Layer-by-layer desquamation of corneal epithelium and maturation of tear-facing membranes

A method to devitalize single layers of apically exposed rabbit corneal epithelial cells through the use of digitonin is described. Devitalized cells exfoliate spontaneously as loosely cohesive, trypan-blue-stained layers, exposing underlying viable cells. Repeated application of this devitalization-exfoliation methodology results in the gradual elimination of each of the epithelial cells. The generation of corneal surfaces composed of the tear-facing membranes of all intraepithelial cell types - subsurface, wing, and basal - is thus attainable. Exposed surfaces were studied with respect to microanatomy, the binding of lectins, and the adherence of Pseudomonas aeruginosa. Microprojections (microvilli or microplicae) were absent in the basal cells but were present in all suprabasal layers, and increased gradually in density as cells approached the surface position. Wheat germ agglutinin and concanavalin A were found to bind to the tear-facing membranes of all suprabasal cell layers. The tear-facing membrane of the basal cells, in contrast, was not labeled. Within each labeled layer, the magnitude of lectin binding differed markedly from cell to cell; lectin binding decreased as the cellular area exposed to the tear surface increased. Pseudomonas were found exclusively at microprojection-free cellular areas, suggesting that inhibition of attachment is linked to the ontogeny of these microprojections.