Following injury, whether it be the result of trauma, surgery, or infection, the cornea utilizes a variety of cellular mechanisms to repair itself. These events include reepithelialization, collagen repair via keratocytes and endothelium, and repopulation of the endothelium in areas in which it has been denuded. This process can be quite effective and often leads to complete healing of the cornea with no associated visual impairment. Unfortunately, the wound repair process can also result in stromal edema, scarring, ulceration, or in the case of refractive surgical procedures and penetrating keratoplasty, unpredictable and possibly fluctuating refractive errors. To better understand and possibly to better control the wound healing process, an understanding of the basic cellular mechanisms of corneal wound healing is essential. Ion channels in cellular membranes have been shown to be important mediators in events such as cell activation, mitogenesis and cell proliferation, migration, volume regulation, secretion, and responses to growth factors. Because all of these events are involved in the response of keratocytes and endothelial cells during the corneal wound healing process, the understanding of the interaction between ion channels and these events is crucial. Preliminary data shows that surgically wounded rabbit corneal endothelial cells have different ionic currents than those in the non-wounded control eye. Interestingly, these currents are similar to those seen in cultured endothelial cells. In keratocytes, preliminary data shows that fetal bovine serum stimulation, a known "activator" of this cell type, causes a rapid increase in the amplitude of the membrane currents. This effect appears to be associated with both activation of a new channel type as well as the voltage sensitive Na+ channel. A similar current activation is observed when 8-bromo-cAMP is added to the bath. The specific aims of this grant are designed to specifically address the biophysical nature and physiological relevance of many of these preliminary findings. Initial studies will utilize whole cell and single channel patch clamp techniques to characterize and determine the mechanisms of injury induced changes in endothelium and keratocyte ion channel activity. In addition, the physiological and pharmacological significance of these changes will be examined using in-vivo wound healing studies as well as in-vitro perfusion studies.
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