In Vivo Detection and Modulation of Reactive Oxygen Species in a Mouse Model of Retinal Ischemia/Reperfusion

Purpose: Ischemia results in deprival of oxygen and metabolic substrates, energy depletion, and ultimately cell death. As a result, there is a significant and detrimental increase in free radical formation, mediators of oxidative stress. Our study aims to establish a novel method for noninvasive in vivo detection of reactive oxygen species (ROS). Methods: Retinal ischemia/reperfusion (I/R) was induced in left eyes of C57BL/6J mice. They were cannulated in their anterior chamber, and intraocular pressure (IOP) was raised to 120 mmHg for 60 minutes. Right eyes served as internal controls. Detection of ROS was conducted by a chemiluminescent compound L-012. At indicated days after I/R, L-012 (75mg/kg) was injected intraperitoneally. Pupils were dilated using phenylephrine HCl 2.5% and mice were placed in the small animal In Vivo Imaging System Lumina XR 15 minutes after L-012 administration. In some studies, ROS scavenger TEMPOL (100mg/kg) or NADPH oxidase inhibitor apocynin (50mg/kg) was injected 30 minutes prior to L-012 treatment. At day 14, eyes were harvested and paraffin embedded for H&E staining. Retinal morphological changes were evaluated. All measurements were conducted in Living Image software (Caliper Life Sciences) and statistical analysis was performed using SigmaPlot (Systat). Results: L-012 chemilluminescent signals were successfully detected in the I/R-injured eyes following systemic L-012 administration. Over a 14-day time course, only 24 and 48 hours post I/R were statistically significant (p<0.05) signals detected for greater than 1 hour after L-012 injection. No toxicity or gross inflammation was observed throughout the eye. Treatment with both TEMPOL and apocynin caused a statistically significant (p<0.01) reduction of L-012 radiance at both 24 and 48-hour time points. Conclusions: Our studies indicate retinal I/R causes a transient and significant induction in ROS production. L-012 appears to be a reliable and nontoxic tool for noninvasive detection of ROS in mice. Furthermore, we showed TEMPOL and apocynin successfully reduce chemilluminescent signal through the removal of excess ROS. Previous detection of ROS was possible only in post mortem samples; however, our method does not require euthanasia of animals fulfilling a largely unmet need in the study of oxidative stress.