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Melanie MARIE, Karine Bigot, Coralie Barrau, Pauline Gondouin, Delphine PAGAN, Claire Angebault-Prouteau, Thierry Villette, Denis Cohen-Tannoudji, José Sahel, Serge A Picaud; Blue light induced oxidative stress in an in vitro model of AMD. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4256.
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Blue light is an identified risk factor for age-related macular degeneration (AMD). Using a custom-made illumination system delivering 10 nm-wide illumination bands within the blue-green range, we recently showed that the narrow range 415-455 nm was the most toxic for A2E-loaded RPE cells (Arnault et al., 2013). To further understand the mechanisms involved in this phototoxicity, we investigated the induction of several oxidative stress markers in this blue-green range of the visible spectrum.
As an AMD in vitro model, we used a primary culture of porcine retinal pigment epithelium cells (RPE) incubated for 6 hours with A2E. Cells were then exposed for 15 hours to 10 nm-wide illumination bands centered from 390 to 520 nm in 10 nm increments (one additional band centered at 630 nm). Light irradiances were normalized with respect to the natural sunlight reaching the retina after being filtered by the ocular structures. Oxidative stress markers were quantified at the end of light exposure.
At the end of light exposure, reactive oxygen species (ROS) levels were assessed using ROSGLO and Mitosox assay kits. High levels of both hydrogen peroxide and superoxide anion were detected in cells exposed to blue light between 415 and 455 nm. Glutathione, which is as an antioxidant molecule, either exists in a reduced (GSH) or in an oxidized form (GSSG). A decrease of the GSH/GSSG ratio, which reflects an increase in oxidative stress, was observed in the blue range of the visible spectrum. Mitochondria respiratory activities, as measured by oxigraphy, decreased when cells were exposed to light in the presence of A2E with a maximum at 440 nm. Finally, mitochondrial intracellular localization, as studied by confocal microscopy, showed a peri-nuclear clustering upon light illumination at 430 and 440 nm.
Using our in vitro model of AMD and consistently with our previous study, oxidative stress markers were greatly increased in similar blue illumination bands as for phototoxicity. These findings provide us with more precise tools to study oxidative stress generated by blue light in AMD.
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