We 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 initial microscopic corneal changes occurring with surfactants of slight, mild, moderate, and severe irritation. Surfactants were directly applied to the corneas of rabbits (6/group) at a dose of 10 μl. Eyes and eyelids were examined macroscopically and scored for irritation beginning 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 epithelial cell size, epithelial layer thickness, total corneal thickness, and depth of keratocyte necrosis. The average macroscopic scores at 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 vivo CM revealed corneal injury with the slight irritant limited to the epithelium, resulting in reductions in epithelial cell size and thickness 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 to 157.6% of controls (p < 0.001), and necrosis of keratocytes extended to an average depth of 4.3 μm (0.8% of the corneal thickness); these parameters were essentially normal by Day 14. For the moderate irritant, at 3 hr the epithelium was markedly attenuated, corneal thickness was increased to 155.8% of controls (p < 0.001), and keratocyte necrosis extended to an average depth of 19.0 μm (3.6% of corneal thickness; statistically greater than with the mild irritant, p < 0.001); these parameters were essentially normal by Day 14. For the severe irritant, at 3 hr the epithelium was significantly thinned, corneal thickness was increased to 165.9% of controls (p < 0.001), and keratocyte necrosis occurred to an average depth of 391.1 μm (70.1% of corneal thickness). These findings demonstrate that significant differences in area and depth of injury occur with surfactants of differing irritancy. The data suggest that differences at 3 hr can be used to distinguish different levels of ocular irritation. Data such as these will be important in the development and evaluation of future mechanistically based in vitro alternatives for ocular irritancy testing.
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