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The cornea is routinely exposed to the environment and is susceptible to damage from mechanical abrasion and infection. Ocular complications associated with diseases or other stresses such as dry eye, changes in blinking, or toxicity may cause decreased corneal sensitivity, epithelial thickness, and defects in barrier function. The epithelial disease may become more pronounced with age and may affect the severity of corneal neuropathy. Persons with these corneal epitheliopathies are seen on a frequent basis for months at a time and the lengthy treatments culminate in loss of jobs or lower job productivity and ultimately large monetary costs to society. In both rats and mice corneas there is a decrease in density of sensory nerve terminals with age that is accompanied by reduced corneal sensitivity and changes in nerve pattern. Our lab has demonstrated that we can perform ex vivo and in vivo live cell imaging experiments to detect calcium mobilization between corneal epithelium for two to three hours. For this proposal the experiments will involve extensive live cell imaging to examine communication between cell layers in various regions of the cornea (basal, wing and apical in central, nasal and limbal) to determine if the age related changes in nerves reported by several groups alter cell communication. Corneas from mice over the age range 2 to 24 months will be examined. Our previous imaging experiments have revealed that the cell-cell communication after injury is required for cell motility. The preliminary data imaging data indicate that there is a decrease in responsiveness with age and that there are subpopulations of cells (conductor cells) after a wound that initiate the signal, and then recruit and promote mobilization between cells. Our preliminary machine learning analyses from in vitro experiments predict that the later long lasting response after an injury recruits cells and is driven by P2X7R. The goal of Aim I is to test the hypothesis that cell-cell signaling after injury depends on association of epithelial cells with high-signaling conductor cells and proximity to “active” sensory nerves. These active nerves display mobilization along it. Our goal is to identify the conductor cells, understand their relationship with nerves and determine if intensity of signaling is correlated with age. In Aim II experiments are designed to elucidate the role of the conductor cells and determine the direction of cell-cell communication from the wound, how additional cells are recruited, if the conductor cells have enhanced intensity and frequency and if their presence depends on distance from the wound. We will use machine learning to determine if we can make predictions from the signals regarding the wound response and if so these will give us important insights.