Integrity of Epithelium and Endothelium in Organ-Cultured Human Corneas

To examine components of the junctional complex and the actin cytoskeleton and the incidence of apoptosis in epithelium and endothelium of organ-cultured human corneas. Human corneas, either organ-cultured for 1 to >28 days or excised directly from eyes stored in moist chambers, were stained with antibodies to ZO-1, vinculin, and caspase 3 coupled to FITC-conjugated secondary antibody. These markers were combined with rhodamine-phalloidin staining for F-actin and DAPI labeling for DNA. The corneas were examined by confocal microscopy. The depth of the epithelium was reduced during organ culture, but no changes were observed in the distribution of ZO-1 or vinculin, or in the F-actin cytoskeleton. The appearance of apoptotic epithelial cells positive for caspase 3 or with condensed DNA increased with time after 14 days in organ culture, but there was no correlation with donor age. ZO-1 and F-actin staining patterns in endothelium were similarly undisturbed by organ culture, but apoptotic endothelial cells were only rarely seen and then only after >28 days in organ culture. Organ culture maintained the integrity of tight junctions and the actin cytoskeleton in epithelial and endothelial cell layers. Apoptosis was evident in epithelium but was observed rarely in the endothelium and then only after extended periods in organ culture.