Evaluation of a Novel Fluorescent Probe for the Assessment of Retinal Oxidative Status

Purpose: To evaluate the utility of a novel profluorescent nitroxide (PFN) probe that selectively detects superoxide radicals in live cells, as a reporter of retinal oxidative status in models of retinal metabolic challenge.Method: (i) Cultured RGC-5 cells were transformed with trichostatin A to stimulate a “neuronal-like” morphology. Following treatment with 0.01 μM methylester rhodamine nitroxide probe, cells were exposed to the mitochondrial complex III inhibitor, antimycin (0.01−100 μM) to stimulate superoxide production. Fluorescence (556 nm/590 nm) was quantified via flow cytometry with statistical analysis based on 10,000 cells. (ii) Following intraocular injection of the PFN (2 μM), unilateral acute retinal ischaemia was induced in rats by elevation of intraocular pressure (IOP, 120 mmHg 60 min). After restoration of normal IOP, retinal fluorescence was assessed during reperfusion with a Micron III rodent fundus camera.Results: Antimycin caused a dose-dependent decrease in fluorescence in non-transformed and TSA-treated RGC-5 cells. The PFN probe detected a significant (p < 0.05) change at the lowest concentration of antimycin tested. (ii) Restoration of blood flow after retinal ischaemia, which stimulates free radical production, induced at marked decrease in fluorescence. Fluorescence intensity declined during the first 60 minutes of reperfusion. Fluorescence intensity in non-ischaemia/reperfusion eyes did not change.Conclusion: PFN probes can detect changes in retinal oxidative status in both cell culture and in vivo models, under pro-oxidant conditions. Because the probes are reversible and react to both reducing and oxidizing conditions, we can look for the first time at anti-oxidant treatment effects in real-time for retinal diseases like glaucoma that involve oxidative stress.