This study seeks to determine the mechanisms of intense light-mediated photoreceptor cell degeneration. The hypotheses to be tested are: (1) that there is a sensitive period within the circadian cycle which results in enhanced susceptibility to retinal light damage and which is mediated by the expression of intrinsic factors in the retina (aim 1) and (2) that susceptibility to light damage is affected by genetic inheritance, which can enhance the rate of photoreceptor cell loss from environmental insult in the form of dim cyclic light exposure or acute intense light treatment (aim 2). This proposal will focus on identifying the circadian expression of intrinsic factors in the retina that enhance or prevent light-induced cell damage leading to apoptotic cell death. The investigator will study the interactions of light-and dark rearing conditions and the timing of intense light treatments in vivo that produce synchronous photoreceptor cell damage in normal albino rats and in transgenic rats with mutations in the rhodopsin primary amino acid sequence. Photoreceptor cell loss will be assayed using rhodopsin and photoreceptor DNA measurements, electrophoretic analysis of DNA fragmentation, and light and electron microscopy of light-damaged retinas. The circadian expression of melatonin and dopamine will be measured and Western and Northern analysis will be used to identify potential modulators of retinal damage. These measurements will be complimented by 2D gel electrophoresis of retinal proteins and ordered differential display of mRNA from retinas at different times of the day and night. The synthetic antioxidant dimethylthiourea will be used as a probe to uncover early events in the mechanism of cell death, including mitochondrial involvement and the potential for oxidative stress to induce apoptosis. In addition, the time course of the appearance of cytoplasmic cytochrome C and the activation of cellular caspases will be determined by antibody-based techniques, immunocytochemistry and enzymatic analysis.
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