RESUMEN
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.
Asunto(s)
Antioxidantes/uso terapéutico , Suplementos Dietéticos , Luz/efectos adversos , Traumatismos Experimentales por Radiación/prevención & control , Retina/efectos de la radiación , Degeneración Retiniana/prevención & control , Animales , Western Blotting , Supervivencia Celular , Evaluación Preclínica de Medicamentos , Proteínas del Ojo/metabolismo , Masculino , Traumatismos Experimentales por Radiación/etiología , Traumatismos Experimentales por Radiación/metabolismo , Traumatismos Experimentales por Radiación/patología , Ratas , Ratas Sprague-Dawley , Degeneración Retiniana/etiología , Degeneración Retiniana/metabolismo , Degeneración Retiniana/patología , Rodopsina/fisiologíaRESUMEN
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.
Asunto(s)
Luz , Degeneración Macular/patología , Retina/patología , Retina/efectos de la radiación , Animales , Arginina/metabolismo , Atrofia , Proteínas Portadoras/metabolismo , Movimiento Celular/efectos de la radiación , Coroides/patología , Coroides/efectos de la radiación , Glutamina/metabolismo , Humanos , Neuritas/metabolismo , Neuritas/efectos de la radiación , Fenotipo , Células Fotorreceptoras de Vertebrados/patología , Células Fotorreceptoras de Vertebrados/efectos de la radiación , Epitelio Pigmentado Ocular/patología , Epitelio Pigmentado Ocular/efectos de la radiación , Ratas , Ratas Sprague-Dawley , Ácido gamma-Aminobutírico/metabolismoRESUMEN
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.
Asunto(s)
Ritmo Circadiano/efectos de la radiación , Luz/efectos adversos , Epitelio Pigmentado Ocular/metabolismo , Traumatismos Experimentales por Radiación/metabolismo , Rodopsina/metabolismo , Animales , Adaptación a la Oscuridad , Espectrometría de Masas , Fosforilación , Ratas , Rodopsina/químicaRESUMEN
PURPOSE: Clusterin has been associated with active cell death in several different model systems, including animal models of retinal degeneration. Clusterin is also expressed in normal tissues, a finding that leads to the question of how it could then play a cell death-specific role during tissue regression. To address this paradox, we have examined clusterin expression during light-induced retinal damage in rats. METHODS: Normal albino rats were reared in darkness and then exposed to intense visible light to induce retinal degeneration. Clusterin expression was then examined at various times after light treatment. Standard molecular techniques including Northern analysis, immunohistochemistry, and Western analysis were employed. RESULTS: Northern analysis established that the largest increase in clusterin expression occurs after a decrease in interphotoreceptor retinoid binding protein, IRBP, expression (an indication of a photoreceptor cell dysfunction) and after an increase in heme oxygenase 1, HO-1, expression (an oxidative stress inducible gene), suggesting that induction of clusterin expression is an oxidative stress response. Immuno-histochemical analysis with two different clusterin-specific antibodies, anti(SGP-2) and anti(301), localized distinct forms of clusterin to Müller cells and degenerating photo-receptor cells. Western analysis demonstrated degeneration associated isoforms of clusterin in light treated retina that are not present in normal retina. CONCLUSION: Clusterin over-expression is characteristic of a retinal degeneration phenotype and we propose that clusterin action may be defined by the nature in which it is modified. We hypothesize that alternate processing leads to retinal degeneration-specific forms of the protein (65, 61, and 50 kDa) that are not present in normal retina.
Asunto(s)
Proteínas del Ojo/metabolismo , Glicoproteínas/metabolismo , Chaperonas Moleculares/metabolismo , Traumatismos Experimentales por Radiación/metabolismo , Retina/efectos de la radiación , Degeneración Retiniana/metabolismo , Animales , Northern Blotting , Western Blotting , Clusterina , Adaptación a la Oscuridad , Proteínas del Ojo/genética , Expresión Génica , Glicoproteínas/genética , Hemo Oxigenasa (Desciclizante)/metabolismo , Hemo-Oxigenasa 1 , Técnicas para Inmunoenzimas , Luz , Masculino , Chaperonas Moleculares/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/metabolismo , Traumatismos Experimentales por Radiación/etiología , Traumatismos Experimentales por Radiación/patología , Ratas , Ratas Sprague-Dawley , Degeneración Retiniana/etiología , Degeneración Retiniana/patología , Proteínas de Unión al Retinol/metabolismoRESUMEN
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.
Asunto(s)
Ritmo Circadiano/efectos de la radiación , Luz/efectos adversos , Traumatismos Experimentales por Radiación/etiología , Retina/efectos de la radiación , Degeneración Retiniana/etiología , Tiourea/análogos & derivados , Animales , Northern Blotting , Daño del ADN/efectos de la radiación , Fragmentación del ADN/efectos de la radiación , Adaptación a la Oscuridad , Electroforesis en Gel de Agar , Proteínas del Ojo/genética , Proteínas del Ojo/metabolismo , Depuradores de Radicales Libres/farmacología , Perfilación de la Expresión Génica , Hemo Oxigenasa (Desciclizante)/genética , Hemo Oxigenasa (Desciclizante)/metabolismo , Hemo-Oxigenasa 1 , Masculino , Traumatismos Experimentales por Radiación/metabolismo , Ratas , Ratas Sprague-Dawley , Retina/efectos de los fármacos , Degeneración Retiniana/metabolismo , Proteínas de Unión al Retinol/genética , Proteínas de Unión al Retinol/metabolismo , Rodopsina/metabolismo , Tiourea/farmacologíaRESUMEN
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.
Asunto(s)
Luz , Degeneración Retiniana/fisiopatología , Rodopsina/química , Secuencia de Aminoácidos , Animales , Espectrometría de Masas , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fosforilación , Ratas , Ratas Mutantes , Ratas Sprague-Dawley , Rodopsina/metabolismoRESUMEN
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.
Asunto(s)
Daño del ADN , ADN/efectos de la radiación , Oscuridad , Luz , Retina/efectos de la radiación , Animales , Antioxidantes/farmacología , ADN/efectos de los fármacos , Masculino , Ratas , Ratas Sprague-Dawley , Retina/efectos de los fármacos , Retina/metabolismo , Tiourea/análogos & derivados , Tiourea/farmacologíaRESUMEN
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 reached its maximum 2 days later. Pretreatment with DMTU completely eliminated DNA ladders in these rats. Alkaline gel electrophoresis revealed a pattern of single-strand DNA breaks, with relatively high molecular weight fragments, 6 h after light exposure of dark-reared rats. Single-strand DNA breaks in cyclic light rats corresponded with the onset of apoptotic ladders, but peak values preceded by 12 h the peak of DNA ladder formation. The quantity of 8-OHdG in retinal DNA remained close to control values in all samples with the exception of a peak of twice the control value 18 h after light exposure in the dark-reared rats and a value 60% higher 16 days after exposure in cyclic light animals. Dimethylthiourea had no effect on the amount of oxidized purine in any of the samples. The differences between dark-reared rats and rats reared in dim cyclic light in the kinetics of DNA fragmentation and in their response to treatment with DMTU is consistent with previous observations of fundamental differences in retinal cell physiology in these animals. In dim light-reared rats, the pathway to apoptosis may be qualitatively different from the pathway to net photoreceptor loss in rats reared in darkness. The lack of effect of DMTU on 8-OHdG formation suggests that the oxidation of DNA bases is not a causal factor in light-mediated photoreceptor cell death.
Asunto(s)
Retina/lesiones , Retina/efectos de la radiación , Animales , Antioxidantes/farmacología , Apoptosis , Supervivencia Celular , Fragmentación del ADN , Luz , Masculino , Células Fotorreceptoras/efectos de los fármacos , Células Fotorreceptoras/metabolismo , Células Fotorreceptoras/efectos de la radiación , Ratas , Ratas Sprague-Dawley , Tiourea/análogos & derivados , Tiourea/farmacologíaRESUMEN
PURPOSE: To determine the effects of genetic background and light rearing conditions on intense-light-mediated retinal degeneration in young RCS rats. MATERIALS AND METHODS: Albino rats, homozygous or heterozygous for the rdy gene were bred and born in dim cyclic light. At P7 they were moved to a dark environment, and maintained there until exposure to intense visible (green) light at P18 or P25. Other rats remained in the dim cyclic light environment. At various times between P11 and P40 rats were killed for determinations of rhodopsin and photoreceptor cell DNA levels, western transblot analysis of retinal S-antigen (arrestin) and alpha-transducin, or northern slot blot analysis of their respective mRNA levels. RESULTS: At P18, unexposed dark maintained homozygous RCS rats and their phenotypically normal heterozygous counterparts have nearly equivalent rhodopsin levels and photoreceptor cell DNA. Intense light exposure at this age, to 8 hours of continuous light or 3 hours of intermittent light, did not lead to a loss of either rhodopsin or retinal DNA when compared with their respective unexposed controls. At P25 rhodopsin levels were higher than at P18, while photoreceptor cell DNA was essentially the same as in the younger rats. However, intense light exposure at P25 resulted in substantial losses of rhodopsin and photorecptor cell DNA and the losses were greater in homozygous rats than in heterozygous animals. Light damage of P25 rats maintained in dim cyclic light was essentially the same as in dark maintained homozygous rats, but no damage was found in the heterozygous animals. By western analysis, alpha-transducin levels in the retina increased with time in darkness, while retinal S-antigen levels either remained the same or decreased during the period P15-P35. For rats in the cyclic light environment S-antigen expression was greater than alpha-transducin at all ages. Slot blot analysis of mRNAs for the two proteins generally followed the patterns seen by western analysis. S-antigen mRNA was expressed at an earlier age and at higher levels than alpha-transducin in both types of rats from both light rearing conditions. Peak expression of S-antigen most often occurred at P18 in both the heterozygous and homozygous rats. CONCLUSIONS: The relative expressions of S-antigen and alpha-transducin in P18 and P25 rats correlates with their relative resistance to retinal light damage at P18 and their enhanced susceptibility at P25. Rats homozygous for the rdy gene also exhibit more damage than heterozygous animals when photoreceptor cell DNA is used to estimate the extent of retinal light damage.
Asunto(s)
Células Fotorreceptoras de Vertebrados/patología , Células Fotorreceptoras de Vertebrados/efectos de la radiación , Traumatismos Experimentales por Radiación , Ratas Endogámicas/fisiología , Degeneración Retiniana/etiología , Degeneración Retiniana/patología , Envejecimiento/metabolismo , Animales , Animales Recién Nacidos/crecimiento & desarrollo , Animales Recién Nacidos/metabolismo , Arrestina/genética , ADN/metabolismo , Heterocigoto , Homocigoto , ARN Mensajero/metabolismo , Traumatismos Experimentales por Radiación/metabolismo , Traumatismos Experimentales por Radiación/patología , Ratas , Ratas Endogámicas/genética , Ratas Endogámicas/metabolismo , Retina/metabolismo , Degeneración Retiniana/metabolismo , Rodopsina/metabolismo , Transducina/genéticaRESUMEN
Induction of apoptosis in the retina leads to cellular death by molecular mechanisms that are not well understood. Clusterin expression is increased in tissues undergoing apoptosis, including retinal neurodegenerative states, but the causal relationships remain to be clarified. To gain insight into clusterin's role in photoreceptor apoptosis, the cellular distribution of clusterin mRNA was compared with the pattern of apoptotic nuclear labelling in a rat model of light-induced retinal degeneration. In control retinal sections, clusterin mRNA was localized to the retinal pigment epithelium cells, photoreceptor inner segments, inner nuclear layer, and ganglion cell layer. Clusterin expression decreased in photoreceptors and retinal pigment epithelium cells, which progressively degenerated, and increased in preserved inner nuclear layer, in proportion to the duration of light exposure in both cyclic light- and dark-reared animals. These results suggest that clusterin is not causally involved in apoptotic mechanisms of photoreceptor death, but may relate to cytoprotective functions.
Asunto(s)
Apoptosis/fisiología , Glicoproteínas/metabolismo , Luz , Chaperonas Moleculares , Células Fotorreceptoras de Vertebrados/fisiología , Traumatismos Experimentales por Radiación/fisiopatología , Degeneración Retiniana/fisiopatología , Animales , Núcleo Celular/fisiología , Clusterina , Hibridación in Situ , Etiquetado Corte-Fin in Situ , Masculino , Traumatismos Experimentales por Radiación/patología , Ratas , Ratas Sprague-Dawley , Retina/metabolismo , Retina/patología , Degeneración Retiniana/patologíaRESUMEN
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.
Asunto(s)
Daño del ADN , Retina/efectos de la radiación , Animales , ADN/genética , ADN/efectos de la radiación , Luz , Masculino , Fotobiología , Ratas , Ratas Sprague-Dawley , Retina/químicaRESUMEN
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.
Asunto(s)
Envejecimiento , Luz/efectos adversos , Traumatismos Experimentales por Radiación/metabolismo , Retina/efectos de la radiación , Degeneración Retiniana/metabolismo , 8-Hidroxi-2'-Desoxicoguanosina , Envejecimiento/fisiología , Animales , Muerte Celular/efectos de los fármacos , Muerte Celular/efectos de la radiación , Fragmentación del ADN/efectos de los fármacos , Fragmentación del ADN/efectos de la radiación , Adaptación a la Oscuridad , Desoxiguanosina/análogos & derivados , Desoxiguanosina/metabolismo , Electroforesis en Gel de Poliacrilamida , Depuradores de Radicales Libres/farmacología , Hemo Oxigenasa (Desciclizante)/genética , Hemo Oxigenasa (Desciclizante)/metabolismo , Hemo-Oxigenasa 1 , Masculino , ARN Mensajero/metabolismo , Traumatismos Experimentales por Radiación/etiología , Traumatismos Experimentales por Radiación/prevención & control , Ratas , Ratas Sprague-Dawley , Retina/efectos de los fármacos , Retina/metabolismo , Degeneración Retiniana/etiología , Degeneración Retiniana/prevención & control , Rodopsina/metabolismo , Tiourea/análogos & derivados , Tiourea/farmacologíaRESUMEN
PURPOSE: To determine the effect of acute, intense, visible light on the activities of all-trans retinol dehydrogenase (t-RDH) and glutamine synthetase (GS), two oxidatively sensitive enzymes located in the photoreceptors and Müller cells, respectively. METHODS: Male albino rats, previously maintained in a weak cyclic light- or dark-rearing environment, were exposed to intense light (490-580 nm) for as long as 24 hrs. One-half of the experimental animals were pre-treated with the antioxidant 1,3-dimethylthiourea (DMTU), at 500 mg/kg, i.p., 24 hrs before and just before light exposure. Upon sacrifice, retinas were excised for the determination of t-RDH and GS activity, or for the preparation of rod outer segments (ROS). Other light-exposed rats were maintained in darkness for 2 weeks before sacrifice, for rhodopsin determinations. Retinal homogenates were also treated in vitro under oxidizing conditions to compare enzymatic inactivation with the in vivo effects of light exposure. RESULTS: In cyclic light-reared rats 24 hr light exposures resulted in a significant loss of t-RDH activity in retinal homogenates and in isolated ROS. In both the retina and ROS, pretreatment of the animals with DMTU completely prevented the loss of t-RDH activity. As measured by rhodopsin levels 2 weeks after light exposure, DMTU-treated rats exhibited no loss of photoreceptor cells, whereas those not given the antioxidant lost over 50% of their photoreceptors. Retinal GS activity was unchanged by 24 hr intense light exposures. In dark-reared rats 4 hr light exposures did not alter retinal t-RDH or GS activity, despite the loss or approximately 70% of the rhodopsin content or the eye, measured 2 weeks later. When 4 hr light-exposed rats were held in darkness for an additional 20 hrs, a significant loss of retinal t-RDH occurred, but no change in GS activity was measured. In these rats DMTU treatment also prevented the loss of t-RDH activity. In contrast to the lack of an in vivo light effect on retinal GS, oxidation in vitro completely inactivated the enzyme after only 1 hr. CONCLUSIONS: The light-induced loss of t-RDH in both cyclic light- and dark-reared rats is an oxidative and time dependent process that is not strictly photochemical in nature. The loss of rhodopsin and t-RDH activity, but not GS activity, following intense light exposure are manifestations of light's effect on photoreceptor cells without a comparable effect in the adjacent retinal Müller cells. Additional work will be needed to understand the differences in light damage susceptibility between retinal photoreceptors and glial cells and between cyclic light- and dark-reared rats.
Asunto(s)
Oxidorreductasas de Alcohol/metabolismo , Luz , Retina/enzimología , Retina/efectos de la radiación , Segmento Externo de la Célula en Bastón/efectos de la radiación , Animales , Antioxidantes/farmacología , Familia 2 del Citocromo P450 , Adaptación a la Oscuridad/fisiología , Glutamato-Amoníaco Ligasa/metabolismo , Masculino , Oxidación-Reducción , Fotoperiodo , Ratas , Ratas Sprague-Dawley , Retina/citología , Segmento Externo de la Célula en Bastón/efectos de los fármacos , Estereoisomerismo , Fracciones Subcelulares/enzimologíaRESUMEN
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.
Asunto(s)
Ácidos Docosahexaenoicos/metabolismo , Luz/efectos adversos , Traumatismos Experimentales por Radiación/prevención & control , Retina/efectos de la radiación , Segmento Externo de la Célula en Bastón/metabolismo , Animales , ADN/análisis , Grasas de la Dieta/administración & dosificación , Grasas Insaturadas en la Dieta/administración & dosificación , Ácidos Grasos/metabolismo , Masculino , Células Fotorreceptoras/patología , Células Fotorreceptoras/efectos de la radiación , Traumatismos Experimentales por Radiación/metabolismo , Traumatismos Experimentales por Radiación/patología , Ratas , Ratas Sprague-Dawley , Retina/metabolismo , Retina/patología , Rodopsina/metabolismo , Segmento Externo de la Célula en Bastón/efectos de la radiación , Ácido alfa-Linolénico/deficienciaRESUMEN
The nature of the oligosaccharides of rhodopsin from normal rats and from the Royal College of Surgeons (RCS) rats was examined by chemical, enzymatic and chromatographic procedures. This report is the first description of the structures of oligosaccharides of rat rhodopsin. The major oligosaccharide isomer of rat rhodopsin was shown to have the same structure as that from cow, human and frog. No neutral galactosylated species were detected. Although the site of the dystrophy in the RCS rat has been shown to be located at the retinal pigment epithelium, the possibility was examined that alterations in the glycosylation of rhodopsin might also be present. No differences were observed in either the amounts or structures of the rhodopsin oligosaccharide chains from young or adult control rats and RCS rats, although some differences were observed in the relative distribution of some oligosaccharide isomers between the RCS and controls. In addition, no differences in the amino acid content or SDS-PAGE patterns were detected.
Asunto(s)
Oligosacáridos/química , Degeneración Retiniana/metabolismo , Rodopsina/química , Secuencia de Aminoácidos , Animales , Secuencia de Carbohidratos , Cromatografía de Afinidad , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Electroforesis en Gel de Poliacrilamida , Datos de Secuencia Molecular , Ratas , Ratas Sprague-Dawley , Rodopsina/aislamiento & purificaciónRESUMEN
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.
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Hipertermia Inducida/efectos adversos , Luz/efectos adversos , Traumatismos Experimentales por Radiación/etiología , Retina/efectos de la radiación , Animales , ADN/metabolismo , Adaptación a la Oscuridad , Calor , Metabolismo de los Lípidos , Masculino , Traumatismos Experimentales por Radiación/metabolismo , Traumatismos Experimentales por Radiación/patología , Ratas , Ratas Sprague-Dawley , Retina/metabolismo , Retina/patología , Rodopsina/metabolismo , Segmento Externo de la Célula en Bastón/metabolismo , Factores de TiempoRESUMEN
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.
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Ácido Ascórbico/fisiología , Retina/metabolismo , Ácido Tióctico/análogos & derivados , Vitamina E/biosíntesis , Animales , Cromatografía Líquida de Alta Presión , Ácido Deshidroascórbico/metabolismo , Espectroscopía de Resonancia por Spin del Electrón , Radicales Libres , Glutatión/farmacología , Masculino , Oxidación-Reducción , Ratas , Ratas Sprague-Dawley , Retina/efectos de los fármacos , Retina/efectos de la radiación , Ácido Tióctico/farmacología , Rayos UltravioletaRESUMEN
The effect of intense visible light (light damage) on the expression of heme oxygenase 1 (HO-1), a protein induced by oxidative stress, was investigated in the rat retina. A sensitive reverse transcription-PCR assay demonstrated the expression of mRNA for HO-1 as well as HO-2, the noninducible HO form, in the normal retina. As analyzed by Northern blotting, however, HO-1 mRNA was barely detectable under normal circumstances. After exposure to intense visible light, retinas had markedly higher HO-1 mRNA levels than unexposed controls, with increases up to 52- and 98-fold at 12 and 24 hr of exposure, respectively. Intense light exposure also resulted in an increase in HO-1 protein. In contrast, no appreciable change in HO-2 mRNA or protein was observed. The increase in HO-1 message was more pronounced in rats previously reared in the dark than in those reared in a weak cyclic-light environment. A marked decrease from the high level of HO-1 mRNA induced by light insult was observed when the animals were allowed to recover in the dark for 24 hr after light exposure. Most important, treatment of animals with 1,3-dimethylthiourea, a synthetic antioxidant, prior to light exposure effectively blocked the increase in HO-1 mRNA. Thus, HO-1 is a sensitive marker for assessing light-induced insult in the retina. Since increased expression of HO-1 is thought to be a cellular defense against oxidative damage, its expression may play an important role in protecting the retina against light damage.
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Antioxidantes/farmacología , Hemo Oxigenasa (Desciclizante)/biosíntesis , Luz , Retina/efectos de los fármacos , Retina/efectos de la radiación , Tiourea/análogos & derivados , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cartilla de ADN , Inducción Enzimática/efectos de la radiación , Hemo Oxigenasa (Desciclizante)/genética , Masculino , Datos de Secuencia Molecular , Estrés Oxidativo , ARN Mensajero/genética , Ratas , Ratas Sprague-Dawley , Retina/enzimología , Tiourea/farmacologíaRESUMEN
Prolonged periods of high-intensity visible light exposure lead to photoreceptor cell degeneration, but the mechanism of damage is not understood. Increased clusterin mRNA levels have been found in several models of apoptosis, as well as in neurodegeneration. We report here that changes in clusterin mRNA levels are also associated with light-induced retinal damage in adult male albino rats. Animals previously maintained in darkness or a weak cyclic light environment were exposed to intense visible green light for up to 24 h. Some rats were pretreated with a synthetic antioxidant, dimethylthiourea (DMTU), which reduces photoreceptor cell degeneration. Clusterin mRNA steady-state levels increased with the duration of light exposure in both cyclic light and dark reared animals, suggesting that an apoptotic mechanism may be involved. Animals pretreated with DMTU showed a delay in the initial increase in clusterin mRNA levels, suggesting that oxidative damage is involved in the damage mechanism. However, the incomplete suppression of increasing steady-state clusterin mRNA levels by DMTU suggests that either oxidative damage triggers a second pathway or multiple damage mechanisms are induced in the retina by light exposure.
Asunto(s)
Glicoproteínas/biosíntesis , Luz/efectos adversos , Chaperonas Moleculares , Retina/metabolismo , Degeneración Retiniana/metabolismo , Animales , Clusterina , Masculino , ARN Mensajero/análisis , Ratas , Ratas Sprague-Dawley , Retina/patología , Retina/efectos de la radiación , Degeneración Retiniana/etiología , Degeneración Retiniana/patología , Tiourea/análogos & derivados , Tiourea/farmacologíaRESUMEN
The retinotoxic drug diaminophenoxypentane (DAPP) was administered to rats to determine its long term effects on rhodopsin levels, retinal morphology and the retina's susceptibility to damage from visible light. In rats given 2 intraperitoneal injections of DAPP at doses of 65 mg/kg body wt, there was a dramatic and sustained loss of rhodopsin. One wk later visual pigment levels were 41% lower than in comparable dark maintained rats injected with saline. Rhodopsin levels in the DAPP treated rats remained lower than in control animals for the 13 wk period of the study. Morphologically, the ROS of rats 1-2 wks after DAPP treatment exhibited some disorganization and shortening; the RPE was unremarkable. Seven wks after DAPP treatment an occasional focal area of damage was seen in the RPE. Similarly, focal areas of degeneration were seen in the outer nuclear layer between the rows of photoreceptor cells. As determined by photoreceptor cell nuclear counts, the retinotoxic effect of DAPP persisted long after drug administration. In the treated rats the loss of visual cell nuclei was 11% at 7 wks; it was 22% 13 wks after DAPP treatment. Immediately after exposure to intense visible light, damage was seen in both the photoreceptor cells and RPE of DAPP treated rats. However, the effects of exposure in the treated rats were less dramatic than in retinas from rats without DAPP treatment. Following a 2 wk dark recovery period, the DAPP treated rats had a normal appearing retinal morphology and an intact RPE layer. The retinas of rats without DAPP treatment showed extensive visual cell and RPE loss.(ABSTRACT TRUNCATED AT 250 WORDS)