Apoptosis of Murine Corneal and Limbal Epithelial cells and Keratocytes Is Induced by Dispase Digestion of the Epithelial Basement Membrane

Abstract

Purpose: : To determine whether the enzymatic digestion of the basement membrane (BM) by Dispase without debriding the corneal epithelium may trigger apoptosis of the basal epithelial cells and the underlying stromal keratocytes.

Methods: : Sixteen mouse eyeballs were enucleated and divided into two groups. Control Group (n=6) that were studied either immediately (n=2) or stored in a serum–free KSFM medium at 4 ^oC/18h (n=2) or at 37 ^oC/1h (n=2). Dispase Digestion Group (n=10) that were incubated in 10 mg/ml Dispase in KSFM at 4 ^oC for 2h, 8h, and 18h (n=2 each) or at 37 ^oC for 1h and 2h (n=2 each). Following incubation, eyes were cryosectioned for HE staining and TUNEL staining.

Results: : All eyes in Control Group showed intact morphology, and no TUNEL–positive cells in the epithelium and infrequent TUNEL–positive cells in the stroma when studied immediately or following 37^oC/1h storage, but occasional TUNEL–positive superficial epithelial cells following 4^oC/18h storage. Following 1h and 2h Dispase digestion at 37 ^oC, there was no TUNEL–positive cells in the corneal and limbal epithelia nor in the stroma although there was peripheral corneal/limbal epithelial detachment. In contrast, following Dispase digestion at 4 ^oC, a marked increase of TUNEL–positive basal epithelial cells and stromal keratocytes was noted at 8h and 18h treatments while partial and total epithelial detachment was observed respectively.

Conclusions: : Simple digestion of epithelial BM by prolonged incubation of dispase is sufficient to induce murine corneal and limbal basal epithelial apoptosis, and accompanied by corneal stromal keratocyte apoptosis. This observation indicates that cautions should be exercised in designing protocols for isolating limbal epithelial progenitor cells and stromal keratocytes. Further studies are underway to investigate whether anoikis is the mechanism responsible for such apoptosis and whether epithelial apoptosis triggers keratocyte apoptosis.

Keywords: apoptosis/cell death • cornea: epithelium • cornea: stroma and keratocytes