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A. Pauly, F. Baudouin, H. Liang, J.-M. Warnet, C. Baudouin; Confocal Fluorescence Microscopy Allows 3D-Reconstructed Corneal Epithelium (SkinethicTM) Multi-Analyses: Application to the Toxicological Evaluation of the Eye-Drop Preservative Benzalkonium Chloride. Invest. Ophthalmol. Vis. Sci. 2008;49(13):2547. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To analyze the effects of benzalkonium chloride (BAK), a preservative involved in ocular surface apoptosis and inflammation, on cellular viability, tight-junction integrity, cellular activation, apoptosis and proliferation, in human reconstructed corneal epithelia.
PBS, 0.01% BAK and 0.1% BAK were applied on reconstructed corneal epithelia (SkinEthicTM) for 24 hours. The cytotoxicity was evaluated using the MMT test. Frozen sections were analyzed using fluorescence confocal microscopy for the presence of occludin, ZO-1, E-Cadherin, TUNEL, activated caspase-3, p53, CD95, Ki67, ICAM-1, HLA-DR.
BAK at 0.01% and BAK at 0.1% were found cytotoxic according to the MMT test. BAK induced ZO-1 and occludine disappearence, cell apoptosis with an increased number of TUNEL-, activated caspase-3- and p53-positive cells. Its effects on CD95, ICAM-1 and Ki67 expression was dose-dependent, with Ki67 expression being stimulated at 0.01% and decreased at 0.1%. E-cadherin and HLA-DR were not expressed.
Fluorescence techniques conjugated with confocal microscopy on 3D-reconstructed corneal epithelia were well suited for the investigation of toxicological markers such as cell junction alteration, apoptosis, cell activation and proliferation and gave relevant results compared to the known human data. They constitute sensitive complementary tools to the MTT test and improve the operating potential of this new unavoidable 3D model in ophtalmotoxicology.
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