Extent of initial corneal injury as a basis for alternative eye irritationtests

Based on studies that have characterized the extent of injury occurring wit h irritants of differing type and severity, we have proposed that extent of initial injury is the principal mechanism underlying ocular irritation. We report here our efforts to apply this hypothesis, as a mechanistic basis, to the development of an alternative eye irritation assay using an ex vivo rabbit corneal model. Rabbit eyes were obtained immediately after sacrifice or from an abattoir and 8.5-mm diameter corneal buttons were removed and c ultured overnight at an air-liquid interface under serum-free conditions. B uttons were exposed to materials of differing type (surfactant, acid, base, alcohol and aldehyde) and irritancy (slight to severe) that had been previ ously characterized microscopically in the rabbit low-volume eye test. Expo sure was accomplished by applying 1.5 mul of an irritant to a sterile, 3 mm diameter, filter paper disk and then placing the disk on the center of the corneal button for 10 s. After removal of the disk, buttons were washed an d cultured for 3, 24 or 48 h. Buttons were then evaluated for extent of inj ury using 3 Live/Dead staining kit and fluorescent microscopy to measure ce ll size of live surface epithelial cells, area of epithelial denudation and depth of stromal injury. Ex vivo exposure to slight irritants generally re duced surface epithelial cell size (i.e, erosion) while exposure to mild ir ritants produced epithelial denudation with variable injury to the corneal stroma. Severe irritants generally produced extensive epithelial denudation and damaged the corneal stroma and endothelium. Overall, ex vivo extent of injury significantly correlated with in vivo extent of injury as measured in previous animal tests (r = 0.81, P < 0.001). These findings indicate tha t extent of corneal injury, as shown to be associated with ocular irritatio n occurring in vivo. can be applied to the development of a mechanistically -based alternative eye irritation model. We believe that this approach may ultimately lead to an alternative assay to replace the use of animals in oc ular irritation testing. (C) 2001 Elsevier Science Ltd. All rights reserved .