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Toshio Narimatsu, Seiji Miyake, Shunsuke Kubota, Manabu Hirasawa, Norihiro Nagai, Kazuo Tsubota, Yoko Ozawa; 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(15):6077.
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To elucidate the possible influence of light-induced oxidative stress on age-related macular degeneration (AMD), we analyzed the changes of retinal pigment epithelium (RPE) after light exposure focusing on the cell-cell junctions, i.e. tight junction (TJ) and adherens junction (AJ), actin cytoskeleton, and cytokine induction in mice.
BALB/c mice (6-8 weeks old) were exposed to the white fluorescence lamp (3000 lux, 3h) after complete dark-adaptation for 12 hours. One day after light exposure, flat-mounted RPEs were stained with the antibodies to beta-catenin, N-cadherin, ZO-1, and with phalloidin, respectively. The reactive oxygen species (ROS) was measured using DCFH-DA. To elucidate the involvement of ROS and Rho/Rho-associated coiled-coil forming kinase (ROCK), the mice were intraperitoneally injected with either an antioxidant, N-Acetyl-L-cysteine (NAC) or a ROCK inhibitor, Y-27632, respectively. The protein level of monocyte chemotactic protein-1 (MCP-1), chemokine (C-C motif) ligand 11 (CCL11) and ROCK activity were measured by ELISA assay. The RNA expression levels of MCP-1 and CCL11 were analyzed by the quantitative real time PCR.
In the mouse RPE, excessive light induced ROS, and disrupted TJ, AJ and actin cytoskeleton. NAC effectively reduced the ROS level and prevented these structural changes. Elevated ROS induced Rho/ROCK activation. Administration of Y27632 prevented the light-induced structural changes. Furthermore, the levels of AMD-associated cytokines, MCP-1 and CCL11 were increased by light exposure, but reduced by NAC treatment, both at the RNA and protein levels. The light-induced MCP-1 levels were also suppressed by Y-27632.
The results indicate that light exposure induces ROS and Rho/ROCK activation, which cause pathological changes in the RPE. These changes would lead to disruption of RPE’s barrier function and cause inflammation, therefore, they may be involved in the pathogenesis of AMD progression. Although further studies are required, these findings will contribute to the future development of new therapeutic approaches for AMD.
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