Real-time in vivo visualization of induced retinal oxidative stress and the effect of antioxidants

Purpose

To quantify in real-time the antioxidant properties of novel nitroxide compounds administered after an in vivo pro-oxidant stress.


Methods

We employ a novel mitochondrially-targeted profluorescent nitroxide (PFN) probe that is reversibly responsive to general cellular redox status. Following intraocular injection of the PFN probe (2 µM in DMSO), unilateral acute retinal ischaemia was induced in anaesthetized Sprague-Dawley rats by elevation of intraocular pressure (IOP, 120mmHg 60min). After restoration of normal IOP, retinal fluorescence (556nm/590nm) was assessed at various time-points (up to 60 mins) during reperfusion with a Micron rodent fundus camera. Nitroxide antioxidant compounds (5-carboxy-1,1,3,3-tetramethylisoindolin-2-yloxyl, CTMIO or 5,6-dicarboxy-1,1,3,3-tetraethylisoindolin-2-yloxyl, DCTEIO, 100 µM in 2 µl, or vehicle DMSO) were injected intravitreally after 30 mins of reperfusion and retinal fluorescence monitored for a further 30 mins. Changes in fluorescence, which reflect retinal oxidative status, were quantified and compared with those measured before antioxidant administration. Control fluorescence time-course data were obtained from the non-ischaemic contralateral eyes. The effects of the novel nitroxide antioxidants (n=6) were compared with the known antioxidant compound, resveratrol (100 µM, 2 µl, n=6).


Results

We previously showed that post-ischaemic reperfusion (I/R), which stimulates free radical production, induces a marked decrease in retinal PFN fluorescence (ARVO 2014). Here, we show that intraocular administration of CTMIO or DCTEIO reverses the I/R-induced decrease in retinal fluorescence, as reported by the PFN probe: CTMIO induced a 66±14% reversal, DCTEIO 90±18% reversal. In comparison, the recognised antioxidant resveratrol reversed the I/R-induced decrease in retinal fluorescence by 50±12%. Neither DMSO nor the nitroxide antioxidants significantly altered the fluorescence intensity in non-I/R retinas.


Conclusions

We have demonstrated in vivo, that two potent nitroxide antioxidants effect a real-time reversal of oxidative stress in the retina. Through the use of our novel, reversibly-responsive probe for oxidative stress, we have provided the first evidence that antioxidants such as these hold potential to protect the retina from the deleterious consequences associated with diseases that involve oxidative stress.