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E. van Kuijk, N. Tirgan, P. Gupta, Y. Jiang, B. Godley, M. Motamedi; In-situ Spectroscopic and Morphological Analysis of Chronic Oxidative Stress Induced ‘Lipofuscin-Like’ Fluorophores in Cultured Retinal Pigment Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):1407.
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© ARVO (1962-2015); The Authors (2016-present)
Lipofuscin is a conglomerate of many molecules with characteristic autofluorescence and are found to accumulate in pathological conditions in the retinal pigment epithelial cells (RPE) both in ageing and in various diseases. Unfortunately, we lack knowledge on the nature of the composition of lipofuscin and on the mechanistic pathways that lead to such formation. We investigated single "lipofuscin-like" autofluorescent granule that accumulated at chronic oxidative stress with the goal of resolving them spectroscopically for insights into its components and mechanistic pathway.
Herein, first we present a daily oxidative stress model to induce artificial lipofuscin material in RPE cells in culture and secondly, we analyzed the morphological and emission spectroscopic properties of individual lipofuscin granules in-situ using a Olympus Fluoview FV 1000 Multiphoton Laser Scanning Microscope system. Briefly, ARPE-19 cells were exposed to 40uM H2O2 and/or leupeptin (lysosome inhibitor) and/or (+/-) peroxidized rod outer segments (POS) for a chronic 7 day treatment.
We found that there is an increase in the accumulation of autofluorescent compounds under all the treatment groups when compared to controls after a treatment of 7 days. Artificial lipofuscin ranged from small granules in the H2O2 group versus a "salt-pepper" dusty appearance in the Leupeptin + H2O2 group and were coalesced into larger granules in the combination group that received H2O2, Leupeptin and POS. Spectroscopically, there was a variation in the emission spectra observed between different treatment groups. In the untreated group there was a peak at 520nm, and the peak shifted to 540nm with the treatment of Leupeptin and H2O2. Interestingly, the emission spectrum was broader in the granules from the RPE cells that were exposed to a combination of H2O2, Leupeptin and UV-POS with shoulder peaks at 530nm, 560nm and 610nm.
These data suggest that, though, RPE-lipofuscin may look similar morphologically in various disease states in-vivo, spectroscopically they may be composed of different fluorophores. These also suggest that the molecular events that lead to such deposition of lipofuscin are probably different under different conditions of stress.
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