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Anser Abbas, Jennifer J Bu, Khadija Agsalud, Dominic Fakhir Hakim, Takako Noguchi, Peter Shaw, Natalie A Afshari; Anti-inflammatory effect of prescription eye drops on porcine corneal endothelial cells with induced oxidative stress. Invest. Ophthalmol. Vis. Sci. 2022;63(7):2738 – A0227.
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© ARVO (1962-2015); The Authors (2016-present)
To assess inflammatory markers in a porcine model of smoking-induced oxidative stress in corneal endothelial cells after treatment with three commonly used eye drops: cyclosporine (0.05%), lifitegrast (5%), and tacrolimus (0.1%).
The corneoscleral rim was separated from eight harvested porcine eyes and treated with 0.5% trypsin for 15 minutes. Corneal endothelial cells were scraped from Descemet membrane, plated into one well of a 12-well plate, proliferated to confluency in a low growth factor medium, and passaged three times until reaching 12 wells of a 12-well plate for treatment. Hydroquinone (HQ) was used to induce oxidative stress. Cells were exposed to medium containing 250 µM HQ and one of the following nine eye drop treatments: 10%, 5%, or 2.5% cyclosporine (Restasis 0.05% eye drops diluted ten-, twenty-, and forty-fold respectively); 10%, 5%, or 2.5% lifitegrast (Xiidra 5% drops diluted respectively); and 5%, 2.5%, and 1.25% tacrolimus (0.1% drops diluted respectively). All treatments were two hours and tested in biological triplicate. Control groups were: each eye drop condition without the addition of HQ (N=3 each), HQ alone (N=5), and untreated (N=7). Total RNA was extracted using the RNEasy Mini Kit and converted to cDNA using the iScript Reverse Transcription Supermix. Expression of inflammatory markers IL-6, TNF-alpha, and the housekeeping gene GAPDH, were quantified using real-time quantitative PCR. The delta-delta Ct method was used to calculate relative gene expression, and statistical tests were calculated using delta Ct values.
Cells with hydroquinone-induced oxidative stress had significantly reduced IL-6 expression when treated with cyclosporine (p<.05), lifitegrast (p<.01), and tacrolimus (p<.05), compared to cells with hydroquinone alone. Single-factor analysis of variance (ANOVA) did not reveal significant differences based on each level of eye drop concentration. In the absence of hydroquinone-induced oxidative stress, eye drop treatment did not significantly impact inflammatory marker expression.
Cyclosporine, lifitegrast and tacrolimus can protect against inflammatory marker expression in corneal endothelial cells in an induced oxidative stress model. More research is needed on these effects at various eye drop concentrations.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.
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