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

Nigel L. Barnett, Cassie Rayner, Glen A. Gole, Steven E. Bottle

Research output: Contribution to journalMeeting AbstractResearchpeer-review


Purpose: To evaluate the utility of a novel, reversible, profluorescent nitroxide (PFN) probe that selectively detects superoxide radicals in live cells, as a reporter of in vivo retinal oxidative status in a model of retinal metabolic challenge.

Methods: Following an intravitreal injection of PFN (2 µM), unilateral acute retinal ischaemia was induced in anaesthetized Sprague Dawley rats by elevation of intraocular pressure (IOP) to 120 mmHg for 60 mins. After restoration of normal IOP, retinal fluorescence (556 nm / 590 nm) was assessed at various time-points (5, 10, 15, 30, 45, 60 mins) during reperfusion using a Micron III rodent fundus camera. Changes in fluorescence were quantified with Image J and compared with the fluorescence intensity measured before the ischaemic insult. Control fluorescence time-course data were obtained from the non-ischaemic contralateral eyes. The effects of known antioxidants, lutein (0.2 mg/kg i.p.) and edaravone (3 mg/kg i.p.), either alone or upon the ischaemia/reperfusion-induced fluorescence response, were quantified. The effect of intraocular PFN on retinal function was assessed by electroretinography (ERG).

Results: Restoration of blood flow after retinal ischaemia, which stimulates free radical production, induced a marked decrease in retinal PFN probe fluorescence intensity (59.8 ± 4.3 SEM % of the pre-ischaemic value, n=5, at 15 mins reperfusion). Administration of lutein or edaravone ameliorated the ischaemia/reperfusion-induced decrease in retinal PFN fluorescence: lutein (increased to 85.5 ± 6 %pre-ischaemic, n=7), edaravone (increased to 88.5 ± 3.1 %pre-ischaemic, n=4). The antioxidants did not significantly alter the fluorescence intensity in non-ischaemia/reperfusion retinas. The intraocular injection of the PFN probe did not adversely affect the ERG a- or b-waves.

Conclusions: PFN probes can detect changes in retinal oxidative status in real-time in vivo, under pro-oxidant and anti-oxidant conditions. Because the probes are reversible and react to both reducing and oxidizing conditions, we can look for the first time at novel anti-oxidant treatment effects in real-time for the myriad retinal diseases that involve oxidative stress.
Original languageEnglish
Article number1916
JournalInvestigative Ophthalmology and Visual Science
Issue number13
Publication statusPublished - Apr 2014
Externally publishedYes


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