A rapid technique to quantify retinal oxidative stress and the protection provided by novel nitroxide-based antioxidant / anti-inflammatory compounds

Purpose : To develop a rapid screening technique to evaluate the capacity of novel nitroxide-based antioxidant compounds to reduce oxidative stress in cultured retinal cells, and to analyse the neuroprotection offered by a candidate compound in an in vivo model of retinal injury.

Methods : Cultured 661W mouse photoreceptor cells were incubated (45 min) with a novel mitochondrially-targeted fluorescent probe (methyl ester tetraethylrhodamine nitroxide, ME-TRN) that is reversibly responsive to cellular redox status. Oxidative stress was induced with antimycin (AMC, 1 µM) following incubation (30 min) with a nitroxide (5,6-dicarboxy-1,1,3,3-tetraethylisoindolin-2-yloxyl, DCTEIO; 2KT109A or 1KT141D) or lutein antioxidant. ME-TRN fluorescence was quantified by flow cytometry. The effects of DCTEIO were then assessed in vivo. Unilateral retinal ischemia-reperfusion (I/R) was induced in anaesthetized rats by increasing intraocular pressure (110mmHg for 1hr). DCTEIO (20 mg/kg i.p.) was administered 1 hour prior and 1 hour after I/R. An additional intravitreal injection (2µl, final conc. 100 µM) was given 30 mins into the reperfusion phase. Animals recovered for 8 days prior to assessment of retinal function by scotopic flash electroretinography (ERG).

Results : Untreated 661W cells converted the probe from a non-fluorescent (oxidized) state to a fluorescent (reduced) state. Oxidative stress decreased mean fluorescence intensity by 50%. Antioxidant data were normalized to the maximal effect of AMC and expressed as the % amelioration of the AMC-induced change in mean fluorescence. Lutein (10 µM) significantly blunted the effects of AMC by 82±23%, p=0.0002, n=8, ANOVA; DCTEIO (nitroxide antioxidant, 500 µM) by 62±13%, p=0.0002, n=5; 2KT109A (indomethacin-nitroxide hybrid, 10 µM) by 97±12%, p=0.0003, n=5, and 1KT141D (aspirin-nitroxide hybrid, 100 µM) by 92±10%, p=0.0004, n=5. A significant reduction in ERG a-wave amplitude was induced by ischemia-reperfusion, which was ameliorated by DCTEIO (I/R 265±48 µV, I/R+DCTEIO 451±43 µV, p=0.0011, n=6).

Conclusions : Flow cytometry with a reversible fluorescent probe for oxidative stress is an effective tool to assess antioxidant compounds in retinal cells. Novel nitroxide antioxidant compounds significantly ameliorated the effects of oxidative stress on 661W cells and may provide an effective neuroprotective strategy in vivo.