Glaucoma is a major cause of blindness in which optic nerve fibers die in a characteristic pattern. Although many factors may influence this process, elevated intraocular pressure (IOP) is the best documented and all glaucoma therapy is directed at controlling eye pressure. In spite of this, relatively little is known about the neuronal and glial cell response to elevated IOP, due in large part to the lack of a reliable, inexpensive animal model of pressure-induced optic nerve damage. Such a model has recently been created in rats. Selective injection of sclerosing agents into ocular surface vessels using a specially designed microneedle causes scarring of the aqueous humor outflow pathways, producing measurably increased IOP and identifiable optic nerve fiber death. This proposal will refine the technique to produce mild and severe pressure rises in separate groups of animals. The associated nerve damage will be studied using histology, automated nerve fiber counts and immunohistochemistry to demonstrate its relationship to human glaucomatous optic neuropathy. Efforts to analyze the cellular response to elevated IOP in the mammalian optic nerve and retina will begin by studying changes in protein presence and distribution using immunohistochemistry for a variety of well characterized neuronal and glial cell components, most of which have been shown to change in response to injury. Initial studies will concentrate on eyes with severe IOP elevation. Protein changes detected will then be confirmed and clarified using in situ hybridization with either synthetic oligodeoxynucleotide probes, or riboprobes, to detect evidence for cellular changes in message production. Northern blot analysis will also be used to provide semiquantitative analyses, where indicated, although their primary use will be in verifying probe specificity. Once reliable changes in specific proteins are defined in eyes with severe IOP elevation, these markers will be used to clarify the exact role of IOP in causing nerve damage by comparing eyes with mild and severe IOP elevations and comparing the effects of early versus prolonged IOP rise. These studies win serve to establish these changes as markers for future studies of the other factors that influence the glaucomatous process, how they affect optic nerve susceptibility to IOP and how they might be altered to protect the optic nerve in glaucoma.
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