Antioxidants protect against reactive astrocytosis-induced sensitization to oxidative stress

Abstract

Purpose : Clinical evidence suggests a pathological role of cellular oxidative stress in glaucomatous tissues, including the optic nerve head. The purpose of this study was to determine whether antioxidants exert glioprotective effects against reactive astrocytosis-induced sensitization to oxidative stress.

Methods : Primary adult rat optic nerve head astrocytes (ONHAs) were pre-treated for one hour with 100 mM Trolox, 20 mM resveratrol or vehicle. ONHAs were exposed to hyperbaric pressure (25 mmHg above ambient) to induce reactive astrocytosis and subsequently to exogenously-applied chemically-induced oxidative stress using tert-butyl hydroperoxide (tBHP; 0 to 1000 mM). Oxidative stress was quantified using 2',7'-dichlorodihydrofluorescein diacetate (H₂DCFDA) or CellROX^®. MTT uptake and lactate dehydrogenase (LDH) release cell proliferation and viability assays were performed to determine glioprotective potential.

Results : Reactive astrocytosis resulted in significantly elevated levels of cellular oxidative stress. Specifically, CellROX^® fluorescence increased from 10.4% to 47.9% of nuclear area; Trolox completely prevented generation of oxidative stress (2.9%). Similarly, levels of oxidative stress measured by DCF fluorescence increased two-fold after a 2 hr exposure to hyperbaric pressure; this increase was similar following exposure to 100 mM tBHP. Accordingly, reactive astrocytosis shifted both the IC₅₀ for tBHP in the MTT assay and the EC₅₀ for tBHP in the LDH assay by > 80 mM. Pre-treatment with Trolox resulted in a proportional right-shift of the IC₅₀ value for tBHP under ambient (73.0 mM to 135.2 mM) and hyperbaric pressures (50.0 mM to 99.3 mM; n=3). A similar effect on IC₅₀ values was observed following pre-treatment with resveratrol under ambient pressure (79.9 mM to 119.9 mM). However, the glioprotective effect of resveratrol on reactive astrocytes was significantly attenuated (64.5 mM to 76.3 mM). This differential effect was attributed to a different mechanism of action of Resveratrol.

Conclusions : Our data suggest that reactive astrocytosis in ONHAs is associated with significantly elevated levels of oxidative stress. Furthermore, antioxidants can exert potent glioprotective effects on ONHAs, supporting the continued preclinical development of antioxidant approaches for glaucoma.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.