Ocular Irritation: Microscopic Changes Occurring Over Time in the Rat with Surfactants of Known Irritancy

Abstract

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.

Use of In Vivo Confocal Microscopy to Understand the Pathology of Accidental Ocular Irritation

“…The correlation of dead keratocytes to depth of keratocyte damage was 0.79 (12). The mild and moderate irritants did not affect the endothelium (12, 17). With the severely irritating surfactant, we demonstrated there was complete loss of the corneal epithelium, with damage of keratocytes extending deep into the stroma and marked corneal thickening (12,13,17 (19).…”

“…To date, attempts to validate in vitro alternatives as replacements for the use of animals in assessing ocular irritation have been generally unsuccessful (12,17,19). The general approach taken has been to correlate in vitro test results to scores derived from macroscopic examination of rabbit eyes in either the Draize test or LVET (19). This lack of success can be attributed to the fact that the current in vivo scoring system provides very descriptive and subjective information (13,17,19 (12,13,(17)(18)(19).…”

“…The general approach taken has been to correlate in vitro test results to scores derived from macroscopic examination of rabbit eyes in either the Draize test or LVET (19). This lack of success can be attributed to the fact that the current in vivo scoring system provides very descriptive and subjective information (13,17,19 (12,13,(17)(18)(19). And second, changes in types and/or levels of biomarkers, cytokines or extracellular factors, will reflect differences in the extent of initial damage (i.e., area and depth of injury) ; the initial responses (e.g., edema, neutrophilic infiltrate) ; and the eventual repair processes (e.g., neovascularization, epithelial regeneration, collagen deposition) (17)(18)(19).…”

“…This lack of success can be attributed to the fact that the current in vivo scoring system provides very descriptive and subjective information (13,17,19 (12,13,(17)(18)(19). And second, changes in types and/or levels of biomarkers, cytokines or extracellular factors, will reflect differences in the extent of initial damage (i.e., area and depth of injury) ; the initial responses (e.g., edema, neutrophilic infiltrate) ; and the eventual repair processes (e.g., neovascularization, epithelial regeneration, collagen deposition) (17)(18)(19). Further, these biomarkers can be applied to in vitro systems as predictors of eye irritation (17)(18)(19) (12,13,17).…”

“…And second, changes in types and/or levels of biomarkers, cytokines or extracellular factors, will reflect differences in the extent of initial damage (i.e., area and depth of injury) ; the initial responses (e.g., edema, neutrophilic infiltrate) ; and the eventual repair processes (e.g., neovascularization, epithelial regeneration, collagen deposition) (17)(18)(19). Further, these biomarkers can be applied to in vitro systems as predictors of eye irritation (17)(18)(19) (12,13,17). Additionally, this approach allows us to conserve animal use (13).…”