The long-term objectives of this research are to define how the rat cornea metabolizes arachidonic acid (AA) and the pathophysiological roles of AA metabolites and protein kinase C (PKC) in corneal inflammation, neovascularization, and reepithelialization.
Specific aim 1 will determine whether flurbiprofen diminishes corneal neovascularization by mechanisms independent of inhibition of corneal cyclooxygenase activity. PGE2 levels will be measured using gas chromatography/mass spectrometry (GC/MS) as a reflection of corneal cyclooxygenase activity. Another study will determine if flurbiprofen inhibits corneal neovascularization in leukopenic rats. These studies will advance our understanding of corneal angiogenesis, a clinically important problem due to reduction in visual acuity.
Specific aim 2 will determine whether inhibition of arachidonate 8- or 9-lipoxygenase activity results in delayed reepithelialization or organ-cultured rat corneas with 3 mm diameter epithelial abrasions.
Specific aim 3 will test the hypothesis that PKC and lipoxygenase inhibitors delay reepithelialization by preventing actin polymerization which is necessary for epithelial cell migration. F-actin will be assessed by image analysis of epithelial cells stained with rhodamine phalloidin and correlated with inhibition of PKC and arachidonate lipoxygenases.
Specific aims 2 and 3 will contribute to our understanding of mechanisms regulating epithelial wound healing.
Specific aim 4 will determine whether calcium induces membrane translocation of rat corneal 12-lipoxygenase. This may ultimately suggest new pharmacological approaches to reducing corneal inflammation since 12(S)-HETE, the major metabolite of AA in the rat cornea, is a potent chemotactic factor for neutrophils in this tissue.
Specific aim 5 will test the hypothesis that normal, cauterized, and hypoxic rat corneas synthesize only 12(S)-HETE. The stereoisomer of 12-HETE will be determined using radiolabeled AA and by GC/MS. Determining which stereoisomer of 12-HETE is produced under different circumstances is critical since 12(R)-HETE may influence corneal turgidity, while 12(S)- HETE is a leukocyte chemoattractant.
Specific aim 6 will determine whether PKC differentially regulates cholinergic activation of phospholipases C and D in the epithelium. these studies may help clarify how acetylcholine exerts a beneficial effect on corneal epithelial wound healing.
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