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A. Pauly, F. Brignole-Baudouin, A. Labbé, H. Liang, J.-M. Warnet, C. Baudouin; New in vivo Confocal Microscopy Scoring of Toxic-Induced Corneal Patterns. Invest. Ophthalmol. Vis. Sci. 2007;48(13):3867.
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To propose and assess a new in vivo confocal microscopic grading scale of irritant-induced corneal changes.
Benzalkonium chloride (BAC) at 0.01%, 0.1%, 0.25%, and 0.5% was applied to rat corneas for 11 days. From D0 to D52, the eyes were examined under slit-lamp and with the use of the HRT-II/RCM confocal microscope in vivo to calculate corneal thickness and compare a large amount of high-resolution corneal images. We precisely characterized the patterns of corneal damage and defined four main evaluation items for the superficial epithelial, basal epithelial, stromal and endothelial layers. Four criteria were considered for the cellular layers (presence of the layer, shape/size of the cells, reflectivity patterns, and inflammatory cells) and for the stroma (disorganization, inflammatory cells, intrastromal neovascularization and preendothelial neovascularization). Depending on the severity observed, a given score was assigned for each item. All item scores were added to obtain a total score that could reach a maximum of 64.
Mean HRT scores showed significant differences between high dose- and low-dose treatments from D3 to D11 with a dose-dependent effect of BAC. The low 0.01% and 0.1% BAC concentrations induced damage mainly restricted to the epithelium extending from cell border loss (0.01%) to epithelial erosion (0.1%). They were thus characterized by a low but significant total HRT score, except on D3 and D8 for 0.01% BAC. The highest 0.25% and 0.5% concentrations caused epithelial denudation, stromal inflammation and neovascularization, loss of endothelial visibility and fibrosis. The HRT-derived scores were thus high. The incomplete corneal recovery after 0.25% and 0.5% BAC treatment strop was associated with a decreased but still high HRT score.
HRT is a new tool for ocular surface toxicity evaluation in animal models avoiding killing large number of animals and so being in growing use. Therefore, it has to be standardized for improving reproducibility and reliability between examinations. The scoring proposed in this study allows standardization of damage at the cellular level even when assessing irritating compounds at low concentrations, where the Draize-derived test fails to be discriminating.
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