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The purpose of this study was to assess the ability of in vivo confocal microscopy (CM) to provide noninvasively derived histopathologic correlates of surfactant-induced eye irritation from which specific pathologic mechanisms can be identified and later evaluated in alternative in vitro models. Rats and rabbits, divided into groups of 5, received 10 μl of an anionic or cationic surfactant in one eye with the other eye used as a control. At specified times, eyes were examined and scored for ocular irritancy using a penlight and slit-lamp. Subsequently, corneas were evaluated by in vivo CM to evaluate epithelial layer thickness and surface epithelial cell area, corneal thickness, depth of necrosis, inflammation, fibrosis, and endothelial injury. At 3 hr, the anionic surfactant produced slight irritation with peak scores of 12.4 and 8.0 out of a possible 110 in the rats and rabbits, respectively. In vivo CM revealed changes limited to the corneal epithelium that decreased in thickness to 78% in rats and 81% in rabbits at 3 hr. This decrease in the thickness correlated with a significant decrease in surface epithelial cell area from 2,061 & p l u s m n ; 395 μm 2 to 567 & p l u s m n ; 330 μm 2 in the rats and 1,523 ± 185 μm 2 to 934 ± 71 μm 2 in the rabbits (p < 0.005 and 0.005, respectively). The cationic surfactant produced severe irritation in both the rats and rabbits with peak scores of 85.4 and 80.2 occurring at day 2, respectively. In vivo CM in the rats showed complete loss of corneal epithelium, lysis of keratocytes, and loss of corneal endothelium. In the rabbits, injury appeared limited to the anterior cornea with complete loss of epithelium and loss of keratocytes extending to 52% of the corneal thickness. These findings establish the application of noninvasive, in vivo CM to qualitatively and quantitatively characterize the pathobiology of ocular irritation in situ. This information will be important in the development and evaluation of mechanistically based in vitro alternatives for ocular irritancy testing.

Limited information exists on the pathologic changes occurring with surfactant-induced ocular irritation in the context of accidental human exposures and animal tests used to assess for such irritation. The purpose of this study was to begin to characterize the pathologic changes that occur with surfactants in the context of standard animal tests and compare the response in rats to that in rabbits. Representative anionic, cationic, and nonionic surfactants causing slight to severe ocular irritation were directly applied to the corneas of rabbits and rats at a dose of 10 μl. Eyes and eyelids of each animal were macroscopically examined for signs of irritation beginning 3 hr after dosing and periodically until recovery or day 35. Eyes and eyelids from animals in each group were collected for microscopic examination after 3 hr and at recovery or day 35. Microscopically, all of the surfactants caused erosion, denudation, and/or necrosis of the conjunctival and corneal epithelium in rabbits and rats. Necrosis of keratocytes was observed in rabbits and rats treated with the severely irritating cationic surfactant and in rats treated with anionic surfactants that were mildly irritating and moderately irritating. Corneal endothelial changes were observed in rabbits and rats with only the cationic surfactant. Changes in eyes of rabbits and rats that had not recovered by day 35 included decreased prominence of goblet cells, conjunctivalization of the corneal epithelium, neovascularization and fibrosis of the cornea, and presence of devitalized stroma. Overall, the changes in rabbits and rats were similar and suggest that the rat may be used as a surrogate for the rabbit in studies to understand better mechanisms of surfactantinduced eye irritation.

The pathology of surfactant-induced ocular irritation, especially in the context of accidental human exposures and animal tests used to assess a surfactant's potential ocular irritation, is not well understood. The purpose of this study was to characterize the microscopic changes in rats at 3 hr and on days 1. 2, 3. 4. 7. 14. and 35 following treatment with anionic. cationic, and nonionic surfactants of differing irritancy. The right cyc of each rat was-treated by placing 10 pI of a surfactant directly on the cornea. Untreated left eyes served as the controls. At each time point, eyes and eyelids were macroscopically examined and collected for microscopic examination. Macroscopically. the differing levels of irritation were characterized by differences in incidence and magnitude of scores, reflecting involvement of the cornea, conjunctiva. and iris, as well as by the incidence of neovascularization and time to recovery. Microscopically. differences in the area and depth of injury, paralleled the differences seen grossly and the relative irritancies of the various surfactants. All surfactants affected the corneal and conjunctival epithelium. All surfactants, except the slightly irritating anionic surfactant, caused corneal stromal changes, with this involvement being proportional to their overall level of irritation. Corneal endothelial cell effects principally occurred with only the severely irritating cationic surfactant. Over time, responses to surfactants of differing irritancy were qualitatively and quantitatively different. and these differences correlated with the extent of initial injury. Qualitative differences in response included presence of keratocyte regeneration, corneal neovascularization, and conjunctivalization of the corneal epithelium with all of the surfactants except the slight irritant. Quantitative differences in response occurred in the extent of epithelial regeneration, edema. and inflammation f6r surfactants of slight to severe irritancy, and with neovascularization, keratocyte regeneration, and conjunctivalization for surfactants of mild to severe irritancy. These results suggest that by defining initial area and depth of injury associated with an ocular irritant, it may be possible to predict the subsequent rcsponsc and final outcome. Such an approach would be applicable to the development of mechanistically based in virro assays.