Abstract
Abstract: :
Purpose: Previous studies have demonstrated that epithelial injury triggers keratocyte apoptosis in mouse, rabbit, monkey, and human corneas. Recently, however, it was reported that tear exposure to bare stroma after removal of epithelium caused keratocyte death in the mouse cornea. The purpose of this study was to probe whether tears are necessary to trigger keratocyte apoptosis in the mouse or rabbit cornea. Methods: The eyes of four rabbits and four mice were enucleated and rinsed several times in beakers of phosphate buffered saline solution (PBS) to remove all traces of tears. The corneal epithelium of two rabbit and two mouse eyes was scraped with a #64 beaver blade. The remaining rabbit and mouse control eyes were only washed with PBS. Both control (unwounded) and experimental (wounded) eyes were placed in a humidified chamber at 37 oC for an hour. After one-hour, mouse eyes and excised rabbit corneas were cryopreserved in optimal cutting temperature (OCT) compound. Keratocyte apoptosis in the rabbit and mouse corneas was evaluated by TUNEL (terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick and labeling) assay and transmission electron microscopy. Results: No apoptotic keratocytes were detected by either TUNEL assay or transmission electron microscopy in the rabbit and mouse corneas where there was no epithelial scrape. Conversely, both rabbit and mouse corneas with epithelial injury after removal of all the traces of tears showed numerous TUNEL-positive keratocytes in anterior stroma. In electron microscopy, several keratocytes showed characteristic features of apoptosis such as chromatin condensation, DNA fragmentation, cellular blebbing, apoptotic bodies in all corneas of either animal that had epithelial scrape, but not in the controls. Conclusion: These results confirm that injury to corneal epithelium primarily activates keratocyte apoptosis. Tears are not necessary, but likely contain epithelium-derived modulators released from epithelial cells. EY10056 and Research to Prevent Blindness, New York, NY.
Keywords: 323 apoptosis/cell death • 341 cell death/apoptosis • 372 cornea: epithelium