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LAMMP Seminar Video
Sub-lethal photodynamic stress as a model of chronic oxidative stress to RPE cells in vitro
Tadeusz Sarna, Ph.D., D.Sc.

Exposure to intense visible light from focal irradiation and high oxygen tension, and accumulation of the age pigment lipofuscin increase the risk of oxidative stress in the retinal pigment epithelium (RPE). Chronic oxidative stress in this retinal tissue is viewed as a contributing factor to the pathogenesis of age-related macular degeneration (AMD). To study sub-lethal oxidative stress in RPE in vitro, human RPE cells in primary culture and cultured ARPE-19 cells were subjected to photodynamic stress mediated by human RPE lipofuscin or selected dye photosensitizers. Motility of endogenous lipofuscin and melanosome granules was measured by real-time imaging during sub-lethal blue light irradiation. Effects of photodynamic treatment of ARPE-19 cells on their activity to phagocytize fluorescein-5-isothiocyanate-labelled photoreceptor outer segments isolated from cow retinas, and fluorescent polystyrene beads, were analyzed by flow cytometry. Changes in morphology and nanomechanical properties of ARPE-19 cells, induced by sub-lethal photodynamic stress, were monitored by atomic force microscopy and by fluorescence imaging. Lipofuscin granules and melanosomes or black latex beads showed motility slowing with mild photic stress, but slowing was greater for lipofuscin. Specific phagocytosis of ARPE-19 cells was reversibly inhibited by photodynamic treatment, indicating that the activity of this biological function is a sensitive measure of sub-lethal oxidative stress to RPE cells. Mild photodynamic stress to ARPE-19 cells significantly reduced formation of the actin stress fibers and modified nanomechanical properties of the cells, which become much softer than control cells. The results demonstrate that sub-lethal photodynamic stress mediated by endogenous or exogenous photosensitizers is a convenient model for studying effects of chronic oxidative stress in RPE cells in vitro.

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