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E. Warcoin, Jr., D. Godefroy, C. Clouzeau, A. Grise, C. Baudouin, F. Brignole-Baudouin; Trabecular Cells Apoptosis in a Toxic Induced Model. Invest. Ophthalmol. Vis. Sci. 2010;51(13):3232.
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Glaucoma is one of the most common cause of blindness caused by an alteration of the trabeculum. This disease is actually treated by several topical treatments that often contain benzalkonium chloride (BAK) as preservative. the toxicity of BAK has already been demonstrated on conjunctiva, showing apoptosis, inflammation and fibrosis. However, the toxicity on trabeculum has not been well studied yet. The objective of the study was to investigate further the toxicity of BAK on trabecular cells by exploring the cell death mechanisms of trabecular cells in vitro using a BAK toxicity model.
Human trabecular meshwork cells (HTM3) were stimulated with two dilutions of BAK for 15min followed by a 24h recovery period. AnnexinV-7AAD assay, sub-G1 peak and Bcl2 were assessed by flowcytometry to evaluate apoptosis. Microtitration fluorescence assays on cells were used to evaluate viability (Neutral red, Alamar Blue) and apoptosis (YO-PRO-1, Hoechst 33342). Immunofluorescence staining was performed in order to analyze the expression of active caspase-3.
HTM3 cell viability decreased in a BAK-concentration dependant manner. We reported a lethal concentration 50 around 0,005% and less than 10% cell viability at 0,01% while, in the same time, all the apoptotic markers increased. BAK 0,005% preferentially induced early apoptosis as confirmed by AnnexinV-7AAD and YO-PRO-1/Hoechst33342 assays while BAK 0,01% induced late apoptosis and necrosis.
This study confirmed that the trabecular cell apoptosis could be induced by low concentrations of preservative. This cell death could play an active role in the course of the glaucomatous disease because of the key role of the trabeculum in this pathology. Therefore, its consequences in terms of inflammatory cell attraction and tissue remodeling deserve to be investigated.
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