PURPOSE: To examine the protective effects of antioxidants in cultured trabecular meshwork (TM) cells exposed to oxidative stress. METHODS: Primary porcine TM cells were pretreated with 50 μM resveratrol, 0.2 mM urate, 1 mM ascorbate, 1 mM reduced glutathione (rGSH), or 1 mM ρ-coumarate followed by exposure to hydrogen peroxide (0.5-4 mM). Cell metabolism was determined by mitochondrial enzyme activity and cell viability by uptake of the vital dye calcein, a fluorescent calcium binding dye. Reactive oxygen species (ROS), which may reflex oxidative damage, were determined by 2'7'-dichlorofluorescein diacetate. RESULTS: Trabecular meshwork cell metabolism was reduced to 72 ± 5% of control levels with 1 mM hydrogen peroxide (H2O2) treatment. TM cells that co-incubated with ascorbate (85% ± 5%), ρ-coumarate (98 ± 11%) or rGSH (103 ± 17%) had significantly increased metabolism compared to 1 mM H2O2 treatment. Resveratrol significantly increased TM cell metabolism at both 2 mM (102 ± 14% live) and 4 mM H2O2 (27 ± 12% live), with H2O2-treated cultures containing mostly metabolically inactive cells (3% at 2 mM; 2% at 4 mM). Similar results were obtained in cell viability assays. Ascorbate and resveratrol, but not ρ-coumarate or rGSH, decreased ROS levels in TM cells exposed to a sublethal dose of H2O2 (0.5 mM). Urate had no protective effect against H2O2 damage in any of the assays. CONCLUSIONS: Increased oxidative damage was demonstrated in the TM of patients with primary open angle glaucoma. The antioxidants (resveratrol, ascorbate, ρ-coumarate) and the antioxidant enzyme cofactor (rGSH) protected TM cells from H2O2-induced damage. TRANSLATIONAL RELEVANCE: Future experiments are needed to determine whether addition of antioxidants may maintain TM cell viability in vivo. Antioxidants could be applied either topically or coupled with extended-release vehicles for intraocular injection to reduce free radical formation leading to enhanced therapeutic outcomes. Ultimately, studies using animal models could determine whether application of antioxidants can ameliorate progression in diseases such as glaucoma and macular degeneration.
PURPOSE: To examine the protective effects of antioxidants in cultured trabecular meshwork (TM) cells exposed to oxidative stress. METHODS: Primary porcine TM cells were pretreated with 50 μM resveratrol, 0.2 mM urate, 1 mM ascorbate, 1 mM reduced glutathione (rGSH), or 1 mM ρ-coumarate followed by exposure to hydrogen peroxide (0.5-4 mM). Cell metabolism was determined by mitochondrial enzyme activity and cell viability by uptake of the vital dye calcein, a fluorescent calcium binding dye. Reactive oxygen species (ROS), which may reflex oxidative damage, were determined by 2'7'-dichlorofluorescein diacetate. RESULTS: Trabecular meshwork cell metabolism was reduced to 72 ± 5% of control levels with 1 mM hydrogen peroxide (H2O2) treatment. TM cells that co-incubated with ascorbate (85% ± 5%), ρ-coumarate (98 ± 11%) or rGSH (103 ± 17%) had significantly increased metabolism compared to 1 mM H2O2 treatment. Resveratrol significantly increased TM cell metabolism at both 2 mM (102 ± 14% live) and 4 mM H2O2 (27 ± 12% live), with H2O2-treated cultures containing mostly metabolically inactive cells (3% at 2 mM; 2% at 4 mM). Similar results were obtained in cell viability assays. Ascorbate and resveratrol, but not ρ-coumarate or rGSH, decreased ROS levels in TM cells exposed to a sublethal dose of H2O2 (0.5 mM). Urate had no protective effect against H2O2 damage in any of the assays. CONCLUSIONS: Increased oxidative damage was demonstrated in the TM of patients with primary open angle glaucoma. The antioxidants (resveratrol, ascorbate, ρ-coumarate) and the antioxidant enzyme cofactor (rGSH) protected TM cells from H2O2-induced damage. TRANSLATIONAL RELEVANCE: Future experiments are needed to determine whether addition of antioxidants may maintain TM cell viability in vivo. Antioxidants could be applied either topically or coupled with extended-release vehicles for intraocular injection to reduce free radical formation leading to enhanced therapeutic outcomes. Ultimately, studies using animal models could determine whether application of antioxidants can ameliorate progression in diseases such as glaucoma and macular degeneration.
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