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M. Hammer, S. Richter, K. Kobuch, D. Schweitzer; Lipofuscin Accumulation in an Organotypic Perfusion Culture of Porcine Fundi Under Oxidative Stress and Blue Light Irradiation . Invest. Ophthalmol. Vis. Sci. 2005;46(13):251.
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Purpose: Lipofuscin accumulation in retinal pigment epithelial (RPE) cells is a continuos process ongoing over the whole life–span. It is shown to be related to light and free radical exposure of the cells. The goal of the presented research was the observation of lipofuscin accumulation in an organ culture model within a few day. Methods: Porcine fundi (choroid, Bruch’s membrane, RPE, and retina) were explanted in toto not later than four hours post mortem and transferred into a perfusion culture chamber. The explants were perfused with DMEM, supplemented with 15% porcine serum, 25 mM HEPES, and 1% penicillin–streptomycin, at the retinal as well as the choroidal side and were kept at 37°C. Free radical stress was induced by supplementation of 300 µM H2O2 and the specimen were exposed to blue light (1.2 mW/mm2, peak wavelength: 467 nm). The tissues were cultured up to six days. Thereafter , the retina was removed and the RPE was observed by fluorescence microscopy. Fluorescence spectra were recorded by micro–spectrophotometry as well as fluorescence lifetime imaging (FLIM). The morphological integrity of the tissues was examined by histology. Results: The RPE in the culture treated with H2O2 and blue light showed a fluorescence as 40 times as high as that of the untreated control after six days of incubation. The fluorescence of the control was the same as that of native RPE. The fluorescence spectra and lifetimes suggests the fluorescence to originate from lipofuscin. Conclusions: Even young porcine eyes show lipofuscin fluorescence. This fluorescence was not altered by the organ culture. Excessive lipofuscin accumulation was observed after incubation with H2O2–supplementation and blue light exposure. Thus, procine ocular fundus organotypic perfusion culture may be used as an in vitro model for particular aspects of AMD–genesis.
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