Application of In Vivo Confocal Microscopy to the Understanding of Surfactant-Induced Ocular Irritation*^{Oryzias latipes}

Abstract

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 &mu;l 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 &mu;m 2 to 567 & p l u s m n ; 330 &mu;m 2 in the rats and 1,523 &plusmn; 185 &mu;m 2 to 934 &plusmn; 71 &mu;m 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.

Light Microscopic Comparison of Surfactant-Induced Eye Irritation in Rabbits and Rats at Three Hours and Recovery/Day 35*1

“…Alternatively, this species difference may reflect the relatively higher dose given to the rat and the fact that the rat cornea is much thinner than the rabbit cornea, allowing for deeper penetration of the surfactants. In vivo confocal microscopic measurements have revealed the cornea to be approximately 131 and 377 /-Lm thick in the rat and rabbit, respectively (28). This contrasts to a greater similarity of the corneal epithelial cell layer thickness, with this layer being approximately 30 and 40 >m thick in the rat and rabbit, respectively (28).…”

“…In vivo confocal microscopic measurements have revealed the cornea to be approximately 131 and 377 /-Lm thick in the rat and rabbit, respectively (28). This contrasts to a greater similarity of the corneal epithelial cell layer thickness, with this layer being approximately 30 and 40 >m thick in the rat and rabbit, respectively (28). Furthermore, radiolabeled detergent has been found to penetrate the cornea, to accumulate in ocular tissues, and to be released slowly (9).…”

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

“…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).…”

“…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).…”

“…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).…”