Proliferation of corneal epithelium and apoptosis of keratocytes in the rabbit cornea after treatment with 20% ethanol

Abstract

To study the proliferation activity of corneal epithelial cells and the apoptosis of keratocytes in the rabbit cornea after treatment with 20% ethanol. The results were compared to the cornea treated with mechanical scraping of the epithelial cells. The experimental group consisted of 42 rabbits. One of the two corneas of each rabbit was incised by 8 mm epithelial trephine using in LASEK and was exposed to 20% ethanol (in distilled water) in the trephine for 40 seconds. In the other eye, the corneal epithelium in the central area was mechanically scraped. The rabbits in the experimental group were randomly divided into seven sub-groups. Each sub-group had six rabbits and the rabbits were killed at 0, 4 hours, 1, 3, 5, 8 and 30 days after the surgery. Three rabbits without any treatment were used as blank controls. Immunohistochemical staining (Ki-67 antigen) was performed to detect the proliferation of corneal epithelial cells. Apoptotic cells were detected by TUNEL assay. Number of keratocytes in the central anterior stroma of cornea was determined by counting the total number of cells under 400 x magnification field in hematoxylin-eosin-stained corneal sections. In the ethanol-treated eyes, the number of Ki-67 positive cells peaked 5 days after the treatment in the central corneal epithelium and 1 day after the treatment in the peripheral corneal epithelium. TUNEL-positive cells were detected in the anterior central stromal keratocytes under the epithelial incisions and the number of TUNEL-positive cells reached a peak 4 hours after the treatment. There was no statistically significant difference in the number of central anterior stromal keratocytes between the ethanol-treated group and the controls (P = 0.68). In the mechanical scraping group, the number of Ki-67 positive cells in the peripheral corneal epithelium peaked 3 days after the treatment. The number of Ki-67 positive cells in the peripheral corneal epithelium of the scraping group was greater than that of the ethanol-treated group. TUNEL-positive cells were detected in the anterior central stromal keratocytes and reached a peak 4 hours after the scraping. The number of central anterior stromal keratocytes was decreased maximally 1 day after the scraping (P < 0.01). The cornea injury caused by treatment with 20% ethanol for 40 seconds is milder than that caused by mechanical scraping. The wound-healing process in the ethanol-treated cornea is faster than that of the cornea treated with scraping. Corneal epithelium treated with 20% ethanol for 40 seconds can protect the stromal keratocytes.