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R. Joseph, R. R. Pfister MD., C. I. Sommers; Keratocyte Apoptosis and Persistent Epithelial Defect in Alkali Injured Cornea. Invest. Ophthalmol. Vis. Sci. 2007;48(13):1489.
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To investigate the role of keratocyte/myofibroblast apoptosis in persistent epithelial defects of alkali injured cornea. Stromal - epithelial interactions are key determinants of corneal functions. The bi-directional signaling between the two plays a significant role during the normal development, homeostasis, and wound healing. However after alkali injury this interaction between the two is severely altered due to the loss of basement membrane, denatured collagen and stroma deficient in GAGS.
Rabbits were divided into 2 groups of 30 each. In group 1, the cornea was exposed to 1N NaOH in an 11.0 mm well for 35 seconds. In group 2, the central cornea was marked with a 11.0 mm trephine, then abraded. Rabbits were then randomly assigned to 5 time periods to be sacrificed at 48 hours, 72 hours, 84 hours, 96 hours and 114 hours post injury. At the designated time, 6 rabbits from each group were killed and the globe removed. The terminal deoxnucleotidyl transferase-mediated dUTP-nick end labeling (TUNEL) assay, which measures the fragmented DNA of apoptotic cells by catalytically incorporating fluorescein-12-dUTP at 3'-OH DNA ends as an early event in the apoptotic cascade, was performed with a DeadEndTM Fluorometric TUNEL kit. The fluorescein-12-dUTP-labeled DNA was visualized directly by fluorescence microscopy. Fluorescent stain, 4',6-diamidino-2-phenylindole (DAPI), visualizes nuclear DNA.
Keratocyte apoptosis was significantly greater in alkali injured corneas when compared to abrasion injury as seen with the TUNEL assay. The apoptosis in the alkali injured cornea was present until 114 hours (last time period tested) when compared to the same time point post abrasion injury. The nuclear DNA was counter stained with DAPI and it showed that the TUNEL positive region corresponded to the nuclear region of the cell. All DAPI positive regions were TUNEL positive in alkali injured corneas when compared to abrasion and control. During alkali injuries only the anterior region showed presence of nuclear DNA when compared to abrasion injuries where nuclear staining was present both anteriorly and posteriorly. There was a significant decrease in nuclear staining of alkali injury when compared to abrasion injuries at all time points.
In contrast to the beneficial effect of apoptosis, the inappropriate activation of apoptosis alone, or common denominants may contribute to the persistent epithelial defect as seen in alkali injured corneas.
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