Nigel L Barnett, Glen A Gole, Cassie L Rayner,Kok Leong Chong, Steven E Bottle

Purpose:
To evaluate the utility of a novelprofluorescent nitroxide (PFN) probe that selectivelydetects superoxide radicals in live cells, as a reporter ofretinal oxidative status in models of retinal metabolic challenge.

Method:
(i) Cultured RGC-5 cells were transformedwith trichostatin A to stimulate a “neuronal-like”morphology. Following treatment with 0.01 ?Mmethylester rhodamine nitroxide probe, cells wereexposed to the mitochondrial complex III inhibitor,antimycin (0.01?100 ?M) to stimulate superoxideproduction. Fluorescence (556 nm/590 nm) wasquantified via flow cytometry with statistical analysisbased on 10,000 cells. (ii) Following intraocular injec-tion of the PFN (2 ?M), unilateral acute retinal ischae-mia was induced in rats by elevation of intraocularpressure (IOP, 120 mmHg 60 min). After restoration ofnormal IOP, retinal fluorescence was assessed duringreperfusion with a Micron III rodent fundus camera.

Results:
Antimycin caused a dose-dependentdecrease in fluorescence in non-transformed and TSA-treated RGC-5 cells. The PFN probe detected a signifi-cant (p < 0.05) change at the lowest concentration ofantimycin tested. (ii) Restoration of blood flow afterretinal ischaemia, which stimulates free radical pro-duction, induced at marked decrease in fluorescence.Fluorescence intensity declined during the first 60minutes of reperfusion. Fluorescence intensity in non-ischaemia/reperfusion eyes did not change.

Conclusion:
PFN probes can detect changes in retinaloxidative status in both cell culture and in vivo models,under pro-oxidant conditions. Because the probes arereversible and react to both reducing and oxidizingconditions, we can look for the first time at anti-oxidant treatment effects in real-time for retinal dis-eases like glaucoma that involve oxidative stress.