Integrity of epithelium and endothelium in organ-cultured human corneas

PURPOSE. TO examine components of the junctional complex and the actin cyto skeleton and the incidence of apoptosis in epithelium and endothelium of or gan-cultured human corneas. METHODS. 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 antib ody. These markers were combined with rhodamine-phalloidin staining for F-a ctin and DAPI labeling for DNA. The corneas were examined by confocal micro scopy. RESULTS. 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 positiv e 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-acti n staining patterns in endothelium were similarly undisturbed by organ cult ure, but apoptotic endothelial cells were only rarely seen and then only af ter > 28 days in organ culture. METHODS CONCLUSIONS. 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 t hen only after ex tended periods in organ culture.