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Toshio Narimatsu, Yoko Ozawa, Seiji Miyake, Shunsuke Kubota, Manabu Hirasawa, Norihiro Nagai, Shigeto Shimmura, Kazuo Tsubota; Disruption of Cell-Cell Junctions and Induction of Pathological Cytokines in the Retinal Pigment Epithelium of Light-Exposed Mice. Invest. Ophthalmol. Vis. Sci. 2013;54(7):4555-4562. doi: 10.1167/iovs.12-11572.
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© ARVO (1962-2015); The Authors (2016-present)
Toelucidate the influences of light exposure on the retinal pigment epithelium (RPE) in vivo that may be involved in the pathogenesis of AMD.
Six- to 7-week-old BALB/c mice were exposed to light at 2000 lux for 3 hours. Flat-mount RPE samples were immunostained with anti-ZO-1 antibody for evaluating tight junction, anti-N-cadherin, and anti-β-catenin antibodies for adherens junction, and stained with phalloidin for actin cytoskeleton. The reactive oxygen species (ROS) level was measured using DCFH-DA; Rho-associated coiled-coil forming kinase (ROCK) activity was by ELISA. Cytokine expression was analyzed by real-time RT-PCR and/or ELISA in the RPE-choroid, and macrophage recruitment was by real-time RT-PCR and immunohistochemistry. Either an antioxidant, N-Acetyl-L-cysteine (NAC), or a ROCK inhibitor, Y-27632, were administered to analyze the roles of ROS and ROCK activation, respectively.
Light exposure disrupted staining patterns of tight junctions, adherens junctions, and actin cytoskeleton in the RPE, where ROS was elevated. However, NAC treatment avoided the RPE changes, reducing ROS. ROCK activity increased after light exposure was suppressed by NAC, and the structural disruptions were suppressed by Y-27632. The levels of MCP-1, CCL11, and IL-6 increased after light exposure were suppressed by NAC. Light-induced MCP-1 and IL-6 were suppressed by Y-27632. Macrophage recruitment after light exposure was also suppressed either by NAC or Y-27632.
Light exposure induced ROS and Rho/ROCK activation, which caused disruption of cell-cell junctions (tight junctions and adherens junctions) and actin cytoskeleton, the RPE's barrier structure, and induced AMD-associated pathological changes in the RPE-choroid.
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