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Yoko Miura, Gereon Hüttmann, Regina Orzekowsky-Schroeder, Philipp Steven, Márta Szaszák, Novert Koop, Reginald Birngruber, Ralf Brinkmann; Investigations of Two-photon Excited Bright Autofluorescent Granules in the RPE Cells Around Photocoagulation Spots. Invest. Ophthalmol. Vis. Sci. 2011;52(14):926.
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Detection of biochemical cellular responses to photocoagulation in living retinal pigment epithelial (RPE) cells using porcine tissue culture model.
RPE-choroid tissue was isolated from freshly enucleated porcine eye. The RPE was irradiated by frequency-doubled Nd:YAG laser radiation (=532 nm, t= 0.1 sec, d= 300 µm, p= 80 mW) and cultivated in a perfusion culture system. Autofluorescence (AF) was observed by two-photon microscopy (ex=710-920 nm) in combination with fluorescence lifetime measurements 1, 3, 24, 48, 72 hrs after irradiation. Intracellular reactive oxygen species (ROS) were detected with chloromethyl-2'7'-dichlorofluorescein diacetate. Lipid peroxidation was induced by exposing cells to ferrous sulphate heptahydrate (FeSO4.7H2O). For the staining of lysosome and mitochondria, Lysotracker® and Mitotracker® were used respectively.
From 1 to 48 hrs after irradiation, punctated and bright AF appeared in some RPE cells around the coagulated area. The excitation maximum of the AF was found at710 nm and the emission peak was between 450 and 500 nm at ex= 750 nm. Fluorescence lifetime is best-fitted with a three-exponential decay curve (t1= 0.19 ns, t2= 2.58 ns, t3= 5.23 ns; mean 0.655 ns). Intracellular ROS were detected in those cells demonstrating bright AF. FeSO4 exposure also induced the appearance of similar AF granules dose-dependently. Some AF granules co-localized with lysosomes.
The results suggest that sub-lethal hyperthermia by photocoagulation generates oxidative stress and leads to the appearance of bright AF granules in RPE cells, whose spectral characteristics is different from that of A2E, a main fluorescence component of lipofuscin. It is assumed that this AF might result from heat-induced oxidation of phagosomes, which include undigested photoreceptor outer segments, leading to peroxidation of unsaturated fatty acids.
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