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D.E. Possin, J. Huang, R. Ambrosio Jr., S.E. Wilson; Activation of Caspase-3 in the Cornea after Epithelial Scrape Injury . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3820.
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Purpose: To begin characterize activation of caspases that participate in keratocyte apoptosis following corneal epithelial scrape injury. Methods: Six-week old New Zealand black mice had corneal epithelial scraping in one eye. Mice were sacrificed and the eyes enucleated at 0, 2, 5, 10, 15, and 30 minutes, 1, 2, 4, and 24 hours after scraping. Uninjured control eyes were also evaluated. The sclera was pierced at the limbus with a hypodermic needle and the eye fixed with PIPES buffered 4% paraformaldehyde for 4 hours at room temperature. The eyes were dehydrated and embedded in paraffin for sectioning. Fourteen µm thick paraffin sections from the center of each cornea were mounted on glass slides. Antigen retrieval was performed by immersing the slides in boiling 0.1 M Sodium citrate solution for 15 minutes and allowing them to remain in the buffer as it cooled for another 15 minutes. Anti-active caspase-3 PAb, 1:1000 (R&D; Systems, Minneapolis MN) was used as a primary label, followed by an anti-rabbit IgG conjugated Alexa 488, 1:200 (Molecular Probes, Eugene OR) secondary. Propridium iodide, 1:50000, was added to provide a nuclear label. The slides were cover-slipped with DABCO-glycerol and viewed with a Zeiss 510 confocal microscope. Keratocyte apoptosis was detected with the TUNEL assay. Results: Caspase-3 activation was marked in residual epithelium, keratocytes, and endothelium within two minutes of epithelial scrape injury. Activation was noted in keratocytes throughout the full thickness of the stroma and was similar in the anterior stroma compared with the posterior stroma. Caspase-3 activation persisted until 15 minutes after epithelial scrape injury in keratocytes. Endothelial caspase-3 activation was only detectible at 2 minutes after injury. Keratocyte apoptosis was restricted to keratocytes in the anterior stroma to approximately 50% stromal depth. Conclusions: Caspase-3 is activated in cells throughout the cornea within two minutes following epithelial injury. Only the anterior keratocytes undergo apoptosis. Therefore, anti-apoptosis pathways likely inhibit apoptosis in posterior keratocytes and endothelial cells. This study also demonstrates rapid information transfer from the epithelium to the endothelium following surface injury.
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