The stress of dry eye on corneal barrier function

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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.