CONFOCAL MICROSCOPIC CHARACTERIZATION OF INITIAL CORNEAL CHANGES OF SURFACTANT-INDUCED EYE IRRITATION IN THE RABBIT

dWe have previously demonstrated with slightly and severely irritating surfactants that the new technology of noninvasive, in vivo confocal microscopy (CM) can be a useful approach to a better understanding of the pathobiology of ocular irritation in situ. In this study, in vivo CM was used to qualitatively and quantitatively characterize the initi al microscopic corneal changes occurring with surfactants of slight, m ild, moderate, and severe irritation. Surfactants were directly applie d to the corneas of rabbits (6/group) at a dose of 10 mu l. Eyes and e yelids were examined macroscopically and scored for irritation beginni ng at 3 hr after dosing and periodically through Day 35. Concurrently, the corneas were evaluated by in vivo CM; 3D data sets extending from the surface epithelium to the endothelium were assessed for surface e pithelial cell size, epithelial layer thickness, total corneal thickne ss, and depth of keratocyte necrosis. The average macroscopic scores a t 3 hr for the slight, mild, moderate, and severe irritants were 6.0, 39.3, 48.5, and 68.7, respectively, of a possible 110. At 3 hr, in viv o CM revealed corneal injury with the slight irritant limited to the e pithelium, resulting in reductions in epithelial cell size and thickne ss to 59.0 and 82.4% of controls (p < 0.001 and p < 0.01, respectively ). These parameters returned to normal by Day 3. For the mild irritant , at 3 hr the epithelium was absent, corneal thickness was increased t o 157.6% of controls (p < 0.001), and necrosis of keratocytes extended to an average depth of 4.3 mu m (0.8% of the corneal thickness); thes e parameters were essentially normal by Day 14. For the moderate irrit ant, at 3 hr the epithelium was markedly attenuated, corneal thickness was increased to 155.8% of controls (p < 0.001), and keratocyte necro sis extended to an average depth of 19.0 mu m (3.6% of corneal thickne ss; statistically greater than with the mild irritant, p < 0.001); the se parameters were essentially normal by Day 14. For the severe irrita nt, at 3 hr the epithelium was significantly thinned, corneal thicknes s was increased to 165.9% of controls (p < 0.001), and keratocyte necr osis occurred to an average depth of 391.1 mu m (70.1% of corneal thic kness). These findings demonstrate that significant differences in are a and depth of injury occur with surfactants of differing irritancy. T he data suggest that differences at 3 hr can be used to distinguish di fferent levels of ocular irritation. Data such as these will be import ant in the development and evaluation of future mechanistically based in vitro alternatives for ocular irritancy testing. (C) 1997 Academic Press.