H Ren1, G Wilson. 1. Department of Opthalmology, University of Texas SW Medical Center, USA.
Abstract
PURPOSE: To determine the viability of corneal surface cells and to determine whether apoptosis is present in the corneal epithelium of the rabbit. METHODS: The viability of epithelial surface cells was examined using a calcein-ethidium assay. In this two-color fluorescence assay, viable cells fluoresce green, and the nuclei of nonviable cells fluoresce red. The number of nonviable cells on the normal corneal epithelial surface was quantified. Surface and shed cells also were examined from shear-stressed corneas. The TUNEL technique (TdT-mediated dUTP nick-end labeling) was used to detect DNA fragmentation characteristic of apoptotic cells. RESULTS: The calcein-ethidium viability assay revealed that the normal epithelial surface is composed mainly of viable cells, with nonviable cells making up a small percentage of the whole. There was an increase in the density of nonviable cells from the periphery of the epithelial surface to the center, In flatmounts of the cornea, TUNEL labeling demonstrated that a small percentage of surface cells exhibited DNA fragmentation, whereas cross-sections revealed that DNA-fragmented cells were found exclusively on the epithelial surface. Epithelial cells from shear-stressed corneas showed an increased number of apoptotic cells. CONCLUSIONS: The normal epithelial surface consists mainly of viable cells, with only a small percentage of nonviable cells and apoptotic cells. The results suggest that nonviable epithelial cells are shed after terminal differentiation, whereas viable cells can be shed by classical apoptosis with the formation of blebs. Thus, there appears to be more than one mechanism for removal of cells from the corneal surface.
PURPOSE: To determine the viability of corneal surface cells and to determine whether apoptosis is present in the corneal epithelium of the rabbit. METHODS: The viability of epithelial surface cells was examined using a calcein-ethidium assay. In this two-color fluorescence assay, viable cells fluoresce green, and the nuclei of nonviable cells fluoresce red. The number of nonviable cells on the normal corneal epithelial surface was quantified. Surface and shed cells also were examined from shear-stressed corneas. The TUNEL technique (TdT-mediated dUTP nick-end labeling) was used to detect DNA fragmentation characteristic of apoptotic cells. RESULTS: The calcein-ethidium viability assay revealed that the normal epithelial surface is composed mainly of viable cells, with nonviable cells making up a small percentage of the whole. There was an increase in the density of nonviable cells from the periphery of the epithelial surface to the center, In flatmounts of the cornea, TUNEL labeling demonstrated that a small percentage of surface cells exhibited DNA fragmentation, whereas cross-sections revealed that DNA-fragmented cells were found exclusively on the epithelial surface. Epithelial cells from shear-stressed corneas showed an increased number of apoptotic cells. CONCLUSIONS: The normal epithelial surface consists mainly of viable cells, with only a small percentage of nonviable cells and apoptotic cells. The results suggest that nonviable epithelial cells are shed after terminal differentiation, whereas viable cells can be shed by classical apoptosis with the formation of blebs. Thus, there appears to be more than one mechanism for removal of cells from the corneal surface.