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H. Youn, N.C. Bols, V. Bantseev, R. Tchao, J.G. Sivak; In vitro Study of the Effects of UV–B on Cultured Human Repinal Pigment Epithelial Cells : UV – Blocking Efficacy of Intraocular Lenses . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2861.
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© ARVO (1962-2015); The Authors (2016-present)
To evaluate UV–B radiation – induced damage of cultured human RPE cells in an effort to develop an in vitro model that can be used to evaluate the efficacy of IOL material in protecting the retina from UV radiation.
Human RPE cells, the ARPE–19 line, were cultured in 4 groups: 1 control group and 3 irradiated groups : IOL covered (two thicknesses, 9T and 15T, respectively.) and IOL uncovered. The treated cells were then irradiated with broadband UV–B radiation at energy levels of 0.05, 0.1, and 0.2 J/cm2 (0.089, 0.045, and 0.022 J/cm² in the biologically effective energy levels). After irradiation, all 4 groups were further incubated for 48 hours while they were analyzed at predetermined intervals (0–hour, 24–hour, and 48–hour) using 4 different assays; Alamar blue assay (Medicorp Inc.) for cell viability, confocal microscopy morphological assessment (Carl Zeiss LSM), Hoechst assay (Molecular Probes) for DNA fragmentation, and phagocytotic activity assay using microspheres (Invitrogen). Confocal analysis concentrated on the study of the cell nuclei and mitochondria using the following fluorescent stains: Hoechst 33342 (staining double strand DNA), Rhodamine 123 (staining mitochondria), and Acridine Orange (staining DNA and RNA, respectively).
The Alamar blue assay results for UV–B – exposed cells at all three energy levels (0.05, 0.1, and 0.2 J/cm2) clearly showed energy level – dependent decreases in cell viability in comparison to non–irradiated control cells, while both 9T and 15T IOLs blocked UV–B radiation very effectively showing similar viability as control cells. Morphological evaluation using confocal microscopy and fluorescent–dyes also showed that increasing the energy level of UV–B radiation induced progressive morphological changes, showing the degradation of mitochondria and nuclei (both DNA and RNA) while IOL covered cell groups showed very efficient UV blocking ability of both 9T and 15T IOLs. These findings correspond to the Alamar blue assay results and to measures of apoptotic DNA damage and phagocytotic activity in RPE cells after the UV–B irradiation.
The findings suggest that UV–B radiation–induced cultured RPE cell damage can be evaluated by assays that probe cellular and morphological viability, DNA fragmentation, and phagocytotic activity, and that these four assays together provide a valuable in vitro model for evaluating the efficacy of IOL material in protecting the retina from UV radiation.
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