Apoptosis expression in preserved human corneal endothelial cells

Abstract

Abstract: : Purpose:To evaluate the apoptosis pattern of human corneal endothelial cells in vivo and in vitro preserved at 4 C at different time intervals. Methods: Corneas affected by keratoconus, which distorts the architecture of corneal stroma leaving a normal endothelium, removed at the time of penetrating keratoplasty were harvested. Each cornea was immediately cut in two halves. One was fixated in O.C.T. with liquid nitrogen vapour; the other half was placed in corneal storage medium and refrigerated at 4 C. The preserved corneal halves were subsequently fixated in OCT under liquid nitrogen vapour at 1, 2, 3 and 7 days intervals. All tissues were stained using the in situ cell death detection TUNEL kit. Positive apoptotic cells were counted as cells per field. Comparisons between fresh and preserved tissues at varying intervals were performed. A total of 178 preparations were processed. To minimize mechanically induced artefacts, all fields within 1 mm from the cut edges were excluded from analysis. Results:Some degree of apoptosis was already present in fresh tissues. After two days of preservation mean values of positive cells were 10 % and increased to 25% at 7 days. Conclusions:Our data show an increase in the number of apoptotic cells with time. This is in agreement with the decrease of endothelial cells observed with vital staining during refrigerated preservation. This work supports the following hypotheses: that human corneal endothelial cells are regulated with the apoptotic mechanisms and that with commonly used in vitro preservation techniques the time–dependent decay in cell density is due to apoptosis rather than necrosis.

Keywords: cell death/apoptosis • clinical laboratory testing • cornea: endothelium