The Existence of Dead Cells in Corneal Endothelium Preserved With Storage Media

Abstract

Purpose: : In the United States and Japan, donor corneas are currently preserved in Optisol GS storage medium cooled to below 4°C, yet few reports exist regarding the viability of corneal endothelial cells preserved in storage media. Here we present a report on the viability of storage-media-preserved endothelium by histological examination.

Methods: : Twenty-eight donor corneas were obtained from an eye bank (SightLife, Seattle, WA), and redundant peripheral portions of those corneas were used for histological examination after removal of the center corneal graft for transplantation. To assess corneal-endothelial cell viability, biostaining experiments were performed using propidium iodide, calcein-AM, and Hoechst 33342. Endothelial cell density was determined by counting the number of blue nuclei cells, and the dead-cell rate (DCR) was determined by dividing the red-nuclei cell-count by the blue-nuclei cell-count, with the resulting number multiplied by 100. These endothelia were examined histologically with vimentin V, anti-vimentin antibody, and toluidine blue. The tissue fragments that were cut from the center to peripheral area from 4 whole donor corneas for research (SightLife) were then used for the endothelial-cell viability test.

Results: : Histological analysis of the endothelium showed that the cytoplasm of the dead cells had low-intensity fluorescence and that their nuclei stained red, while almost all living cells had green cytoplasm and blue-stained nuclei. The average DCR in the 28 donor peripheral corneas was found to be 0.6%-10.5%, 4.9+/-3.3%, and in the 4 whole corneas was found to be 1.3%-2.4%, 1.9+/-0.39% at the center and 1.3%-3.0%, 1.9+/-0.7% at the periphery. These PI-positive cells stained positive for annexin V, negative for vimentin, and pale for toluidine blue.

Conclusions: : Our findings showed the existence of dead cells in storage-media-preserved corneal endothelium, suggesting that these cells fall off soon or have a shortened survival time due to a decrease of corneal-endothelial cell-rate after keratoplasty.

Keywords: cornea: endothelium • cornea: storage • microscopy: light/fluorescence/immunohistochemistry