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Morphological and Functional Changes in the Rat Cornea with an Ethanol-Mediated Epithelial Flap

From the Laboratory of Ophthalmology and Visual Science, College of Medicine, The Catholic University of Korea, Seoul, Korea.

PURPOSE. To establish morphologic and functional changes in the rat cornea after 20% ethanol-mediated epithelial flap creation.

METHODS. The epithelial flap was detached using 20% ethanol and then repositioned. On the other eye, the corneal epithelium was mechanically scraped. The morphologic changes were examined by light and transmission electron microscopy (LM and TEM). Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assays and proliferative cell nuclear antigen (PCNA) staining were performed to detect apoptosis and proliferation, respectively. The number of central stromal keratocytes was counted during wound-healing periods. A Y-chromosome-specific gene (SRY) was detected in genomic DNA obtained from female epithelial cells with male epithelial flap. Expressions of matrix metalloproteinase (MMP)-9, aquaporine5 (AQP5), and mucin1 (MUC1) mRNAs were examined by reverse transcription-polymerase chain reaction.

RESULTS. After flap repositioning, damaged basal cells were observed until day 1, and the death of epithelial cells and stromal keratocytes loss peaked at day 1. Cell proliferation and MMP-9 mRNA levels peaked from days 1 to 3. By contrast, after mechanical scraping, the denuded stromal surface was covered with multilayer epithelial cells at day 3, cell death peaked at 4 hours, cell proliferation peaked from 12 hours to day 1, stromal keratocytes loss peaked at 8 hours, and MMP-9 mRNA was widely expressed from 12 hours to day 3. In both corneas, hemidesmosomes were not observed until day 1, and they continued to be present at day 3. The SRY gene was detected in female epithelial cells at day 1 after transplantation, but not at day 3. Expressions of AQP5 and MUC1 mRNAs were unchanged.

CONCLUSIONS. Ethanol-mediated flap creation induces less keratocyte loss and a slower wound-healing process than mechanical scraping. The flap protecting the underlying stromal surface may have epithelial function. In addition, this Sprague-Dawley rat model may be useful in the study of the wound-healing response after LASEK.

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