Long-term objective: to identify--and eventually control--the cytotoxic molecule(s) formed on irradiation of the retina with visible light, causing photosensitive cells to degenerate. The theoretical mechanism being tested is based on the concept of lipid peroxidation and the formation of intermediates that are toxic to the photoreceptor cells and, under certain conditions, to the retinal pigment epithelium (RPE).
Specific aims : to 1) quantitate hydroperoxides and aldehydes of linoleic and docosahexaenoic acids that have been identified in the retina and photoreceptor cells and extend this research to products from other significant PUFAs found in photosensitive cells; 2) synthesize mass-labeled molecules as standards for quantifying peroxidation products by GC-MS; 3) quantitate PUFA change resulting from photic exposure; 4) determine cytotoxicity of primary and secondary products of peroxidation in vitro and in vivo; 5) determine if nutritional vitamins E and A, Se, or carotenoids are able to modulate the production of products of PUFA oxidation; 6) assess the antioxidant-metabolic state of the photosensitive cells by determining their content of reduced glutathione--vitamins E and C, and the level of activity of superoxide dismutase and glutathione peroxidase. To accomplish these aims, albino and pigmented rats will be maintained on the AIN-76A formulated diet as a control diet or adjusted to specific levels of nutrients to evaluate their effect on lipid peroxidation. Rearing room illumination will be cyclical (10L:14D) with an intensity of approximately 25 fc. Exposure regimens will be one of the following: 1) 3-8 hrs at 1000 fc, 2) 2-4 days continuously at 200 fc, or 3) 2-6 weeks continuously at 25 fc. Freeze-dried retinas will be microdissected into: """"""""whole retina,"""""""" neural retina, photoreceptor cells, outer segments, RPE, and choroid. Samples of each layer will be used for the analysis of PUFAs, products of peroxidation or antioxidants by GC or by GC- MS. Traditional biochemical procedures will be used to evaluate specific cellular enzymes and glutathione. Structural analysis will include light and electron microscopy. It may be that virtually everyone is subject to subclinical light damage to their retinas. Daily accumulated light damage appears to be aggravated by nutritional deficiencies and extended exposure to light (i.e., electric illumination). This work seeks to understand the basic mechanisms of retinal phototoxic degeneration in order to better evaluate its importance and to open routes to control it, thus preserving retinal health.
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|Thomas, D W; van Kuijk, F J; Dratz, E A et al. (1991) Quantitative determination of hydroxy fatty acids as an indicator of in vivo lipid peroxidation: gas chromatography-mass spectrometry methods. Anal Biochem 198:104-11|
|Stephens, R J; Negi, D S; Short, S M et al. (1988) Vitamin E distribution in ocular tissues following long-term dietary depletion and supplementation as determined by microdissection and gas chromatography-mass spectrometry. Exp Eye Res 47:237-45|
|Stephens, R J; Negi, D S; Short, S M et al. (1988) Lipid peroxidation and retinal phototoxic degeneration. Basic Life Sci 49:283-9|