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Experimental Oxidative Stress Breaks Down the Barrier Function of the Corneal Endothelium

C Anupama, Abhijith Shettar, ,

Abstract:Purpose: The fluid pump and barrier functions of the corneal endothelium maintain stromal deturgescence required for corneal transparency. The effect of oxidative stress, a hallmark of Fuchs endothelial corneal dystrophy (FECD), on the endothelial barrier function has been investigated. Methods: The endothelium of porcine corneas ex vivo was exposed to (1) membrane permeable oxidants (H2O2, 100 μM, 1 h; tert-butyl-hydroperoxide, 100 μM, 1 h), or (2) ultraviolet A (UVA) with photosensitizers for 15 min, riboflavin (50 μM) or tryptophan (Trp) (100 μM). The effects on the apical junction complex were analyzed by (1) immunostaining the perijunctional actomyosin ring (PAMR) and ZO-1 and (2) assessment of paracellular flux of fluorescein isothiocyanate (FITC)–avidin across cultured endothelial cells grown on biotinylated-gelatin film. The extent of oxidative stress was quantified by changes in intracellular reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) in addition to lipid peroxidation and release of lactate dehydrogenase (LDH). Results: Both methods of oxidative stress led to the disruption of PAMR and ZO-1 concurrent with changes in ROS levels, depolarization of MMP, increased lipid peroxidation, elevated LDH release, and increased permeability of FITC-avidin. The effects of direct oxidants were opposed by SB-203580 [p38 mitogen-activating protein (MAP) kinase inhibitor; 10 μM]. The damage by UVA+photosensitizers was blocked by extracellular catalase (10,000 U/mL). Conclusions: (1) Acute oxidative stress breaks down the barrier function through destruction of PAMR in a p38 MAP kinase-dependent manner. (2) UVA+photosensitizers elicit the breakdown of PAMR via type I reactions, involving H2O2 release. (3) Blocking the oxidative stress prevents loss of barrier function, which could be helpful in the therapeutics of FECD.
Keywords: corneal endothelium / oxidative stress / barrier function / actin cytoskeleton / p38 MAP kinase

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