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Michael-Adrien VITOUX, CRISTOPHE BAUDOUIN, Stephane Melik Parsadaniantz, MARTINE CLARET, guerin camille, Françoise Baudouin, Annabelle REAUX LE GOAZIGO; Proinflammatory and toxic effects in trigeminal neurons induced by a conditioned medium of corneal epithelial cells exposed to benzalkonium chloride. Invest. Ophthalmol. Vis. Sci. 2019;60(9):5102.
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Environmental stresses are known to induce ocular irritation and corneal nerve fibers, originated from trigeminal neurons, stimulation. Benzalkonium chloride (BAK) is a quaternary ammonium compound widely used as preservative in eyedrops and as detergent agent in cleaning sprays. Here, we aimed to evaluate in vitro the proinflammatory and toxic effects in trigeminal neurons induced by a conditioned medium of corneal epithelial cells exposed to benzalkonium chloride.
Human corneal epithelial (HCE) cells were exposed to 0.005% BAK for 15 min, followed by a 5h-recovery period to obtain a conditioned medium (5h-CM). Primary culture of adult mouse trigeminal neurons was then exposed to the 5h-CM for 2h, 4h and 24h. The cytotoxic effects of 0.005% BAK and of the 5h-CM were evaluated. IL-6, IL-8, CCL2 and MIF cytokine secretions were quantified using ELISA. RT-qPCR was performed to determine mRNA levels of cFos (neuronal activation), ATF3 (neuronal injury) and proinflammatory cytokines (IL-6, IL-8, CCL2). Levels of P-Erk1/2, P-p38, P-STAT3 and ATF3, cFos, GADD153 were measured by western blot.
After 5h-recovery period, a 15 min of 0.005% BAK exposure to HCE cells decreased cell viability (58±4 %; p=0.001; n=4), increased mRNA levels of IL6 (3.4±0.7; p=0.02; n=6), IL8 (6.8±1.5; p=0.01; n=6) and CCL2 (4.6±0.6; p=0.002; n=6) and triggered IL-6 (1978±135 pg/mL; p=0.001; n=3), IL-8 (1249±175 pg/mL; p=0.04; n=3), CCL2 (1604±476 pg/mL; ns; n=3) and MIF (2694±451 pg/mL; p=0.01; n=3) release.Concerning primary neuronal culture, 5h-CM decreased cell viability for 2h, 4h and 24h. In addition, a 2h-exposure of this 5h-CM increased ATF3 (2.5±0.6; p=0.05; n=5), c-fos (2.3±0.5; p=0.03; n=5), IL6 (2.2±0.4; ns; n=5) and CCL2 (1.5±0.3; ns; n=5) gene expression. Interestingly, western blot analysis revealed that the 5h-CM significantly increased P-Erk1/2, P-p38, ATF3, cFos and GADD153 levels whereas a significant decrease was observed for P-STAT3 levels.
The conditioned medium of HCE exposed to 0.005% BAK triggers proinflammatory cytokine expression, neuronal damage and neuronal activation. This in vitro culture model offers an interesting tool to better understand the inflammatory and primary sensory neuronal responses observed in some ocular surface diseases caused by environmental or toxic stresses.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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