title: Disruption of cell-cell junctions and induction of pathological cytokines in the retinal pigment epithelium of light-exposed mice.
abstract: OBJECTIVE
  To elucidate the influences of light exposure on the retinal pigment epithelium (RPE) in vivo that may be involved in the pathogenesis of AMD.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Laboratory of Retinal Cell Biology, Keio University School of Medicine, Shinjuku-ku, Tokyo, Japan.
authors.forename: Toshio
authors.initials: T
authors.lastname: Narimatsu
authors.forename: Yoko
authors.initials: Y
authors.lastname: Ozawa
authors.forename: Seiji
authors.initials: S
authors.lastname: Miyake
authors.forename: Shunsuke
authors.initials: S
authors.lastname: Kubota
authors.forename: Manabu
authors.initials: M
authors.lastname: Hirasawa
authors.forename: Norihiro
authors.initials: N
authors.lastname: Nagai
authors.forename: Shigeto
authors.initials: S
authors.lastname: Shimmura
authors.forename: Kazuo
authors.initials: K
authors.lastname: Tsubota
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D002829:Choroid; D016207:Cytokines / Q000378:metabolism*; D003599:Cytoskeleton / Q000528:radiation effects; D004195:Disease Models, Animal; D004797:Enzyme-Linked Immunosorbent Assay; D007150:Immunohistochemistry; D007365:Intercellular Junctions / Q000528:radiation effects*; D008027:Light / Q000009:adverse effects*; D008268:Macular Degeneration / Q000503:physiopathology*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D018384:Oxidative Stress / Q000528:radiation effects; D017382:Reactive Oxygen Species / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D055213:Retinal Pigment Epithelium / Q000378:metabolism / Q000528:radiation effects*; D054460:rho-Associated Kinases / Q000378:metabolism
chemical_list: D015415:Biomarkers; D016207:Cytokines; D017382:Reactive Oxygen Species; D054460:rho-Associated Kinases
keywords: age-related macular degeneration; light damage; oxidative damage; retina; retinal pigment epithelium