Retinal pigment epithelium (RPE) from humans of all ages has been prepared for ultrastructural examination. Now, using morphometric methds we will quantify subcellular organelles, e.g. lipofuscin, melanin, lysosomes, melanolysosomes, melanolipofuscin, etc. in RPE of macular, equatoria and peripheral retina.
The aim i s to estimate the normal composition of RPE constituents at each decade of life to aid in future interpretations of RPE abnormalities. Lipofuscin and other complex granules (above) will be isolated on sucrose gradients from pairs of human Eye Bank eyes and analyzed morphologically and biochemically. Chromatography will be used to separate fluorescent compounds from extracts of granules. Tests will be run to determine if peroxidized lipids, substances with conjugated Schiff bases, retinoyl compounds, etc. can be identified as contributors to observed lipofuscin fluorescence. The role of diet in the formation of components of fluorescent RPE granules will be explored using eyes of chicks or rats maintained on diets deficient in selenium, vitamin E, and other antioxidants. Putative precursors of lipofuscin, i.e. phagocytized photoreceptor outer segments, will be radiolableled and the phagolysosomes/lipofuscin granules, produced in the RPE by either in vivo or in vitro phagocytosis, will be isolated by cell fractionation and sucrose gradient centrifugation. Isolated granules will be extracted and chromatographed; autoradiographs will be made and analyzed for sources of fluorescent and radioactive molecules. RCS rats that have inherited retinal dystrophy and fail to phagocytize outer segments have been shown to also have diminished RPE retinol esterifying enzyme activity (Berman et al. 1980). We will test the postulate that this enzyme deficiency leads to abnormalities in glycoproteins required for phagocytosis. Collaborative studies of monkey maculas and cystic fibrosis RPE will continue. This research is aimed at clarifying cellular aspects of senile macular degeneration, retinitis pigmentosa, and other retinal diseases.
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