Apoptosis in the Rabbit Cornea After Photorefractive Keratectomy

PURPOSE: It is well-known that after trauma to the ocular surface such as that seen in refractive surgeries like photorefractive keratectomy (PRK; laser corneal surface ablation), there is a "dropout" of underlying stromal keratocytes. Recently it was proposed by Wilson et al. that this loss of keratocytes may be the result of apoptosis. This process can be initiated by several cellular factors that result in release of endonucleases, DNA segmentation, and eventual cellular destruction. One protein, bcl-2, has been shown to have a profound role in its inhibition. The goal of this study was to evaluate the mechanisms and level of apoptosis in the cornea after PRK. METHODS: Rabbits were anesthetized systemically with ketamine/xylazine. The corneal epithelium was removed by using phototherapeutic keratectomy (PTK, 100 pulses). PRK was then performed (-9.0 D, 5.0 mm optical zone). Rabbits were killed at 4 days and 4 weeks, and the corneas were prepared for light and electron microscopy, as well as for apoptosis evaluation. RESULTS: The apoptosis assay demonstrated that (a) the normal cornea exhibited a limited level of apoptosis, primarily in the superficial epithelium, very little in the basal epithelium, with none in the keratocytes and endothelium; (b) the entire epithelial layer was found to be apoptotic 4 days and 4 weeks after PRK; (c) an elevated level of apoptosis was detected in both keratocytes and endothelial cells after PRK at the same time points. The immunohistochemical staining showed that (a) the normal cornea had a low level of bcl-2 protein, exclusively in the superficial epithelium; (b) bcl-2 was induced in the basal epithelial cells and anterior keratocytes under the wound bed 4 days after PRK and increased 4 weeks after PRK. CONCLUSIONS: These findings suggest that apoptosis plays an important role in the corneal wound-healing process, which appears to be mediated in part by bcl-2. Bcl-2 induction may protect corneal epithelial cells from apoptosis after trauma such as PRK.