Abstract
Purpose::
To determine whether oxidative adduct formation or heme oxygenase-1 expression are altered in RGC cell cultures exposed to elevated hydrostatic pressure and in a mouse model for glaucoma.
Methods::
Cultured RGC-5 cells were subjected to 0, 30, 60, or 100 mmHg hydrostatic pressure above atmospheric pressure for 2 hours at 37 C in a modified pressure chamber containing 5% CO2 mixed with air. In anesthetized mice, IOP was elevated to 30, 60, or 100 mmHg for 1 hour using a microneedle attached to a height-adjustable fluid column. Recovery experiments were also performed in RGC cell cultures after 1-hour 60-mmHg-pressure treatment followed by 0, 1, 3, 6, 10 hours incubation at normal pressure. Cultures were also directly treated with 1-50 uM HNE for 16 hours. To assess the possible benefit of antioxidants, cultures were pretreated with 20-40 mM resveratrol and quercetin for 30 minutes prior to pressure elevation. Markers of oxidative stress, including lipid peroxidation product (4-Hydroxynonenal, HNE) and antioxidative protein expression (Heme oxygenase-1, HO-1), were quantified using immunohistochemistry and immunoblotting.
Results::
Our data showed that: 1) there is a dose dependent increase in level of HNE-protein adducts (up to 5 fold) and HO-1 expression (up to 2.5 fold) in pressure-treated RGC-5 cell cultures; 2) in recovery experiments, HNE-protein adducts in the RGC-5 cells continued to increase for up to 10 hours, in contrast HO-1 expression decreased immediately; 3) HNE, at a concentration as low as 5 uM, leads to neurotoxicity in RGC-5 cell cultures; 4) the level of HNE adducts and HO-1 expression increased in the mouse retina and optic nerve following acute IOP elevation up to 5.5 fold and 2 fold respectively (P<0.05); and 5) antioxidant treatments can reduce the oxidative stress level in pressure treated RGC-5 cells.
Conclusions::
Oxidative stress is an early event following acute hydrostatic pressure in vitro or IOP elevation in vivo. These findings support the view that oxidative damage contributes early to glaucomatous optic neuropathy. These models provide the opportunity for investigating the effect of antioxidants on oxidative damage and survival of retinal ganglion cells.
Keywords: oxidation/oxidative or free radical damage • neuroprotection • protective mechanisms