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Julie M. Crewe, W. John Armitage; Integrity of Epithelium and Endothelium in Organ-Cultured Human Corneas. Invest. Ophthalmol. Vis. Sci. 2001;42(8):1757-1761.
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purpose. 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.
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 antibody. These markers were combined with
rhodamine-phalloidin staining for F-actin and DAPI labeling for DNA.
The corneas were examined by confocal microscopy.
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
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.
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
then only after extended periods in organ
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