de Massas em Tandem , Tioureia/análogos & derivados , Tioureia/farmacologia , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMO
Transgenic rats with the P23H mutation in rhodopsin exhibit increased susceptibility to light damage, compared with normal animals. It is known that light-induced retinal damage requires repetitive bleaching of rhodopsin and that photoreceptor cell loss is by apoptosis; however, the underlying molecular mechanism(s) leading to photoreceptor cell death are still unknown. Photoproducts, such as all-trans retinal or other retinoid metabolites, released by the extensive bleaching of rhodopsin could lead to activation of degenerative processes, especially in animals genetically predisposed to retinal degenerations. Using wild-type and transgenic rats carrying the P23H opsin mutation, we evaluated the effects of acute intense visible light on retinoid content, type and distribution in ocular tissues. Rats were exposed to green light (480-590 nm) for 0, 5, 10, 30 and 120 min. Following light treatment, rats were sacrificed and neural retinas were dissected free of the retinal pigment epithelium. Retinoids were extracted from retinal tissues and then subjected to HPLC and mass spectral analysis. We found that the light exposure affected relative levels of retinoids in the neural retina and retinal pigment epithelium of wild-type and P23H rat eyes similarly. In the P23H rat retina but not the wild-type rat retina, we found a retinoic acid-like compound with an absorbance maximum of 357 nm and a mass of 304 daltons. Production of this retinoic acid-like compound in transgenic rats is influenced by the age of the animals and the duration of light exposure. It is possible that this unique retinoid may be involved in the process of light-induced retinal degeneration.
Assuntos
Luz/efeitos adversos , Retina/efeitos da radiação , Animais , Animais Geneticamente Modificados , Mutação , Ratos , Rodopsina/genética , Tretinoína/isolamento & purificação , Tretinoína/efeitos da radiaçãoRESUMO
PURPOSE: Dysregulation 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. METHODS: Retinal 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. RESULTS: All 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. CONCLUSIONS: The 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.
Assuntos
Fatores de Crescimento Neural/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Lesões Experimentais por Radiação/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Retina/efeitos da radiação , Degeneração Retiniana/metabolismo , Animais , Western Blotting , Técnica Indireta de Fluorescência para Anticorpo , Fator Neurotrófico Derivado de Linhagem de Célula Glial , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial , Luz , Masculino , Neurturina , Proteínas Proto-Oncogênicas c-ret , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Lesões Experimentais por Radiação/etiologia , Ratos , Ratos Sprague-Dawley , Degeneração Retiniana/etiologiaRESUMO
PURPOSE: To determine relative light-induced retinal damage susceptibility in transgenic rats expressing mutations in the N- or C-terminal region of rhodopsin. METHODS: Heterozygous transgenic rats, including P23H sublines 2 and 3 and S334ter sublines 4 and 9, were reared in dim cyclic light or in darkness before visible light exposure starting at various times of the day or night. Before exposure to light, some rats were given the synthetic antioxidant dimethylthiourea (DMTU). At various times after intense light treatment, rats were killed for determinations of rhodopsin and retinal DNA recovery, DNA fragmentation patterns, and Northern blot analysis of retinal heme oxygenase (HO)-1 and interphotoreceptor retinol binding protein (IRBP). Rod outer segments (ROSs) were isolated for Western blot analysis of rhodopsin using N- and C- terminal-specific monoclonal antibodies. RESULTS: All rats incurred greater photoreceptor cell damage from exposure to light starting at 1 AM than from exposure at 5 PM. Among cyclic-light-reared rats, P23H line 3 animals were more susceptible to light-induced damage than P23H line 2 animals. S334ter rats exhibited retinal light damage profiles similar to those in normal rats. Dark-rearing potentiated retinal damage by light. However, dark-rearing alone prolonged photoreceptor cell life in P23H rats, but had no such effect in S334ter animals. DMTU pretreatment was effective in preventing or reducing light-induced retinal damage in all transgenic rats. S334ter rat ROSs contained the truncated form of rhodopsin. Intense light exposure resulted in DNA ladders typical of apoptotic cell death and the simultaneous induction of retinal HO-1 mRNA and reduced expression of IRBP. CONCLUSIONS: Light-induced retinal damage in transgenic rats depends on the time of day of exposure to light, prior light-or dark-rearing environment, and the relative level of transgene expression. Retinal light damage leads to apoptotic visual cell loss and appears to result from oxidative stress. These results suggest that reduced environmental lighting and/or antioxidant treatment may delay retinal degenerations arising from rhodopsin mutations.
Assuntos
Animais Geneticamente Modificados , Proteínas do Olho , Mutação , Lesões Experimentais por Radiação/genética , Retina/efeitos da radiação , Degeneração Retiniana/genética , Rodopsina/genética , Animais , Northern Blotting , Western Blotting , DNA/análise , Fragmentação do DNA , Adaptação à Escuridão , Suscetibilidade a Doenças , Feminino , Heme Oxigenase (Desciclizante)/metabolismo , Luz , Masculino , Estresse Oxidativo , Lesões Experimentais por Radiação/etiologia , Lesões Experimentais por Radiação/metabolismo , Ratos/genética , Retina/metabolismo , Retina/patologia , Degeneração Retiniana/etiologia , Degeneração Retiniana/metabolismo , Proteínas de Ligação ao Retinol/metabolismo , Rodopsina/metabolismoRESUMO
PURPOSE: To determine relative susceptibility to, and regional variation of, light-induced retinal damage in two rhodopsin-mutant rat models of retinitis pigmentosa, using slow- and fast-degenerating lines. METHODS: Transgenic S334ter (lines 4 and 9) and P23H (lines 2 and 3) rats were reared in dim cyclic light or darkness and then exposed to intense green light for 1 to 8 hours. Sections along the vertical meridian were collected for retinal morphology and photoreceptor morphometry 2 weeks later. Unexposed transgenic and normal Sprague-Dawley rats served as the control. Mean outer segment lengths and outer nuclear layer thicknesses were analyzed as a function of position along the vertical meridian and as averages across that vector. RESULTS: Rapidly degenerating S334ter-4 retinas, reared in dim cyclic light, exhibited no light-induced damage, whereas retinas in the other sublines sustained damage within a sensitive region in the superior hemisphere. Light-induced damage always involved loss of outer segment membrane and photoreceptors. In some cases, the retinal pigment epithelium and inner nuclear layer were also affected. Potentiation of light-induced damage by dark-rearing was increased by at least a factor of three, and in some sublines the sensitive region was enlarged to include the entire vertical meridian. CONCLUSIONS: A complex pattern of light-induced damage outcomes was identified in S334ter (sublines 4 and 9) and P23H (sublines 2 and 3) rats. The relative susceptibilities of each subline to damage by light were different, even within the same transgene, but consistent factors included a sensitive region in the superior hemisphere and potentiation by dark-rearing.
Assuntos
Lesões Experimentais por Radiação/patologia , Retina/efeitos da radiação , Retinose Pigmentar/patologia , Animais , Animais Geneticamente Modificados , Luz , Epitélio Pigmentado Ocular/patologia , Epitélio Pigmentado Ocular/efeitos da radiação , Lesões Experimentais por Radiação/etiologia , Lesões Experimentais por Radiação/metabolismo , Ratos , Ratos Sprague-Dawley , Retina/metabolismo , Retina/patologia , Células Ganglionares da Retina/patologia , Células Ganglionares da Retina/efeitos da radiação , Retinose Pigmentar/etiologia , Retinose Pigmentar/metabolismo , Rodopsina/genética , Rodopsina/metabolismo , Segmento Externo da Célula Bastonete/patologia , Segmento Externo da Célula Bastonete/efeitos da radiaçãoRESUMO
PURPOSE: To identify genes with altered expression levels in the degenerating retina in a light-induced retinal degeneration (LIRD) model. METHODS: Adult Sprague-Dawley rats were exposed to intense green light for 4 hours. After this treatment, the retinas were excised, RNA was extracted, and a cDNA library was prepared. The cDNA library was differentially cross-screened with probes representing 0-hour and 4-hour light-exposed rat retina. Transcripts with altered expression levels were sequenced and expression was confirmed by Northern blot analysis. Gene-specific primers were designed and used to examine the expression levels of other genes involved in protein synthesis. Promoter sequences of the ribosomal-binding protein (Rbp) genes were analyzed for transcription-binding sites. RESULTS: Of the 10,000 clones that were initially screened, 41 exhibited altered expression levels. Six of these corresponded to five known Rbp genes. Six additional Rbp genes were also examined. In total, 9 of 11 Rbp genes exhibited an increase in expression levels in response to a 4-hour light exposure. In contrast, the transcript levels of elongation factor 1alpha1 and 18S rRNA did not increase. The most abundant transcription factor-binding sites conserved in the promoter regions of all Rbp genes examined in this study include AP-1, Oct-1, V-myb, USF, Pax-4, and the FOX family of transcription factors. CONCLUSIONS: The results indicate that light-induced retinal degeneration (LIRD) is associated with increased expression of specific Rbp genes. These Rbp genes may be involved in mediating visual cell loss in LIRD through a translational or an extraribosomal mechanism.
Assuntos
Expressão Gênica , Lesões Experimentais por Radiação/genética , Retina/efeitos da radiação , Degeneração Retiniana/genética , Proteínas Ribossômicas/genética , Animais , Northern Blotting , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Biblioteca Gênica , Luz , Masculino , Estresse Oxidativo , Reação em Cadeia da Polimerase , RNA/isolamento & purificação , RNA Ribossômico 18S/metabolismo , Lesões Experimentais por Radiação/metabolismo , Ratos , Ratos Sprague-Dawley , Retina/metabolismo , Degeneração Retiniana/metabolismo , Proteínas Ribossômicas/metabolismo , Fatores de Transcrição/genéticaRESUMO
PURPOSE: To determine whether dietary-induced alterations in the long-chain polyunsaturated fatty acid content of retinal rod outer segments (ROS) of P23H rats, a transgenic model of retinitis pigmentosa (RP), prolongs photoreceptor cell life. METHODS: Heterozygous P23H and normal Sprague-Dawley rats were fed a standard house diet or a diet deficient in 18:3n-3. Diet-deficient rats were given supplements of either linseed oil (high in 18:3n-3) or fish oil (high in 20:5n-3). ROS fatty acid profiles and serum fatty acids were determined by gas chromatography. Serum cholesterol was evaluated by HPLC. Retinal damage was assessed by measuring whole-retina rhodopsin and DNA content before and after exposure to high-intensity light. RESULTS: The retinas of 60 day old, cyclic-light-reared, P23H transgenic rats contained 50% of the rhodopsin and 75% of the DNA content found in control Sprague-Dawley rats. Eight hours of intense light had little effect on the rhodopsin or DNA content in the Sprague-Dawley rats, but resulted in rhodopsin and DNA losses of nearly 70%, compared to controls, in P23H animals fed either a standard or an 18:3n-3-deficient diet. Supplementation with linseed oil resulted in small, statistically insignificant, increases in the rhodopsin and DNA losses, which occurred after exposure to intense light, in P23H transgenics. In unexposed animals, supplementation with linseed oil or fish oil had no effect on either rhodopsin or DNA levels in P23H rats or in Sprague-Dawley controls. On standard diet, the ROS 22:6n-3 (DHA) content in P23H rats was lower than that of control animals. DHA decreased in both groups when an 18:3-deficient diet was fed. The reduction was greater in controls than in P23H transgenics, but a concomitant increase in 22:5n-6 was nearly the same in both groups. Supplementation of the 18:3-deficient diet with linseed oil or fish oil in P23H rats resulted in a ROS fatty acid profile comparable to that of Sprague-Dawley rats raised on a standard diet. Serum DHA and 22:5n-6 levels were low in both groups. No significant differences in serum cholesterol were observed as a function of genotype or diet. CONCLUSIONS: Heterozygous P23H rats are capable of forming ROS DHA from dietary fatty acid precursors found in linseed oil (18:3n-3) or fish oil (20:5n-3). Under all dietary conditions, P23H transgenics are highly susceptible to retinal damage from exposure to intense light. Although levels of DHA in the ROS of P23H rats could be altered by dietary manipulation, only small changes in photoreceptor cell survival, as measured by whole-retina rhodopsin and DNA content, were observed. The lower-than-normal levels of ROS DHA may reflect an adaptive, possibly protective, mechanism in the P23H transgenic rat model of RP.
Assuntos
Ácidos Graxos Insaturados/metabolismo , Luz/efeitos adversos , Lesões Experimentais por Radiação/metabolismo , Retinose Pigmentar/metabolismo , Segmento Externo da Célula Bastonete/metabolismo , Segmento Externo da Célula Bastonete/efeitos da radiação , Animais , Animais Geneticamente Modificados , Colesterol/sangue , Cromatografia Gasosa , Cromatografia Líquida de Alta Pressão , DNA/metabolismo , Gorduras Insaturadas na Dieta/administração & dosagem , Ácidos Graxos/sangue , Lesões Experimentais por Radiação/etiologia , Lesões Experimentais por Radiação/patologia , Ratos , Ratos Sprague-Dawley , Retina/patologia , Retina/efeitos da radiação , Retinose Pigmentar/etiologia , Retinose Pigmentar/patologia , Rodopsina/genética , Rodopsina/metabolismoRESUMO
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.
Assuntos
Ritmo Circadiano/efeitos da radiação , Luz/efeitos adversos , Retina/efeitos da radiação , Animais , Escuridão , Ratos , Segmento Externo da Célula Bastonete/efeitos da radiaçãoAssuntos
Luz , Doenças Neurodegenerativas/patologia , Neurônios/metabolismo , Células Fotorreceptoras de Vertebrados/metabolismo , Retina/metabolismo , Animais , Ritmo Circadiano , Glutamina/metabolismo , Humanos , Microscopia Eletrônica , Células Fotorreceptoras de Vertebrados/ultraestrutura , Ratos , Ratos Sprague-Dawley , Retina/ultraestrutura , Degeneração Retiniana/patologia , Fatores de TempoRESUMO
By its action on rhodopsin, light triggers the well-known visual transduction cascade, but can also induce cell damage and death through phototoxic mechanisms - a comprehensive understanding of which is still elusive despite more than 40 years of research. Herein, we integrate recent experimental findings to address several hypotheses of retinal light damage, premised in part on the close anatomical and metabolic relationships between the photoreceptors and the retinal pigment epithelium. We begin by reviewing the salient features of light damage, recently joined by evidence for retinal remodeling which has implications for the prognosis of recovery of function in retinal degenerations. We then consider select factors that influence the progression of the damage process and the extent of visual cell loss. Traditional, genetically modified, and emerging animal models are discussed, with particular emphasis on cone visual cells. Exogenous and endogenous retinal protective factors are explored, with implications for light damage mechanisms and some suggested avenues for future research. Synergies are known to exist between our long term light environment and photoreceptor cell death in retinal disease. Understanding the molecular mechanisms of light damage in a variety of animal models can provide valuable insights into the effects of light in clinical disorders and may form the basis of future therapies to prevent or delay visual cell loss.
Assuntos
Luz/efeitos adversos , Lesões Experimentais por Radiação/prevenção & controle , Lesões Experimentais por Radiação/fisiopatologia , Retina/efeitos da radiação , Degeneração Retiniana/prevenção & controle , Degeneração Retiniana/fisiopatologia , Animais , Antioxidantes/farmacologia , Apoptose , Humanos , Fatores de Crescimento Neural/farmacologia , Oxirredução , Células Fotorreceptoras de Vertebrados/efeitos da radiação , Lesões Experimentais por Radiação/etiologia , Retina/fisiopatologia , Degeneração Retiniana/etiologiaRESUMO
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 gamma-hydroxy(or oxo)-alpha,beta-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.
Assuntos
Antígenos CD36/fisiologia , Luz , Fagocitose , Fosfatidilcolinas/metabolismo , Epitélio Pigmentado Ocular/metabolismo , Segmento Externo da Célula Bastonete/metabolismo , Animais , Ligantes , Masculino , Óxido Nítrico/fisiologia , Oxirredução , Ratos , Ratos Sprague-DawleyRESUMO
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.
Assuntos
Cristalinas/metabolismo , Luz/efeitos adversos , Lesões por Radiação/metabolismo , Retina/efeitos da radiação , Animais , Epitélio Pigmentado Ocular/efeitos da radiação , Ratos , Segmento Externo da Célula Bastonete/efeitos da radiaçãoRESUMO
As part of ongoing efforts to better understand the role of protein oxidative modifications in retinal pathology, protein nitration in retina has been compared between rats exposed to damaging light or maintained in the dark. In the course of the research, Western methodology for detecting nitrotyrosine-containing proteins has been improved by incorporating chemical reduction of nitrotyrosine to aminotyrosine, allowing specific and nonspecific nitrotyrosine immunoreactivity to be distinguished. A liquid chromatography MS/MS detection strategy was used that selects all possible nitrotyrosine peptides for MS/MS based on knowing the protein identity. Quantitative liquid chromatography MS/MS analyses with tetranitromethane-modified albumin demonstrated the approach capable of identifying sites of tyrosine nitration with detection limits of 4-33 fmol. Using two-dimensional gel electrophoresis, Western detection, and mass spectrometric analyses, several different nitrotyrosine-immunoreactive proteins were identified in light-exposed rat retina compared with those maintained in the dark. Immunocytochemical analyses of retina revealed that rats reared in darkness exhibited more nitrotyrosine immunoreactivity in the photoreceptor outer segments. After intense light exposure, immunoreactivity decreased in the outer segments and increased in the photoreceptor inner segments and retinal pigment epithelium. These results suggest that light modulates retinal protein nitration in vivo and that nitration may participate in the biochemical sequela leading to light-induced photoreceptor cell death. Furthermore, the identification of nitrotyrosine-containing proteins from rats maintained in the dark, under non-pathological conditions, provides the first evidence of a possible role for protein nitration in normal retinal physiology.