The stress of dry eye on corneal barrier function

描述(申请人提供)：该项目将研究紧密连接(TJ)对实验性干眼(EDE)对角膜上皮细胞的反应，重点是角膜上皮屏障功能的扰动。角膜上皮屏障将被评估为摄取俄勒冈绿葡聚糖，一个高相对分子质量的荧光分子(70kda)，它将与EDE前和后1，3，5，7，10和12天的TJ蛋白和晚期包膜蛋白(LEP)的表达相关。本研究还将评估促炎信号介质在EDE反应中对TJ和LEP表达的作用。应激相关途径/介体的特定抑制剂在预防角膜屏障功能障碍方面的有效性将得到评估。小鼠干眼症将通过药物抑制泪液分泌，并放置在低湿度和恒定风量的环境控制室中来诱导。实时定量聚合酶链式反应将用于评估促炎介质的基因表达。TJ和LEP的表达将通过激光扫描共聚焦显微镜和免疫组织化学染色进行评估。促炎症介质的表达将通过Western Blot、EL ISA和Luminex Multiplex Assay进行评估。

DESCRIPTION (provided by applicant): The project will study the response of the tight junctions (TJ) to experimental dry eye (EDE) on the corneal epithelium with specific emphasis on the perturbation of corneal epithelial barrier function. Corneal epithelial barrier will be evaluated as the uptake of Oregon Green Dextran, a high molecular weight fluorescent molecule (70kda) and it will be correlated to the expression of TJ proteins and late envelope proteins (LEP) prior to and after 1,3,5,7,10 and 12 days of EDE. This study will also evaluate the role of pro-inflammatory signaling mediators on expression of TJ and LEP in response to EDE. Effectiveness of specific inhibitors of stress associated pathways/mediators will be evaluated in the prevention of corneal barrier dysfunction. The dry eye will be induced in mice by pharmacologic inhibition of tear secretion with scopolamine and placement in an environmentally controlled chamber with low humidity and a constant air draft. Real time PCR will be used to evaluate the gene expression of pro inflammatory mediators. TJ and LEP expression will be evaluated by laser scanning confocal microscopy and immunostaining. Pro inflammatory mediator expression will be evaluated by Western Blot, ELISA and the Luminex Multiplex Assay.