The corneal epithelium is often subjected to mechanical and chemical wounds, and a number of systemic diseases affect its healing and adhesive capabilities. These insults can lead to persistent defects or recurrent erosions. The long-term aims are to obtain information about corneal epithelial wound healing and adhesion that will lead to improved treatment for persistent epithelial defects and recurrent corneal erosions, and to develop procedures for autograft epithelial transplant to humans who have chronic ocular surface disease. Building on progress during the last funding period, the following specific goals are proposed: Epithelial wound healing: (1) Determine by treansmission and scanning electron microscopy which cell layer of the stratified epithelium moves into a wound to form the leading edge of the migrating sheet. Apical or basal cells will be labeled with particles that can be visualized by electron microscopy. (2) Determine, by pulse-labeling with latex spheres that specifically bind to cell membranes, whether the amount of membrane per individual cell exposed to the tear or wound surface remains constant, expands, or turns over during healing. (3) Determine if and how presence or absence of specific molecules (laminin, fibronectin, heparan sulfate proteoglycan) on the wound substrate affect healing rate, expression of cell surface glycoconjugates, and syntheses by the corneal epithelium. Corneal epithelial adhesion: (1) Develop a defined organ culture system for basement membrane synthesis and assembly by the orneal epithelium. Assay for synthesis will be done by TEM, immunohistochemistry, and SDS-PAGE electrophoresis. (2) Determine origin, synthesis, and composition of the anchoring fibril, a fibrillar stromal component of the hemidesmosome (HD) complex whose function is to hold the basement membrane to the stroma. (3) Characterize the nucleation sites for new HD formation that are present on the epithelial basement membrane. (4) Perform in-depth morphometric analysis of the HD complex (including anchoring fibrils) in normal and diabetic human corneas and, if available, in corneas with persistent epithelial defects or a history of recurrent erosion. Epithelial transplant: (1) Determine the feasibility of transplanting oral mucosal epithelial autografts to defects on rabbit corneas, and (2) determine their fate.
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