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Localization of Phosphorylated Proteins Involved in the Regulation of Corneal Cell Proliferation in Response to Wounding

Previous experiments have suggested that the activation of epidermal growth factor receptors (EGFR) plays a key role in the regulation of corneal epithelial wound healing, but it has not been shown which cells are activated and how long they stay activated. This regulation entails controlling two important aspects of wound healing—the proliferation and migration of cells into the wound area—and it is thought that EGF plays an important role in signaling both actions [1]. In order to research these questions, rat corneal debridement (epithelial removal) wound tissue was stained with antibodies against both total EGFR and phosphorylated EGFR (activated or pEGFR). The localization of total and activated EGFR during the immediate stages of wound healing was analyzed via immunofluorescence microscopy with a TCS SP2 confocal microscope (Leica; Heidelberg, Germany). The time points used were 0, 5, 15, 30, 60, 90, and 120 minutes after a corneal debridement wound. To determine whether there exists a different temporal pattern of activation at the edge versus the cells adjacent to the wound, we defined four regions of the cornea, examined their respective rates of activation, and qualitatively compared the different areas.