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Megan Prunty, Moe Hein Aung, Jeffrey H Boatright, Niren Murthy, Peter Thule, Machelle T Pardue; Fluorescent probes for in vivo detection of oxidative stress within the eye. Invest. Ophthalmol. Vis. Sci. 2014;55(13):235.
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© ARVO (1962-2015); The Authors (2016-present)
Oxidative stress plays a major pathogenic role in many ophthalmic diseases but is largely assessed using only ex vivo techniques. In vivo detection of reactive oxygen species (ROS) could enable early diagnosis and tracking of retinal diseases. In this study, we tested the efficacy of using hydrocyanine, a highly sensitive fluorescent probe that binds to hydroxyl radicals and superoxide, to quantify ROS generated in vivo within the retina after light induced retinal damage (LIRD).
After dilating the pupils with atropine sulfate ophthalmic solution (1%), 129SV mice were placed in bright light (60,000 lux) for 6 hours to induce LIRD. Immediately after light exposure, ROS800CW hydrocyanine dye (LI-COR, Lincoln, NE) was intravenously injected at 2.5, 5, or 10 mg/kg. Mice without light damage also received a similar dosage of ROS800CW dye to serve as controls. After 48 hours, animals were examined under the indocyanine green (ICG) filter of a scanning laser ophthalmoscope (SLO). Fluorescence of the reacted ROS800CW dye was quantified by normalizing to the intensity of the optic nerve head in each fundus image. Visual acuity measured with optokinetic tracking 6 days after light exposure was used to confirm LIRD. On the seventh day, animals were euthanized and retinal tissues were processed for immunohistochemistry. Retinal cross-sections and whole mounts were labelled with a primary antibody against Heme oxygenase-1 (HO-1), a marker of oxidative stress.
Punctate hyper-fluorescence was visible in the fundus of mice exposed to LIRD. Fluorescence increased significantly by 25% for intensity (One-way ANOVA, p<0.05) and 32% for area (One-way ANOVA, p<0.05) as dose of ROS800CW dye increased from 2.5 to 10 mg/kg. Fluorescent intensity inversely correlated with visual acuity (R2=0.611). Immunohistochemistry revealed that ROS800CW fluorescence was localized to the outer nuclear layer and colocalized with HO-1 staining.
Retinal ROS can be visualized in vivo using ROS800CW dye to meaningfully detect different levels of oxidative stress in the retina after LIRD. Forthcoming experiments will confirm hydrocyanine’s sensitivity to track the progression of specific ocular diseases, such as diabetic retinopathy and glaucoma. In the future, ROS800CW may be used clinically as a biomarker for oxidative stress in vivo because it can be visualized with an SLO.
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