Multicellular regulation of corneal wound healing

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DESCRIPTION (provided by applicant): The cornea is the most densely innervated tissue in the body. Damage to corneal nerves in the epithelium can originate from trauma including surgery, disease, infection and chemical injury and can lead to dry eye, a keratopathy, that is exhibited by photophobia and pain, corneal ulcers, and, ultimately, blindness. In all of these conditions, signaling molecules are released by both corneal epithelium and nerves, creating potential for auto- and cross-stimulation. However, relatively little is known about this crosstalk during homeostasis or regeneration. To evaluate these potential interactions, we developed a co-culture of nerves and epithelia. Using this model, we demonstrated that Ca2+ mobilization occurs after stimulation of either cell type. Both epithelia and nerves release ATP in response to injury or agonist stimulation and activate a family of purinoreceptors that play a role in Ca2+ mobilization, cell migration, and pain. The mobilization is divided into two phases with an initial rapid increase and decrease followed by a second set of slower oscillations. Pretreatment with apyrase, an ectoenzyme, removed the initial response, whereas pretreatment with N-methyl-D-aspartate (NMDA) inhibitors prevented the second response. Nerves release glutamate, and both epithelia and nerves express NMDA receptors. Preliminary data indicate that environmental stress causes a decrease in the Ca2+ oscillations, a change in localization of the NMDA receptor away from the membrane, and a change in cell-cell signaling. Analyses using MATLAB demonstrate distinct patterns in cell-cell signaling. Since our co-culture system does not capture the complexity of the native connections that exist between sensory axons and corneal epithelial cells, we used the Thy-1-GFP homozygous rat. Preliminary in situ imaging studies performed ex vivo reveal that we can image cytosolic Ca2+ mobilization and fluorescent sensory nerves. Our goal is to determine mechanisms by which sensory nerves and corneal epithelial cells signal to coordinate the response induced by trauma and its impact on migration. We hypothesize that release of ATP by nerves and epithelium and release of trophic factors by nerves regulates the activation of specific purinergic and glutamatergic receptors that mediate wound repair.