Quantitative measurement of acute corneal injury in rabbits with surfactants of different type and irritancy

We have hypothesized that differences in ocular irritancy are related to di fferences in extent of initial injury and that, regardless of the processes leading to tissue damage, extent of injury is the primary factor that dete rmines the final outcome of ocular irritation. In previous in vivo confocal microscopic (CM) studies we identified quantifiable differences in the ext ent of corneal injury occurring with four surfactants (three anionic, one c ationic) known to cause different levels of ocular irritation and demonstra ted that extent of initial corneal injury was related to the magnitude of c ell death. The purpose of this study was to assess the applicability of thi s hypothesis to a broad sampling of surfactants. Specifically, initial corn eal changes induced by seven different surfactants (one anionic, three cati onic, three nonionic) were measured by in vivo CM and cell death was measur ed by an ex vivo live/dead assay. The right eye of each rabbit was treated by placing 10 mu l of a surfactant directly on the cornea. Eyes were examin ed macroscopically and scored for irritation at 3 h and 1 day. At 3 h and 1 day, in vivo CM was used to examine the corneas and quantitate epithelial cell size, epithelial thickness, corneal thickness, and depth of stromal in jury. At 3 h and/or at 1 day, corneas were removed and excised regions were placed in culture media containing 2 mu M calcein AM and 4 mu M ethidium h omodimer. Using laser scanning CM, the number of dead epithelial and/or str omal cells in a 300 x 300 x 170-mu m(3) (xyz) volume of the cornea was dete rmined. In vivo CM and live/dead assay findings revealed three surfactants to affect only the epithelium, three surfactants to affect the epithelium a nd superficial stroma, and one surfactant to affect the epithelium and deep stroma. Extent of initial corneal injury reflected level of ocular irritat ion, and magnitude of cell death was related to the extent of initial corne al injury. These findings are consistent with those for known slight, mild, and moderate to severe irritants, respectively. They suggest that our hypo thesis is broadly applicable to surfactants. Additionally, we believe these surfactants should be included as part of a new "gold standard" for use in developing and validating in vitro tests to replace the use of animals in ocular irritancy testing. (C) 1999 Academic Press.