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A.M. Timmers, J.H. McDowell, I.A. Elder; Biosynthesis of Lipofuscin–Like Inclusions in Rat RPE . Invest. Ophthalmol. Vis. Sci. 2004;45(13):1808.
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Purpose: One theory on the pathogenesis of Age–related Macular Degeneration (AMD) is that an excessive level of lipofuscin (LF) in the RPE initiates the disease. LF, and one of its constituents, A2E, have been described as photocytotoxic and as inhibitors of phagolysosomal degradation. In an effort to induce LF accumulation, a series of potential precursors of LF were introduced to the RPE in vivo. Methods: Phosphate–buffered saline suspensions with varying concentrations of either isolated bovine rod outer segments (ROS), egg–phosphatidylethanolamine (PE), A2E with or without ROS, A2PE with or without ROS, all–trans–retinaldehyde (RAL) incubated with ROS (RAL–ROS), RAL incubated with PE (RAL–PE), or ROS with covalently bound texas red (ROS–TR), were injected into the subretinal matrix of one eye of Sprague Dawley rats, the contralateral eye served as control. Scotopic and dark–adaptation ERG was measured over time on both eyes simultaneously. Samplings of eyes were checked for A2E and A2PE by HPLC or fixed and sectioned at 12 microns for examination by bright field and fluorescence microscopy. Results: No significant impact was observed in average scotopic and dark–adaptation ERG responses for eyes injected with A2E or A2PE suspensions without ROS or PE. Average scotopic ERG responses for eyes injected with materials containing ROS and PE were diminished. HPLC analysis of experimental eyes showed no significant A2E or A2PE accumulation. Microscopy demonstrated that ROS–TR was cleared from the RPE around post–injection day 7. No significant differences in morphology were observed for any experimental group with the exception of RAL–ROS, which gave a strong and persistent lipofuscin–like fluorescence in the RPE. Conclusions: Sprague Dawley rat RPE is able to phagocytize and degrade high doses of subretinally injected bovine ROS. A2E and A2PE subretinally injected, with or without ROS, are apparently cleared from the eye within hours of introduction. The biosynthesis of significant amounts of autofluorescent LF–like material in rat RPE was only observed with subretinal injection of RAL–ROS. Our findings question the physiology of A2E accumulation in vitro and that reactions unique to ROS are critical for the formation of LF. This in turn could lead to redefining the role of LF in AMD.
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